Confirmation Number:334284
Event Started: 3/15/2005
To extend a welcome to all of you, our guest speakers, RAC members and the public participants to be -- the safety symposium sponsored by the NIH RAC. I wanted to start the meeting off, not only with the welcome to you but to give you a thumbnail sketch of today's agenda and what we hope to accomplish during the wrap-up session and provide you with a little bit of context. The goal for today's meeting arely to promote not only scientific understanding but public awareness of the latest research findings regarding treatments for the combined immunodeficiency disorders and findings of genesis and its potential ideology. When you look at the agenda, the meeting is divided into three sessions. The first is aimed at providing a comprehensive overview of current U.S. and international trials. Looking at Gene transfer size a possible treatment for SCID. The second is focusing and drilling down on the latest research into retrovirus integration and mechanisms of insertional mutogenesis and the third and very important session explores the use of bone marrow and some cell transplantation as an alternative treatment for SCID. Each of these you will find in your packets a set of handouts. They're on lavendar-colored, purple-colored paper. There are a list of questions that the Ajjs -- agency and consultation with the moderators for today's meet having put together. Not only to facilitate the progress of today's discussion, but also to lead us to the wrap-up session where we'll be revisiting the recommendations that the committee made in February of 2003 on this very same topic. Certainly today's meeting is intended for a form of public discussions of more recent findings regarding the emergence of the case of the T-cell pro-liferation in the French X-SCID trial and I wanted as a backdrop for today's discussions to provide you with an overview of the recommending as, the findings and recommendations the RAC reached in 2003, and I think our keynote speaker will talk in some are specifics on this. The RAC reyou're -- reviewed the clinical and molecular data were at the time were two series of adverse events of studies conducted in France on SCID. We met in December of 2002 and February of 2003, and the February 2003 meeting was done in conjunction with the FDA biologic advisory committee. Several members of that committee, and the group conducted an analysis of the then-available data from the French studies, as well as all the other Gene transfer studies for the combined immunodid fishiencey disorders, and the RAC recommended on the basis of this analysis that pending further data or extent -- ex10iating data, studies should be reviewed on a case-by-case basis and that SCID should be limited to patients who have failed, identical or halfway identical cell transplant identification or for whom no suitable transplant -- stem cell donor can be identified. This case-by-case review would include an analysis, accompanied by implementation of appropriate informed consent and monitoring plaps. They also concluded that at the time there were not sufficient data or reports of adverse events directly attributable to the use of retroviral victors to warrant su -- other retroviral human jeep transfer studies, including studies for non-X-link sced. Such studies might be justified coptimplent upon the analysis of implementation of appropriate conformd -- informed concept and monitoring plans. So this is a backdrop of today's discussion. At the end of it, we'll have a wrap-up discussion, which is not only a summation of today's findings, but a very concrete revisiting of the 2003 recommendations. They need to be treated, to dothey need to be revamped or totally redone. So that's the specific task of before on the meeting participants today. I also wanted to take this opportunity because today's discussion is very imageatic of long-standing tradition of the committee to bring forward new scientific findings that fixes the problems for very open scientific deliberation and public discussion. I'm very happy to report and Dr. ZOHONI will come toward to -- tomorrow to present the Award on. March 10th, the NIH RAC was Awarded the scientific freedom and responsibility Award from the American Association for the Advancement of Science. We're thrilled the committee has been recognize inside this way. It was Awarded to the NIH RAC and here I'll read the citation. In recognition of the principles of transparency, public and ethical discourse that underpend the activities of this committee for the last 30 years and for its role in the advancement of specific understanding and progress. And the NIH firmly believes that this type of activity is exactly what scientific progress and public trust are predicated upon, and today's meeting is very exemplary of this Ideal open public process. With, that I will end and turn the proceedings over to the RAC chair, Diane Wara .
thank you, Dr. Patterson. Good morning, everybody. On behalf of the RAC membership, I want to welcome everyone who has come today to participate in and to listen to the planned symposium on Gene therapy in X-link combined immuned deficiency. A Pecal thank you to those of White House have -- a special thank you to those of White House agreed to come and speak today. It always takes time to prepare a thoughtful presentation, and because we're looking forward to hearing the scientific backdrop, the background for the field of jeep transfer in X-SCID. It's only in the context of that background that we as a group, meaning those of us who participate in Gene transfer and in the care of children with X-link severe combined immuned deficiency can make thought plans and proposals in terms of how we should move forward. A special thank you to those of White House have agreed to come to speak to us today. Our goal is, as Dr. Patterson mentioned is at the end of the day to revisit, carefully revisit, the statement that was put forward by the RAC in February 2003 on our perspective of how to proceed with Gene transfer in X-SCID. It's possible that that proposal will need no revision whatsoever after careful thought. It's a contained proposal and it may need no revision. On the other hand, it may and it's our job today, especially the members of the RAC, to make that decision and to another endorse or revise our statement. So that's our job and I am looking forward to the day in a great way. I should say on a very personal note, that I'm a pediatric immuneologist. I have spent the last 35 years as many others in this rule -- room have as Ltaking care of children, and which were not only with this inheritable nun -- immunodefish ynsy but others. It's a complicated issue that presents itself to me as a care provider and the issue in a straight-forward way is, these are children, babies with X-SCID who will die if they don't receive receive some form of immune reconstitution. And the end point in this disease is death, and it's death very early in life. Traditionally, we have used as those experts in the audience will tell us, we have used forms of bone marrow transplant and more recently stem cell transplant in efforts to reconstitute these babies. When the first experience was reported out of France of immuned reconstitution with jeep transfer, we were all thrilld -- Gene transfer, were all thrilled. We will learn all about today, the early message was it was a home run. And as we waited and watched, the news that Dr. Patterson has summarized for us a moment ago came forth and then the issue was what should we do now. Should we proceed with Gene transfer sending our patients to sites where Gene transfer occurred or should we retreat a bit and continue with our standard of care, which was and remains bone marrow transplant and/or stem cell transplant of various types what, we will hear about this morning. We still don't know the answer, and that's the delamy we're faced today. We're face -- dilemma we're face ised with today. We're faced with it as a community but as care providers with these children with a devastating and fatal disease W.that introduction, I'm going to turn things over to Dr. Ted Friedmann from the university of California, San Diego, a colleague of all of ours and a crept past chair of the RAC. Dr. Freedman. -- Dr. Friedmann .
Well, first of all, let me thank Dr. Patterson and Dr. Wara for having me back and I guess we're going to continue to do this until I get it right. So nice to be back. Thank you. So this symposium, safety symposium is really designed to, for members of the RAC to examine the issues surrounding the problems in the French X-SCID study and to help the RAC arrive at recommendations for investigators in the field and for the general public and for the other interested parties. I think Amy patterson has already told you of the presentation by the triple AS by the freedom and scientific freedom and responsibility Award, and I think that that indicates the 30-year history of the RAC in providing this kind of expert and much-needed advice to the scientific community in terms of, in the areas of recombinant DNA, work, and certainly over the last decade or two in gene therapy, and the RAC always seems to have the opportunity to rise in its responsibility to get over some of these problems that Arise inevitably in new and difficult science. So the development of the problems in the French X-SCID study is obviously a very challenging and very difficult period in the development of this field and it's the responsibility and the opportunity now of RAC to help sort out the difficulties. It's a critical time for the field because it comes, I think, at a time when we've had our first, or the field has had its first really robust clinical therapeutic effect and at the same time, it's most impressive and treatment-related difficulties and seems to me now this is very critical, pivotal time for the field and for the RAC now to give its best advice to investigators on how to proceed with this problem Okay. Okay. Well, I would like to spend these few minutes giving you sort of a personal view of the issue at hand and I am going to try to aim at some conclusions. I'm certainly going to run out of time, so I'll give you my conclusions at the beginning and the conclusions are these, and probably others. The emphasis on the first, that is gene therapy research is experimental and still is, and will be for some time and the technology is at a very immature and early stage. The technology is at a very nrption ACENT period of its development and the -- NACENT period of its development and the techniques are still refined and we can agree on all those points. On the other hand, we have to agree, I think, that the X-SCID Gene transfer study has really provided the first strong proof of principle for the therapeutic efficacy of Gene transferring and to some of us, at least, the phrase that we emphasize over and over again that what we're concerned with is Gene transfer research, rather than Gene therapy research. That's, that phrase and that waffling is a little bit harder to defend now, and really, it's clear that these children consider have had a treatment offered to them. They've been treated and they certainly have had a very powerful clinical response. It's certainly no question that the criteria of work in this area should, in fact, be related, should be examined with the same risk benefit analysis as other experimental procedures. Certainly no less and the same calcueulous of the risk to benefit ratio should be applied. Now, the point is, however, that with this study, I think the field has had the opportunity and the luxury, finally, to have a risk-benefit ratio calculable. There is a robust and effective therapy and benefit. For the first time, I think we have aical cowable risk benefit Asia -- calculable risk benefit ratio that is available for this field. The conclusion from that is that the field needs not less emphasis on problem-solving but more. More research and more applied research to overcome the scientific and medical obstacles. Obviously, a great emphasis on the development of solutions to these difficulties and rather than, rather than acquiesce in a way to what is very often kind of a strong negative response to many of these reversals in the field and setbacks, I think it's very clear that there has been a tendency to not minimize the problems, but to certainly emphasize the trees rather than the forrest to see the dif cu89 -- forest to see the difficulties and at the same time, not see what has happened in the field. That there is a forest here and that that forest is, I think, for the first time, a robust, therapeutic effect in the gene transfer experiment. So, we're here because of this study. And the whole discussion revolves, really, around the events related to this work by the French group Fisher and his colleagues and with similar work being carried out by Adrian Issue thatter and his group in -- Adrian Thrasher and his group in London and this was 2002 that Fisher reported a sustained correction of an X-link scud with Gene transfer approach -- SCID with Gene transfer approach . That result came as a wonderful blessing and a surprise to the field. Finally, we had something to grasp on to, and was consistent with the biacies that, and knowledge that existed in the field related to retroviral Gene transfer. As stated by really one of the fathers, maybe the original father of retroviral victorology, howard -- vectorology, the late howard TendOM, that stated this. Can you read it as well as I do, that retroviruses have an enormously valuable place of the development of Gene transfer studies and with the assumption that once the safety issues and thentgration issues were solved retroviruses would play a role in these kinds of studies. Nope understood integration at the time as well as howard 10 a.m., so this was a powerful impetus to those interested in retroviruses. The ORA, in fact, was the retroviruses have problems that relate to an integration, but those problems were sol uble. This is from something that I followed up with and, in '89. Again, reiterates the recognition that the retroviruses have baggage. That there are going to be problems related to integration. Again, concluded on an optimistic note that these are solble problems and -- soluble problems and retroviruses continue to be one of the major candidates for clinical application. Well, then, of course, came the, vents in 2002 with the two treatment-related leukemias and the knowledge that the integration events were probably responsible for the disease. Possibly because of the presence of the viral enhancer in the provirus, and potentially some have begun to see change itself in the development of the leukemia that was not known, still isn't very clear, but with the, also the knowledge that these two children responded well to the cancer chemotherapy and went into, rapidly went into clinical remission. And Dr. Patterson described to you to the NIH recommendation in February 2003. I won't reiterate those, but just to highlight the recommendations that the X-link SCID be a clinical targ carget -- target be for the retrovirus media Gene transfer, for those patients who failed or have failed identical stem cell transplantation or for whom no suitable donor is available, and with the caveat, too, that the date available for other retroviral models, including non-X-link SCID did not justify a restriction on those studies. And that's the, those are the criteria that one worked under until this winter, until three months ago when one child of the two original with leukemia died. A second is in remission and the third case of leukemia rose with somewhat different molecular characteristics. We'll learn more about these issues later on in the morning, but with a leukemia cell that has three integration sites in it. None apparently in Alamo II, and with no overexpression of Alamo II as far as one knows in the third case. Again with the complete and rapid response to chemotherapy and with the report that the remaining children in the study, in the French studdet and study remain well. Immunologically reconstituted and some going into their sixth year post-treatment, and so the summary, overall summary is that there is something like 18, the number's a little bit difficult for me to pin down, but something like 18 patients altogether and with one original non-response, 17 immune reconstitutions, three life-threatening SAEs and one debt. The regulatory response to the third leukemia by the French is a clinical hold at the request of Dr. Fisher and his colleagues with the intention of developing safer vectors to reduce this integration of the insertional mutogenesis effect and better time to understand the vectorology at the U.S. FDA as you all know, several weeks ago put a clinical hold on X-SCID application protocol, but also recommended that ADA SCID and other retrovirus space protocols perceived under the conditions that they had been evaluated until now. I -- I don't know, perhaps someone here knows better than I, what the events have been in the Italian and Japanese studies. Both countries have studies both with ADA SCID and with X-SCID, I think. That we would like to know about. Their regulatory response and in England, the English regulatory agency has responded as it did originally with the first two leukemias, that is to say that in their experience and in their view, there is far more robust clinical benefits than there is risks in this study, and so they're proceeding to Wtheir study into accruing new patients into their X-study, X-SCID study . So here's the dilemma in front of the RAC and in front of us all. Obviously, there is very severe risks in the contexts, now different from other gene transfer and gene therapy protocols and that these risks have Arise -- Arisen in the context of a robust clinical success. At least a robust clinical phenomenon of therapeutic effect. -- affect. There is little doubt that there has been a treatment for these which were, the treatment, they're living normalized lives, not normal but normalized lives with the possibility even in some patients of what might be called, eventually a cure of this disorder by this procedure and some of this makes it a different kind of phenomenon from other SAE's and other Gene transfer protocol. Gee recognize there is a high risk of SAE, leukemia but the current technology through the mechanism of insertional mutogenesis probably, but this risk is inherent in the biology of the vector system chosen. That is that the integration of cells is responsible for the event and then until the integration mechanisms are maniplable, that's unlikely to change drastically. We'll hear later from Rick Bushman that there is evidence, at least some ideas of how to do that, but we're certainly not there yet. And so that raises the question, of course, of the, the generic question of what is the study? Is it, in fact, a study in human experimentation, is it a human experimentation issue or is it therapy, and when one looks at the alternatives much this protocol, when does this high-risk experimentation become justifiable therapy? I think that will be an issue that the RAC should, should consider in this deliberations today and then make recommending as for the future. While, of course, acknowledging that it's important to develop now and better technology, the question, the dilemma, really, for the clinical application is what one does between now and then what. Does one do until this now technology appears what. Does one do with patients who are rearing treatment? Well, so that's all the obvious issues that you all see as well as I do. I wanted to put all of this discussion in the context of some history and remind us that this is not a new issue. This dilemma is not new in clinical medicine and that there are a number of examples of, in history of the development of new and very difficult treatments which have re, which have gone through this thing, or something like this thinking process, and it's something to remember what we might gleam what we can from the development of these treatments and there are a number of examples that are particularly telling, the treatments of childhood lymphocytic leukemia, Hodgkin's disease, transplantation and endocroneal antibody development. The development of chemotherapy for childhood leukemia in some people's eyes had -- with this work by Sidney Fasher and his colleagues in 1948. The development of chemical methods for manipulating the growth of cells in T-cell leukemia, development of EMINOPTRIN as an antagnist for this disorder and in 1948, between 1948 and 1962 to '66, treatment of which were with lymphocytic leukemia took on this sort of development. There was a very, very poor long-term survival. A very high level of remission, but not very much in the way of long-term survival. And then came the development of chemotherapeutic agents, PNS agents and can you see between 84, between 62 and 66 with the development of CNS to radiation and new drugs, the survival rate increased from minimal amounts to very high levels. 40 to 60% to the point today, I think, that we'll hear better from Dr. Buckly, but certainly expectation of cure, long-term survival and cures up into the 80, 90% range and then some possibly. So let's, let's try to imagine what the discussion might have looked like in no one 62. While we're going through this kind of, this kind of analysis with respect to childhood limbfocytic leukemia. If we imagine the field was here at that time, that expectation, that kind of improvement over the next couple of decades. One imagines the -- might be an equivalent point of this, we might look forward to the history of increasing technology and efficacy if we're look -- if we're lucky. Similarly about with Hodgkin's disease, this is the treatment of Hodgkin's disease, what it looked like in the early days before 1963 or around 1963 with the development of the first ALCOLLATING agents with some improvement in initial with a clinical response, but not until the development by DEVITTA and his colleagues here at the NIH -- with the clinical efficacy increased to this sort of level resulted in something like 60, 70% or better, long-term survival. That survival came, that clinical efficacy came at a cost in that cost, the treatment-related leukemia in many patients and many patients meaning something like this incidents up to 5% or more depending on, on the efficacy of the therapy and, in fact, this is an interesting point here that might be relevant to the discussion of X-SCID and that's the more effective the therapy became in this disorder, the more common was the long-term SEQUELIE of the treatment, the secondary-treatment -- secondary treatment-related leukemias. Similarly for tissue transplantation, for liver transplantation, this is from an article from Tom StarZell. In 1963, the treatment of liver transplantation looks something like this with the early supression provided by spreadno zone and AZIOPIRAZENE, patient survival and graph survival. With the implementation of CYCLOSPORIN in 1980, the survival of graph&and patient survival improved up to 60%. In the decade, the experience of the immunosupression approaches became far grader and drugs, of course, improved. Technology in this case improved to the point where liver transplantation was successful and provided long-term surviveal to many, many patients who otherwise would have died. And, again, that improvement in therapy came at a cost and that cost remains with the field as far as I know until today, and that is certain incidents of renal failure, secondary malignancies in renal transplantation, in liver transplantation . So, what I don't want to imply or state is that Gene therapy field is the same, that there are certainly unique problems in Gene transfer technology, but we might, in fact, learn something from the history of these other treatments, these other developments of the therapies, but the research in human gene transfer and human Gene transfer experimentation has, to some extent, been different from these other therapis. Gene therapy has Arisen, of course, in an Era of far greater visibility and overall, and oversight and scrutiny than these other areas of therapy had, even at its most NACENT stage. The field was born under the microscope and remains under the microscope far more than other areas of research. Other areas of clinical research. Secondly, the field has been burdened, I think, remains burdened a little bit by some of its history, by the history of some exaggeration and hype that we went through in the early '90s. Expectation, elevated expectation of quick clinical delivery and efficacy and, really, undeliverable promises of efficacy. There's been an additional burden provided by the very -- and additional help, of course, provided by the powerful influence of the commercial interest in the field of Gene transfer, gene therapy research to deliver the technology sooner rather than later to clinical, to critical clinical issues, and that really didn't complicate the life of these other therapies nearly as much as it does for Gene therapy . So the issues, I think, facing the RAC and the discussion today, overall general issues are something like this: First and foremost, of course Swhat to do now with the X-SCID study, how to provide -- proceed with current studies and current technology. What to recommend in terms of vector modification, specifically with respect to the insertion inclusion of elements that provide insulator effect, suicide functions and ablation elements to the vector and to the transgene. The question of expectation of additional possibility of additional adverse, vents in this initial X-SCID study raises the issue, the potential for pro -- proof Lakic chemotherapy in some of the which were and at what point they should be treated in the course of the development, the early stages of the clinical leukemia. The question of what are the goals of the clinical hold until when, what is the end-point of the clinical hold and what, in fact, does one do in the meanwhile to the question of accrual and the treatment of existing patients. Admitting that the treatment is difficult and has -- hazardous. Clearly one has to define long-term research goals what, it is about vector designs that needs to be incorporated into future studies, the choice of target cells, the effects of integration sites need to be understood better. The potential role in this study, of, again, the C-chain itself of potential oncology talking with other, other cellular functions, other cellular oncogenes. The potential for targeting the integration event to a safe site or to toan inocular site. We know it's one of the holy grails of the field but is unlikely to be with us for some time. Again, we'll hear from Dr. Bushman about some of that. The general generic question of how one handles a therapeutic approach, therapeutic technique that borders, that sits somewhere between experiment ail -- experimentation and therapy, how one identifies that transition point and how one finally says on which side of the line one is sitting, when does human experimentation become a therapy, and specifically then how the RAC feels about revisiting the pend exam and looking at additional questions to be proposed to investigators. Those are the genEric issues, but I think the specific end points of today's, today's discussion, I suggest might be the following: The most parent is -- important is, of course, to look at and re-examine the February 2003 RAC position. Either affirm it or modify it, but take a strong position on that statement. And, in fact, to determine whether, whether any of the thinking of that statement ought to be extended to other stable transducing systems at the systems such as herpes and adenovirus. I think that in the discussion today when -- one has to anticipate there might be additional SAE's in the X-SCID study and how the discussion today will be shaped by the emergence of those additional SAE's, ought to be included in the considerations for today. Very specifically with respect to the pend exam and we'll see this also in other discussions dug the next couple of days. The question of whether appendix in its current form raises sufficient questions to the investigators with respect to the following issues: One, the minimizeation of the transdug -- transducing, the transduction event. The minimization of the number of integration sites, the minimization of the number of grafts itselfs -- grafted cells with the system of therapeutic effect, but using one as little as one needs to achieve the therapeutic elect -- efect. That's based on the assumption that the fewer integrations one has, the less likely it will be to induce a cellular oncojeep. With respect, also, to the inclusion in opco gene. With respect to the jeep transferring procedures to regulate gene expression, are there methods available to turn the gene up and down, on and off as need be. Other mechanisms included in the study to ablate the phenomenon. To remove the cells, to kill them or to reverse their effect if they go Awry. And also to ask investigators what their thoughts are about archiving and testing cells, either prospectively or post-grafting to ensure that any cells going Awry will be detected quickly and there is a plan in place to deal with that affect. The issues are not all technical for the RAC and RAC has an educated, very important educational role, and I think that also ought to be part of the thinking today that there is a need to discuss in public again in its educational role the question of, and the ethical and policy questions of human experimentation as they relate to this study. When does a high-risk human experimentation procedure become therapy? I think the X-SCID model provides a very good starting point for that discussion, because it's clearly been therapeutic, it's clearly experimental and when do we, when do we accept the experimental, when do we accept the therapeutic aspect of the study as being compelling? And that can be done in the context of the policy conditions, or policy conferences that the RAC might insty gate. Well, I think -- might instigate. LI think that's all I wanted to say as a historical introduction to the discussion there. Is a feeling, I certainly have a feeling that there's very good news in the field in all of this that there is a forest that we have to be able to discern why we're paying, obviously, very close attention to the trees. The trees in this case being very sec little children and, I think, we all -- sick little children and I think we all agree that we extend our best sympathyes and our best wishes to these children. The RAC's responsibility is, of course, to, as it's proven over the past 30 years by the AAAS Award to foster the field and to help investigators swim through these difficult issues to enhance the field, and I think the RAC now has an opportunity today in this discussion to do that, and I wish us all well. Thanks very much. [ Applause ]
Dr. Fried mann. I can't thank you very much -- Dr. Friedmann. Thank you very much. We're going to defer questions until the wrap-up period. I want to refer everyone to the pink packet on the left of your folder. It's called discussion questions for X-SCID. And the questions that we put together for each of the overall sessions are summarized here. I'm not going to review them with you. I would like you to review them by yourself, and then at the end of the day, in fact, the end of each session, we will come back and have a period for questions and answers. And, Ted, I want to thank you very much for putting the dilemma of the X-SCID gene transfer experience in context for those of us who are members of the RAC. Putting it in context for us in terms of our specific scientific and public responsibilities. I'm going ask Dr. Patterson next to read us our conflict of interest guidance and then we'll move forward. Dr. Patterson.
Thank you. For the record and also just as a refresher to the committee members because, again, at the closure of today we'll not only discuss the findings but we'll actually hopefully craft a recommendation and refresh a recommendation on the basis of the new data that is available and because of that, it's important for to you keep in mind whether you have any potential conflicts of interest that need to be taken into account as we come up with the new recommendations. So, for the record, and for to you keep in mind as the day moves forward, I would like to read to you the rules of conduct and conflict of interest. Being a member of the committee makes you a special government employee, and thereby, subject to the rules of conduct that apply to government's employees. The rules and regulations are explained in the report that I know you have all studied. The standards of ethical conduct for employees of the executive branch and you each received a copy of the document when you were appointed to the committee. At every meeting, in addition to reminding you of the importance of following the ethics rules, we always like to review the steps that we take and that we ask to you take to ensure that any conflicts of interest are addressed. As you know before every meeting, you provide us with information about your personal, your professional and your financial interests. We then use that information as a basis for assessing whether you have any real potential or parent conflicts of interest that could compromise your ability to be objective and getting advice during committee meetings. Now, while we wave conflicts of interest for general matters because we believe that your ability to be objective will not be affected by your interested in such which were matters, we rely to a great deal on your judgement to be attentive during the meeting to the possibility that an issue meet come up that could affect or at least appear to affect your interest in a specific way. And if this happens, we ask to you recluse -- recuse yourself from the discussion and to leave the room. As always, if any of you have any questions about the rules of conduct or conflict of interest, our committee management officers will be happy to address them and can you also speak to me during the meeting or during the break. Thank you.
Thank you. Thank you, Dr. Patterson. Our first speaker in session I, which is current SCID gene transfer experience is Dr. Jennifer Puck from the National Human Genome Research Institute at NIH, and Dr. Puck will discuss approaches to the patient with X-link SCID who has failed standard bone marrow treatment. Dr. Puck .
I'd like to thank the members of the committee and the members of the public who have come today and what I would like first to do is give a brief introduction to the disease that we're talking about. Severe combined immunodeficiency includes a spectrum of genetic disorders and the X-linked form is one of these. We don't actually know -- thank you -- the exact incidence because these conditions are rare and it's estimated to be one in 50 to one in 100,000 births. The definition of this syndrome is that patients have a very profound lack of both their T cell and B cell immunity leading them to suffer excessive recurrent and opportunistic infections very early in life when their maternal antibodies wane. They then develop failure to thrive and this condition is fatal in infancy unless an immune system can be provided for the infants . As an estimate of how common this disorder is, I can give you my personal lab's experience because I run a molecular diagnostic mutation detection lab for anyone who wants to enroll and sudden samples and, of course, this is your tax dollars at work, so we don't charge for the testing, but I'm sure I don't have every sample from every SCID patient sent to me. This is a typical year in which I received 34 samples and 19 had genetic defects in the X-linked gene. Which is about half or about what one would expect from published studies. If you look at the incidents just comparing this number to the number of beferghts in the U.S. which, is four million, then you would say that I diagnosed X-link SCID in one and 210,000 infants. If you accept that X-link SCID is half of all SCIDs, that means the incidents would be 1 in 100,000 if I were support all the X-SCIDs in the whole country, so I'm sure this is an underestimate. Certainly we also know SCID is diagnosed at autopsy and some infants who died of infections before the disease was considered and in 1997, NIH held a conference for SCID families in which they were brought to the NIH without having to pay and even so, it was noted that of the participants whose babies had been diagnosed with SCID, their median income was nearly twice the national average. This suggests to me that SCID is diagnosed much more readily in families with resources and, perhaps, missed in families without resources. As we talk about this disease X-linked SCID, should bear in mind the genetics illustrated in 24 Pedigree. Only males are affected with this condition because they have one X chromosome, and females who might be carriers are themselves unaffected. On the other hand, this is actually the most common presentation of SCID. These days, our family sizes are small in the U.S., and also, X-linked conditions have a high, spontaneous new mutation rate. This is a patient with SCID showing oral CANDIODISIS. One of the early signs but not a specific sign so the condition might be missed, and this pie diagram shows some of the genes we'll hear about ADA deficiency shortly, but this is the X-linked form of SCID caution certainly more cases than any other form. Because of the rapid advances in the field, we can diagnose molecular cause of SCID in the great majority of patients today. This was not the case a decade ago, and research has been rapid in this area. Certainly knowing the molecular diagnosis not only enables us to do carrier and prenatal diagnosis in families who have been identified. It may help us predict responses and tailor the standard bone marrow transplant treatment and it certainly is a prerequisite for gene therapy. The gene we're talking about includes this protein shown in yellow in this slide, which is the commongamma chain because it's common to -- common gamma chain because it's common to many different CYTOKYNEs. They signal cells to undergo activate -- activation. And the jeep is encoded in a -- the gene contains eight X-ONS and they span the entire range of the gene but there is little clinical diversity, most of the pacifics are very severely affected. The great majority. SCID is treatable by bone marrow transplant and the best results are obtained as will hear this afternoon, if the transplant is done early in life before serious infections set in. By far, the best results are obtained if a patient has an HLA-matched sibling to be the donor. Even without a matched sibling, some patients might be completely or substantially cured by bone marrow transplant. However, many patients require long-term Emoono glob ulip replacement after a transplant, and some are only -- imuno globulin and some are partially reconstituted and a few left. Gene therapy has been thought of as a good alternative for SCID because transplant patient is not perfect and the method is to take hemapoetic Tim cells out of the patient to transduce them with the retrovirus and reinfuse them and the reasons why we think that this particular gene is an excellent pilot for gene therapy is, first of all, the gene product is expressed in all blood lineages and it itself is not teetly regulated whereas the partner chains in the receptors that it's matched up with do have much more exquisite regulation. The immunes elimination of corrected cells is the BANE of many gene therapy approaches. But, of course, in this disease where the immune system is lacking, that's not a problem and very importantly, there is an in vivo selected advantage for cells expressing the common gamma chain so that they can expand. The NIH X-SCID gene therapy trial which, you will hear a little bit more about from Dr. MALECH in a minute is a salvage treatment protocol. Our idea from the start is that a risky new therapy should be considered first for patients who don't have other options so we have decided to focus on children who, despite in many cases repeated bone marrow transplant attempts, have unsatisfactory immune immune reconstitution. This is a summary of some of our patients who have been considered at the NIH in this regard and you can see that their ages are from 6 to 19 at the time they were evaluated. They have had from one to four bone marrow transplant taes.. Everyone over age six -- attempts. Everyone over age 6 has had growth failure with very low height and weight. They have many chronic conditions including skin infections, respiratory infections and asthma. And chronic gastrointestinal disease. And I would just point out that some of the patients are on I have a nutrition because they don't -- on IVY nutrition because they gopt eat properly. Some get GASTROSTITY feedings and many get chronic conditions, sianosis and fibrosis of the lungs are seen with increasing frequency as the age of the patients increases and the oldest of the patients is a victim of cancer. He has a hepatocell color -- hepatocellular carcinoma, possibly related to a virus but one we have probably not been able to identify. So in the patient 245s we have studied, we have looked for evidence of -- we have studied, we have looked for evidence in grafted cells. This is an artificial mixture mixing maternal cells into a patient's cells to show by PCR how we detect a maternal contribution so can you see that down to 0.1%, we can see some sign of these cells and if we look at the different separated lineages from a SCID patient in this case, the patient has engrafted T cells but not at all B cells, myelloid cells, the CB 34 stem cell enriched population. Those are all hosts derived completely compared to the T cells and some of our patients have minimal or no maternal T cells. The T cells they have are not, in many cases, doing the proper job. Here is a T cell inflammatory response in the dud on numb of the patient, which is -- DUDONEUM of the patient which is causing the intestinal disease there. Is -- the limbfo cites that they do have, and when we -- lymphocytes that we did have. When we look for new production of cells, this is an analysis of T cell receptor excision circles or treks compared to adult controls and those are CD-8 and CD-4. Cytotoxic and helper phenotype cells being produced in the THYMUS. The levels in our patients are extremely low to even undetectable. This is an indication that whatever transplant they might have had has run out of steam and no new production of T cells or very low production of T cells is taking place. Nonetheless, our patients have beenably to respond to GCSF treatment to mobilize their CD-34 cells, whichy that do in a way that is compparable to healthy adults. So, our trial has beenably to use mobilization and poriferal Harvest of still cremes to -- of stem cells to treat with the gene therapy agent. Okay and I think I will stop there and turn yet over to Harry maherb to tell but the -- Harry MALECH to tell you about the actual trial. [ Applause ]
Thank you very much, Dr. Puck. Dr. MALECH, also from NIAID is going to talk with us about preliminary results of an NEH trial of retroviral receptor gene transfer treatment for older SCID patients. Thank you, Dr. MALECH .
Dr. War ADr. Patterson, members of the RAC and guests, thank you for inviting us to speak today and I am the second half of the of a tag team here. Dr. Puck and I have, together as co-investigators began a protocol at NIH which you see the title there. The protocol number is there also. Doctor Puck has provided introduction to you which provides the justification for the study. Some of the things I say will repeat a little bit of what Dr. Puck has said, but then -- Dr. Puck has said but then we'll go forward from there. Just to remind you, the patient characteristics of our trial at the NEH is that this is a protocol that is designed to be salvage therapy for patients without an HLA match sibling who continue to have:ically significant impairment of immune function despite previous treatment or treatments with a identical donor. With that, we mean a pear antiwill -- parental lymphocyte depletd -- depleted bone marrow graft. The patient characteristics as Dr. Puck alluded to is the trial was designed to treat up to six patients with X-asked. They all had to have a previous history of Hepplo identical bone marrow transplant and hey had to be two to 22 years of ageing. Hey had to have lower limb 230 -- lemfo sight and T epgrafts. They had to about IVIG depend and have evidence of recurrent bacterial and viral infections and in addition they had to have some clinical evidence of severe chronic disease and in the case of the two patients I'm going to describe today, they both had growth failure, chronic diarrhea and malabsorption, progressive lung disease, eczema and hair loss and a variety of other problems. Two patients have been enrolled in our clinical trial and their clinical status before gene therapy is noted below. As I indicated, they have growth failure of less than 3% of heightened weight, diarrhea and so forth. They have had frequent scoog absences. In fact, patient number two has been mostly home schooled and not very well at that because of his chronic condition. Patient number one is 12 years of age, patient two is two years of age. Patient one, I think is important at this juncture to say something very specific about patient one and patient two with respect to their sort of genetic background. Patient one is what I would term kind of a variant SCID that. Is to say Dr. Puck has shown this patient has a defect in the poll-8 tail region such as the RNA is not, is not processed properly and so that patient makes a very tiny amount of actual normal common gamma chain, and so that patient is well enough to have rejected every attempt at transplant. So this patient has no donor cells at all whereas patient two is what I would call a more conventional X-SCID part who has a trunkation near the the termination of the protein leading to the protein actually being present on the cell but having a trunchtated tail so there is no signaling. It's a functionalist protein but antigen positive. This patient has had two at the same times at bone marrow transplant and as I'll show further on, actually has a, a partial T cell graft from the mother but with very low numbers of total T cells and no evidence of engasting -- epgrafting either in CD-34 cells or any other of the patient's lineages. This patient has no NK cells. This patient at times is a tiny number of NK cells, patient one as expected, B cell numbers are normal but the B cells are functional of thes in that no immunoglobulin is made and the IVA entravineousglobulin depend. This patient, despite having some of his own T cells has very poor MITOGEN or antigen proliferation as does this in despite the modest graft from the mother. So I think one of the things that I actually want to, I would like to go back to this again and make one statement relating to say slide I probably should have put in and probably didn't put in. Many of you probably know that both in the study in the united kingdom and in France, there were some -- in each case, an older patient was treated and 92 that situation, the gene therapy sort of failed and they published a paper recently in the Lancet and so you meet come Away with the idea that the, that treating older patients is a waste of time. I would like to, I would like to at least throw out a possible hepossibility sis and I would like you to keep it in mind whole you see the outcome in those two patients. I believe that in general older patients probably are enriched in those who are variants. That is patients who survived anyway despite the fact that they get very poor engraftment. And then there are others who just plain have very poor epgraftment, have classic disease and get sicker and sicker and probably don't make it very old. And I would also postulate that possibly the outcome of gene therapy in those two types of patients mean wife rent -- different, and I'm anticipating the results of my, of our study. So just to move on to the protocol characterestics of our NIH trial, the X-ZEBO transduced trial was the stem progenitor cells obtained by GCF immune -- mobilization and enig matic B purification. The vector we used is the MFGS which, is derived from the malining marion leukemia virus and is very similar, perhaps, three or four base pair of changes relative to the MFG vector that was used in the study in France. It contains the interliken II receptorgam gam Aopen reading -- receptor gam Athe pseudoframe and the leukemia envelope. Culture conditions for our gene therapy were [ Indiscernible ] With stem cell factor from -- flit through LYGAN at 50 millimeters, interliken 6 at 25 per millilighter and a low dose of interlucken 3 of 10 per milliliter in the RETRINEKIN -- [ Indiscernible ] Gas peermiable plastic bags. Our transduction conditions are actually not so different from that used both in the study in France and in the united Kingdom that we had an 18-hour prestomach lesion followed by seven our transductions each day for four days and an MOI in the rainfall of probably two or three. Just -- range of probably two or three. Just to remind you, we use GSF mobilization we collected by Afore easis and for both of these patients, we did not a singleby several collections of CD-34 cells done at sevon done at several months to at one case more than a year Apart. And the cells were collected by Aforeeasis, -- APHORESIS, frozen and thawed on the day of use. We had about 500 million CD-34 cells from each of the patients paying for the X-ZEBO transduction from both part one and two. To remind you, 100% of the CD-34 cell when is done in our laboratory, when tested in our laboratory using the PCR-based assays for cimerism, we're at host origin. There were no donor CD-34 cells in there. This gives you an idea for those of White House may not be familiar with the technique, and this is a, the transduction is done in the flexible plastic bags. Which simplifies handling, allows handling of a lot of cells and increases the surface area in which the cells and the virus can enter face with the fiberNECTIN. In the case of patient one, wereably to use slow cytoRemtri on the product to demonstrate the product was 40% expressing gamma chain and PCR analysis showed that that also correlated. We also knew the average copy number of what went into the part was a little more than one, about 1.4 or '5. That is of the 40% that were transduced. This is an example what have the cells looked like after they have been grown in culture for four days. There is about a 3.5fold proliferation and if you remember in the French study, there was quite a bit of variation among the patient and in the case of the two young of the patients, there was speculation that because proliferation was almost 6fold in those patients -- sixfold in thathose patients, that should had some role to play. With the third patient and the amount of proliferation, it was sort of average to the group and in this range, that probably is not, or may not be a critical cash -- critical issue. 80% of the controls were still CD-34 positive at the end of culture, at, as I mentioned, 40% transduction efficiency and coincidentally, it was 40% for both patients in what went in to the patient so when you add all that up, each patient got in the rage of 30 to 32 million cells transducing the CD-34 cells per coolo gram body weight. A fairly robust number of cells. So this is the, this is the bottom line and in this table, we're, we're -- the vector copies and blood cell lip yams are expressed as a percentage relative to single copy control, equaling a hundred percent. When you look at the numbers, for example, down here, 137% really means a copy, more conventionally, a copy number of 1.37. Just an tooryent you. Type iss it gets confusing, whether you're talking about percentages or copy numbers. Part one at six months had, if one assumes about one copy per transduced cells B4.3% of his -- about 4.3% of his CD-3 cells were positive for vector, at 12 months it still remains at 2.6% even though this is a relatively low number, it does demonstrate a level of selective advantage because these cells here are very low and CD-15 meaning the myelloid lineages are also very low. In the case of patient two, cho is -- who isol at six months, we had a -- is only at six months, we had a copy number of 1.37 which suggests that most of his circulating T cells probably now have a, a vector insert. Interestingly, even his B cells showed a lot of marking consider whereas his CD-15 granuular sites, meaning myo -- mieloid lineage, there is detectable marking which is low showing a similar advantage, similar to what was seen in the French and in the studies in France and the united kingdom. So right Away, you can see this quite a dramatic difference between the outcome in the two patients and I need to remind you of the sort of genetic difference between the two patients. So one of the other things -- so in patient one who had a, an abnormally-low, but significant number of CD-4 cells, those cells initially rose and then sort of modestly stayed where they are and we're sort of -- it's interesting but we remain disappointed that there was not any sort of robust increase in CD 4 kills. CD-4, CD-45 RA, meaning the new cells processed by whatever sign is left, is still almost undetectable in patient one. Patient two, however, has now added six months and has been at six months a, sort of a robust increase in the number of CD-4 cells, almost a tripling. Still a very low number, abnormally low, but a significant increase. What is most important is that we have gone from no detectable CD-4, CD-45 RA cells to actual detectable, slow but detectable numbers of nowly processed cells. What is also interesting in patient two, if you remember, patient one has no donor cells at all. That patient's working on its own cells. Patient two started out with a relatively poor graft, meaning low numbers of cells but, but, but we're about 50%. We're maternal, which is this bend corresponds to the, this is shared, this one is -- donor is host-specific. This is donor-specific. So this is the important one we're looking at here, and this donor-specific band, PCR band, has gone down at six months so that 86% of recipient cells are now 86% of the CD-3 cells are recipient cells what. Is happening is that as these numbers are going up, the patient's new cells are replacing the donor's cells what. Is important, and I think it's important to emphasize, it's not a sudden disappearance as the patient's donor's cells. The patient isn't suddenly having a cell come in and they're reacting to or killing the donor cells. They're just sort of outgrowing and replacing. I think that's important when thinking about whether such patience might be appropriate for performing gene therapy and you might be worried well, if they're sort of living on their maternal or paternal HEPALO paternal limbfo cites, could you risk a situation where some cells came in, killed all the donor cells but you didn't do anything much for the patient. Admittedly, we're talking about the end of one. I think it's an important end of one. So what happened clinically? -- interestingly, part one during the first six months of gene therapy had a number of Curious clinical changes. This patient suffered from lifelong alopecia and that sort of normalized out. Remember what we believe is happening here is the patient from biopsies as Dr. Puck had shown and, remember, it's not graft versus host disease, the patients have no donor cells. Like many patients with immuned deficiencies, it's important to think of one as an immuned disregulation than a deficiency. This patient had a lot of the autoimmune-type phenomena. This is improving disboot the fact that we didn't see any robest increase in Legg Masonfo seats. Perhaps we're seeing mild correction of regulation before we see any sort of robust overall correction. Just to summarize. Patient one had improved well-bing, improved school days. This can be a placebo affect. Resolution of abdominal -- abdominal pain. One can't Dean the fact that this patient had a sudden growth and after a growth and two centimeters in six months, there was some new cervical lymph nodes and topsiles that appeared, reduced abdominal distinction and improvement in the patient's ecswrema. Part two suffered from a lot of headaches, which is, this again, plasheeb -- placebo or something real, hard to say, but had future makes and reduced frequency of his daily dearrhea. Had a significant growth in height suddenly and in weight and those -- in those six months. All right, I won't dwell on. This I want to move long, but we have know about -- have been as required by regulation, we have been doing this land PCR and we find that in both patients, the marking is definitely polycleanal in fact richly polycleanal in both of the patients with no evidence of any prominent band. Sometimes when people present the slides they forget to note and I will note it here so when others of you look at slides like this, you remember the technique is amplifying an internal fragment inside the vector. Unless you do something to make that go Away, all of these gels will result in a prominent, what we sometimes call control band, but it's an internal fragment of this method. So this is not a, not an abnormal abnormal prominent clone band. All I can say is there is nothing particularly interesting about that relative to what others have already talked about, presented or published. So we conclude there were no adverse affects to date and to preadolescents with X-SCID who received gene therapy, post infusion peripheral blood shows polycloneal etof the reduced cells and testing in the products and patient cells. In terms of efficacy, there is multilineage marketing of blood cells with evidence of selective marking of T cells, Mod nest patient one. Quite robust in patient two. There is -- modest in patient two. There is provirus marking as I indicated and in part two at six months, we observed an increase in CD-4 T cells, an increase in naive T cells and new substantial proliferation response, particularly to Canada which was absent and now is quite robust. I will acknowledge the fact that this is a collaboration between Dr. Puck's lab and my lab, and I particularly want to note -- [ Indiscernible ] Who not only provided most of the day-to-day care for the patients but prepared a lot of the slides you saw. So, thank you very much . [ Applause ]
Thank you very much, Dr. MALECH. Are there questions from the audience or members of the RAC or any clarifications that you would like to have made? From either Dr. Puck or Dr. MALECH. We'll have another opportunity when we finish this session. All right, let's move on then to Dr. Candotti, also from NIH, who will discuss retroverag -- retroviral gene transfer for treatment of X-SCID and ADA-SCID. Dr. Candotti .
good morning and thank you for inviting me here this morning. I also need to thank Dr. Thrasher, Ian Thrasher from London who agreed share his data with the committee and asked May to present them to you this morning. I will start with this data that regards to X-ling for combined immunodefish ynsy but the collaborators believed that patients who have no match sibling donor or R-related sibling donors should be eligible for treatment of the trial after their diagnose sis confirmed molecularly 1c3, this trial is similar to the French trial you that will hear about later -- used in Paris, mean a little different in the side but that should be not very relevant. One different is that this vector is packaged in the -- [ Indiscernible ] Leukemia virus envelope. Other differences in terms of protocol are the fact that the pressure protocol called -- called for -- [ Indiscernible ] Culture conditions and uses as a lower level of therapy compared to the French trial, but the rest of the procedure -- [ Indiscernible ] There is a pre-activation for almost two days and then a transduction that goes on for three consecutive days and the cells after safety that is our injected and the patient will follow . This is a -- [ Indiscernible ] Of the details of the first severed classical SCID patients. In this trial, I also treated a 20-year-old patient who faild -- [ Indiscernible ] Reported in the blood paper by the French and the investigators. As can you see the first seven patients are with different age but most are at a young age of four months up to three years of age. There are differences in terms of perhapses or absence of the engraft -- engraphment. The changes of the gamma chain and those were compatible more or less with residual expression of the gamma chain. Using the number of the infused C-34 cells, the total number. I don't have the data for that tells me what a percentage of T cells where transduced and expressive gamma in the -- [ Indiscernible ] That was presented and published by the group. There is a, between 78 and 200 million cells per kilo were injected here. And this is the reconstitution in the first six patients, seven patients and on the next slide as can you see, after gene therapy, 20 to 40 weeks after gene therapy, there is a robust increase of C-3 numbers, the solid black line and that is composed by increase of both C-4 and CD-ity republican republican -- CD-8 numbers so the dash line is CD-8. The dash line is CD-4. For the first six parts that have a degree of follow-up and this was patient number seven recently treated also. Shows a similar part from early increased of the cell numbers. And this is the followup summary slide. Can you see there are at least three patients that are above the 30-month threshhold that seem to trouble zone period in the French trial, there are three patients below the threshhole, they're all Alive and well, and four of the seven are off chemoprophylaxisics and AG prophylaxisis. Later -- [ Indiscernible ] Discuss the integration cites in these patients, so I will skip this slowed but physically, there is the conclusion that there is a difference in the terms of the decrease of the cites compared to the French trial and any other information available for integration compared to the treatment -- [ Indiscernible ] And genomic sequence for each of the genes. I will -- a compatibility problem here. They're talking about ADA efficiency and I will start by saying that this is a forest, another form of immunocombined de -- efinish yepsy combined between 15 and 20%, according to published supports. It affects both males and femaless. What this slide show tells -- tells you is that this is a metabolic disease of old cells and the lack, the immunodeficiency are suffering from the lack of the enswrom and the patients also have the effects of -- [ Indiscernible ] Skeletal system, so it's a global disease that can present as a severe form with a classical presentation of combine -- combined immunodeficiencies with a setback of infection or a more delayed and milder form of combined deficiency and patients are later on diagnosed in life. The conventional treatment for the -- [ Indiscernible ] Deficiency, based on the -- [ Indiscernible ] Cell transplantation and the results from a European sorry have a, recent European survey, indicates if they have an identical sibling donor, that part is very successful in survival of the patient. The results of parental transplantation are much less sattis factory and actually is -- sattis factory and should also look at this survival rate. We'll hear more about transplantation for ADA deficiency, I believe, in the afternoon for what concerns American experience, but based on those results, many patients are treated with a second therfy -- therapy for -- [ Indiscernible ] Replacement therapy providing ADA -- [ Indiscernible ] Increasing the stability and using the gen -- JENOCITY. [ Indiscernible ] Presented in the meeting in 2002 showed that there were more than 100 patients treated with this therapy, which can provid protective immunet of normal in four out of five patients. However, these patients have really a correctional -- [ Indiscernible ] That all development antibody and gapes. As soon as their immune system improves and in 10% of the cases, the antibodies become utilizing, complicating things. And overall in the patients that respond, there is a mortality of 25% soreo. So, because of the imperfect results, of course, there has been a lot of work in the developing of the therapy of the disease, and this is around all the trials that have been performed over the years, and I will go through those briefly . Start be -- starting with approaches in the early '80s and '90s, targeting T cells as an easier approaching in those days, it was a technical challenge. I will go through is this quickly to tell you that two patients were treated at NIH in '90 and '91 with a result of treatment that targeted T cells. They were obtaining the EX VEVO from patients on the treatment, expanded transduce with a retroviral vector and infused back into the parts. This is patient number one that -- [ Indiscernible ] After the last treatment still carries around 20% of marked cells and that is simply a data that needs to be considered for the safety of this procedure. So this, this persistencey is -- [ Indiscernible ] And the cells still produce ADA activity, which is around 16 units, which is between 1/5 and 1/6 of what a normal PBMC will produce. This was a T cell gene therapy. The second trial, comes in chronological order, was started -- started intally, where you would take eight hours from the young and combine the T cell and the -- [ Indiscernible ] Stem cell target approach and they had three patients. Two patients with this strategy of being published, I believe, after five more patients telling you of the T cells that data is not published yet. The results of this trial has shown that there was a marking of between five and 18% in the crenical sites and -- clinical cites and looking at conforming units as high as 8% of progenitors by this approach. The next trial is, of course, the newborn trial where Dr. Con and collaborators in collaboration with NIH have treated three newborns that we were diagnosing with ADA deficiency, targeting C-34 positive cells obtained from the core blood of the patients at birth . This is one of the long-term studies,or survey of marking in the cells of their patient number one, the patient that has the highest marking as can you see these are the T cells where in the 10% marketing and myelloid cells with the AXE wherearound in the highest level at 1% at 54 months after birth what. Is more significant is the perhaps now at 12 years after treatment of still a 3% marking in PBMC's and a detectable marking of granulocytes and this is data that they shared at the FDA advisory meeting a couple of weeks ago. So this Wthis approach with the technique that the technology was available now more than 10 years ago, this patient still carries long-term marking. Now in the same time, a collaborative effort teen Dr. Fisher, Dr. LEWINSKI and Dr. Capped lairia targ -- [ Indiscernible ] Format cells from ADA patients to girls from UK and one patient from France to do with this approach, which I was not successful. There was a very short mashing in all. Three patients and based on the -- short marking in all. [ Indiscernible ] The very good result the Dutch group observed in the system after myeloablation, however, this was, the orders of this experiment first indicated that maybe success of gene therapy for ADA-SCID would need some kind of myeloablation. And then in 1995, our colleagues in Japan and Dr. YAKIMA and his partner ran a trial that was a copy of the NIH T cell trials in one part. And I will just say that bite end of the trial, the overall impression of the overall conclusions were that gene therapy for ADA efficiency had not been very successful for the results of -- [ Indiscernible ] The improvement were not clear and none of these patient his been corrected enough to be able to suspend ADA treatment. It was a very important background information, but there was no clinical benefit that one could really point to. And thises started to change after -- [ Indiscernible ] And his group probably subscribing to the theory generated by the Dutch colleagues decide to introduce a significant steps of non-myelograding of gene therapy in patients. These have been the results of -- [ Indiscernible ] Being published early in 202 N.science, where they depleted -- [ Indiscernible ] In the absence of concomminant treatment with ADA buzz the -- because the patients had no access to the treatment. They had shown very, very important results. Can you see he had for patient number one as soon as 120 days after gene therapy, this patient had a normal number of T cells, B cells and next&K cells. Other investigational -- investigation of the group showing anything they could measure became normal in this patient and based on these results they went on and so far they have treated six patients and this is based on personal communications by Dr. ONCAROLA and Dr. BIONIONE and he'll see summary slides of these six six patients at different age and therapy and they're followed for a different amount of time and, again here, the three patients at 30 month thrrbhold. that we always hear about. The first five patients were followed long enough for the investigators to say that the immunodoppler -- constitution had occurred for all patients except patient number two, that was an older patient receiving less cells per kilo. All patients at home and well without need of it. There is a result definitely, a clinical result that can be measured in these patients and that is difent from -- different from what we have seen in our trial, and when we study a trial and, one, we don't describe to the theory of chemotherapy, we caught that improving expression by the use of vectors was the way to go and so we tried it on vectors that were expressing -- expressing continue to 15 times higher ADA activity compared to the vectors used in the the early clinical trials. This is just a presentation of, or a summary of what our patients look like. This is a 5-year-old female that received more than 10 million, 74 cells per kilo, roughly half of them were used looking at the presence of ADA by takeman or colony BCR and there was a software in transfused that was a ADA activity that was similar to normal or both normal in normalcy, the four cells we expect to have on the hundred unit of-from-ADA. So what we saw in these parts was an initial increase in cells and lymphocytes. However, did not reach the low-range of normal and I will just take a minute to say that these values are very low and those are the values that you see in a patient on the treatment. Just to say that the ADA, although it keeps the patients off the risk of the infections, clearly doesn't give you a normal number of lymphocytes. And over time, however, the cell number to turn to biasline and held with the eye of hope can you see a trend here. But, obviously, the other numbers are very low and goes, reflects the numbers, low numbers, reflect the low, the percentage of markings that.
seen in this place next all of our patients of what we have seen N.this trial, I failed to mention we use two VECS because we have selected over the years two very good vectors. We didn't know which was better. We didn't know how to decide which one was better. We decide to test them in enreef iso, one against the other -- test them in vivo one against the other. Each part would receive both vectors and we would be able to detect each by a specific PCR and this is a vector that uses the M&D, -- [ Indiscernible ] Mosted to resist the inactivation in ES and -- [ Indiscernible ] As can you see from the square sim bowl -- symbols indicate cells or unseparated -- [ Indiscernible ] Earlier in the trial, we had three to five percent markings in the cells but that marking went, obviously, down, and now at 30 months after yet, we have a two in 10,000 cells marked for lymphocytes and no marking for myelloid cells. In this vehicleor, this carries the unmodified myelo -- [ Indiscernible ] And the marking as you see is a log lower throughout. We have marking in the, a little more the one cells per 10,000 cell in unseparated granulocytes at 30 months in this patient. But the summary of our four patients with this approach show that we had low level of Len yaj marking that was long-lasting for more than one year and two patients and should last in the other two patients. We treated two patients that were one, five years of age, and two others were 15 and 20 years of age, and we definitely saw a higher marks in a yonger part, which is similar to the experience in the X-SCID trial, if you compare the chafckal X-SCID patients with the adult parts described in the blood pavement as was referring to before. There was high markings compared to the myelloid cells and that is expected since we know that ADA positive, ADA expressing have a select advantage and we didn't see one in the three-year followup. But obviously we have not penetrated. We can really spook up. Our colleagues in Japan visit have taken the next logical step. Our conclusion from our trial is that in the presence of peg ADA and without myoAblag, the strategy, the jeep transfer -- [ Indiscernible ] Leads to a low marking and no benefit. Again, our colleagues in Japan were not totally in using myeloAblag. They decided to take one step and remove the ADA from the equation and stop the ADA before gene transfer but did not yield chemotherapy, and there have to be two patients so far and they used our vector whole our vectors did so and desire. The data from that trial is the 4-year-old and the 12-year-old that enrolled and one million cells per kilo and roughly half transdoused and very good activity in the south of the world. Unfortunately as you see, the number of lymphocytes remained below the low rainfall of normal, again, you know, if you're an optimist me, can you see a trend in it, but obviously there is nothing that is similar to the Italian style where the normallization of a T cell, the lymphocyte number occurs bee 120 days and here we are out one year for both parts. There is marking in these patients, this is patient number one and you can see there is marking in PBMC's and in the granulocites. It seems to increase over time. Patient number one and in patient number two. Again, this is any quantative PCR there. Is no tap month -- [ Indiscernible ] No realtime. Quant Tative PCR a date available for this trial. But in a 10 months after treatment, there is clear presence of the hygiene, the transfer vector. That brings us to the last trial of the series and that was started by Dr. Thrasher again and collaborators in London. They, they believedma thamyeloablation is to be used and used a full dose -- [ Indiscernible ] Instead of a low dose. There is no data published from this trial. Patients were teg triggered in November 2003. But it's been used from the local newspapers that indicates that this one patient is doing very well and is being called as a revolution -- [ Indiscernible ] Before the disease as well follows. Following the success on the same group for the X-SCID troll. -- trial. So if you follow the progression of the results of this therapy, I think it's interesting that now we have a almost full array of approaches from our trial where we started from the safest of the position, or where we didn't do anything to the current group of patients. We're adding gene therapy to patients on the ADA. The marking that is the results there were poor and then there is an intermediate position where the ADA was moved but no chemotherapy was given and in those two patients where there was that approach, the results are potentially promising but obviously not as exciting as the results that come from two trials where myeloablation was used and ADA was stopped in this case and not available from the start in the Italian case. So we have, you know, at wide spectrum, almost all responsible variation, and I think this is the case for gene therapy to work for ADA deficiency, we'll have to most likely go through some meelo ablation before the -- myeloablation. In summary, we have for gene transfer into stem cells, the most relevant issue for this meeting today, there have been 22 patients treated, 19 were infants of children, two and one adult. Eight patients are Alive and in the absence of adverseIVANT with gene marking, very viable gene marking from our parts, that are one in 10,000 cells to 100% of cells in the Italian trial.
gene marking for more than 21, eight patients. Overall, 7 error error parts -- 7 patients though these markings by this approach there. Is no complication by any of these patients and the available data on the -- [ Indiscernible ] Does now show a difference between the pattern of ADA vector integrations compared to the extra trials. That seems to be a different I would like to offer this is a safety for this disease as it might be indicating ADsa, a different disease and potentially the risks involved with jeep therapy in that disease are from the ones that gra are obvious for X-SCID. And I will stop there. I will thank Dr. Thrasher again for sharing his results and my collaborators at the NIH, Dr. -- [ Indiscernible ] And the infection of -- [ Indiscernible ] Helping with the trial and the collaborators in Los Angeles and east France. Thank you. [ Applause ]
Dr. Candotti, thank you very much for walking us through the jeep transfer experience and ADA definishience -- deficient SCIDs. It spans a long period of time, it's complicated and for me, at least, it was presented in a very coherent manner for us. So, thank you. We're going to move on to Dr. Friedmann again from uC San Diego, who will give us a summary of the recent safety data emerging from the French X-SCID trial. Dr. Friedmann . [ No Audio ] -- including elevated levels of the expression. The -- the child had a complicate family history with the existence of some family history of cancer ands child responded well to bone marrow, to chemotherapy and eventually to bone marrow transplantation. Eventually relapse and that was the child that died in October last year, five years after the treatment. Patient number five was treated at three months of age to help clinical leukemia more or less at the same time, the same kinetics as the first child 34 months after the treatment had three leukemic T cell clones with the same single integration site upstream from, not in, but upstream from Alamo II. That child did not have a family history of leukemia, but did show chromosomeal aberrations in the Lou cim -- leukemic cells. Again, responded well to chemotherapy and is under arrest Kently Alive and well -- and is currently Alive and well. After the two cases, the criteria for inclusion in the study were modified to some extent and included age greater than six months because of the leukemias and the two younger children who were much young her they were treated. The inclusion criteria was elevated to include children on the over-six months of age and with a history of at least one serious past infectious episode and the absence of family history of cancer, and no sign of genetic abnormality in the cells and with the reduction in the number of transduced cells to less than 10 times 10 to the six per kilo to be infused. The third leukemia developed in the child who was the patient, was in good health until November of last year. In January of this year, developed a round of larn.
This and wheezing with progressive cervical adenopathy with respiratory distress from enlarged mediaSTYNUM. That child was found to have single proliferated cortical clone. Some blood cells in the blood and in the marrow and NODES. The leukemic cell in that child contained three integrated proviruses. The integration cites as of last week when I heard from Dr. Fisher, those cites are unidentified but they're not -- cytes, they're not Alamo 2 II. This is not anentgration into or near Alamo II and there is no increased expression of Alamo EI there. Is obviously something different -- Alamo II, so there is something different from this event and the first two events. The kinnets of the development of clinical disease, similar to 30 to 34 months after treatment is when the clinical disease became manifest. This child shows no cyteogenetic abnormality in the proliferating cells and responded accurately and well to corticosteroid treatment and is currently in complete remission. I heard from Dr. Fisher several days ago who was very kindly letting us know what his thinking is in this study. And in form he feels that the study for now remains on clinical hold. He has the feeling that one of the major Avenues they're going to pursue in the near future is the development of the virus vector system, -- [ Indiscernible ] Self--inactivated vector. We'll hear about about that later in the day, and we'll hear some of the details of how the antivirus system integration mechanism is different from the, from the current leukemia-based retroviral integration events. In any event, this is the last I heard to professor Fisher, allen Fisher's current views about the gene transfer methodology, a shift to the virus system, rather than the leukemia-based vectors and I think that's all that I have in terms of report of the thinking and the progress and the French study, and I think we will hear more about that from later speakers, from Dr. MANKALA and Dr. Bushman. [ Applause ]
Thank you very much. We're going conclude the first session. I've lost it. But presentation with Dr. Carolyn Wilson from the food and drug administration and she will talk with us about the U.S. FDA perspective. Dr. Wilson .
Thank you. Okay. And I wanted to thank the committee for an opportunity for the FDA to present an update on what we've been doing from a regulatory perspective. I wanted to start by giving an overview of what I am presenting to you today. I apologize. I'm just getting over a cold. I hope can you hear me all right and hopefully my voice will last. Anyway, I want to start, actually, two years ago as you know, as you already heard, when these events first began. The actions we took in response to the first two children that developed leukemia in the French clinical trial and also bring you up to date in terms of how IMD's placed on hold at that time have responded. Then I will come to the present time and discuss what actions we took in January of this year in response to this notification from the French of the third child and then I will finish with a discussion of our advisory committee that we copveeped on March 4th -- convened on March 4th and reviewed for you what was discussed, what questions we asked the committee and what their recommendations were. So, going back to January of 2003, we support -- actually three different flavors of a letter to sponsors of retrovectors IND. Okay. So the first cat gorry is shown here. This was sent to sponsors that had active IND's that used ex vivo transduction wit rethvo -- retroviral vectors with hamatopoetic stem cells and INDs and any other that uses this. They received a letter placing them on hold until that were able to revise their concept reform documents as well as methods to monitor the clinical et -- [ Indiscernible ] As you heard from Dr. MALECH. The second category went to sponsors who had IND's who were at that time inactive but were taking the same approach in terms of ex vivo transduction and they were basically notified if they were turnover reseem a clinical trial under that IND, they needed to satisfy the same requirements and then all other clinical trials using target kills other than hamatopoetic stem cells, receive a -- received a letter informing them and suggesting that could revise the informed consent documents and methods to the cloneal et. These were recommendations. They were not in the form of a clinical hold. In addition, all sponsors were asked to provide a risk benefit analysis and SEBER has evaluated each response as it's come in. So to give you a snapshot in February of this year of where we were with administrative and IND status of the 28 IND's placed on hold on January of '03, we can see then in response to that letter four and subsequently withdrawn, an additional four responded to the hold letter and became in affect but then subsequently inactivated another IND holder just went ahead and inactivated at that time. Nine IND holders have responded adequately to the letter and are currently in affect and then 10 are on clinical hold. I wanted to break that down for you. Of these 10, three are actually in the SCID clinical indication, and I won't go into the details in today's talk, but those IND's actually had responded to the hold issue in January of '03 and between those, in their two-year increment had come off the hold and replaced back on hold in January of '03. They're capturing here the February timepoint. Six of these IND's never responded to the January letter. Those are mostly older IMD's and are probably no longer accruing patients, and one responded to the letter but was insufficient in his continued on hold. Just to let you know since January of 2003, we have also received an additional 15 new submissions using retroviral vectors in the breakdown of the target cells as shown here. So, now to come forward two years to the present, in response to the recent developments that we just heard about from Dr. Fried onmann, in January of 2005, the -- of Dr. FriedMann, the FDA put two on clinical hold, two for X-SCID, one for ADA-SCID. The letter sent to the sponsors was to request they revise the innocent documents and notify their IRB's, and also to let them know that we were having an advisory committee to discuss these events. We also support a letter to all sponsors of retroviral victors -- vectors to inform them of the new, vents, as well as notifying IRB's that regulate the clinical trials again to just make sure they were aware of the new events. As I mentioned previously, we convened a meeting of our advisory committee and mentioned the new names for those not familiar. It's got the catchy title. Cellular tissues and gene therapy's advisory committee, CTGGAC. This is formally the BURMACK committee, you might be familiar with. Now, I want to go through the information that was presented at that advisory committee meeting. I want to also mention that at this time, the transcripts are not available but at some point in the future, I think by the end of March, all the transcripts and 42 slide presentations will be ac -- and some slide presentations will be accessible to the public on the website as indicated. The meeting began by a presentation by the FDA where we reviewed the mechanism of rethvo ryeral immunogenesis, we updated the committee on the SCID trial in France and the regulatory actions taken. And we also reviewed what had happened when they -- at the advisory committee in February 2003, what their recommending as were and what subsequent actions were taken in response to the recommending as and finally, we, like Dr. FriedMann, were fortunate and having cooperation from professor TREVAN in collaboration with Dr. s Fisher and KELVO -- [ Indiscernible ] From the hospital to be able to provide a more detailed update on the events in France. So, in addition to that presentation, we had three speakers to provide updates from animal models that we thought spoke to the issues of insertional mutogenesis. Dr. Cindy Dunbar presented information from a non-human primate study using ex-reef iso transduction of hemapo at the time -- hemapoetic stem cells where six years later, one primate developed a myelloidsaur coma positive for vector cells and the exact link ang -- linkage of that event with that victor is still under investigation. But we thought that was important Dade -- data to make the committee aware of. Dr. BALDAVE spoke to his data from a mouse model using wild-type rethvo retro viruses. You will be hearing today from Keepland similar information and Dr. BANAN using ex vivo transduction with hemapoetic stem cells with the retroviral vector where he reported observation of leukemias where these animals, when they were performed at high dose of vector. And then finally, we also heard an update from the Dr. Donaldcon on the human experience with ADA-SCID. I wanted to finish by going through the questions we asked our committee and what their recommending as were and I also wanted to give the caveat that our interpretation of the recommendations found on these slides are based on rather cumulative comparison of individuals notes within the FDA, but the Trents have not been made available, so hopefully these are fairly accurate capture of what was said. So, I know this is a long question and I apologize, but I'm going just read it to just make it sleer. The code of federal regulations part 312.42 defines the base ease for FDA to place the study on clinical hold. Subpart B-4, also known as insufficient information was seated previously as a base for replacing IND's on clinical hold in response to the development of leukemia and subjects in X-SCID clinical trials. However, we note CFR 312 part D-1 that states FDA may place a study on hold if it finds a human subjects are or would be exposed to an unreasonable and significant risk of illness or injury. We asked the committee with this requirement in mind to discuss the current incidents of leukemia in depth of one subject from leukemia reporting the clinical trial in France relative to the potential benefit of retroviral victor -- vector media gene transfer X-SCID. We asked them to consider risk benefit of gene therapy versus bone marrow transplantation. We asked them to also consider how they would view these risk benefit issues should another leukemia develop in this trial or in trial. Their recommendation was to say that -- to change the risk benefit in a more favorable manner. Rethvo -- retro viral should be used under the following divisions. Bone marrow transplant or those that have no reasonable alternative therapies. For example, patients precluded from transplantation because of an acceptibly high risk of previous infects. Question two, we asked them to comment on what changes, if any, would reduce the risks to subjects in clinical trials using retroviral immune O' -- Imeanogene transfer in SCID, asking them to reduce the number of transduced cells, the total number of transduced cells going back to basics and altering the retroviral design. We should not consider the number or ways to address the risk in gene therapy for X-SCID but strongly encourage investigating alternative approaches including new retroviral vector products to lessen risk, suicide vector systems were mentioned as one where they considered feasible and in all cases, they asked that adequate testing in the model should be performed in order to validate any novel approach. And the third question: Please discuss the impact if any of the severe adverse event X-SCID combined with the development of myelloid sarcoma in the single monkey administered hemapoetic stem cells on the vector of the use of retroviral vectors on the clinical -- we asked them to discuss the risk issues in ADASCID as well as X-SCID in other clinical -- [ Indiscernible ] The committee recommended these trials should be allowed to proceed. They wanted to make clear the risks are still present and investigators and patients should be informed of strong and clear communication of these risks. They also noted if a retroviral were to be administered in the future, that the FDA be advised to -- [ Indiscernible ] Likewise, in other non-SCID, they allow cripical trials to proceed but, again, acknowledging the risks are present. Then finally, question 4 which was something more forward-thinking because of the potential use of retroviral vectors to transduce a much higher efficiency than the traditional gamma retroviral vectors, we asked given the transduced cells reducing in copies per cell, up to 10 have been reported, please discuss other restrictions on vector copy number per cell warranted for the vectors and ex vivoduction clinical protocols and if so, what limit would you advice. The committee didn't recommend a specific number, but acknowledged that it is an important issue. They suggested that we assess each IND based on available data and animal models should be used by investigators to assess the relative risk of leukemia induction with increased copy number. So, with that, I just want to finish that from the FDA's perspective, I think we're really at a crossroads in terms of trying to find just the right balance as really it's been mentioned by multiple speakers this morning between what we now see as an actual benefit in the treatment of X-SCID at the very minimum versus these actual risks from the treatment as well. So with that, I thank you for your attention. [ Applause ]
Dr. Wilson, thank you very much. I would like to take just a minute before we go into the general discussion. Are there questions either from members of the RAC and those at the table or from those who are in the audience for either Dr. Friedmann or Dr. Wilson, regarding the third child with leukemia in the French study or regarding Dr. Wilson's summary of the recent FDA stance. Dr. DOLUCA.
Dr. Friedmann, I don't know if you mentioned what was the ageing of the third patient in the X ? Nine months?
Trying to find yet on my slide. I'm told it was nine months of age of treatment.
That was the 35 months ago. So he's --
Two years.
Is he the third youngest in the group?
Pardon?
Is he the third youngest in the group of 12?
I don't know that. I will get that information for you.
Could you tell me if the Cynthia Dunbar result with the one monkey, how many other monkeys were there in that study?
So what she's doing is collecting, if you will, non-human primates that have been involved in various retroviral vector transduction protocols and trying to get them transferred to do long-term surveys for just these types of events and if I recall the numbers correctly, I think she has a total of 78 and the median times of follow-up will be four years, so this was one of the longer ones and I forget the longest time point, but it's not like it's a coherent single study of sort of an aggregation of all the serious monkeys used in a variety of different trials by investigators throughout the country even.
So they represent a lot of different retrovirus vectors, a lot of different doses itself?
Dr. lo.
I have a question for Dr. Wilson, please, a clarification of the recommendations on question number one. I won't try, for the March 2005 meeting, I won't try to pronounce the new acronym. With, this was this a narrowing of the indications for X-SCID trials from the January 2003 and could you explain exactly what --
Yes, actually committee hadn't taken astringent a stance on the use of gene therapy for X-SCID at that time as the RAC did .
Yes. Dr. Friedmann. Or is it Dr. Rosenberg -- it's Dr. Friedmann.
I just wanted to mention that there are these three other patients, maybe four, but certainly three others in the French fighting -- [ Indiscernible ] Younger treatment so well. Six months, seven months and eight months.
Dr. Friedmann, could you clarify weather those three younger patients have whether those three younger patients have reached a three-year time post-gene transfer .
Yes. One is 3.1 years. One is -- another is 3.1 years and the third one is 5.8 years.
Those are still --
Thank you, doctor Patterson.
I have two questions for Dr. Wilson. First in the slide when you recap the administrative and IND status of the 28 IND's replaced on hold on January 2003, you noted that among the quote, unquote, active IND's that six had no response and my question is: Howe have you followed up with those sponsor -- have you followed up with those sponsors in their readdressing the potential risk and need for monitoring of those patients, nonetheless, and with regard to all of the IND's addressed with the sponsors, their plans for long-tomorrow monitoring and could you speak to that. And then my second question is: You have presented the answers to the questions as recommendations of the committee, but do they represent the views of your agency?
The first part of your question in January of '03, the letter that we sent to all, those sponsors that were on hold did tell them that they did well -- as you probably already know, actually from many, since early 1990s, we asked life-long surveys of subjects on clinical trials using retroviral vectors and the policy was still in place, so we also looked for that information through the sponsor's annual report. So, even if they're on hold, they still need to submit an annual report. We're still monitoring that information. And then secondly as far as following up, we feel that the information we sent in the January '03 letter actually lays out very clearly. I mean remembering the 10 in active status are still on clinical hold so they're no longer treating. They have been told very clearly they need to notify patients, the ERB's and so on. That was all in the -- IRB's and soa on. That was all in the January letter. The second question you had as far as the FDA stance is is that right now as you know, this is still very fresh. We haven't had a chance to review the transcripts in detail, so we're actually, at this point, waiting for the Trents of it, you know, so we can be -- the transcripts so that we're not misinterpreting what the committee members they Mai have said and probably toward the end of March, we will internally sit down and assess our own position. It's really too soon to comment.
I just wanted that destchingz made. Yeah.
Thank you for making that distinction .
[ Indiscernible ]
I thought that at one of the prior meetings there meet have been mention that even though there were two children that had gotten leukemia, there might have been a third where there was not clinical disease but there was some suspicion. Is that the same as the third child who now had --
I will answer that. I don't believe we have that information. Dr. Win -- Dr. Wilson.
I think we can dedeuce it's a different child. All previous time points on the child were different integrations and the child you're calling from the previous meeting had an element or two of integration . [pausing to switch captioners ] don't know.
Any questions from those of how are in the audience? Doctor?
Question for Dr. Friedmann or anyone. This first patient who died, sounds like he had acute graft versus host. As a terminal event, is it clear that his death was directly related to the leukemia or to the treatment or -- but in any case it sounds like his leukemia did not respond to the therapies.
Did you want to comment? [ Microphone, please ] .
My understanding it was it was not so much the transplant-related issues but that he basically had reflak try leukemia. .
He I hadn't either, Dr. O'Reilly that this was graft versus host.
No.
It was conveyed to me as a relapse, not graft versus host.
Since during the first session, the serious adverse event in the third patient was defined. I have a question which is I think a very obvious one but I don't have the answer. The third patient for me confuses the issue much more than clarifying it because everything about the third patient seems quite different from the first and second. Except the kinetics. The kinetics are within the three-year time point. But the third patient to my understanding has a proliferation of TCR V-beta five, it's a single clone, it is immtur cortical thymus-related. There is no increased expression of LMO2 which seems to be one of the driving forces for subjects one and two. And there was no cytogenetic abnormality so it confuses the issue for me of the relationship of the serious adverse events, that is, the development of leukemia in this particular patient group following gene transfer. And it makes it less clear that LMO2, not clear that LMO2 is not central and not clear that, uhm, the gamma chain transgene is central. I'm just -- if someone could clarify that for me, yes? .
I'm not sure I can completely clarify that. But I think some of the retrovirology experts that we have could certainly comment that based on studies of retroviral integration, there isn't a single pathway or single target gene that we can point to in some circumstances, you might say, there is a particular target gene that's frequently involved. But there are other examples and maybe Dr. Copeland, particularly, might want to comment further.
Maybe we should hold that question until after the retrovirus talks because I have some data that --
No, I'll hold it. The problem that I see is that moving into the discussion segment, which we'll do in one moment, for the first session, the specific relevance of subject 3 is the underpinnings for that discussion. And so I find it a bit complicated to complete the discussion without that. Can you give us some sense, Dr. Copeland?
Well, the -- I mean, naomi said I think right and that's that I mean, my take on it is that when you put IL2RG in a retrovirus that's an oncogenic event and when you give it to a patient that's one mutation. It integrates all over the genome and it can deregulate. LMO2 is one strong cooperating partner but there are probably lots of other cooperating partners so I would think that patient number 3 could have an integration in another Gene that could substitute for lmo2 or it could have another spontaneous mutation with another gene that could cooperate with IL2RG. Does that make sense?
Absolutely. But it broadens, let's see -- it broadens the significance of the leukemias observed in the first two subjects.
I guess I don't no what you mean by broadens the significance.
Hm [ Pause ] .
In my mind, the first two subjects although not identical, have very similar patterns. And so there was for me, at least, one could look at that as a fairly narrow series of events, specific -- I have to be careful about what I'm saying, but specific Orelively specific -- or relatively specific for this disease and this vector. Dr. Rosenberg?
I think -- and I think, uhm... Neale would -- Neal would probably agree with this, that long history of experience with the way these viruses Clint to tumor development in model systems, really predicts that we can't to a single event and to me, the importance, if you will, with respect to the issue you're addressing of the third patient is that that reinforces the fact that what we know for from these model systems -- what we know from these model systems is going to be of real consequence for individuals that participate in trials such as this. So I think it's really just showing us that we can't rely on looking for insertions near LMO2 as prognostic of what may --
I agree. If you base it on the model systems like in mice there are these preferred integration regions like LMO2 but you also hit other genes, too. It's more broad. And this is exactly what you would predict. You would certainly not predict all of the integrations are going to be in lmo2.
Right. Thank you. Other questions or comments before we move on? .
Dr. O'Reilly?
Yes. The critical variable here remains, though, that's these are tcll, and in this age group it's an unusual mode. The other issue that comes up is the unique biology of is he severed combined immuno-deficiency because these kids have a thymus that's fetal and nonlittle foid. So you basically the first population to reconstitute is going to be the T-cell population within that thymus and in fact even in the nonreduced pads for SCID that's where you see the selective growth so if you are going to talk about a really major growth emphasis, that's where it's going to be and the possibilities for altered recombinations would certainly be at the highest level. .
Dr. Friedmann?
Couple of questions that have occurred to me in discussions with some of the members of this panel and also Dr. Fisher and others. Two things that might be approached by some of the later speakers. One is whether this third case sounds fast it might be something different from the first two in terms of its clinical presentation. And I have heard this third case described more or less as lymphoma-like sort of rather than leukemia. Now, I don't know where the differences lie. But is there significance in that differentiation? And also, do the clinicians have any feelings about this kind of monotonous 34-month window between treatment and clinical disease? Does that suggest anything in terms of whether this is -- which hit this is in the pathogenesis of the disease? 33, 34 months for all three children, and does that suggest anything to you? It might be not the time to discuss it but bring it up later.
Dr. O'Reilly.
There have been several studies looking at thish eye of the pathogenesis in -- issue of pathogenesis in leukemia children particular from studies from the University of Vermont and you know, in terms of the issue of level of production and rearranged clones, this is a peak period at about three years to five years of age in normal kids. But it's usually what we're look caught in the context -- looking at in the con teiveghts rearrangements in the b cell. Now, that niche for most of these kids with SCID may be relatively filled. So it's both sides of these in terms of little foid development, that's a sort of peak time and it's been looked at and thought about as one of the reasons for the pathogenesis of leukemia in normal kids. .
We are going to take a 15-minute break and then we'll begin a more broader discussion of the first session. Thank you. [ Short break ] Please stand byfor realtime transcript. Stand by, please. [ Standing by ] Stand by, please. [ Standing by for audio connection ] Standing by for audio.
Thank you all. Dr. Bernard Lo and I will be chairing the discussion of the first section. We decided to divide it in two. The questions that we structured for this section are from the RAC, are primarily database questions. There are a second group of questions which were framed by the FDA during their discussion. And what Dr. Lo and I would like to do is to quickly and I mean quickly, uhm, go through the database questions, be certain that everyone agrees to a response which we do want to agree on. And then move to the FDA questions and answers and ask simply are we in green or is there something we would like to add or change? So how do we move forward with these? Do I go up there? I guess I do. .
I can do it for you.
Oh, good. So the first set of questions we were posing is, what data is available regarding the research participant who developed the third leukemia and it's divided into four sections, I think the answers to these sections are fairly clear. Is the third patient described at the February '03 RAC meeting, is this the same patient as having two integrations in the LMO2 gene and the answer is no. Second, has to do with clonal expansion and this all relates to again the third subject, the cell type, TCRV beta 5, the methods for early detection of clone at appear to be AAMPCR and tack man PCR. No question about that. Time course of expansion. Three years.
Dr. O'Reilly?
34 months. That's right. And time from vector administration to malignant transformation in comparison to the events in the two previous participants.
Sorry, claudio.
To qualify to the expansion, this patient was negative for oligo clo national in December and then came down with the onset of the leukemia in January. So it's not 34 months. It's 3 months. The time course of expansion I suspect should be three months and the next question which asks for the time from vector administration to malignant transformation is what should be 34 months. The next set of questions really don't have good answers because they involve vector integration. As I understand it, the site or sites of integration are unknown. The detected expression from cellular genes due to integration is an open question. Unknown. Function of the cellular genes EG okay he could gene again unknown. -- oncogene, again unknown. The nature of cellular gene activation, these all depend on definition of the integration sites and the biology that will ensue following that definition. Other incircumstances observed in the -- insertions observed in the participant cells prior to clonal expansion... don't believe we have information about that either. Dr. Von Kalle do you have information regarding the section on vector integration that you could share with us?
I think what I have I'll -- I will present if my talk.
All right. Then we will defer these, the vector dose, the number of transduesed cells administered, appear to be 11 times 10 to the 6th per kilogram. Relatively high but in the same range really as patients 1 and 2 with leukemias. How does -- question 2, so that the entire database for question one really is open to further definition but does expand beyond patients 1 and 2 where there appear to be a definite link with the LMO2 gene. Question 2. How does the data from the third participant compare to that from the two previous participants who developed leukemia? And what we can say is, that the data is different, the site of integration is unknown. The expanded clone cell type is TCRV beta 57. There is no increased expression of LMO2. The timing from receipt of the vector to the development of leukemia was 34 months right around three years. So that is relatively the same but the biological consequences that followed are significantly different. -- from patients one and 2, other than T-cell leukemia. I agree. And I think that's really important to emphasize and Dr. O'Reilly did during the earlier discussion period, that is T-cell leukemia is rare in young children. All three of the subjects in the French x SCIDs trial developed p cell leukemias not the more common d cell leukemia that we see in young children and then I'm going to skip to question 4 and go back to three as the final discussion point. Question 4, what data is available from the other clinical or preclinical studies regarding either maintenance of polyclone national or the range of integration sites. This a very broad question for which we really don't have a good answer. We are going to hear more about this later today. If we go back to question 3, this is where we should spend a few minutes from the lessons learned after the first two leukemia cases after the fren k skids trials it was apsyched that they could be minimized by limiting enrollment to research participants older than three months of age and lowering the number of transduced CD34 cells administered to participants to 3 million CD34 gamma c cells per kilogram of body weight. Dozen the development of a third case of leukemia call for change in these safety precautions? I'm interested to hear if there is anyone who feels -- I would POSIT that the correct answer is yes. It calls for change in the safety precautions. That safety precautions that are limited to age of the participant and dose of cells are likely not adequate to address the scope of this serious adverse event. Is there anyone who disagrees with that perspective? Okay. Dr. Puck.
I might just add that infants diagnosed with X-linked SCID who are within the first three months of life are unlikely to have had as serious infections and other adverse environmental insults that make them poor bone marrow transplant candidates, and so if you're looking at the risk benefit equation, I think you need to also consider that the standard transplant treatment for these patients might be more successful. There is published data from Dr. Buckley's group and others that underlined the relative Ben fifth early -- benefit of early bone marrow transplant. .
Dr. Somari and this Dr. O'Reilly.
This it says these were assumptions but they weren't explicitly stated in our recommendations from February 2003 nor do I see them in any of the FDA things.
Correct.
So where did the assumption come from and where was it explicitly stated?
I don't think it's state police thely stated -- I don't think it's explicitly stated. It came out of a conference call from a number of us looking at the commonalities between subjects 1 and -- the first two subjects who developed T-cell leukemia. And these were commonalities. Dr. O'Reilly?
It came out of an article that -- it actually came out of an article that Fisher wrote suggesting that these were the way that they were going to go and this article was the most stringent one where they dropped it down to 3 million but I think the French FDA had actually recommended 10 million as being the dose.
Dr. O'Reilly.
I have to say that bz you know, the -- the scientific rationale for giving the limited dose to a degree escapes me. I understand that because you are going to quote, unquote give fewer pro general tores but in if you are going to drive a single cell to multiple replications, you give a limited dose of stem cells. You know, it would seem to me that actually if you gave a larger dose of stem cells the likelihood of having this kind of a transvehicle AT&T would be largely reduced this would go along with some of the studies in [ Indiscernible ] models where if you give limited dose stem cells you can have mice that are cruising along on one single stem cell or two single stem cells from the studies of lumishka for a period of years so my sense of it would be that you would rather give -- if you are going to do this, do it with a -- a large dose because you are like -- your like lie hood of developing -- your likelihood of developing a clonal population would be reduced. I don't know exactly what the -- the readout was or why should you give 3 million. Last time we did this was when we were doing, Diane can remember ago, you do matched transplants for severe combined immune deficiency and the whole idea was you give a small number that way you are going to get less problems and GVAs. The fact of the matter is when you gave the small number those are the only patients I know of with SCIDs who developed chronic GVH who got problems. .
Dr. Copeland.
Yes. I mean, I would think just the opposite actually. So if you believe what's going on is this virus is oncogenic and it's integrating all over the genome essentially at random and had some probability in a cell of integrating at LMO2, the more cells you have and the more integrations, the more cells that you transplant are going to have LMO2, which is a cooperating event with IL2RG. So if you transplant two cells, then the probability of having an interdiscrimination and a cooperating gene is essentially zero. And therefore, you would have -- you wouldn't have the cooperating event. So what I would argue is if you give them a large amount of infected cells, say 10 or 100 might have LMO2 integration, what's happening over that three-year period is the cell or cells that have the cooperating mutation are selected and they grow out over time and that that's what you see. So the -- I would -- I would argue that the more cells you transplant, the bigger the problem.
Now, I think if that's been the premise on which the cell number issue was based.
Right.
Who -- Dr. Malech.
Yes. I would actually like to try to mud dit water a little bit rather than cla -- muddy the water a little bit than clarify things. Number one, those of how attended the FDA committee meeting and heard Cindy Dunbar's presentation about her monkey, what was special about that animal actually was that it was extraordinarily Oligo clonal, maybe even monoclonal in its en graftment and it probably was that monoclone that eventually transformed. Bringing up the issue that when you -- that there is -- that there may well be a Lou keemgenic effect to running marrow cells through a narrow window in itself. Which is not to say that Dr. Copeland is incorrect. It almost certainly is correct that the more chances you give to hitting the wrong place, you probably have a problem, as well. And that's why I say I'm not clarifying but rather, muddying the water. And to muddy it further, there -- you know, I remember that one wit said the safest thing to gene therapy is not to give at that time all and, of course, you can work your way down to doses that are not therapeutic whatsoever. And there is a lot of evidence to suggest -- and I would draw your attention to some nice studies done by Peter fellsberg with his dog model of X-SCID in which he gave limiting doses of CD34 cells and found that there really was a threshold effect and that what you ended up with if you gave too few cells was -- was engraftment -- actually curiously a little bit like the Patient 1 that I presented this morning where you get evidence of selective engraftment but you don't get a take-off and it's a little like having a cell culture that's split too thin. There are probably auto crine factors and you have to reach the level and the problem is I don't think anybody has any idea what that is what the right number is and it may vary depending on how old the patient is, how the patient comes to the table, what's been done before. So therefore, you know, I think before you take a chance on regulating dose, you need more data on what is the sufficient dose in a specific situation, which is not to say it isn't a good idea. We just don't have the data.
I could make another comment? And address --
Yes, go ahead, Dr. Copeland.
So Cindy Dunbar and I just two hours ago gave the grand rands talk down at nci and she talked about this data. So basically the monkey that you're talking about that got leukemia and was kind of monoclonal early on, early on I don't know how many months that was because -- uhm, actually has two integrations and one of the integrations is in the BCL2-like gene. She can't prove that that is -- that that insertion is cooperating but it's very likely, it's a pro -- it's an anti-apoptotic gene. The idea is you are giving the monkeys a lot of stem cells and what was selected out early was a stem cell where the integration occurred into the BCL2 gene. That was selected but not enough to get leukemia and then over time, you acquired other presumably spontaneous events that cooped cooperated and then you got this massive expansion in the leukemia. I don't see how that really muddles the water. Maybe I'm miss understanding something.
Go ahead.
One more comment, Dr. O'Reilly.
I mean, I think the biologic evaluation of this, which is always the one that sort of gets me, is the experience in aplastic anemia where you are basically dealing with a limited pool of progenitor cells. We can drive those systems, in some instances, with cytocions or with ATG but the fact of the matter is you have a hematopoietic system that's under stress and the studies that have been done indicate again that it's relatively oglioclonal and about 30 percent of those individuals if followed over the course of around 15 years will then go into an overt mild displastic syndrome and AML. The only sensible mode of this has always been the idea that basically you have single clone under stress requiring multiple divisions, the chance with each division of having one of these events becomes stronger. That's basically one of the reasons why I get out of this. And the other reason why I'm at this also is because as I said before, these are all T-cell ALLs and the one thing that you do know is that in these kids with SCID, their thymus is fetal thymus about the size of the end of this pen. And that's it. And it is basically a lymphoid. It has no lymphoid cells whatsoever. Somewhere around 3 to 4 months after you give these grafts, that thymus fills in and you can literally watch it grow. And basically, the more pro general tore cells it would seem to me that you cede to the thymus, the more likely it would be that you are going to have sort of continuing normal growth of cells not in the con telephones integration. We don't have evidence that that integration caused leukemia. Rather what we have is a series of kids with intergrants where they have required second or at least demonstrate second or third hits that are involved. And those second or third hits are secondary events that are occurring in the context of division. So that's my really major concern about limiting dose. That if you do so, you put stem cells under stress because if you give three million of those cells, quote, unquote, that are transinvestigated, the actual population is likely to be a true stem cell is probably not much more than 1 and 10 to the third or fourth at max. .
Dr. Rosenberg?
I think for me there needs to be a middle ground in what Dr. O'Reilly is saying and what Dr. Copeland is saying but the problem that we face is that we don't know what the middle ground is. Certainly, there is I think compelling evidence that having one single stem cell clone contributing places incredible stress on that particular clone and that's not a good situation. However, I also agree with Dr. Copeland that if there are large numbers of cells, particularly with large numbers of integrations, I think that maybe a missing piece that is not emphasized in what we started here, multiple hits per cell, large numbers of cells, that that also greatly increases the risk that one of those or more than one of those integrations will be near a gene that either influences growth apoptosis, some other process, and really the problem that we're facing is that we don't know how to modulate those two things so we don't wind up with monoclonality of a normal cell that then accumulates events, nor do we wind up with outgrowth because there have been so many retroviral hits. And the chances of that is very high because the cell dose is huge.
Just one last comment. I grow with you completely and I think it really can be boiled down to the fact that you -- that you need efficacy and you need safety.
Right.
And the safety issue you don't want too many. And you don't want too few or the get monoclonal or oligoclonal factors that establish cancer itself which is well established as Dr. O'Reilly put and Dr. Copeland is right if you give enough cells you constantly increase your chance but if you have no efficacy you can take the whole thing and go home. And what we don't know is we don't know how much we need and what setting for efficacy. And that's why more work needs to be done.
I think we all agree that paradigm of merely limiting the number of cells giving given to the subject and limiting the age of the subject is not adequate to control for the risk and Dr. Malech from my per perspective you have targeted the issue. How much and what kind do we need for efficacy and how can we reduce the risk of that to make it acceptable? I want to leave this discussion by adding that I hope it strongly suggests to everyone that additional work needs to be done on the vector itself to improve the safety profile of the vector and we'll have some of that discussion this afternoon. Dr. Lo, you were going to take the discussion in a somewhat different direction for us. .
Right. I wanted to lay out some issues that have already been touched on by our -- this morning's speaker that I think will be important as we come to deliberations. Dr. freed -- it's a double comparison so that on the one hand we're weighing risks and benefits in that final slide that Dr. Wilson shows has the balance we are trying to reach in our minds but it's really a double comparison. You know, it's a comparison of the risks and benefits of Gene transfer versus the risk and benefits of the alternatives. I think that's where the next session comes in with the evidence with transplantation. But as a nonsort of pediatrician, I just want to make sure that we all are clear on sort of some of the more call tatety of aspects of risk and benefit. .
I As I understand it I want to raits issue of quality of life, those kids who get x link SCIDs and undergo a successful gene transfer intervention and have some immune system reconstitution I want to sort of make sure we automatic understand what that achievement means in terms of their daily lives. They are off Indiscernible ] these people in the not all but most 5 out of --
Most are off -- they are not taking prophylactic antibiotics, is that right? And they're back in regular school, playing, rolling around in the dirt and getting the scuffles on the playground all that stuff. So they are living fairly unrestricted lives without concerns about limitations on on activity or avoiding activities because there is concern. That's the goal. But I guess I'm trying to understand how successful is that goal achieved in these gene transfer protocols that seem to reconstitute immunologically. That's something I think we need to compare to the other alternatives we'll be hearing about in the next session.
So comments?
What I think of this issue, I go back to the table of all the patients in the yellow sheets here, uhm, and it's clear to me that out of the four -- out of the four patients who got over 10 to the 7 kilograms per cell, three of them, three out of of four of those have a high risk of getting cancer -- okay, I got cancer so under those conditions, the risk are very high. And, of course, the benefits nonexistent. However, if you go down to one times 10 to the 6th, then you are of course very safe but as you can see, one of the -- it didn't -- the -- one of the immune -- one of the -- didn't respond well so only one out of two of them benefitted. However, in between there, around three times 10 to the 6th to 35 to 7 times 10 to the 6th there are no adverse events and they are all reconstituted. I wanted to put that question in light of the previous discussion about the number of cells. It's true that of three out of the four that got over 10 to the 7th cells, three of them are the ones who got the little no proliferative disorder. The fourth that got the highest is at the 3-year limit right now. .
That's I meant about this likely being the basis for the comments about cell number that the French had put forth. It almost an unintended Phase I... Which I -- I think we should look at from that perspective. I also believe that that perspective is too simplistic to guide the overall work that needs to move forward.
I agree.
It is --
I agree but it's real data.
Yeah, it's obvious data. Doctor?
let me ask a question from somebody who knows cancer itology. Let's assume that we drop the number of retrovirus integrations so it's less than one so you don't have double hits on average. So you are not going to inactivate or activate two different cancer-causing genes. So that's not the issue. Now you have a -- an unlimited number of these cells and you are asking the question, if you go in with a large number, your chance is greater of getting cancer than a small number. So imagine you go in with a large number and you have polyclonality. If you get that second hit by natural spontaneous mutation, isn't that cell going to have a very high selective advantage? Does it really matter whether you are going in at high or low under those circumstances?
Dr. Copeland?
I guess it depends on what's the spontaneous frequency of cancer mutations versus retroviral insertional.
But that was what were you were assuming was happening, right?
Yes. I didn't understander point. You are going to give a cell less than one so you don't get a hit into a cooperating gene?
That's correct. We don't have double hits by the retrovirus --
I'm confused by what you mean by double hits.
No I understand. You are not hitting two different genes for -- that might cause cancer. You have a high probability --
So you only have let's say LMO2 and IL2RG.
You have less than one. Yes.
IL2RG in some cell integrated LMO2 you have two hits presumably that's not enough to cause the cancer so then you are saying the rest will be spontaneous, right, versus --
Well, in principle that must be what's happening, right? Under those circumstances does it really matter whether you are going with a high or low number of cells?
I think -- I don't know. That's a -- I think it's an impossible question right now in the current state-of-the-art of knowledge to, you know, knowledge to... To answer. But clearly the more cells you give them the higher probability you have of an integration into a cooperating gene. Now, how much of a selective advantage is that? So that that cell --
No. Let's be clear. We're -- we have set up the initial transducktion so that we have less than one integration per cell.
But there is some cells that have an integration.
Correct.
And in those cells you could have an integration in LMO2.
But presumably LMO2 integration alone is what I'm hearing is not sufficient to give you --
It's LMO2 plus I would argue IL2RG that's in the virus.
You think that's enough.
No, I think that that's a strong -- I think that those the -- model based on our mouse data which I'll show you is that that would be a strong -- so it's two events. The IL2RG virus, our data would argue it's not proof, but would argue that's an oncogenic virus so that you are giving one cancer mutation by giving him the virus without any integration in LMO2. Then if you have a cell, where it integrates in LMO2 that's a second hit. So that cell then we would argue that is a strong selective advantage to persist following transplant so that it can acquire other spontaneous mutations to build up the number of hits that it would take to give a leukemia.
Dr. O'Reilly.
I mean, in answer, see, I get opposite side. I would say from the standpoint of the mathematics, the second hits are acquired in the context of additional divisions to the degree that you select for limited number of clones. That cell must go through a higher number of represent complications and, therefore, would be at higher risk for developing an additional hit. So my sense of it is particularly with the thymus, if you can repopulate with large numbers, you would have less problems. Now, the issue with regard to the high numbers shown here in the take and I think it's -- table and I think it's an important point, you know, this is where we're really caught, in terms of what is the true, you know, progenitor populations that you have in that particular realm, and, uhm, query actually would come up what would happen if you have a log order higher? My suspicion would be that you would have less. But anyway, that's where my sense of it is.
Can I make another comment?
Go ahead.
So I mean, so what you're saying is if you give them less cells, then you have a cell that's going to have more cell doubling so it would have more chances of picking up spontaneous cooperating mutation, right? Which I agree. However, if you give them more cells, then you have a higher probability of the virus integrating into a -- and mutating a cooperating gene. So it's ain yang thing. More cells you have a higher probably of yarn serbsal mutation, less cells you have a higher probability of picking up 1307B tain just mutations because of more cell doublings and how they weigh against each other and again it has to do with what's the frequency of on? Tain just mutation of cancer genes and he is ever what that effect is versus the effect of insertion in the cooperating gene, I think it's hard to know. I think there is benefit to both. Giving lower cells you are going to have higher spontaneous mutations, giving more cells you are going to have more insertion mutations so it's like you're damned if you do or don't and which is the better approach.
Dr. Rosenberg is going to bring some closure.
I have my instructions. I think this last piece brings us back to Dr. Malech's point. We don't know where too little is not good, too much enhances the chance that you are going to have multiple clones that might have an integration into a cooperating gene and we need real information to let us know what the correct middle ground is. I think.
Agreed. Dr. Wilson?
I wondered if I could just make one additional statement even though that was supposed to be the last. I didn't have time to present this today you buy -- but I presented it at our advisory committee. Dr. Steve Anderson, who is in our office of biostatistics and epidemiology, used a Monte Carlo simulation model to actually ask if you have a single vector copy number per cell, what is the likelihood of as one example integration in LMO2 per one million cells which is obviously I think we would all acknowledge is probably a lower dose than what most recipients get. And in that random model, you get 20 cells would have one integration in LMO2, if you use the gene bias model which is based on the data from WU where he shows there is a bias towards transdescription Nally active genes for MLV you would actually have 50 cells in a million that would have an intergrant in LMO2. Now you can hand calculate if you make an assumption if you know that en graftment is very low only one in 10,000 cells, say, would actually own graft, that if -- engraft, if for example you add that 20 in a million cells what's the likelihood that one of those cells ends up being en grafted? That's one in 5,000 patients in a random model would have that cell en graft. Which I think I'm only saying this, it's just a mathematical way of saying what I think everybody else is already saying which is there is already a strong selective pressure that if you have an LMO2 integration, that you are asking that cell to -- that that has a positive force towards engraftment and proliferation. So... just to reinforce those comments.
It's nonrandom. Let's see. Dr. Friedmann.
Just a quick question about -- I think we can all agree we don't know, right? (Laughter).
And in light of that, does it make sense for this committee to take the position that we go back to -- the committee goes back to the investigators and asks for -- for information? Have you looked at -- in your animal models, have you looked at the effect of varying dosages of transduced cells? And do you know anything about this issue? I think more to committee to take this stronger position given the data that we have now, the fact we simply don't know which side of the fence we are going to fall, uhm, seeps to me that the -- seems to me that the enlightened position is to go to the investigator and say, what do you know? And have you looked at it? And are you planning to look at it?
Well... .
Yeah, I just want to comment and then you may have the last word. I agree that Dr. Friedmann, but in the -- during the time that it will take to gather that information I also believe that this committee is obligated to deal with data we have that Dr. Deluca focused our attention to, which strongly suggests that if you give too few cells you get no good, and if you give too many cells, you... Derive risks so I think we're forced in the absence of better data to use the information provided to us through the French X-SCID study. Dr. O'Reilly?
The only other question I would have and fed, you may have -- Ted, you may have the information on this and it would be really worthwhile knowing this the amount of marrow that you are going to get from each of these kids is still relatively limited and will be less than maybe it could be one to twofold so the other question that comes up for me is, Why is it that you have a tenfold difference in the proportion of cells or -- and the actual dose of cells of gamma C receptor positive? I mean, were there subtle alterations in terms of how they infected? Because I would, you know, that's a very, very large difference unless they were truly limiting the dose and as far as I know, if they were not limiting the dose of marrow cells they gave, but rather they were doing this multiviral vector infection the question would obviously come up there would be is it possible that certain of the SCID variants are more susceptible to retroviral infection than others? It may be that it is dose but I don't know that there was any dose consideration in terms of how much marrow they took or how much they actually did.
We don't have that answer available here. Other comments before we close? This section? Dr. Malech?
At the risk of annoying people by keeping this going, I think there is some data out there that would suggest very strongly that the dosage ranges that have been using may well be at the threshold of efficacy. And therefore, we know that we actually have real events that occur that way. So this is a bit like a chemotherapeutic agent or whatever that has a very narrow therapeutic to -- to, you know, to problem ratio so that you -- you know, you don't have a lot of room there. And I would again argue that in different patient populations, both the risk of leukemia and the dose that's efficacious may vary in ways that aren't clear until you do the studies. Just to throw out a number, we know -- we know from studies that we've published that match what many others do in nonSCID animals that we know that the kinds of [ Indiscernible ] NFL retroviral vectors when they you are hitting around 40 to 60 percent bulk transducks, you are probably only get about 1 to 2 percent or less not SCID repopulating cells. Somebody may jump up and say nonSCID repopulating cells aren't in the patients but it's the best we have as some kind of model. And therefore, if you run the numbers, you are really talking about a very low number of real stem cells. We also know from both regular transplants and from the dog studies again that -- of X-SCID that were done by Pete Salszburg that you need at least a million total c34 cells which means in that setting whatever that real number of true stem cells is, there are 100 percent normal. And whereas in the gene therapy what I'm arguing is, given the ranges of transducktions that we used, the French study used and that the UK study are using, we are probably only hitting somewhere around one to two percent of actual true stem cells. So that's why I believe if you run all those numbers we are just barely at the threshold right now and that's problematic obviously because we have events with the threshold of efficacy.
And again, you have nicely framed the issue for us. There is ee northern must tension -- enormous tension between what is enough to confer benefit and what is excess and will confer not risk but excessive risk and that's the struggle. Dr. Lo, do you have a final comment?
No.
No final comment. Other comments especially from the audience? Folks haven't asked anything. All right. Other questions from those around the table? Then thank you very much. We will take a Break for lunch and we are scheduled to reconvene --
No, we're not! I'm sorry! That's right. We are going to session 2. Thank you for stopping me! (Laughter).
Good try, I'm hungry. Retrovirus integration and insertional mutagenesis. And doctors naomi Rosenberg and Marina O'Reilly will guide us through the next three presentations. .
We're going to have a series of three presentations that are going to focus on the retrovirus aspects and inform us about both the integration in these individuals and some basic research that speaks to how these events may be mediated and affect the patients. We are going to start with Dr. Von Kalle, gene transfer and insertional mutagenesis.
Thank you very much. I would like to thank you for the opportunity to present our data and would like to thank the organizers for the invitation. I can very well feel how Carolyn Wilson must have felt. I have also a very bad case of the flu. I hope you can still hear me at least. So I wanted to talk a little bit about gene transfer and insertional mutagenesis. And if I follow the discussion earlier and I think Dr. Malech very elegantly summarized the topic just earlier, if you look at the time axis on how retrovirus gene transfer into hematopoietic repopulating cells developed over the course of time then it's pretty much in parallel compared to the success rate of carrying genes into those cells and of establishing those modified cells in the organism and then obviously, with almost exponentially increasing success rate of the whole system has hit a therapeutic window and, of course, at the top part of that window, we're now more than as we've just heard a million cells per kilogram of modified cells are being en graft in patients, obviously the potential for side effects is now also rising significantly. And we have of course asked ourselves what are some of the factors that may be contributing to this issue. We had earlier found interest in insertion site analysis because it leaves a molecular marker in the hematopoietic stem cells and other types of stem cells but as you have all realized it has also turned into a dose issue for the toxicity and efficacy of gene therapy. So insertion side analysis tells us about the stem cell clonality of which there is very little other data from other systems earlier. So in fact, a lot of the numbers of stem cells or modified gene cell clones that we are now looking at have not been labeled or visible in any other previous systems so it is sort of new territory.
Then also the distribution analysis of retrovirus vector or even retroviruses has made great advances and with the paper of wu and Burgess and Dr. Copeland's elegant work, has of course found new and very interesting interpretations and I think I'll try to present a little bit of additional data in terms of what that means for the clinical applications.
Then of course there is the question of cell type dependence of insertions which needs to be studied and has been an open question in the earlier discussion. Genomic side effects of insertion, and what I wanted to do here is focus your attention a little bit on the fact that we are very much discussing now rare side effects in rare cell clones that may lead to gene activation of particular genes. But, of course, we have to keep in mind that all of these insertions in the vicinity of actively regulated genes have the potential to deregulate or regulate the gene locust that they are inserting in. Now some of those may not be relevant at all for the cell type they are hitting but what I'm trying to say is that there may be some sort of Jean regulation oo -- gene regulation event in many of the insertions we are actually looking at and then the question of course finally, bearing on future development, is the active modification of such insertion behavior. I think Dr. Bushman will probably talk about that. We'll try to show you some of the elements that the retrovirus vector may be involved in.
So we look at integration site analysis as a summary again this is a sensitive method to amplify the fusion sequence Joenomic information -- genomic information retrovirus information into the genome and it generates a picture which is a restriction length polymorphism basically you see these size separated bands on the gels and these size separations indicate that different insertion sites represent different bands and then if you have complex mixtures you may have complex patterns of bands and if the analysis has been conducted successfully, there is no background am play -- amplification in each of these bands, represents a retrovirus insertion event. So one of the things that we have done is done this analysis on the SCID gene therapy trial. You can see here data from the first there's successfully treated patients, patient 4 was the one who unfortunately later went on to develop the leukemia. You can see that basically the CD3 cells are full of these bands. There are literally hundreds and probably thousands of cells that have been gene-modified. The myeloid population that you can find in these patients only very rarely carries retrovirus insertions about .1 percent of these cells. However, together with a French group by isolating colonies in highly purified myeloid cells we could prove and have just recently published that there are some myeloid cells that you can find in these patients that have insertions that are common to lymphoid clones, meaning that the insertion event occurred at a level of a potentially multipotent hematopoietic stem cell. Of course, the vast majority of insertions may or may not have occurred more at the level of a bone marrow-based lymphoid progenitor that has potentially then also a very high proliferative capacity upon assuming the expression of the gamma c receptor co-chain. This is from the ADA gene therapy trial of Don Cohn and colleagues in Los Angeles, I just put this in here as a reminder that in contrast to the polyclonal Pat answer that we have seen earlier that ADA trial that has seen 10 percent of marking but no therapeutic activity has shown very ol go clonal correction without the tendency to proliferate in two of the three patients over a course of what must now be beyond 10 years. Up to 10 percent of t lymphocytes are carried mostly by one particular clone in both of these patients. Clonality is not always an indication of problems in itself but as we have discussed earlier in some systems, very low numbers of clones can support surprising amounts of "Heat" -- of hematopoieticis. We have gone with hanover analyzed a mouse leukemia they had observed in 2002 the first indication that actually retrovirus vectors which previously hadn't been seen and those may be dose issues, can actually lead to activation of pro leukemic or proto oncogenic genes and then can eventually drive together with other factors a malignant transformation of a clone. And as we have discussed earlier and I'll just go over the data here again to make a couple of remarks that relate to the earlier discussion that we had in the French X-SCID trial we had the patient 4 and patient 5 development from a polyclonality from the CDC3 fraction via on al go clonal stage into -- oligoclonal stage into a monoclonal lymphoid proliferation. You can see the quantitative data what you can basically see and we have plod that as a curve -- plotted that as a curve but I don't have that slide here with me is that can see that these clones are basically detectable as early as 13 months after transplantation. In both of these patients. And then have slowly increased in the contribution over time. So... there may be a two sort of tier development of these clones in the sense that the insertion plus then may be the expression of the gamma c chain itself may be contributing to clonal proliferation. [ Please stand .
It is actually Cynthia Dunbar's who has shown some very interesting and elegant work using high through-put analysis of the primate models you can basically see these are tabulations of the different clonal insertions present at time points before and after treating with bibusulfin marked the hematopoietic system. I find this remarkable for tastes in terms a of points, of all, these animals had about to percent gene in the peripheral blood and even very could that level of is carried from between on 90 clones in these animals. to give you idea of the amount of clones needed to repopulate certain sections of the hematopoietic system and then going on to show that if this is submitted to a nonmyelooblative condition that some of these clones tend to disappear from the circulation and then regrow and is also a section of additionally activated clones that haven't been at in the peripheral blood before that time. So... This indicates that insertion analysis and clonal contribution of the hematopoietic system is very relevant to address issues of those finding and likelihood of toxicity in gene transfer. of course, we -- also went on to analyze retrovirus insertion sites in the X-SCID trials. We have done that both for the French well for the you heard earlier from Adrian's lab in London. is the analysis status last December of Dr. Thrasher's trial. The total number of insertion sites by now were approximately double that number the percentages haven't changed. Findings are fairly representative -- fairly well by the earlier published data by Wu and Burgess in cell leans can see that insertion in genes occurs in about 40 of cases. However, if you define a window of plus minus 10KB around those genes which thought may be indicative of some regulatory interaction, almost two-thirds of insertions are within the close vicinity of such genes and what said if you keep in mind that potentially each of these events could be influencing the of particular gene, say that about two -- you could say that about two thirds of all insertions have the potential to deregulate some the gene. And there of course insertions about 40 percent are of further away from it's also very interesting to keep in mind that these cluster the start of transcription. So of the 59 percent within plus minus 5KD of the transcription -- kb of the transcription site again almost third of them are clustered. can see that the distribution over the chromosome set and chromosomal distribution is not all to the chromosome 5 but much more to the gene content and then we also think especially the numbers of expressed genes in hematopoietic cells, in these target populations, and are currently putting together a publication to show this in greater detail.
I mentioned earlier, the transcription start side is a focus point of insertion within the gene insertions. can see that plus minus 5kb about third of all insertions related to indians occurs right where the yens are started to be transcribed course, are regulated. Then if you the types of that are and lock at a gene ontology analysis of such genes, can see that very relevance activities are at higher-than-expected levels. These are Fisher exact test comparisons comparing the number of hits compared to the number of actively transcribed genes in these cell can see there is highly significant includes steferg insertions in genes with kinase, transfer ase activity, phosphor transfor's activity and protein Indiscernible ]. So the hits that we find, is certainly a very relevant pattern of again cell regulation events. We also find -- that is that can't show you yet today but can mention we also find there is clustering in particular patients sets around particular genes and we not completely understand that is derived from we also differences between the different -- between the different trial populations where we for example, I mentioned earlier that insertions in the vicinity of LMO2 are relatively frequently observed in the French data set and within the about 500 we now looked at the British population, we haven't found a so far. But do not completely understand whether that is of statistical the that we currently have, but we're up on that. So what we should be looking for in the data insertional of genes, the relevant parameters we should be looking for in in vivo samples of course the insertion to the expression pattern of the target cells. And we find that inof in certain sub populations of the integration set is a very significant correlation between that are actively expressed in early hematopoietic cells and target that are hit. We also are assuming that in the en graftment process, insertions that are relevant to gene regulation should prevail over insertions that disrupt genes in 2 and beyond. And we have indications -- preliminary data that may be the case. And we then also think and are currently looking at whether the increase of contribution to the hematopoietic system of certain insertions is adding on
So of course, we have only asked ourselves where these vectors go, but we also went ahead and did some studies together with Cathy Anderson who is person who is most knowledgeable about the LMO2 promoter and happens to at the same institution that I'm working at in Cincinnati, and she together with us has cloned some constructs in of the LMO2 promoter that mimics certain aspects. These insertion events to further under -- aspects iners is events to further understand which the retrovirus vectors may be to these events with caveat that these are transient -- transvehicletion systems -- transvehicletion systems, we work from a working high six that because there is this symmetric insertion patterns with pointing the Indiscernible ] the LMO2 we were assuming that the enhancer is the one that activates the LMO2 promoter. We were also thinking that the position within the LMO2 locus then also of the other Jones may affect act -- other genes may affect activation. Also may to be overcome to see activation of the locations may be inert because are also negative regulatory elements.
And the hypothesis is that potentially a competent transcription or complex LTR may be stabilizing or may be stabilized by of the RNA processing machinery that there is a transcriptional complex you wouldn't see activation. So what Kathy did she basically took the LMO2 promoter and cloned it in front of a gene and put various fragments the MFGGC vector that the French group used into this location and this was also made for patient for where the insertion happens in the Inchon one the that the promoter is sitting up here and the of race was cloned the of the second to see this activation can see that if the prime LTR with part of the transgene still intact so sort of you chop the vector in and it in there in either direction, you will see some significant degree of LMO2 promoter activation. And five prime LTR if it has basically placed donor and accepter, well as the first half of the transgene intact, will actually not lead to a significant amount of activation. However, if it's deleted down to being close to identical to a three prime LTR, will see the same effect. So the question then was if you put a vector that is sort simple floor a syn vector configuration where the five prime ltr is intact and the three prime ltr is deleted to no longvert promoter enhancer activity, you can see that cloned in either direction into the patient for locus configuration, can clearly see there is not the same amount of that you see if the 3 prime LTr is intact.
So in summary we think MLV based syn at least in terms the gene activation issue itself have advantages the Indiscernible ] configurations. So to what just presented, I would like to summarize that preferred interdpraition of MLV-based investigatetors have observed in roughly percent of these insertions in patient materials about 40 percent were within the Joan itself. And 50 percent -- within the gene itself and percent of those again within the first of the gene that's transcription active in that about 30 of insertions occur within regulatory active regions of relevant genes. Insertion peaks the start of transcription. We do see in common insertion sites that have completely nonrandom and that are much more frequent than any likelihood analysis suggests and Carolyn earlier pointed out these Monte Carlo simulations. We I at the second RAC meeting in 2003 presented data that weather thought the likelihood having an insertion in LMO2 is statistically speaking about one in 100,000 insertion events and we found that to be much more clustered for several different genes. are differences before and after engraftment the distribution of insertion sites meaning we have to analyze in detail what exactly are those differences, there are differences, are in the use of insertion sites among en graft cells that may have course with the of the gamma-C but may also have of to with the activation of the gene locus that you see. then also, last but not least, I mentioned that are differences between trials and trials subpop lags that we still need -- subpopulation is we still to interpret. And as an outlook insertion site analysis advancing to next level, the stem cell clonality that we see really in a Indiscernible ] data quality because I said earlier stem cell activity had been looked at this closely a clonal level we are very excited about the possibility to follow this further in animal models and in the human clinical trials. We that for the insertion side distribution of the Lynn of clinical right to -- the clinical trials, the type and gene expression dependence and the potential for genomic side are course the most relevant questions that now to be looked at and then we think insertion analysis allow control for active modifications of insertion behavior that many groups now trying. And we for the next generation of gene vectors for clinical use sort of have a lot more dependable statistical on where insertion sites fall in certainly cell types so with I would like to close and thank the collaborators I have mentioned and also of course the -- my groups at Indiscernible ] University and Cincinnati children's. Thank you. Applause ] Thank much I we will again hold questions until we have heard the presentations. I there is going to be really information in each that will help frame the discussion coming. So we'll move to the next presentation by Dr. Copeland. Gene transfer insertional mutagenesis insights.
Thank you. have to do in presentation today is the beginning briefly describe the current state of knowledge regarding replication, competent retrovirus induced mutagenesis in mice what we know then going to turn to some unfun data from our lab which might provide additional insights into why T-cells leukemias so in the French therapy trials.
So have to show this slide to this audience but as now retrovirus is induced disease mice by insertional mutagenesis. I want to make here is that now that we have lamb PCRs we have very powerful methods for have the sequence the mouse genome where we can blast the flanking sequences, will a tool for idling new disease genes I think the the tool can be illustrated here. So in our we developed what we call the retroviral tag cancer gene database this database we have deposited the genes identified by investigators worldwide using replication-competent retroviruses mice can see should that the database contains all the published at least far able to obtain plus some unpublished data from our laboratory and can when you look at is that we now have 482 candidate cancer genes have been identified by insertional mutagenesis. That's amazing if you think about it. That's about percent the in the mouse genome and more than all the human cancer genes have been identified so currently I think are around genes have been shown to be mutated in human cancer we have now identified potentially more genes have been identified worldwide in human cancer.
Now, of course, cancer doesn't result from mutation in one gene. It results from mutations in multiple genes that cooperate to induce disease is slide from a that was published by Doug han and Bob weinberg years ago in this review they speculate are 6 signalling bat waste that must be deregulated that cooperate to induce disease. So you might not be surprised to know that we look in one of these mouse that are induced by replication-competent retroviruses that we more one integration and depending on the model we might see anywhere 2 to 8, or even more independent retroviral insertions. course, of these insertions are background insertions have of come to realize that a enough. Insertions are into indians that cooperate to in-- genes cooperate to induce disease to able to -- want mention before I show you some of the data this the way we think that these cooperating insertions occur. So what we envision is when a young mouse that's infected by a are the you a bunch of hematopoietic in white and you millions of infected cells before by chance a retrovirus ingreats and deregulates a chance gene that cell has a growth vant so that outcompetes the white cell. not enough to a chance so what we think is that these blue cells become reinfected we more integrations until by you have an integration into a that cooperates with the first mutation. That cell then has a selective growth over the blue and white that's the yellow cell, that's not enough give a tumor and thisYou have integration, mu takes, clonal expansion, integration, mutation, clonal expansion until you the number that you need to get a metastatic cancer. .
Now, able to identify these Poe tintser actions and to look for cooperating genes we build into our mouse retroviral tag cancer gene database what we call the interaction search screen if you go into our database to this screen can type in of the 482 candidate cancer want to identify maybe some potential cooperating genes, and then you hit if do that for this gene mice of the genes in our database, you would see. 17 genes in the database have retroviral induced mutations into this gene. In of these 17 tumors you have independent retroviral integrations upstream the hox a7 or hox a9 don't gene so that provides very strong evidence that mutations at a7 or a9 cooperation with mutation at mice one and leukemia induction. I'm not show you the data Indiscernible ] laboratory in Montreal using transplant studies has confirmed that hokz a9 cooperates with mice one in induction of acute myeloid leukemia you can get not only the genes this approach you can also identify cooperating cancer genes.
A case -- I to turn to our unpublished data that pertains to the T-cell leukemia problem in the French gene therapy trial. you know, we published this paper in "seens" last year so -- -- in the journal last year. He realized he into the database and asked, we have any mouse tumors that have integrations at LMO2? And what we found is we had the time this was over year time do had two mouse that had integrations at the mouse LMO2 this the site of one integration, just upstream of nontranslated Exxon three and another I think about 12kb upstream of untrans late xon one and are not too dissimilar what was found in the French gene therapy trial. Now, also looked to see if we had integrations the IL2RG gene, the gene in the therapy, and very interestingly and this is what we in our science there were two integrations upstream of IL2RG gene. The really important result was that this more to 98031 had integration at IL2RG and in the same tumor an independent integration of LMO2. So what we speculated in our science is that mutations at LMO2 and IL2RG are cooperate to induce leukemia in SCID patients. When you put and this retroviral and express it the other, that's an oncogenic because you can't regulate properly the expression of the that might be an oncogenic event. are all these according to the at least an oncogenic mutation. Now, what we would envision is this thing integration all over the genome until it occasionally hits LMO2 and you put in the patient that that has the cooperating event in LMO2 is selected and probably after several other spontaneous mutations or even retroviral induced mutations you finally get a leukemia. .
So argue this bodes well for future gene therapy trials involving other genes because it's unlikely that most you put it in a retrovirus are going to be oncogenic. to describe some new unpublished
During last we have actually more mouse tumors we have more mouse data and we you into our database today and how many mouse tumors we have the integration to LMO2, see we have five that have integrations in LMO2 and again, they in same regions, either indiscernible ] Or clustered upstream of xon one. Interestingly we have identified a tumor that has an integration at IL2rg these integrations are clustered. is the new one tumor this is in the science paper. if you look, tumor 7107 also has a retroviral integration at LMO2. So we have three tumors with integrations at LMO2 gene of three have integrations at LMO2. We have if the integrations at LMO2 are clonal if are present the in the tumor. And have that by southern analysis as we can tell, all of these integrations and these five are clonal. have to see if the integrations rat IL2RG are clonal and southern analysis indicates they are clonal. .
have also to see Indiscernible ] we did that by realtime pcr and the realtime PCR data indicates that the integrations at LMO2 upregulate expression of this is similar what was in the French therapy trial. However, integrations at IL2RG do not upregulate expression of IL2RG which believe what was what in the French gene therapy trial. And what speculate I certainly can't prove that happening is that the virus is integrating Indiscernible ] of IL2RG and preventing its expression being downregulated perhaps in some time and that that's the oncogenic event, not upregulation but deregulation, in there other are hit by retroviral of mutagenesis like I think slide one where that's been showed ever to happen. So it's not a novel idea.
So... the next question we asked is to be a common problem? If in another gene therapy if you put gene you think is not oncogenic, when you put it the retrovirus it turns it is, how is that gene therapy trial are those patients going get disease? Is this to be is this unique, this problem unique to this gene therapy trial with this or happen over over again? So we decided see we could answer that using our mouse models so basically is of what we might be the gene therapy trial. Now time in mice. we took the Muir reason stem cell virus vector and put it socks a known oncogene. Okay? And what we did is infected that into say 100,000 marrow cells and then [ Indiscernible ] stem cells and translaid in a radiated mouse and waited until got leukemia and they get in this case myeloid leukemia and we took 146 these myeloid leukemias generated by infected bone marrow with socksThey all clonal or composed of two populations of cells, which could be consistent with insertional mutagenesis. You infection 100,000 cells occasionally the virus will a that cooperates it then you will a mouse and select event out like we might envision halves in the gene therapy -- in the therapy trials so we to if insertional mutagenesis was happening in these 14 leukemias we cloned out of the insertion as we could from the 14 leukemias and we asked, we have any common of integration? the results turned to be interesting. It's kind to here but what we found is in of the 14SOX4 induced leukemias had integrations the coating of the called FFPI1, it's an important transcription in myeloid cells in it's been to be a tumor suppresser June. When -- you predispose to myeloid leukemia. are intervaiting the coating region and realtime PCR analysis shows the integrations are downregulating SFPI1
In another two leukemias, we found integrations a MAB box called MEF2c. There was no it was a leukemia gene but two of the 14 leukemias had integrations there and realtime PCR showed they were upregulating MEF2 expression, it might be acting as an oncogene so this might what you in the gene therapy so then we decided to ask, and if we could prove directly that MEF2c cooperates with SOX4 in induction of leukemia. So we put MEF2c in a retroviral vehicle ton took mouse marrow cells and infectedththe them the MFCV Cyrus alone and transplanted though the. We infected with sox4 virus alone tran planned those and infected the marrow cells viruses together transplanted this the results. If you transplant mice with bone that are overexpressed MEF2c we only had one animal die post transplant not from leukemia an unrelated cause. So that express alone do anything. If put socks in it induces leukemia, by 230 days maybe of mice dead of myeloid leukemia. But we transplanted bone marrow cells that received both can see that of the animals are dead by like 140 days of age, same exact disease, it's a granularcytic disease, the starts earlier. This nicely shows that mef2C deregulations with sox 4 in induction of myeloid leukemia. this also proves that replication defective retroviruses that cannery oncogene -- carry oncogenes can in induce leukemia by insertional mutagenesis of cooperating during process of integration. a number of other cell lines that I haven't showed that have other integrations in genes that aren't common yet but look lie very good can'tidates for cooperating genes so could that of the tumors may only a subset have only integrations of sox into cooperating genes. Won't -- the to but certainly we these 4 I'm showing here have integrations the cooperating genes.
so question is, why didn't all SCID patients develop T-cell leukemia? We have heard a lot talk that each SCID patient received multiple cells with an IL2RG gene integration LMO2. why didn't they all develop leukemia? Well, obvious answer is that because you require other cooperating mutations a chance to occur. have had lot of talk here about they could be spontaneous mutations you how many you how long that take to happen. So we decided to try and get a on that from our mouse models and we how many additional mutations does it take to induce a tumor with an integration at LMO2 or IL2RG or both together and can our mouse models provide any insights and maybe identify of the genes that might be involved in this? So what we did, remember we had five mouse tumors that had integrations at LMO2, two those also had integrations at IL2RG but one only had an integration at IL2RG but not LMO2 so we took the tumors used this lamb-mediated PCR a very high through-pout approach for cloning out integration sites to clone out as many integration as from each of these six tumors. Okay? And this is result for just one tumor. Okay? So these all the we cloned out integration sites in one tumor. is the tumorThis the new I showed not in our science paper that an integration at IL2RG and LMO2. Now, first thing we noticed is, should the LMO2 integration in this tumor is clonal, it's present all the tumor cells, first we noticed in the lamb PCR data is that we didn't have one we had two independent integrations at IL2RG which we hadn't recognized on the southern blots. In we have an integration in x on that doesn't disrupt the coating reach and an integration at Indiscernible ]. So what we envision can't prove this, but what we think might be happening here is that LMO2 initiates the tomb the cells expand, there is a very strong selection for cooperating integrations at IL2RG and that happened twice in this particular tumor. can we also have two integrations of another gene called insolent response receptor substrate plus lot other genes. you look over here, if you see a this is gene that's in our retroviral CAD cancer gene database of those genes in our database. can see lots of yeses here so a lot the genes we culling out of this tumor are candidate cancer genes identified look if see that means the cancer gene a number of yeses here, too. So if you take the this is for the tumors that have integrations at LMO2 you at we have now cloned 57 integrations from these five -- 75 integrations from these five tumors on average 15 integration sites per leukemia and we have to see if how many common integrations sites IE candidate cancer have pulled out per leukemia and average we see 7 common integration sites, they are hit per leukemia. turns out that out of these 75 integrations are common integration site genes have been identified in the database. This is 40 percent, the integrations. If you calculate based on fact that 2 of the genes the genome in the database, you would predict that by random we would only see one or two integrations at a common site of the 75 integrations just by chance. And we see 35 instead of to two. we think there lot of selection going on here lot of these genes are are -- are in cooperating cancer genes. I have just mentioned the we pulled out. see of the tumors, three of five tumors, have an integration at this sox 4 gene that just you about, one of our validated cancer genes. Very strong argument for cooperativety this and this. could also see that of genes have integrations at mess 2c. Who ha? is the gene told you about. Three of five very strong evidence for cooperativety. Indiscernible ] This is not a in the retroviral cad cancer gene database but it's hit and among these five tumors again strong for cooperativity. Genic but very strong evident for cooperativity. Genetic.
We think multiple are required to produce a tumor that has deregulated IL2RG and integration in LMO2I can't tell how many cooperating mutations we don't all of those mutations in the same cancer cell the way these tumors are generated. But think our data that is you a lot. So can cancer gene therapy complications be limited by transplanting less cells? Perhaps. We talked that perhaps not. Another issue that's brought are the cancer-free SCID patients leukemia prone? Because if our mouse models and the data I'm showing you today are real and, you are explaining what's goes on, I prove that, then would argue that all of the SCID patients received one oncogenic mutation because they received the -- retrovirus that carries IL2RG that's being deregulated. I think very interesting now that the third patients development leukemia and will be interesting see what happens as the other ones progress. Then is the part of my talk also, genes are identifying Indiscernible ] mouse LMO2 tumors relevant to human tumors that have deregulated LMO2? I want to show a slides to close out. The way we to look at that we went into a microarray database maintained by Tom look at Medical School, the Dana what they published paper a years ago in cancer cells what they did is Indiscernible ] 30 T-cell human Lou leukemias and they published the data put it a website so my bioInformatics person beamed the data, this is blind, their data notous and she how many of these tumors have high LMO2 how many have low LMO2 expression? it it about and half, 19 tumors had high LMO2 expression and had low. So what we did is we -- we -- we stratified the two tom more types and then -- tumor types and then we asked which are the genes whose up regulated consistently give us a list of genes whose express is up regulated in the tumors with high LMO2 expression. could a list of 50 genes that have P use of .001 or greater so there were 50 genes consistently up regulated the tumors with high LMO2 expression. and wanted to show you the next slide that are the very of that this is the Jeans from their data -- genes from their database at top the list. this is another -- must be a PC/MAC problem is the p values for the top 10 genes. can see that the P values are incredibly high. This is the fold overexpression of theThis is the gene products. there lot of interesting things in this data the we asked so we got at the the list. asked how many of 10ers . in a retroviral cafd cancer gene database? and can see the indians in red are genes the database. percent these genes in our retroviral tag cancer gene database. What are the genes here is mess
This is a partial subset the genes. So one. LMO2 tumors has a retroviral integration in this gene LAP this is the second gene the top. Okay? As I said have three integration at MEF2C. Now, didn't an integration at this one gtpase but had an integration in an LMO2 tumor this gene. It's a gaw Nene exchange factor of this that turns this gene. We [ Indiscernible ] you can see number of genes in common and some the genes are Pat waste either upstream or of the genes here in the Pat ways. account for the genes in same pathway the that are at the top the list we hit in those five LMO2 tumors so we think that the mice data is very is going to be very relevant, informative with respect to the French therapy trials. I should say that I told you we have one IL2RG tumor that has integration IL2RG, that doesn't an integration in LMO2. Now, in that tumor we cloned a lot of integration sites. of the integration sites is in notch one as now, notch one is present mutations in notch one are now been recognized more than percent of all human p cell leukemias so could be in this particular case, notch is the gene that with IL2RG in induction that leukemia. would very interesting to see in that third patient if of the mutations are in notch or in the notch pathway or alternatively genes the LMO2 pathway. don't think we know a lot the LMO2 pathway.
So just to conclude, based on this data, we would speculate that the French SCID trial with the IL2RG jibe may represent a special case in therapy and isn't predictive of most other gene therapy trials, know you in cases where -- often we would predict most when you put in a retrovirus wouldn't be oncogenic and this have talking so might can we correct problem? You know, how could we change the protocol to make it of a problem? And, of have talked about the potential of transplanting less cells, maybe using syn vectors. But these -- Indiscernible ] but even if you use syn vectors still have to drive expression of IL2RG with the vector. you going to use as a promoteer? If you use something again that's ubiquitous promoter, the gene is still being deregulated, so it's could still be an oncogenic but the virus itself would have less probability of being insertional general so you would you know, alleviate lot of that problem but you would still presumably perhaps be giving the you know, one oncogenic hit. you get around that maybe by using the endogenous IL2RG promoter if knew of what that was. And could drive expression of IL2RG within the of maybe syn or Lenny virus off of an endogenous promoter. Those someSo just the last slide, this the people did the work. All the work was done in collaboration with Nancy Jenkins. Paul did the -- the gene therapy type stuff. Indiscernible ] our bioinformattics person. [ Giving credits ] .
With that I'll and you your
Thank very much. We're going to move to Dr. Bushman's presentation. Retroviral integration site preferences. After this we'll have for the presenters and also a general discussion of this session. Japan thank you for invite
you for inviting me to speak. I'll briefly summarize we know about integrate in the
known. -- in the human genome. Initially HIV integration targeting then retroviral targeting including MLV some of we heard about and lastly speculations on the mechanisms controlling targeting some on to try to manipulate the targeting mechanism. I'll skip some introduction. DNA and joining Indiscernible well understood now. And the key the viral encoded intergrace enzyme for the first DNA and joining steps and I'll be mentioning intergrace enzymes was go along. So the of the sequence the human genome it became possible to study integration targeting a fun way by infecting cells waiting until reverse transcription and interfrags was complete and cloning out large numbers of integration sites made by infection the human genome and so we have embarked on a systemic campaign to study eat mechanism the phenomenology and mechanism this and I'll be mentioning data on HIV, ASLV and MLV. So we wait hours until integration is complete then isolate and do essentially a ligation mediated PCR protocol to capture junctions between the host cell DNA and viral DNA. Then map those on the genome and the distributions.
we are probably north of 5,000 sites for different viruses and different cell types that give a pretty detailed picture now of where integration takes place some these viruses so this summarizes data for about 3,000 sites. This the human chromosomes one through 11, x and then 12 through 22. The cyan colored lollipops show the HIV integration in cell types. The lavender the Burgess data on MLV and aslv the bird retrovirus so what's shown along the of the chromosomes is gene density. So more red means more gene dense regions. so of the story I'll be has to do with relationship between retroviral integration sites and genes. So let me focus on HIV. can see for example in chromosome 19 very gene-dense. Lots of integration sites. Chromosome 18 relatively gene-sparse, relatively this is of making the point that HIV favors integration in transcription units. So we see that in several different cell types. This shows the analysis. of what I'm showing you is condensed. If you in several we look at HIV infecting sub t1 T-cell line, primary lung fibroblasts you can integration is a of the -- the time in transcription units and the of the genome the whole genome is probably about a third transcription units. There is a strong bias in of integration and transcription units. So one question raises is it about transcription units that's for integration? Is it that they are active or something else about being transcription units that's favorable? We have done match transcriptional profiling of the target cells to measure the gene activity in the cells compare that, ask how that influences the distribution of integration sites so have used various AFI chips and just the simplest conclusion is if take the median signal or average difference used be called the genes that hosted integration events and admired all the genes the chip, it's about as high. are seeing a in favor of active genes you can do kinds of comparisons. For example, can compare random integration sites to the experimental population get the same of answer. Here it's broken out if detail we have the on the chip and them into 8 bay their where is the lowest expressed and the highest expressed then we the that hosted integration events and distribute them in these categories and them up. see that there is you greatly less frequent ingreat lakes in the low expressed genes and lot in the most highly expressed genes and it's very statistically significant.
The point about cell type transcription came up earlierWe broken out these data asking whether we see tissue-specific transcription patterns and we in the AFI data and then that influence the distribution of integration sites? We can see modest but nevertheless we see favored integration that are particularly active each type have looked at. So this translates into quite strong clustering of integration as you go along the chromosomes so we have product out transcriptional intensity versus ingreat lakesal in-- integration al intensity. added and it into bins. Along chromosome 11 or can see the human genome is just remarkably heterogeneous with regions of clustered genes and highly expressed genes clustering together with other features like high GC content certain classes reef petted elements and -- classes of repeated elements and similarly chromosome 22. you the ingreat lakes can see the strong peaks corresponding the regions of hygiene density and high transcriptional intensity. There are lots of little areas good clustered this area interspersed with relatively less favorable regions.
So just to summarize briefly the conclusions for HIV, HIV favors integration in active genes. This is seen in cells and in cell lines. I should have been working Indiscernible ] carrying over a gene therapy trying using vectors and we have at the cells they have tran duesed from patients we have seen the picture numbers lower there. see influence of tissue-specific transcription. We see clear trust stirring of integration sites and we see the favored regions are composed of relatively small regions, interspersed with unfavorable regions. And so this leads to the of this targeting in the HIV life cycle. Most HIV-infected cells have only short lifetimes, two so we imagine it may be targeting integration into active genes helps promote rapid gene expression and of new viruses making hey while the is shying essentially -- making hey while Sun shuns I was very surprised to find other retroviruses, he could retroviruses are very different targeting physical tis so this allows me to summarize that. What's shown here are four HIV integration data sets about 500 integration sites. This the Burgess MLV data this is our data on ASLV. so this is fairly detailed statistical analysis, basically even Jean -- all -- each Jean or genes were combined or broken into ten intervals. .
Similarly disfavored outside genes. for MLV this is the Burgess effect can see favored integration near prime ends of transcription units. For ASLV, avian sarcoma Lou coast Sys can see very little pattern really. you can see a slight favoring of transcription units you really you see slight favoring of active transcription units for these viruses. But really all three are quite from one another. ASLV doesn't show this favored prime targeting and HIV shows this strong favoring for transcription units in other viruses. this is very thought provoking. Just to summarize this bit, mlv favors integration in gene start regions and CBG islands from Sean Burgess. MLV recent work [ ] MLV niendz of is more effective gene trapping than HIV. If you have a gene without its promoter gets turned on effectively by nearby transcription signals so that speculates that MLV owe voofld this way to take advantage of nearby transcriptional signals to boost its own output. ASLV is weaker. It's worth about a possible vector for therapy because of its favorable targeting. All tret row -- retroviruses studied far have mechanisms different from each other which really leads you into wondering the mechanism for of the earliest ideas in the retroviral field was that maybe having active transcription open chromatin be favorable because it would expose DNA for integration. and think that's probablyI think the systems been looked there is a bit of favoring of integration and active genes but because MLV and HIV are so from each other, it really seems there more to than that. it seems likely to me that there is specific interactions between integration complexes of retroviruses and locally bound proteins on target DNA which influence integration targeting. is work yeast that this very can take at for those systems. ma that provides a thinking about retrovirus,
Artificial tethering, we able to show a ago that if you joined intergrace enzymes to sequence specific ending binding domains you see integration too preinvestigated locations in tra we admitted tipped everything to maximize the specificity seen here. It's not been far to translate these into effective retroviral vectors at least it provides some for the idea that tethering could work to direct integration naturally. So I'll conclude by mentioning on going experiments. are trying to figure out the viral determinants. Collaboration with Mike and his colleagues and the cell of immigration targeting. So with have been studying Kimism. been asking how that affects integration targeting again this of building up towards the idea to what extent can manipulate targeting by retroviruses and so I'll just you one event. is all pretty early and I'm a little uneasy saying things that not hold true indefinitely but... uhm, This we have enough now I think this is going to stay true that is if you move the intergrace of MLV into an HIV back bown, we have favored the transfer of it Indiscernible ]. So that's what shown here. Here's transcription start sites. Here's control HIV. Here's with the MLV intergrace and here's one with MLV gag in it actually and here's CPG islands which are CBG islands are regions in the genomes probably landing sites for binding proteins. can see the strong preference of MLV in this HIV the MLV intergrace. I should this isn't the whole story we still see considerable favoring of active transcription uses by this modified drivity . have doesn't exact of you to do for gene therapy. have transferred the undesirable members Indiscernible ] while tank the undesirable features of this is a towards trying to manipulate specificity general we are excited about it for
So this just is what I said before. Targeting in other great lakes systems -- integration systems. My feeling the retroviruses are the best game in town. SUVs look like -- SIVs look like and those vectors may be of interest. Others worry me because they rearrange the DNA at their integration sites which is probably very bad. DNA transposeons. I used it was too dangerous to put an enzyme that creates double strand breaksCells gene therapy. It might too toxic actually was telling me he has some specific data coming along that may suggest that this isn't the case. Obviously mom ol gas -- whom ol gas Indiscernible ] efficiency seems to be an obstacle. Of course, if it became would be the best that doesn't seem like it's coming anytime soon. [ Homologous recombination
I'm optimistic that we can make progress too long. So obviously, switching integration systems to something like ASLV might to you leave behind that are unfavorable and MLV or in HIV. you to be careful because even you go to random integration in the human genome, remember I that the human genome something like 33 percent transcription units you certainly wouldn't be dodging genes, would just be hitting a little bit less. see it shouldn't be possible too much longer to identify these kinds of interactions. We and in field are working this quite actively. if really knew about what these interfaces looked like you might able to mutate the intergraces so they don't as well anymore so diminish undesirable tethering interactions. in the long term you would do like this, generate or engineer these kinds of interactions or make these kinds of fusions work. It's worth mentioning the yeast system I mentioned the TY elements previously the voytas lab has done beautiful work reprogramming these kinds of targeting interactions to make TY5 integrated new locations the yeast genome. don't think it's unreasonable to imagine this thing might be possible soon but what's likely is that we'll what the interactions are driving MLV tethering or HIV tethering able to modify those so we might at least to sort of random levels of integration.
So having made some provocative claims, let May stop there and answer any questions. are the did the work in my laboratory. [ Names .
very much.
would to do now is open for discussion starting I think with questions that specifically address points raised by our three speakers and then gradually moving toward discussion of the general discussion questions for this session.
Doctor?
Do Von Kalle mentioned in of the ovens it in the British trial there doesn't to be the incidence of leukemia that's in the French group. And so... The question I have is any in the vectors in those two trials rore there differences in the transduction efficiency between those two trials far anybody knows?
I you can summarize to say vector except appear. No microphone
I guess my it comes of we the the first session. that is that is a window between benefit and developing leukemia, it becomes very important to consider the actual of the virus or the transduction techniques being used buys minor or differences between these might make big in the number of stem cells ultimately transduced the number of vector copies per cell.
I think that's certainly good point. But think of the things have heard from these presentations in a way that we know from the third participant that has developed leukemia in the French study, that integration into LMO2 itself while it's certainly not a thing doesn't ensure that tumor will develop and Dr. may want to comment more on that he presented about hitting in a common pathway that might be used for example by LMO2 or other oncogenes and the potential importance there.
I I don't really new to than I presented in talk I think it's a very real possibility that of LMO2 might be another gene the LMO2 pathway or... the notch or notch pathway. the data from our models suggests that notch might also cooperate with IL2RG.
Dr. I have a question for Dr.The experiments you did with sox I'm that in a replication defective retrovirus? I guess the with regard to SCID trials is not what's the LMO2 pathway the gamma c pathway. ever in your experiments put gamma in a replication defective virus? picked out insertions?
Not the going on a of labs tried to induce leukemia with an IL2RG virus in mice. don't know has succeeded. .
That's my understanding.
are also doing studies we are taking an IL2RG and LMO2 virus and co-infecting bone marrow to so cooperativity that either -- show cooperativity. Those studies are on don't have any real data. .
be a very interesting to do. .
Dr. Von Kalle presented elegant studies looking at these in the context of T-cell integration sites and this real massive pathway different integration sites. But noted there are relatively few in the myeloid period. you look the b cell periods since were supposed be
we also looked a couple of b cell samples but the b cells are marked and around percent. They actually stay recipient origin I think of the Indiscernible ] transportation trials also... and there is few insertions in there. It's not to the T-cell population..
We able to induce T-cell leukemia in mice with the LMO2 in the manner with did with SOX4. We are now cloning the integration sites from some of those leukemias to see if LMO2 in of those leukemias intergrade at IL2RG. I would be very interesting if we found it. I was getting at the point that the real relevant experiment would have the gamma-c and the infecting virus as in the gene therapy
Sure, sure. To see what other
I said we're doing that but...
Okay. I have a question for Dr.I'm wondering if say a bit more on what you feel may control even when you exchange the intergrace still the HIV preference for transcribed and how you think that may or may not to using lengthty viruses in -- lenti viruses using gene vectors the
The data is early but may see little bit of effect with -- by transplanting MLV gag. So there may be some mechanism that helps it integrate into active genes. So one speculation this is pure would the point be great lakes could have some influence. They are supposed to have integration during the cell cycle and targeting could be a secondary consequence of that that's one the idea are investigating the moment. Regarding toxicity, I guess can well as I if you integrate the region of a gene, there is a good chance it's to be disruptive particularly if in the forward orientation the splicing and polydentlation signals bringing in disruption transcription of the cellular gene. If you are with a tumor suppresser that's bad there is Indiscernible ] and something to happen that before you see another event. Aldini's experiment is interesting. MLV were better trappers than HIV. They may be distant from mo motorers that I may be generally positive for the lenti-based vectors than the MLV-based vectors. It's speculative and you from the I showed and form your own opinion. .
Dr.
to the or not oligoclonality is a predisposing for leukemia these cases, were you able to compare Indiscernible ] 4 5 they became frankly leukemic to patient 1 and 2 to perhaps to the UK patients and see there was in terms of the clones that able to recover those patients? .
Yes. That's part an ongoing study are together the insertion sites the patients who turned leukemic and compared to those haveThere are no striking differences. the things that were notable is that we found several insertions into the vicinity the LMO2 gene in patient more than the other patients. Another thing that we're currently close to looking at is is a difference between tee relationship to gene expression in patients the others. was mentioned earlier that transplantation was of these patients always had very cell numbers. of the issues be that high cell numbers indicate a propensity to proliferate that it's higher than the other patients there are that were counted were Indiscernible ] and it could be there is a there also. .
Dr.
Two quick comments on in terms of table 2, the that is you have namely 3 times 7 fifth gamma-c positive c34s per kilogram I'm presuming that's not gamma-c it's just dd34. actual dose of mar Roy you would out of children, the dosing are seeing the and is usually you see with a peripheral blood mononuclear cell graph after sub stimulation. The the bone marrow be somewhere in the range of the one to five times to the per KG as the maximum that you can get of the amount of that can of a this size. So my don't know what the actual number of gamma-c positive populations actually was.
I would -- table there from does in the footnote that the number of transduced cells by mu no fluorescence -- immunofluorescence but I can't speak...
Yeah.
-- further to that. don't if someone here can.
Well, I know it can't be right. Looking just at immunofluorescence, it doesn't take too account that some of these patients were expressing truncated gamma-c to start with so I think that table needs be checked with the publication and corrected.
Good point. Are there other specific questions that would ask the speakers before move a general discussion of these particular issues?
Yes?
One other brief point and that that Dr. had raised the or not kids with SCID are in fact unwe come leukemia-prone -- quote, unquote leukemia-prone. To knowledge if we look at in the of marrow transplant experience these kids normally would not more than about in the absence of some therapy in the transplant group, we can say that don't of any instance a TALL, ALL in itself not in the skids this is looking back over the last I have been watching the SCID transplant they were first down I was an on case in '68. know of any instance in which that's occurred. And terms of other types of leukemia -- of malignancies in the context patients who have received conditioning with boo sulfate and indiscernible ] is a striking dearth of any secondary malignancies Thank you. I we are ready to Move to our discussion questions. That were set and want to invite everyone to participate as we go through those. A number of these questions have already touched upon before. And they are here the screen. One important thing been discussing relates to factors that may to the potential for insertional mutagenesis including dosage number of vector insertions per percent average et cetera. I think we have cussed this in large measure -- I have discussed this in measure already. We have certainly heard too much is probably not good and too little is equally not I believe we've heard that we don't know where the point is in that range for efficacy. And safety, despite the fact that as Dr. has we do have data to that. hand, I we have perhaps heard some confounding information that the subjects in the British trial don't necessarily or add another dimension to that. And I'm wondering if there are further that speak to that particular
I was about one of the differences with the British trial is the the growth factors they are used that brings the of larger of how you culture the prior to transducing them. And I guess I'm more looking for than you that would make a significant difference potentially to the integration site, for example? And a semi-related believe interests that the envelope should difference the integrationBut it make a difference the cell population being transduced which in turn potentially would influence integration sites. So I'm really just looking for some comments or from the speakers.
have compared HIV entering with using the HIV envelope or VSVG pseudotypes and don't see any differences between so far. It could always turn out downstream find something for moment they seem simple clear just car will -- just to clarify, though would be a in us population of cell culture lines?
Rather than the CD34s which are clearly quite heterogeneous.
Okay. Regarding growth factors, is there too have compared Indiscernible ] versus growth arrested by serum starvation and there, too, so we haven't seen any major differences. Genes seem to be transcription units are favorite in both cases with lenti viral vectors so that's what have specifically to those questions.
Is there someone the that might able to peek to the differences in the cytokine regimens in the infection that is have heard about? -- protocols have heard about? There is no to is that my interpretation of that is I see? So, yes... Dr.
Not regard, the cytokines but regarding the envelope, is work from the Bodine's in NIL that shows that the more primitive -- niThat shows that the more primitive stem and the number of receptor Indiscernible ] [ accent coughing low microphone .
I thought Von said that the in the two trials was identical. Is it?
I think the envelope is different.
Okay.
The vector backbone is identical.
Given that we have heard and then Dr. about transcriptional predilection for Maloney based vectors, I'm wondering if there is anyone who able to comment on the different transcriptional profiles that might be seen the various cells in the CD34 starting population, particularly given that perhaps with the GALV envelope, there is a predilection to infect a slightly different population within that mix. don't know if anyone has information on that. Dr.
I don't have information on that point specifically. But it's certainly clear that most the that are being transduced are committed progenitors already they are not true stem cells. And the of those cells isn't understood well and it's that they the that are giving rise to the leukemia so if we understood and could purify true stem cells better, then we make some real advances. But it's a stem cells are clearly 1 or of the infected and reinjected populations.
Dr. launch
So perhaps from a naive perspective is fair to that this population in these studies both amongst individuals and comparing the studies may be different based upon the regiments, how are pro cured and how are treated during the time or this a fairly although heterogeneous, well defined population of cells that's reinfused? Since we know they are, we certainly don't know how are from patient to another. .
But using markers that are known have not been well
The markers that know don't really help, I believe.
Dr.
However, I would once again reiterate, the point that the patients who these very high doses appeared to have higher propensity, I just -- again, the doses are looking at are doses that are really very high in terms of what you can pull out of kids at that in terms of a dose kill gram of CD34s particularly they have operated them. So we think what Dr. Von Kalle said before is of in this regard because you could have some selection in that regard.
Dr.
on comparing two trials. Dr. Con valley allude -- Von Kalle alluded to the trial where they were induced with fewer cells per kilogram and I'm wondering in the British trial. the UK trial and I'm wondering if in the to the to the to the range where the French trial saw efficacy but no adverse events.
I'm not really sure. I said -- I'm not really I there a between the cell numbers. I think the cell numbers per kilogram are fairly comparable for the occasional Indiscernible ] patient in the other
of the article in the Lancet said that they the order of 8 to per kilogram. So that actually puts them at the high-end of the French study. you use the microphone, please. .
Table in Lancet that gives you the of those cells. Those numbers All right. you have to multiply by a percentage they are around what, give or yes. On the high-end I guess. So I guess you're right, Neal, are little bit lower.
I think we have actually touched on many of the sub questions of question do want to particularly ask about and that's we have heard particularly from Dr. about the nature of the transgene and Dr. Copeland's idea that there are certainly some transgenes that may provide a first hit or allow preferential expansion of infused cells. question that I have in this regard, one of your studies you actually identified a gene you wouldn't have to be a cooperating gene in your SOX4 study. And I wonder if can give us any sense of how we can think about that in real terms because we might think that we would know which genes could be he could genes but you suggest that are others we wouldn't have suspected.
Right. the system we at that as a plus obviously because are used in interesting this technology to interested in using this tech to find newThere wouldn't be any evidence that a chance gene. you know, there reason to think that if what we think on in a therapy trial is happening we are going to hit other genes sometime instead intefd LMO2 there is no in the literature to suggest it's an oncogene but nonetheless it to still be an
Are any criteria, loose that we could think in terms of what kind of genes that might be used in gene therapy in other situations should raise questions?
You at the lakes site or the in I was of the in the vector.
I mean, one thing I think for sure people do is look in the retroviral tag cancer gene database you a therapy trial. We have genes in there now, the in the genome and I there is a common site I question using that, you know strategy. Or about it more anyway. There are latest lots of homologs the genes which haven't been hit yet. You a in the database that's in the database but it's a homo log. a lots of in the pathway. It's hard to, know, to really put a answer on
Right. I realize that. I think it's an important issue.
think the issue are going to be a inform Jones we haven't identified that don't look like cancer genes. see these a lot. And then we do these mouse studies to find out they really are cancer genes and uv wouldn't have predicted -- MEF2C is a myocite cancer factor thought to be involved in muscle but there isn't data would be a leukemia
Dr. Will some.
So -- Wilson.
I guess to expand on your comments beyond looking in your database you suggest is there good preclinical model that if you have a novel transgene you wanted to do therapy with to assess its potential could generalistty what model would you use? .
Well, again, you could put in a most virus like we and put in a mouse if you get leukemia. Again, the IL2RG virus my understanding is don't get leukemia. you know, it's not -- is not perfect model system you could transplant it in monkeys or something I guess but sure you get a different answer. there is a perfect assay system.
To just finish question 2, there is one particular that I think we haven't touched on and that's host factors. have listed here participant and other issues that have discussed. But I certainly and I'm going to ask Dr. Copeland to comment the genetic of the which is going to be something very difficult to really get a handle in this case, can certainly big impact in the mouse models he studies and I if you could on
That'sSo the background have major effect. So can have two mouse models of myeloid leukemia basically the same leukemia, we use insertional mutagenesis to pull out genes. And in one model of the time hit a gene say EVI1 is a gene we cloned and the other mouse model never it and we really know why. So it's clear that can have a major impact that we clone out and so that's obviously going to I would to be in the human well. .
Clearly we have no real way of assessing how that may affect the outcome patients that might be treated in these protocols.
Right. But you would certainly predict should be important. And could of why the third patient might not have an LMO2 integration.
Right.
Yes, doctor.
. I was hoping to hear more about vector design is a current vector this retroviral we are be asked to make a recommendation should that vector be allowed to be used. part of that at least to would be how close are we to having better vectors that could get into patients relatively quickly? So do insulators work in do these syn vectors work? we months away from having those to go into patients? Maybe it's five years and we neat to -- to use the retrovirus. was hoping for how long should we use the model t Ford before we move to something bigger better. like Fisher made his decision already. not going with that. Should we say that should be the here also? don't have enough Is there someone here who can comment? Rick?
Before Rick don't want to put words in Dr. Fisher's so what reported today about his to move to SYN vector that's I understand from him and the regulatory people in France. know whether that's firm sort of time limited decision or if it's on his part maybe no, sir. That's certainly the is going. We were a and they were talking about lenti viruses everything put in beforeThey weren't even considering retroviruses.
Well, I can just briefly comment on work from Karl union. As sort of a pilot for lenti viral vector studies, they have been putting an anti-HIV gene essentially an Indiscernible ] into x planted T-cells from aids patients and putting them back in again. And to be working pretty well. The cells don't last very long being mature cells and there is every reason to it will be reasonable forward with those vectors based on they have seen. San Andreas?
--
Christof?
I would briefly like to add a that the syn vector for the retroviruses are like the square that are lot better store in certain places but a lot harder to produce, also. (Laughter).
But... It's very difficult to make retrovirus in vectors especially at large-scale. At sufficient titre. And maybe I can add a little question to there also, considering a syn would be used in a retrovirus and that those antivirus wrecktors integrate within the genes in about 306 to of the times once the gene in the retrovirus is taken care do you really see a biological safety advantage to lenti versus retroor do you think that an open this point?
don't know. don't think there is a major safety for lenti viral vectors. hard know because we can't directly compare the syn of one versus the the other. I'm surprised -- just one comment. somebody knows about that I'm surprised there hasn't been more animal model studies comparing sins to nonsins vectors. might able to do side by side comparison and how much safety benefit you get. Seems like that would be helpful for these kind of discussions. .
Dr.
I just going to we are actually collaborating Chris Baum through the national toxicology program, are just started with designing a large-scale animal study to those kinds of comparisons sin versus nonsin, lenti versus gamma retroviruses effective MOI on vector well as also looking at insulator elements. that hopefully, it will not be immediately forthcoming but long-term of that will come to light.
We thought about the with the SOX4 where you can induce tumors Indiscernible ] via insertional mutagenesis we about it, also, compare a sin SOX4 a regular SOX4. The problem -- it's hard experiment control because when you use a sin virus, you to drive the expression your from an internal promoter. are not really comparing apples and alles. are comparing apples and oranges and the effects you see might not be of the effect of the promoter effect or both. So... you know, probably are to see differences but what's the difference due to? .
I be sure we time to have discussion our remaining two questions. Although we may return to some of these perhaps later. But our second question really whether or not there are modifications that we need to in the current safety monitoring schema. We have seen the power of the current methodologies used look at integration sites. But I think questions concerning how we might use that there is a way to use that data in a more -- a better way to identify potential individuals who might be at risk of developing tumors, if are people particularly Von Kalle speak to that point.
This be regards the screening or potential prophylactic of insertion
I think both of these. the charges that we have is to make a recommendation as to whether screening or other measures need to be pod fight in light of what we now understand with this third event. And certainly some the screening relates directly towards looking at the insertion in these individuals which is, I think, why this question in this particular
you will allow me I make sort of too tired statement to this. The first statement is don't we have any data that proves that we could predict any outcome of clonal proliferation based on insertion site data prospectively because most of these studies have been either of added realtime or in a way retrospectively. is also quite amount of work to prospectively screen that. I said, I think we will have data to this year where are attempting that for one particular gene therapy trial. I think that again is more of a scientific question. The other question of course also what type of a consequence could that have that even if an insertion were observed in say oncogene or oncogene-like structure and then a clone would start proliferating some degree without however causing any objectively notable sign of such as an elevated Luke he could sight -- he could sight County node swelling or of what consequence would be. he could sight.
Although are probably one the plays that terrorize this -- tries this intensively we are still fairly -- mixed feelings about what and cannot be at this
Dr.
I to on the fact that this third patient a very good case to think about how monitoring can be done. As I said before, on the -- mid-November this patient went through the annual follow-up. It was Indiscernible ] [ sheriff heavy accent
[ Chevy accent, low Mike
Pardon me
[ Heavy accent ] [Indiscernible ].
Good point.
If could comment, it's a numbers game f millions of integration sites are never sure to there aren't any hits in LMO2 or other that you might concerned about. that you looking at clinically when a bad event develops. don't short of single cell cloning, then expanding out from cell putting into patient or a small handful, it doesn't seem likely that you could either look beforehand what the integration sites are and put back unless your material can tolerate single cell
think these are actually correct and very helpful points to have raised for discussion. We're certainly approaching the time scheduled to for lunch. don't want to truncate the discussion. Dr. you have you would like to add the discussion? Or things may have missed ?
Uhm... I mean, I think that discussion covered probably the sort of don't think need to further there. So...
Are there other comments, comments from the public, Dr.
Just a quickie. show this later on this afternoon of things that might put the X-linked SCID at particular risk for as well which hasn't been adequately that to my is the fact the majority of these kids are absolutely NK deficient and often times remain NK deficient thereafter. question that hasn't been addressed so far in the retroviral vector mode but clearly in several other types of -- of -- of viral modes, would be the fact that NK cells in fact can distinguish between viral versus those that are not certainly those cells can't also distinguish between leukemic versus normal hematopoietic cells. These would be at particular risk by virtue the fact they have no such capability.
Are there other comments? I'd like to thank the speakers, again, for their very informative presentation. And have arrived at lunch. [ LuncheonPlease standStand by, please. Please stand transcript. Stand by, please. Please stand by for realtime transcript. Stand by, please.
Beef a quare run. we have a quorum. This are going to shift our focus to bone marrow transplantation for severe combined immune deficiency the reason being an obvious one. Bone marrow is the of care for babies newly diagnosed with SCIDs we wanted to obtain baseline knowledge about the efficacy and the risk inherent in BMT for this patient population. And we have invited speakers who are viewed nationally and internationallying toed spubdz you believe to be the experts in their field. Dr. Rebecca Buckley from Duke University is going to with an overview of HLA identical and haploidentical BMT, the Duke experience. Dr.[ Pause ] [ Standing by for audio .
Thank you, Dr. the organizers of the symposium for inviting me to come. As Diane said, the charge I is to give you update bone marrow transplantation at our place, but also, to sort of summarize of other studies that have been published. And just to again reiterate what this condition is, is really a diverse set of genetic defects and one thing that is unique all them is they have no T-cells, and some of them have cells and some of have natural killer cells and so there are are different no types. Just -- no types. to put this into perspective, there are at least abnormal genes that were mutated causing human SCID. even hearing about this one right here, but, uhm, the major signal transducer from the GM ma c when the for that is mutated you also get SCID. this is another cytokine recent their when mutated gives you SCIDs. There are five genes that when mutated give you SCID. And we have 88 efficiency and CD45 Very few the reports break this according to GENOTYPE. Most of these broken down into b positive and b negative types of SCID. It includes x link SCID, and others. Also CD gamma I'm sorry, delta and epsilon deficient patients have b cells. And patients have a few b so it's really helpful to refer to these b positive and b negative in terms of trying to evaluate outcome. Just to give idea about the relative frequency about these conditions, these are all the SCIDs we seen at Duke over my career, can see that of these are X-linked we learning now that after 88 deficiency the third most common SCID is il7 receptor Alpha chain deficiency. There still up here that we call so many al recessive our unknown but have a known mutations -- unknown mutations as yet. .
To talk about transplantation, it's been known since Dr. Goode did the first transplant in 1968 that all types of SCID be treated successfully bone marrow transplantation and they have any T-cells they don't need any pretransplant chemotherapy but until the early 1980s unless a baby had an identical sibling transplant wasn't possible. I still that of the most important developments the treatment of SCID over the past almost 23 years now has been ability to use parent as a donor. order to do that would have to remove post Indiscernible ] T-cells the donor bone marrow cell suspension and do you that also is best way to prevent GVH you don't have to give immunosuppressive drugs after the transplant you do, then that sort of defeats the of your transplant which is to give you -- give the baby immune reconstitution. this is what happens if you try to use anything than identical section and when you give an HLA identical sibling unfractionated marrow can see this is graft versus host this is caused the T-cells the graft recognizing the foreign transplantation and the of the baby and causing GBH so the that been using is the method was first published in 1981 by Ricener and his group New York and it takes a lighter bone marrow cells -- a liter of bone narrow from the donor, usually a parent, getting a buffy coat and [ Indiscernible ] that aglut natures of the cells and not others. These are mainly mature marrow cells these are immature cells which still contain T-cells so to get of those we take of the fact that sheep right row sights bind to human T-cells and by doing [ Technical termYou get rid the once that don't react the row are used for the There are essentially no lymphycytes there at least aall immature cells. There are cd34 cells or dedritic cells. are not cb3 positive cells. The remember is all of these different ten different molecular types of SCID they all have thigh must and as rich -- thymus and rich said earlier it's the size of of end your finger.We know the thymus is long you put good in the garden garden grows. So this doesn't have any lymphycytes in it it doesn't have any corpusles. This is a picture of a SCID thymus show you the epithelium that's present but lacking them mustites. But if you -- thymusites. if you put cell suspension like I showed you last slides neat vein of a child with and you to what happens, can of our earlier transplants we were very worried for the first 90 days because nothing seemed beAll of sudden we saw CD3, CD4 and CD8 positive cells appear same time, we saw the phenotype appear, we also saw the fraction appear. this was a child who had Candida meningitis with no siblings we used mother as a donor we to make sure these were donor and we found that 100 these were females cells knowing even though we hadn't given any T-cells and had not given anything that looked like a little no sight -- lymphycyte, that this had in the microenvironment that can mature these sim cells into functioning T-cells this what happened with his B-cell function. It took about two years before we saw good B-cell function.
But we have seen this pattern over and over for 3 years that it takes about 90 to 120 for stem cells to mature and to normal mature T-cells that can function as T-cells. So able to apply this in a number of transplants over the last 22 years, and the thing to remember that most of these children not have a HLA identical sibling this is why I able to use a mother or as a donor was of the most important developments. The other advantage that this has types of for example matched unrelated donor cord blood is that we the donor available immediately and we don't have to wait. also don't have to wait for chemotherapy and with a baby on a ventilator you can do a T-cell depleted half matched bone marrow transplant because you don't to chem MOA blags so the results -- chemo the results of our efforts the last two decades shown here currently 77 percent of these children are surviving, all of the HLA identical recipients are survival of striving and of half matched are surviving. And it doesn't really matter which genetic type, there is a 79 percent survival in the x link, in the -- Tad -- ADA deficient, this is because these 2 have had gene therapy in Italy five these are on peg ADA. The percentages of surviving about the same in the different molecular of SCID. what did they of? Well, they of things they came in with. can see that the most died from CMV, next most was adeno-virus. And have had four children this past 23 years developed EBB lymphoproliferative disease, at least are rotoer enterovirus infections. Others, term ] herpes simplex, two with pulmonary disease, one was unrelated mitochondrial defect this was a child who been misdiagnosed with a malignancy been given chemotherapy and developed nephrotic syndrome and died shortly after transplant this was a child had received a cord blood transplant there was no immune reconstitution and died of veno collusive disease. can see what's missing from this of causes of death. No one died of graft versus host disease. Even though these children received half matched transplants did not have lethal graft versus host that doesn't mean we didn't see GBH because many times these children present with trans[ technicalterm ] maternal cells and will present with some GBH. We have seen GBH to a milder -- two three cases severe degree in about 35 of our recipients. Okay, so one of the things that comes up relative the gene therapy trial what is the incidence of lymphoproliferative difficulties in patients with SCID? This was study that was done by I this is Elaine jaffe's group here where they had reported the incidence as estimated at to five percent. At our institution 2.8 percent had this disease. The mean age at diagnose was was 1.6 years this is approximately 74 percent nonhodgkin's lymphoma,5 percent hodgkin's disease, carcinomas are less frequent and these are not always EBB positive. I might add, though, that I agree with Rich I have heard of any cases of leukemia in SCID This is a survey that I did years ago just to show you the worldwide experience with haploidentical donors at this point, out of 417 haploidentical transplants were successful. Overall for all different types of immune deficiency about half them were surviving. Now, the survival rates differ vastly report to report. And if you use a nonidentical donor which is N stands for here, versus an identical donor, can see that in most series, the survival rate is much if you have an identical donor. And these are both from the European society for primary immune deficiency, can see the overall survival rate is around 50 this is from L.A. children's where the overall survival rate was 46 this I think is from UCSF, it was 55 the non-identical and for the Europeans term ] latest which was the report in 2003, the nonidentical survival rate -- these are basically three-year survival rates and most of these reports, was percent versus 77 the identicals.
So are differences from center to center. Now, the other factor that is of crucial importance is diagnosing this condition before they come in with EBV, CMV, adeno-virus, Parra INFLUENZA is a report of Newcastle by cane, et al., where there were 13 who were transplanted in the newborn period and 100 of them were surviving. Laurie Meyers and our group published in there were 28 neo-nates that were transplanted and percent of these survived. And then currently, we have transplanted in the first months of life and 95 of these are surviving. So they can be transplanted before they become infected, there is a very high probability of survival. this is to show you some graphs from the European report by Antoine where they have graphed here the results related HLA mismatch, unrelated donors, no typically or gino typically identical and obviously the genotypically are the best they had some who had related -- unrelated matched transplants who a little bit better Than the half matched transplants in that series.
The thing that of that report was that are getting better with time. can see from 1968 to 1985, results were pretty abysmal with an overall survival rate of less than 40It was better here in 86 to 90, 91 to 95, and then 96 to 99. These are all types of SCID, not just for combined immuno-deficiency it was broken out that way not just for X-linkedThis shows you the transplants we have in the neonatal period. We have lost two babies, to CMV and to EBV. But all the rest are alive and and one of them is 23 years old. And then if you look all the ones have done the first 1/2 of life, it's still just those two that have lost, one from CMV and one from EBV. So early diagnosis, of course, crucial of the outcome.
And terms of immune this certainly not perfect treatment. Whereas were hoping that gene would be perfect treatment, uhm, we have 100 out of cells who are Kim ras. Nine these are not sky Merrick. of the ADA patients underwent successful treatment in Italy. We one who still not chimeric but it's early after transplant. The other is that we don't do chemotherapy so many these patients retain their own B-cells, and if their B-cells have abnormal cytokine they not going to work. However, have donor b cells in about, uhm, to percent of these They have B-cell function. The other group with B-cell the ones with normal b crelz like the cdc epsilon, and the il7 receptor Alpha whose B-cells to be normal.
Okay. So one thing to remember about all of these patients is that they are lymphopenic and the other caveat is that they may have persistence of term ] maternal T-cells. I personally this happens in most of these children, they a few maternal T-cells and then in some patients we a very large number these cells. So this could as the basis of for condition at birth -- screening for condition at birth. Dr. had a about screening she didn't show, both of us really that newborn screening should be done for SCID if you could do that, then could save virtually all of these children. So if you found on the cord that the lymphycyte count was under 2,000 then you would do sigh tomorrow try and confirm the diagnosis. This is the lower limit of normal for the lymphycyte these molecular types of SCID had lymphycyte lower than lower limit of normal. We had one patient who had transplacental transfer of like around 8,000 maternal T-cells. So would be one caveat that could give you a false negative screen just using lymphopena.
this is a X-SCID pedigree that Dr. has identified the carriers and this was a mother who a known carrier who was carrying twins. And she didn't want to know whether the boy was or not because for religious reasons, they not going to terminate the pregnancy anyhow. So the question was, you know, does he have SCID? And were also hoping that the girl could be a donor of cord blood the little boy. When the twins were born at our institution did stat of white can that the boy had a low white count, total white count, whereas the girl had a normal white count. The little boy had a low lymphycyte County little had a normal limo sight County. So we did stat flow cytometry can see that the diagnosis could made immediately by this method. It turned that little girl was a 2 haploidentical mismatch the little boy so we were to do a cord blood but we use the the donor can see that these children are healthy. They actually 4 years now and is one of our links who doesn't require RVIG. Approximately one-third the x links have donor B-cells and don't require ivig.
The other reason for doing the transplant early is shown in these figures here showing that the group are transplanted in the newborn period have T-cells much sooner and to a higher degree those that are transplanted they have already been infected. They also have naive T-cells to a much higher degree whereas the activated and memory are the same in both groups. if you look at the T-cell receptor recombination excision circles can see that those who are transplanted in the -- sorry, I this is proliferation here. this is PHa, can see the T-cell function was higher in the neo-nates this is true even several years after the transplant, although at age 10 years, it balanced out. These are the track data showing you that the group that was transplanted the newborn period had T-cell receptor recombination excision circles to a much higher degree than those transplated later -- transplanted later. this is just for Dr. O'Reilly point out that if you put a normal in guard earn the garden grows this was a child had been transplanted at day 18 of life has now at 4 years a normal sized thymus. The also can have normalization of their T-cell repertoire. This is an X-SCID before bone marrow transplantation where you have all these oligoclonal bands here, this is in an immunoscope analysis the T-cell repertoire and then after bone marrow transplantation can see a gossian distribution shown here and and then and here. only do you get thymic processing of these stem cells but you get a normal T-cell repertoire. the number of that are present generally correlates with the polyclonality of the T-cells in these patients.
So are the factors that affect stem cell therapist in SCID? Infections time of transplan take age at transplan statements earlier is better. The genetic types of SCID. There is resistance to en graphment and ADA deficiency and rag SCIDs. function is in the il7 receptor Alpha cd3 Delta and epsilon and ADA deficient SCIDs. The other point that have learned of this is that pretransplant natural killer cells and IL2RG Alpha CD3 Delta and CD3 epsilon deficiency not interfere with engraftment even though we didn't use any
And another point is that these natural killer cells disappear in both X-linked and genetic term ] and I might add that Elaine Fisher tells me after gene therapy even those they are transduced natural cells the natural killer cells disappear in these two types of SCID after gene therapy eventually.
B-cell function is best as I said the IL2RG receptor Alpha where only percent of our patients are on IVIG, ADA DEFICIENCY. then don't have some of other of SCID on but also the CD3 epsilon and Delta patients.
So in conclusion, then, this is a pediatric emergency, the potential exists to diagnose this condition routinely at birth. if you do a stem cell transplant from a relative the first 3 months of life before infections is a very high probability of success. I might when you do these transplants the newborn period, you can do these as outpatients because are not giving chemotherapy. The whole procedure can be for less than $50,000, whereas wait they in 6 months age with PCP pneumonia, your intensive care Bill alone can be to million dollars before you treat the infection. And then, uhm, the initial success with gene therapy offered that the remaining defects in these Kim was could will be -- sky could be correctible. I'll there and glad to answer questions. .
I failed to introduce the moderators for this this afternoon. are Bernie lo from UCSF, Ted from the University of cramped, San Diego. And Dr. Tops Holohan, from office of biotechnology activities, NIL. And there were guide the hour or so..
We have two presentation, Dr. and then we'll hear about umbrella cord -- umbilical cord blood transplant. This is particularly of to me. I believe bone marrow transplanters are the most reasonable of people. And with that introduction, Dr. O'Reilly will present to us the data from Sloan-Kettering. .
So what I'm to do today is present results based on studies of T-cell depleted transplants since the beginning of T-cell depletion. was in my laboratory developed interesting system in mouse which to be completely converted so when we finally got the term ] graft we started to do this initially in in vitro and then in primates and then took into humans. I like to briefly do is discuss T-cell depletion and going to to be really and I'm going to focus primarily on T-cell depleted grafts applied to SCID. agree with the things that Rebecca has to but real terms I disagree with several others. So I'll about those. This is a compendium. can't in the background go through a little bit. is a general aspect of the literature that's quote, unquote a T-cell depleted transplant, therefore it's same but that's not theSo you have here are the log T-cell depletion observed with a series of different techniques of T-cell depletion each of these has been tested up against the lecton term ] which we initiated and presented back in 1989-1990. Suffice it to if you use single or multiple monoclonal antibodies from mouse, basically what we have able to show by limiting dilution analyses of residual T-cells is that those techniques produce a maximum of a log depletion. The closest approach using an antibody based system was actually the cam path system which can actually produce a quite consistent 2 to 1/2 log depletion of T-cells. The electric ton aglut other hand, produces a 2.5 to log depletion. [ unknown term ] been talked b that at max about a 1.5 to log depletion and I know the only positive selection technique that allows for T-cell depletion in excess of where we're is term ] device. The isolex alone produces a 2 log depletion. is we are talking about an that yields a consistent 2.5 to 3 log depletion.
have looked at this a large number of patients with leukemia. We initially introduced it the treatment of a child with ALL, is group of 284 adults with the median age of 41. And with this technique using lecton separation depletion alone without post transplant prophylaxis with cyclosupportry anything what are seeing the overall incidence of grade term ] disease in matched donor recipient pairs of 3 percent an an overall incidence of chronic GBH of 4.6 have found in unrelated term ] or disparate donors the incidence of the grade 2 would be 8 percent and long-term incidence of chronic GBH disease is 9This the dosage. This is to the 5th. As can see to be the threshold dose these patients developed the disease grade 2 here and these are grade 1GBH. of these patients didn't. In contrast with unrelated graphs are able to clearly demonstrate a quantitative difference in fact as look at in terms of peripheral blood cells versus marrow cells where we think is it's not just dose but the of the that are administered. can this in Terps of long-term disease-free survival? These are examples of AML or second remission in adults with the median age of and can see it's a percent long-term disease-free survival for first remission, 64 for second remission, and I always get encouraged because I consider anything younger than to be pediatric is because cloudy for individuals 18 to 40, it's 80 and it's still 62 percent for patients the age of 40 to these types of grafts. And we now look at this also in kids, in the context either matched or mismatched unrelated transplants again and the mismatch is up to 2alil. S, long term disease free survival for kids transplanted with bad acute leukemias in first second is ranging between 74 and 78 .
I'd to discuss the between the States and Europe in the the that certain the centers in Europe basically were trained by us and actually use the same technique we are going to did a review of all the consecutive transplants for SCID that were done between when we initiated this on through 1995. And these were series of studies we looked at 55 done our center, in olm giving a total of 118 cases. And there were in fact a variety different types. time these were worked out genetically but a very large proportion of these patients were not genetically defined but clearly, we had can see 32 X-linked SCIDs definite, of the auto so manile recessive and we have 33 inherited. I'd like to review issues of the role of different components of the system in terms of transplant result. First were these genetically disparate? Yes. can see in the context of HLA alegals unique the host, the patients have three alils, had 1 had 2 and 10 had single alil disparity was their parental donors and in most instances are the mother the donor. .
Where we had no donor B-cells only 4 of patients developed B-cell recons stiewsmghts importantly with this technique 7 patients of the patients valuable developed grade to two graphers sos disease. We did not see grade 3 or 4. We had patients who did die of antecedent maternal GBH that was existing before the transplant was administered.
Now, are the results that we have comparing two groups. 73% for memorial and 57 for olm. were interested to find it was different because groups were schooled in the same technique. The being used the same and the critical is that there were 63 patients in am but clearly 19 of these individuals presented with BCGosis because in Europe they basically Vaccinated against it and they presented with it. you take the b this gosis group out it works out to percent for OLM which not at all.
If look at these patients, our group is different from what Dr. Buckley talked about. We have initial patients and large group of over 50 patients that were transplanted initially with outsider of that had patient who we didn't reduce based on features the of presentation. 56 who didn't receive cytoreduction. Ma we could talk about then what we could talk about then is terms of engraftment. Of that initial 58 patients who not dive early in-- die of early infections, 48 engrafted and 10 failed to. Based on these we we were interested to what was associated with failure of engraftment. we observed clearly and this has been also in the European groups was that engraftment in ADA defirst was quite inconsistent but we couldn't clearly other the fact that the X-linked recessives consistently engrafted we couldn't demonstrate clearly engraftment in of these types of severe immune deficiencies. one thing that appeared to be correlated in our shop we had discussed this it was big concern or not this in Europe, this is an interesting finding because what we did was to look prospectively at NK cells and NK functions looking at percent lies and these are every patients who faileden graft and can see the vast majority of these patients have quite striking NK activities.
These patients lacked NK activity. you see are patients here who had NK who engrafted and there are these four individuals who also had NK and engrafted. say this as something of a homage to the great Gus who initially described the whole concept of NK mediated rejection by F1s of parental strain donors, because he noted in that initial paper the journal of experimental medicine that the one way to overcome that type of resistance was actually to induce a blockade the RE system using BCG and strikingly, the patients who had NK activity who engrafted in the series were basically these patients who also had BCGosis the time. This is appear UV way to achieve engraftment but it can be The the you see these variations there are big differences between our group and Dr. as well, can see, the vast majority the kids with X-linked SCID are absolutely deficient in NK cell populations whereas several of the autosomal recessives high levels of them, clearly the ADA patients consistently do and we this block of unknown this was a as well. . To overcome this, we have had relatively inconsistent results with anything other than that which uses a myeloalative term ]. In those circumstances you see is consistent engraftment of T-cells with full well as consistent B-cell engraftment of donor type and again, with immunologic in of full production of anti-above bodies. The -- antibodies. term of niece that series these patients all have approximately to years of follow-up without conditioning for those patients who have NK cell before the long term disease free survival was 41 with conditioning was 77 percent, in no conditioning group without NK can see it's about 76 percent. But with in certain instances, have a 91 percent long-term disease free survival. These studies to us indicated that fact conditioning for patients with ADA deficiency or conditions of patients with high NK might be associated with a significant improvement in long-term result. And what we looked at this be who were transplanted without primary conditioning who lacked NK cells and can see long-term disease free survival of for patients without primary conditioning who did not get a second transplant can see, it's relatively reasonable but can see, the long-term survival with second grafting, these two are somewhat and not significantly different but it's the 70 to 62 percent disparity. if you at who is do Indistinguishable ] Reduce we're sighter reducing to ensure engraftment there was another that became a critical who didn't receive sat rear ducks, we achieved -- sater reduction, while we did the T-cell function what we found that only five of patients to actually got donor B-cells and can the vast majority of these have little or no evidence of B-cell reconstitution. In contrast, the patients who received either boo cul fan or don't ten in the pregrafting period consistently en with the T-cell, this is associated with full or partial recons constitution of the -- cons constitution of the function. We also see full reconstitution the B-cell function. I'd like to go over our own cases this is now 68 we are looking at. These are patients who have about a 15 -- actually, the first case, these are the cases since we started in so it's 25 years. The first kid is now a shakespearian scholar doing really quite but the of the matter is that these the groupings that have and the distribution in particular of the gamma chain defect. There is a crowd of that particular group. And we also have other populations as well including particular a significant group of term ] as well ADA deficients. These the gbh have seen in these donor recipient pairings the vast majority of of HLA3 alyle, disparities between donor and host that be recognized, there are three with grade and three with in the series.
When we look at the term results of these patients there are 20 patients that we lost, 48 are long-term disease free survivors. There are important difference looking the groups that did not get myeloablations versus those who did. There 40 in the nonmyeloabeltwayed group and clearly terms of early prior to engraftment percent or of the patients died before engraftment was seen. So 7 for the night, are primarily abeltwayed.
There is an important difference down here that is this group again without myeloablation, there is a disturbingish late deaths that we see, late the grafting accounting for percent we have in the context of a patient who has received prior ablative therapy. [ issue of This is long-term disease free survival. This is 72 disease free survival. can see the autosomal recessive in blue which is is over 90 percent. These are patients the which is 75 that are in the percent range of it long term disease free survival. The unknown we looking at with Jennifer Puck these of early cases we
we look these patients again, the patients the age of months versus those age 3 months, see a striking difference. 80 to 90 percent for the patients transplanted very early. The only ones we lost here are exactly as Rebecca said, that the patient who comes you with a severe infection in the infant mode. even those patients who develop infection and to us with severe infections term disaease-free your shrivel is in the 70, 57 percent range. if we look at it in context of prior infection, and these would include particularly viefl infections with CMV adeno-or measles virus, those without them do well. The percent range. Those patients in -- with severe infections including term ] or otherwise we are seeing in this circumstance is around a percent disaease-free survival. Oh, God what did I do. (Laughter) [
.
look at immune recovery we was to at the recovery T-cells, this is gray is basically the normal range. What we see is with sater there is a really relatively minimal period about in terms of reconstitution up to normal of T-cells. The cd8 recovery is pretty much the same in these there is big difference in terms of NK cell and that is the patients who are not sito there a significant proportion of at least half who don't reconstitute NK cell in post drafting and are other patients who develop early evidence of it it drops off. Test are -- these are normal NK numbers and cell
If you look at the B-cell populations large proportion of patients without sitoreduction were in fact X-linked with of host time. There are MHz had B-cell are who had B-cell deficiencies who up to but the point here is function. If you look the patients who have donor B-cells that is are patients who are predominantly patients who have received sito reduction, see is a normalization the serum mu no u which is not in the patients with the residual B-cells. Similar through for id and also I -- IGA and IGM. This from our standpoint suggests that you engraft the donor B-cells functional reconstitution of the system will in fact be consistently observed. The patients left with host B-cells in our there are some who make very low levels of IGM we have no of IGG or IGA antibodies in these populations of patients. The other disturbing feature that I'm concerned about been doing this now since 1980 is that among the group it did not receive cytoreduction there is a falloff in terms of the T-cell and the T-cell this was initially talked about in the context of treks by Dr. that is the case far as able to see it, for those patients who have received cytoreduction, with burathi [Phonetic], we are not seeing this reduction at all. Again in the cytoreduced there is a larger crowd here but these are really long termers. These are patients in the four of our experience can see responses to PHA are normal but not in the patients who are myelooblated is a fraction who dropped in terms of overall functional reconstitution. If look causes death again early period, can see here is prior without engraftment embassy in -- in of overall grouping you are is what Dr. Buckley talked about encephalitis or hepatitis with viral pneumonias and RSV and measles and in one patient who received second graft with cytoreduction we had one EBB lymphoproliferative disease a who failed to engraft with donor cells. Post engraftment lost five of these patients. Again, viral pneumonias. One patients lost to effect of GBH from a party infusion that was given in an outside circumstance. And then had the mainly issue that is these are exclusively occurring in the patients who did not receive cytoreduction, that we have patients who died four with chronic obstructive pulmonary disease, one a chronic hepatitis and one with metabolic seizures who had evidence of chronic obstructive pulmonary disease. I'm particularly also that as we now look at that 20 -- now, 20 years out, in the cytoreduced patients are seeing very in the way of lateWe two who developed the polymyopathy early the grafting which could be an offshoot of a peculiar GVH or metabolic disorder but in the noncytoreduced group the major here is that we are seeing patients with interstitial have frights, one patient with CPOT, they have frights 7 patients with chronic obstructive disease and a very significant proportion of these with problems of warts important to actually engraft the early progenitor cells? These are that were done by Friedrich but more and we looking at this and are bade basically is that among patients are conditioned, you can in fact find populations of in the CD34 frocktion that are donor type -- fraction than donor and that becomes then a critical of the overall functional integrity. And the question that really comes up here how does this fit also with x link SCID? in 18 we done here, those who were not cytoreduced there are 16 of them can see, presented without infection all alive. the patients who had antecedent and these were severe, pneumonias can see, 2 6 were and the death were in fact these complications. Those cytoreduced again, one had RSV, one cryptosporidium but both are avenue look -- aive look and well but when you look the late issues without cytoreduction n X-linked circumstance, without cytoreduction and no infection of them have B-cell function, only 3 of have NK cell function. All of them achieved T-cell but there a late in six of the 13. In in the cytoreduced basically, are the way across. Now, what we need find out is what the difference you know, Beck da is -- Rebecca talking about as have a series of kids with X-linked SCID about of crowd who really look perfectly This is going out through 15 years and they maintain perfectly normal PHA spontses, their T-cell function looks normal and you have this other crowd we have one patient with autosomal recessive we have other patients most of in the context or almost all of in the context of X-linked where you see these late falloffs. And that is now coming up to my mind as follows. That is if we summarize -- well, we say it this way. Without cytoreduction, T-cell chimerism is if you an NK-depleted recipient. term ] you consistently have t function is reconstituted here. B-cell chimerism rare without cytoreduction but consistent with myeloablative conditioning. It's rare to recover any production in absence of donor B-cells it's in the myeloabeltwayive. They have B-cell function. Nk cell function persistent deficiency consistent normal function. Late reductions in thymic output very significant portion the noncytoreduced but in the patients cytoreduced late development of these auto-immune affinities and [ term ] and the myeloablative no. the question is, why? We don't know. are trying determine whether this represents differences in terms of the genetics of the particular transplanted disease. The that comes uhm is -- up you would go about it? Increasingly are looking at if we do a transplant in our series with the primary conditioning, 78 of those patients are long-term survivors so they unknown term in the they are very well. The who didn't have primary conditioning and did not require second transplant, that's a lot. X links, of the doing well but there is this early falloff later the 15-year-old and the patients who required a -- after years and patients who required a to get engraftment obviously around percent
So what we're interested or not in a hematopoietic transplant of this type you are giving with myeloablation a progenitor pool which includes a continuing population of stem cells which can allow for not only reconstitution of the B-cells the NK cells but also potentially can provide a constant source for thymic precursors to allow for both early and late T-cell reconstitution and maintenance of normal T-cell homeostasis. In contrast without myeloablation, what I'm concerned about is that we in fact engraft early thymusite populations completely expand that thymus and these populations then would allow for very nice early reconstitution which may persist for 15 years. After that I'm concerned this drop-off represents the absence of continuing resourcing leashing newly of the thymus of the stem cell pool and a consequence of that late on you lose these thymic populations the T-cell function begins to drop off and potentially what I'm concerned is possibility that you may also have an association with that -- the evolution of auto aggressive disorders simple floor you would see in -- the late adulthood in man. So we would say in conclusion is that we think engraftment of donor NK cells is an point where we think term may be important engraftment of donor B-cells is obviously critical term ] maintenance of adequate pool of thymic precursors may also be important to sustain it up the third, fourth, fifth and 7th decade. And then the maintenance of thymic constituents, from my standpoint, may also be critical for positive selection of antigen reactive cells but also negative selection against auto reactive T-cells and may put a of these patients at risk for late development of these n fell. Fridities -- nefridities term ]. stop there. Applause ]
We probably should go on the third talk in this series unless is some very specific questions for Dr. about some the he presented. Is there anyone a burning question? Okay, if not, next speaker is John wagner from the University of Minnesota. John.
While are waiting seem to have different views about myeloablation. I if you on that. (Laughter).
Dr. Buckley? [ No microphone
My philosophy is do no harm. children are not diagnosed until 5 or 6 of age, you give them poisons on of their infection, it my view that this would result poor survival. And I think that our survival speaks for itself. I know that there are other reports or not you can get term ] function by giving chemo ablation. The series by haddad from France where they compared those who had chemo ablation and those who had not there was no the amount of B-cell function. I said, about one-third of our X-linked SCIDs have donor B-cells we don't know why they got and the other two-thirds did who have donor B-cells have B-cell function. And I think it's just a difference in philosophy.
I think, you know, just to be sure that it's also clear on this, obviously, if a child comes to us with really severe pneumonia and we're in circumstance where, you know, this kid really sick, even with a, you know, uhm, an X-linked SCID or non-X linked SCID we would certainly obviously consider do a transplant without cytoreduction in the x links we would routinely do that. But able to do these grafts and potentially give a secondary graft on to get the and NK cell population in. don't think philosophically terms we with kids that different other to say that if child also comes in with severe pneumonia, and has this phenotype with high-end K -- with high NK function, we basically seen of these are really sick and in general, know, unfortunately, if we sit around on them and give them a -- an unmodified and wait, too have been to funerals. So that's the real problem. So it I think of other issues that comes up is that if we have patients where we know who is coming for example, the in the family, it becomes usually relatively straightforward. It's that first in the family who presents with things like CMV pneumonia, measles, retrovirus natures or otherwise. Where it really becomes a very, very difficult circumstance. I of the things that you see in the European series is because of where these are initially seen. We see very high proportion of kids coming in who are extremely sick with really big infectious complications and so... difference between Europe and us is part that. I also think that, yeah, I think cytoreduction terms really getting the full reconstitution that's going to allow kids I taken care for years become grandpas, to an part of it.
I proceed?
Well, unfortunately, I'm here as more of a oncologist rather than an expert in combined immuno-deficiency. Me area of interest is the area of using umbilical cord blood as an alternative to hematopoietic stemWhat I could pose is haploidentical identical transplants have really addressed all questions and is clearly a strategy that has very compelling data. Then perhaps there is no need address the issue of umbilical cord blood. But is a clear-cut place in literature where the institutions have used unrelated donor sources as a source of hematopoietic stem cells so obviously are choices among, -- before us, this is the one indication where haploidentical identical transplants has a proven track record I would suggest that is this is the only indication where haploidentical transplants is indicated there are two alternative stem cell sources where interest be someplace somewhere the list of treatments that you provide to these kids. Obviously, I'm here talk about umbilical cord blood but me just to you a second the results are with unrelated marrow transplants. Now, again, I'm not an in this particular area. This is certainly a field I have just reviewed the literature the couple of days there numerous reports use of unrelated donor transplants for this particular disease. And the overall in survival is listed in the literature as 50 to percent survival. is complete immunoreconstitution in the of the at in the literature reports, it's limited by donor availability. this is a major benefit of haploidentical transplants. And is certainly the potential for term ] effects. We have addressed long of these like chronic graft versus host disease. Also growth failure some of these patients receiving high doses of poisons as you have said.
I give poisons so I guess I'm in that group.
But clearly, know, unrelated bone marrow transplants are not the end-all. clearly a number of limitations we have address aid couple of those. for those patients that have go on to unrelated transplant particularly if it's -- -- a risk of acute and chronic graft versus host we want to replace one Immuno-deficiency syndrome with another and we have to develop strategies that limit this type of complication. We wanted to strat diswlaiz would to us reduce that for the search process. It takes 3 to months now that requiring matching between the donor and recipient. That's a problem particularly for these kids that require immediate transplant. And we want able get them to transplant of the high risk opportunistic infection even before the transplant takes place. So why even think about umbilical cord blood? This not be the right particular disease group to talk about cord blood as an alternative stem cell source but clearly in other there is a role for considering alternatives. And one the reasons being is there is some differences in immune aspects of umbilical cord blood compared to adult cellWe know there are some in the immune profile. For example, there to be a very robust response to allo the general but toxicity is decreased remarkably compared to adult lymphocytes. [
I would to you that it's dedrived because of circulating trofo blasts or because of t regulatory cells. term there to be some evidence to suggest tolerance of a noninherited maternal alyle. this is all descriptive there is appears to be less HLA restriction as compared to adult stem cell sources. There is no requirement for T-cell depletion there is reduced graft versus host disease. is also differences the amount of hematopoietic properties. Whether it be a toll and genetically modified or alternative stem cell sources but one thing that's surprising when look at umbilical cord blood is considering the number of cells we infuse it's probably shocking that any of them engraft we are employing such low numbers of cells. The only reason why it works properly is because there is something about the amount of hematopoietic as opposed to adult stem cells to us get engraftment in the majority of patients. The underlying hypothesis when we started investigating stem we wanted to figure out if we extend the donor pool that was a limiting factor in marrow transplants. We could hopefully able to cross barriers of an increased of HLA disparity. So what to do is I was asked to present was an overview what we know about cord blood transplant but also then share you some of the data that's New York blood center specifically. In the series of that we took care of pretty representative of others had done as well was that we only limited use of umbilical cord blood to who didn't a suitable matched or unrelated or related donor and then those patients who went on to blood transplants either had a mismatch or had up unknown term ] or antigen level and resolution BR1. When you the results, and comparing to what you might have expected with an unrelated marrow transplant you see able to barriers that we otherwise probably couldn't with marrow from an unrelated source. in terms of the first question not this is rapidly available, I can this is now several years old but it an average of about 13.5 dies have our no to us. This is in con trues 3 4 with an unrelated marrow donor and clearly compares quite favorably with what would have expected with a haploidentical transplant. In terms the probability of finding an adequately matched donor for our patients, at at the University of I have to with you that a skewed population primarily northern European descent we find donors for this one year's worth of searches, patients found the donor out of 122 searches. So all patients can a donor. And we also national referral base for many of the children, and so we have ethnic minorities well but not as many as those centers particularly the coasts but nonetheless, umbilical cord blood able to, you know, find donors for the majority of our patients. in terms of, you know, the of this part and that you can find donors almost and it's rather rapid. Now the second obvious obstacle or not umbilical cord blood contains sufficient numbers hematopoietic stem cells to effect engraftment in the majority of patients. In this analysis at University of in the can see the cumulative en dpraftment is 88This is cumulative incident. What I can is that this compares exactly as what you have expected with a marrow where the overall inches deafens engraftment is 88 percent. However, when you then look at the speed of recovery, however, and in patient population that received -- achieved prophylaxis that didn't consist of methotrexate to is would have predicted something like around 17 days to recovery as compared to it 3 days of core blood so clearly core blood is sloweren graft although ultimately gets there. And terms of looking at the effect of HLA disparity no clear-cut effect. Both in our data well as the registry data set are looking at one antigen versus 2 antigen mismatch clearly there is more rapid there a out of six match that doesn't occur in very many patients. I you can appreciate the impact of cell dose and the time to recovery. There is that linear effect can see the more cells you give the more rapid the recovery. Shown a can see here that when you the patients into core tiles we're tiles with the lowest -- quartaisles -- quartiles, it's more rapid recovery with higher doses. reason you see this you are on threshold of number of stem cells required for engraftment. But what I can also is that have also to clearly define laiftion of patients or cell dose by below which be considered unacceptable for routine transplants. But that's not an important issue with group of patients that you talk about these are small, young, and the doses in effect are quiteThis the that shows there is no effective match there is a dramatic effect of cell don't. In terms of graft versus host, t replete graph no manipulation of the umbilical cord blood it was and the the majority of patients received a three antigen mismatched graft or almost all received degree of mismatched at low resolution a b can see that their overall of 3 to graft versus host to disease is quite low percent. So quite acceptable for this pediatric of patients. When you look at the individual effect of now grade 2 to 4 graft host disease the impact of mismatches individually, can there is no difference. in fact you looked the grade 3 to they would be overlapping with one another. So it is true that graft versus host disease does occur there is some cases in some a result of graft versus host disease in contrast in marrow represents a small of patients by far the majority of cases you find that there is a very low incidence from practical point of view. When you look then in the multiple regression what factors fall out as being predictive graft versus host disease find every mon.
In terms of chronic -- we find none. In terms of cropic, we don't want to create one immune deficiency for another chronic graft host disease occurs infrequently in some patients. It's the rare pediatric that respond to suppression who get
So I think that obviously, the bottom line is that kind we doing for our patients in terms of transplant. This is a major obstacle that prevents the successful use of unrelated donor transplants in general. There is high risk of some that results in the patient for various reasons. One thing we see the slide is the major impact of dose with umbilical cord transplant. The lower the dose the decision particularly if it's less than has a dramatically increased Risk of transplant related mortality. Those patients receiving higher cell doses have a more respectable transplant related mortality approximately I would say. in the terms of leukemia -- in leukemia patients which is this is, you see that that transplant with immortality quite compared to unrelated mar tro transplant. can see also the with age but not surprising because the smaller are the judger you are -- the younger are the higher your cell dose so they go hand to hand.
So in terms of overall survival see those patients receiving the highest doses here have an overall outcome that's quite good this is patients with leukemia. But one thing tell that when we talked about I'll show in a few seconds when talk about patients with severed combined immuno-deficiency you are talking about higher doses compared to the typical leukemia patient because of young age. This an important slide because is an interrelationship between cell dose and HLA, they both are important factors. And think you would observe two things here. so far all the curves are in those receiving a term ] mass matched graft here. Those patients receiving term ] mismatch transplant graphs are all just shifted down are the same general pattern tells us two things. The hire cell dose, the better the outcome Australian the 3 cell doses do better. The lowest cell dose does much poorer and then everything is just worse with the mismatch. It tells us the opposite. In part, the higher the cell dose overcomes at least partially overcomes the barrier of HLA mismatch. any event, what it also tells is that need to strive to have better matched graphs it if we but also to graphs that have higher cell doses and I'll show you a few that is might address that particular issue. Overall this is the survival outcome as related to cell dose initially mismatched so the strategies are trying to employ to make transplants core blood bet remember focused on cell dose issues. Let me just obviously this is a meeting on immune deficiencies particularly severe combined immune deficiency so critical issues that it relates to core blood is most patients are young many come into transplant with infections which you might predict would result in a higher risk of transplant related mortality. And as Dr. Buckley out these are patients would make it more difficult to give a chemo or a chemotherapy to because it makes them further prone to infectious deaths. Many patients don't have an HLA identical sibling donor and are forced to undergo an haploidentical transplant or unrelated donor of some sort. I asked the York blood center what data we could you today. This is Provided from the York blood center so I'm to give you a brief overview. Obviously will have many questions I able to address terms of, with the specific gino types, the of the patients but give you the overview I it today. Of the 57 patients transplanted with core blood from 1995 2004 we data on 52 patients. The median age was 11
the majority of patients received a mismatched transplant. And qiewx that the doses were exceedingly high, far higher than me prior slide the children are so small. The majority patients received Two drugs ] But many received ATG and cyclofos mied and some other chemotherapy alone as immune suppressive therapy to improve affected en graphment. The overall incident of recovery is percent can see the cumulative incidence hoof ever here of 57 This is not as high as with leukemia patients. there is still some that can be done to hopefully improve upon that outcome and show you some ideas although I'm not propp they will And you can see the overall survival here is 57 So it's to you to decide how this compares with the already observed. This the data of all patients as facilitated the York blood center. Now, although not the we are focused on today, at I have a little data to show new terms of term ] another syndrome immune deficiency although the issues are different. There 33 patients. Unrelated mar rose couldn't be found marrows couldn't found theseAlthough not quite as high a cell it was still higher what we observe most our leukemia patients. .
In this next of patients the overall engraftment is high. You can see the overall incidence of graft versus host is 26 Survival is 67 percent. This compares favorably with bone marrow transplants particularly since they are mismatched. I want to share strategies that might overcome some of these barriers. But in conclusion, terms of the immune deficiency can see engraftment what I would believe is suboptimal for patients with combined immuno-deficiency despite high cell dose. I have no data on immune reconstitution although in the literature with marrow transplants they say immune is good don't that to from you data set. And survival is comparable to results to matched -- unrelated matched marrow. We move from 75 to 100 percent, how can we move there? How can we reduce trm and late effects? We to talk about sterility and infertility and the late effects of potential for second malignancy or malignancy. Although no has reported at least what was discussed this morning this afternoon. How can we improve overall survival? , we have improved engraftment. The rate went to here. is only patients over 16 where engraftment is a problem you can to see how this might be useful in patients with severe combined immune if you were going to employ a preparative therapy by adding [ Unknown drug ] Other things might to consider the possibility of doing is he could infusing two units. on surface this may seem a ridiculous way of trying to effect improved engraftment and survival but we developed the strategy we to figure out a strategy of doing ex vivo expansion cultures expanding the number of stem cells that was limit for large number of patients. But we simply mixed two core blood units partially matched each other. One I can you is that what we is that we from the adult population or historical set shows that the overall inches deafens engraftment is approximately 75 By taking the same group afterdult patients and employing a double core blood transplant have effected engraftment in 100 the patients at a medium of 23 days. have markedly shifted it to the left and this again may be strategies that may be useful the that have engraftment problems begin with. Regardless the age. There are two graphs two donors. And we don't have to get into this, it turns out, this is according to a mathematical mod they'll we have created to predict time to engraftment is validated now. What we say is it appears that second unit is purely additive in terms of CD34 positive cells at least what the mathematical model will tell us and this second unit gives us only a transient way of engraftment, speeds early recovery and then disappears. And only will win out over time. Now strikingly and for those of take care afterdults with leukemia, what I can say is you would expect an overall survival rate of approximately about 25 percent maybe if you're lucky. so although you could this is early data, this is still quite striking for those who do transplants in the adult patient population. Particularly of the fact that all these patients received two term ] mismatched grafts. This is intriguing suggest that is perhaps by using two blood transplants not making things worse and we be making things better we have overcome the obstacle of engraftment. So the alternative thing that we might consider because what we are -- Buckley has pointed out that are given poisons our patients and I think we to figure out ways -- how can we effect complete immune recovery and yet not create any long-term side effect from the chemotherapy and radiation or boo cul fin that we use and -- boo cul fin. Giving some immunosuppressive therapy to effect better reconstitution of immune well as term ]. is employed with leukemia patients particularly over for high doses of chemotherapy are not well tolerated. .
We give sigh toxin and other therapy. We have more than 100 in this particular type of therapy and the overall incidence of sustained donor recovery all patients engraft, all patients -- there is recovery
Because those who failen to graft they have autologous recovery so this is a proportion with that of those who have donor drive recovery term it's not 90 and a median of 8 days so we are doing transplants with a very rapid recovery a very period of knew Pena and may be a strategy that will give us T, B and NK immune reconstitution although I can't you for sure in patients combined immuno-deficiency but it does so in patients with leukemia.
it gives you an of what the success has been terms of how we treat patients in the pediatric age group and perhaps the adult group soon. Umbrella core blood your passed use of unrelated marrow. There a tremendous in the use of umbilical cord blood the couple of years. This is in pediatric and I we'll see the not too distance what in the adult population if some of measures of success have to you work out and are replicated to other institutions. Now the population we talking about as a genetic disease and the fact that it's investigated a new strategy of creating an HLA identical donor, we heard today is that HLA identical donors would be at first treatment of choice. So therefore, that's true for leukemia as and it's true for a va right of genetic diseases I take care of. aSo we are about is the possibility of now hyper stem lating the woman or mother of couple with a known genetic disease like severe combined immune deficiency or at least subpopulations with combined immuno-deficiency. And are to be doing is actually then testing each that is then created and then or not the embryo is by the disease, certainly is very easy we are talking about X-linked disease. are also then now testing the embryo as single cell at HLA. Now, the limitation this is the timing. Okay? Obviously this is a patient that requires a transplant this is not the thing to do there are certain circumstances where we then inject the sperm into the egg and then over the couple of days can see that you then create an embryo and over the next three days will be at the 8-cell stage. At the 8-cell stage one of the stow mirs will be removed by embryo biopsy. is laser making anick a pipette to be put into the nick and single be removed and cell now is available for genetic testing. this is obviously not appropriate for many of the we talking about of transplants required now. other hand, it may be amenable to certain patient populations. I can we have employed this strategy in two far with severe combined immune deficiency an HLA matched donor. So the cell is removed the cell is then placed on a slide and then that fixed blast stow mere is then evaluated for specific structural chromosomele abnormalities and so we'll -- chromosomele abnormalities so we'll done spectral testing on the cell. We'll look at the to there are an abnormal number of chromosomes. Presuming that the embryo is would then second step is then looking the disease in HLA typing. is a particular with the very first patient this was ever done in a patient with Fancone anemia but the same strategy be done with any other gene disorder we have the mutation the family. particular case, emBOROeys 2 and -- have the right correct HLA type and in particular case, embryo 10 is normal, can see the mutant alittles here and then the embryos 1, 2, 4 6 are normal but carriers and most diseases or many the carrier state doesn't matter. You will every emBORO,-- embryo 4 was implanted. have hours to have the or of care, have identical doe knows that would be the first choice. Clearly, there is in the field of bone narrow transplant there is a group that very strongly advocates haploidentical bone marrow transplants, others that advocate under related donor transplants. Clearly for those with BMT would not be considered an optimal plan, gene transfer would be investigated.
And then obviously, uhm, IDS, GBD may be useful for some subpopulations.
I was to talk about today where are we with umbilical cord blood. I think we can say that it is an acceptable alternative if you are willing to consider the possibility of unrelated donor transplants to begin with. The second generation I think of studies will hopefully make this a much safer strategy perhaps by using nonmyeloablative therapies or other cell manipulations. Thank you. [ Applause ]
.
Doctor, can I just clarify something? Maybe I mistook it in my notes. I thought you had said that NEUTROPHIL or recovery in engraftment occurred in 88 percent of the leukemiaic patients that you were treating in umbilical cord blood and 81 percent of the combined Immuno-deficiency das data from the New York --
Correct.
Okay. So with the leukemia patients you find there appear to be a -- a higher number... however, in terms of whether or not it was statistically different I can't tell u but nonetheless I that I what it tells us is that there is probably room for improvement in terms of the severe combined immune deficiency. There were some patients that did have graft fail
Your thank you.
I guess we should open it up to general questions from... General questions from the RAC or the Audience. RAC first. Yes.
I wonder if you could just summarize what you think the treatment algorithm should be based on your experience. Which are the population of children that you think would be candidates for gene transfer who -- how many bone marrow transplants should you give a child before you go ahead to that?
Well, I think the -- if you have a patient who does not become immune reconstituted despite say having a T deplete help low transplant from a mother and father that patient would be a candidate for jet stream transfer therapy .
My sense of it you know, gets back to your prior question also. The timeline is one of the central issues. And I think if the child is, you know, in extremist, and failed a trams plant without cytoreduction, failed a transplant, I think this only -- what we can really say is that in soaferlsles this primarily focused on X-linked SCID, what I would say is that it is a rare X-linked SCID who cannot get one of these t doe pleated transplant and not get a graft. Okay? T depleted transplant and not get a graft. From our own experience that's basically what we have seen. Several of the other areas where you have these NKO if you have ADA those particular ones are the ones that have been very, very difficult to engraft without cytoreduction. And my sense of it is if you had a patient who was really sick and is it is now, you know, 4 to 5 months post grafting and then nothing there, my sense of it is that's a patient who would be appropriate for gene therapy manipulation because the next approach is in fact in an x link SCID would be to do some type cytoreduction and get a graft in. On the other hand if the child is in good clinical condition, my own sense of it would be a secondary graft with cytoreduction would be at the present time the surest approach to be -- to get both engraftment and full immunologic recovery.
So under that scenario, not very many patients would be Candidates for gene transfer, then?
I think that's the difference in the European trials and the United States because their criteria for doing gene therapy In Paris, for example, was lack of an HLA identical donor.
Identical donor.
And I think that was true also in England.
But again, as I said before, I tried to summarize, in Europe there are many different techniques of T-cell depletion. If you don't adequately deplete a transplant, you will get very, very severe or lethal GBH and the other issue that you may also get into is in the absence of adequate T-cell depletion, you require sustained immunosuppression following the transplant as prophylaxis against GBH. One of the things that we have felt really strongly about and obviously you heard a skewed view because all three of the centers we are talking about here would be lecton, that depleted grafts n those circumstances we have never required either in the leukemics or in the immune deficient kids or any of the disorders we have treated any prophylaxis post grafting. The other t depletion techniques with the recent exception of the mill ten ye all of them have involved post transplant prophylaxis against GBH and they still have an ambient level of [ unknown technical term ]. There are big differences. The Paris group uses an antibody based system. The British group also uses an antibody-based s the level of T-cell depletion we have done these, these are all the -- studies I showed you there were all based on side-by-side comparisons because when yayir and I were working on this we fully recognized the fact that this was in the premonoclonal antibody era and we made a pact to -- with one another that we would immediately change if we found something that gave us comparable T-cell depletion. And you know, until the [ unknown technical term ] device came on, we have not seen that. So that is a big difference there.
So given the respective long-term survival rates in both of your experiences which is approaching 57 in both cases, why would a group vary from the lectin depletion protocol?
You know, I think that the issue of T-cell depletion systems has evolved, you know, something of a cottage industry among a va right of different University centers working -- a variety of different University centers working under the real stress of trying to develop approaches for kids with lethal disorders or for adults with lethal disorders and from that different approaches have evolved. This approach has worked, I would also say, that it has -- it's not just us too it's been used in San Francisco, Winnipeg, Montreal, Germany, Italy, Britain, Australia, China, japan, they are probably something in the order of over 400 children who received these types of grafts for SCID. As far as night results have been quite consistent. It's not a simple approach. It is a labor-intensive approach but it has been a consistent and transportable type of system. So that's basically all I could say. There are several systems that are simpler. I would say what you are asking for may soon occur because the -- you know, certain of these positive selection approaches do allow to you get in a single step a t depletion to the level that we are talking about or better.
I'll just comment on that. I think that one has to weigh the, you know, the other risk and that would be the [ unknown technical term ] selection. Year only selecting for cd34 positive stem cells so usual missing the ones that are negative whereas in this approach using the [ unknown technical term ] you really are doing a negative selection for the T-cells and you are leaving everything else. And I think that may be one of the reasons why it works so well.
But in real terms, this -- for example, the CD34 selection for example the [ unknown technical term ] that's the only one that actually does it consistently, that is not really applied to SCID. It leads to rapid hematologic reconstitution. .
Before we break, Dr. Puck you could answer the same question for us? Which -- could you answer the say question for us? Which patients with SCID do you consider to be candidates for gene transfer?
I think as Dr. Buckley said, uhm, the patients who come to medical attention very early in life can be beautifully treated by bone marrow transplant treatment. And I think many of the differences in transplant outcomes may reflect the babies going in and when babies are very sick at the time they are diagnosed, then there are risks from any treatment increase. And I -- I don't personally treat those infants and I think that there are, uhm, I think they should be treated in centers with maximum volume and experience. I do think that the poor responders to transplant deserve to be considered for gene therapy. So the question that I'm hearing that I don't think anyone has said it sounds as if from what's been presented today that candidates for transplant would be -- children with well documented X-SCID who have failed at least one haploidentical identical transplant and for whom the second or next is viewed too risky for that child to undergo cytoblags therapy. -- cytoreduction therapy. Is that a fair --
From my standpoint I think that that would be a fair mode. I really think that's the appropriate way to go. And if you looked at the group that we had in our series, the group that -- where we waited for the graft to come in and then, you know, went to cytoreduction and then give a transplant, I mean, there -- it's still in the 50 to 60 percent range but that's the group that's the poorest outcome so I think that would be an appropriate group to think about. And would be appropriate to be done not just late on but potentially, you know, early. That's correct. And I also think Rebecca I would be interested in your feeling on this, but my sense of it is I think that it is absolutely correct that if they are in good shape as the -- when they are really young they clearly do extremely well. But I don't know that it's age per say. I think it's much more what is their antecedent infectious history because some of these patients I don't know that there is something magical that occurs between 3 and 6 months that suddenly puts them in a a different category of transplant other than the problem of infection. And I could even see even in very young kids a child comes in with, for example, congenital cytomegalovirus infection who basically is now developed pneumonia and potentially has I am penting -- impending encephalitis. Are you really going to want to give that kid -- you could give that child a transplant without cytoreduction and potentially no graft but if it failed you would wish to go to the next step to give [ unknown technical term ] or boo cul fin and the answer to my mind is no. I would much rather go with the gene therapy thing and get him back up and -- you know, deal with it from there
In terms of the age between birth and infection I always assumed it was due not fact that they were not infected and it may than viral infections do damage to the thymus. So maybe the ones who are transplanted at 6 months of age you come in with Parra INFLUENZA 3 or adeno-virus don't have as good an outcome because they damage from these infections but... because we have had some patients who were not in daycare and who were healthy who were past six months who have done very well so it may be just the infection problem. .
Dr. Mal election?
I would like to add to that by bringing in another factor [ Malech ]
One. Things I don't think has been highlighted is as these patients age, they probably become less capable being fixed by anything you do. There is a lot of evidence to suggest that -- that transplant is more dangerous unless effective as you get older. There is also evidence not just from the fact that the... united kingdom and France and French groups published about the two older patients who failed their therapy. But other kinds of evidence that suggests that gene transfer less efficient as you get older. What is really needed and isn't really out there even though everybody sort of is thinking about it is a real algorithm. I think there are clearly honest differences between what kind of transplant you do first in what setting depending how the patient presents but at least I see myself a kind of consensus emerging that, you know, with the toxicities we have seen in France and so on that maybe gene therapy under almost any circumstances may not be the first thing that you do. One the things that we don't have an answer to yet is the question of -- it isn't either or but our studies may -- it may not but it may answer the question, can you have your cake and eat it? Can do you some kind of transplant and when things don't work right, do you then do gene therapy? And have you lost a lot. I would suggest that you probably have lost less if you act early and more if you wait until the patient is, you know, 15 or 16 or 18 years old and really has tremendous a end organ damage and so on as has been the case with a few of the patients referred to us at NIH who the older they are, the more damage we see [ Tremendous amount of end organ damage ]
So I'm not here to formulate the exact algorithm but to hint to the direction we ought to go.
When you say act early, what do you mean?
That's somewhat undefined. So in the sense that when you -- most are diagnosed with X-SCID in the first year. The ones who aren't are probably the ones with variant SCID who have other issues that one has to Dell with X-SCID I'm talk -- other issues that one has to deal with. The conventional X-SCID diagnosed in the first year, what I'm saying is I don't think it's an either/or. I think that there are some -- although there are honest differences in exact lit kind of algorithm -- exactly the kind of algorithm you use for the transplant, I think that a consensus is emerging that maybe, you know, the transplants are actually pretty good for a significant subset of patients. But that at some point thereafter, and I don't know whether it should be a year or two years later, whether one try it again and when one should do that but probably between the age of a year and 10 years some decision ought to be mate about what can be done next. And it was nice to have people present something about conditioning transplants, about cord blood transplants, because whether or not one agree that is they should or shouldn't be used, they are the competing technologies. You know?
So doctor waser?
As a transplanter that really has no -- that's an -- no interest one way another whether you use covered blood or -- just looking at the data as I saw today in terms of what stem cells source would be used there was nothing that we heard here that would say we should replace the use of haploidentical transplants. Number one. And certainly if we are not using preparative therapy that's a clear major advantage. Not using immune suppressive therapy is a clear major advantage. And certainly the cost is a clear major advantage. So there was nothing that was suggested to me other than asked to talk about core blood that would make me choose, you know, an alternative cell source. But the second question is, is that, you know, really what is the place of, you know, transplant versus gene therapy? And just what everyone else has indicated, I would be hard pressed to say go right to gene therapy. As a hematologist, if a patient such as, you know, one with severed combined immuno-deficiency came to me first, I would be hard pressed to say go right to gene therapy when we saw results that were quite good with the haploidentical transplant. Now, One thing I didn't hear or may have missed is whether or not there is a specific factor that tells us when there is a higher risk of poor transplant outcome. Maybe you told us. Maybe one of your told us but are there factors now -- for example, when you came back and said those with multiple infections or high risk for bad transplant outcome I would suggest to you also that they are going to be not able to wait for gene therapy, some type of therapy needs to be done right away, to get a prompt immune recoverly, you can't wait -- if you have multiple infections I'm not sure that there will be an alternative approach that you would have immediately available. That you are going to get immune function immediately. So... you know, but if you could identify a specific high risk factor that might be the patient that would go to gene therapy perhaps rather than transplant. But if that's not possible, then I think that you would have to fail transplant first.
Patient 2 in Dr. Malech and Puck's trial was very interesting because this was a patient who is sky America with the doe --ky did I meric the with the donor T-cells. The observation that you are noticing that the host gene-corrected cells are out numbering the others makes the possibility that we may want to consider doing gene therapy for people who have been -- made it. Creme chimeras but they don't have any B-cell function and they don't have any NK function so I think that's something that once we get past this risk factor would be another indication for gene therapy.
I agree but there is a caveat. And that is the nature of gene therapy at the present time. If they are engrafted with donor T-cells and then you have hematopoietic cells there, you want to be very, very sure that in fact you start with the preparation that is extremely enriched for purified CD34s because the relative sensitivity of human T-cells versus CD34 cells to retroviral transduction is 100 to 1,000 to 1. It's very easy to introduce a gene into it. Cells in man. It's very difficult to get it into a true renewable stem cell. And so even if you had minor contamination of the graft with the donor T-cells my expectation would be that you would preferentially reconstitute T-cells that would be of donor type. Or, I'm sorry, not reconstitute but you would preferentially transduce T-cells with donor type in which case you would have long lasting donor type T-cells that are genetically modified.
There was another comment I think that was made briefly that I'd like to hear a little bit more about especially when people are discussing when you intervene and what you do it and that was some of the comment about testing all newborns?
Well, we would like to see that happen. And I think that we have a very compelling argument for that. I mean, we have a treatment for this condition where as many of the conditions that they screen for currently either have I guess only moderately effective therapy or, you know, no good therapy. And, uhm, it's very difficult to get this condition on the list because people think it's too rare to consider. But I think it's probably at least as common as senokitonuria which are screening for [Phonetic]. I if we could test for SCID we could treat them better whether gene therapy, bone marrow transplantation or whatever.
A comment on that point. When ADA deficiency was initially defined, New York State actually put ADA testing into all newborns. And they didn't find it to be horrific. That was a biochemical assay. A lymphycyte seems to be small pickings in comparison and really if you see severe lymphopena in the newborn, unless it's a really strange stressed newborn that's going to be alerting you to look further. .
.
I would just say obviously this group deals also with the issue of gene therapy trials in ADA deficiency and as was stated by Dr. Wilson, those should continue. Around the world the ADA deficient patients -- most of them engraftments are poor. They do very well if you give them cytoreduction and a haploidentical disparate transplant they do no worse than any other groups, in fact they are one of our best crowd. But I can envision as Diane knows you get some patient who comes in to you who is extremely ill, who basically can't tolerate it. I can envision in certain circumstances where could you really consider doing a, you know, Gene manipulation in that kind of a context. I also think that this is my own bias, but I -- I remain quite, uhm, surprised in a lot of ways about the x linked SCID that when you gave the genetically modified cells which they did in France you had this very rapid reconstitution of the lymphoid population. Whereas in the ADA experience with the exception of a patient who got busulfan, they all basically piddle along for pane many years or months to years, with very, very small numbers of ADA-positive cells. Almost all of that experience, however, in the context of patients getting peg ADA. There was a readout that was quote, unquote ethically untenable to give them. You eliminate the selective bias of the genetically modified cells. To my mind it would be genetically unethical to do at this point to do gene therapy if you have peg ADA on board.
Just to car will fight answer to the question, if you have no HLA match, so you are going to use a haploidentical donor for an infant with ADA-deficient SCIDs, unless you use cytoreduction, you will have a substantially worse outcome than in the x links? And I think there is uniform agreement to that, Nick. .
We have had several successful T-cell depleted half match transplants without chemo in AD a defirst but we have five on peg ADA and two with successful gene therapy in Italy so I think that this -- I mean, one of the things you have to think about is when you are talking about SCID you are not talking about one defect. You are talking about 10 different defects. And you shouldn't extrapolate what happened in the X-SCID trial to the other nine types of SCID. And I think that the ADA deficient trials should continue. .
This is I think a very important discussion. I don't want to cut it short but... We were scheduled for a break at 4:00. We are a a little over that. I'd like to at least have one or two more questions. Then I would might suggest we have a 10-minute stretch break. And then come back and recap and tray to make some recommendations.
Thanks. I would just like to come back to the issue of newborn screening. Which sounds very attractive. I don't think X-SCID is anywhere near the frequency of PKU which is probably about one in 10,000 but we screen for things like term ] urine disease and all kinds of rare things and the criterion isn't rarity as much as treatability and I think you made a good case that haploidentical transplants worked very well in the first three or four months. They work well in kids who are not infected. And sole early detext I thill would believe terrific. I feel a my evil incidentally terrible ills girl. He in the senate majority leader -- How do you stay with us respect SCID double howl do you sus respect skilled other than in a patient who has serious infections or multiple infections? Are there any other things that bring tell towel team? .
Noel. Recallle recall [ stand by ] You think about it in the con the accident of that less well is still associated with a 65 percent, 20 to 25-year disease-free survival so you know, this is not -- this is not the circumstances or anything like it as it was before even though the large proportion of those kids can do relatively well. I think the other points to be raised Dr. Friedmann in this regard as we have raised ADA deficiency, the other one which is going to be, you know, some of these patients with [ unknown technical term ] syndrome they are often times also very difficult to engraft without cytoreduction and again some of those patients will come to you with extremely poor clinical circumstances an I have no doubt that you will be entertaining protocols suggesting correction of that disorder. That's also one where, you know, if they flunked an initial one I would go right to it or if they were in certain states I would go right to it.
Before we break, let me just remind you what the language is. We might spend the next 10 minutes or break thinking a little bit about the language in the February 2003 recommendation from the RAC. And it says, gene therapy retroviral gene transfer for X-linked SCID should be limited to patients who have failed identical or haploidentical stem cell transplantation or for whom no suitable stem cell donor can be identified. It doesn't say anything about the relative health of the child, the severity of previous viral infections or other infections. And one might think about the language of that and if one wants to reflect the previous infectious episodes in these children whether that should be put in specifically as language into the RAC recommendation. And perhaps we should have a little break now. Think about what we have just heard. And reconvene in 10 minutes. And try to make some recommendations. [ Short break ]
Thank you. Before we start the last portion The discussion for today, will be band out in front at 7:00 for those of will be joining us for dinner tonight. will be a hotel van. So if people could in the lobby from 5:00 to 7:00 will be transportation. And then we're -- on administrative detail for those of need ground to the airport today, those of how need it tomorrow, please fill in the and the at the front can help you. Okay, thanks. [ Pause ] [ No audio
Dr. Friedmann's going to lead us through a brief series of from last and then do the wrap-up. Kind of as an entry into this these are the recommendations or the issues that at least suggested earlier morning that RAC seriously consider some very specific action. And it might be good starting point for this wrap-up discussion and let me just you those issues were. One is to reexamine either affirm or revise the February 2003 RAC position on retrovirus mediated gene transfer, at least have some idea of how the possibility of future leukemias might affect that recommendation, very specifically to think about it is in appendix N that might need or might benefit from some expansion in the or in some recommendations we make to investigators, and finally the issue nontechnical but equally important issues of how we to grips with this interesting issue of what is therapy what is experimentation and have we moved from to the other in the field gene therapy let me just remind you as a way of underscoring that issue, you the language have been using all day today, is not the language that would have been comfortable using years ago how many of would have comfortable saying gene in a really rigorous clinical years saying day today and I think that's very indicative of something. And it at to my mind indicates really that -- that this field has moved to a different level of -- of, uhm, clinical research and now is -- should not be embarrassed at using the language gene therapy despite the problems and difficulties that know we goingen to in the and already encounter so that as a process for the discussion, let me ask perhaps then for comments about the 2003 statement from RAC. How should that statement -- how should that statement be modified, if at all? Are we happy with it is? We we like add language to it to include the, use we have just been hearing the issues have just been hearing all day? [ ]
Yeah. Uhm, well, we should go to the language. It's way near the top of that -- it there? Okay. All right. So that's the statement from
Just to ask a question make a comment. You know, the practical implications of our recommendations are now put into context of international happenings and recommendations. So question for Dr. Malech was, it sounds like the discussion about when these -- we should limit the of gene transfer to certain circumstances was well agreed upon. But the question really sounds it's almost a recommendation for gene under those conditions, which is another thing that obviously may or not able to make comments on. it was the -- the the consideration that should be offered to individuals with failed haploidentical transplantation or extenuating circumstances if the French study is kept on hold until new vectors are there an believe your study was limited to 6-year-old and above, there be no option in the United States for individuals to receive gene transfer with retroviral vectors until they were
Actually, our protocol was ages 2 to
Okay.
And the reasons for age 2 actually is not a scientific reason. It it has to do patients that NIH is capable caring in the intensive care unit. Where of the dark secrets of NIH that we actually have an intensive care unit can deal with children 2 years of age. we were going extend this to younger children, we would to do in the context of another center. And actually, our -- our collaborator for some of this in the context of -- of the of the protocol was actually Dr. at were actually talking about extending to Duke but that's obviously talk about. [ Pause
In my opinion, in looking at data from the French trial, it appears that they a maximum tolerated dose below which, uhm, below there was no adverse events it was uniform efficacy. And that's almost -- that's a -- you know, a successful trial and possibly warrants a Phase II at some lower dose they haven't -- where they have seen efficacy without -- without an adverse event.
are you saying? In terms of the RAC statement?
Well, I think don't see any -- I that's more to us to recommend to do a II trial. But -- not to us to do a II trial I think it's appropriate right now. All
The I would make is I guess table been questioned to exactly what it means in terms of dose unless I misunderstood. And ee had -- we to be about making recommendations with data that not sure is 100 percent accurate. And are also dose per kilo and know the of those individuals so... I -- I guess I would be concerned about making dose recommendations based upon incomplete information.
Dr.
I would just recommend I think Diane said, and I think she said it better than I would, if you had should be it patients have failed identical or haploidentical T-cell depleted you know, stem cell transplantation who, because of contrary illness cannot receive cytoreduction for a second -- cytoreduction for second try transplant that takes in all comers irrespective of the me because be as early as about 6 after the initial transplant. You want at least wait the three to to at the engraftment issue but... and then... But then any of these would be potentially appropriate particular time. That covers case by case analysis of the overall health of the children and viral and history and so on? think that the -- the key real that you have in the X-linked SCIDs would be that if you take marrow that's autologous you would be not requiring any cytoreduction, the they come think they an initial transplant, unless there were some other extenuating reasons, you would primarily think about using some form of cytoreduction if couldn't tolerate that it was felt that they couldn't tolerate it, would be to go with gene therapy as an alternative.
Dr. Lo? I have question again looking at the 2003 statement, said should be limited. And today I sort of a more positive appraisal of gene transfer. And I'm wondering, we -- we get the conditions correctly and I think been ground than language based on what Dr. said earlier, I I to raise question, we want to say "should be limited" or "should be considered"?
do you recommend?
You recommend is even stronger. But I'm trying to sort of get the level at which we want to be positive about this as a potential approach be considered.
Any discussion of that or comments on Dr. Lo, Dr.
I think the word "limited" is probably appropriate. You still dealing with a 30 right now, it's of leukemia and are worries about how well this is going to respond. I think that these transplants don't carry that type of a risk, and I -- think that the data if you are looking out at 25 years, would be to get the appropriateness of a transplant unless you couldn't do
The query would be the you're saying is you're inferring the possibility that you would potentially consider the idea of gene therapy ab initio certain instances and can't see
no know. I was saying -- no,I was saying for who don't have a identical or haploidentical donor who are too sick or failed a transplant already or are too sick go through the cytoreduction, the characteristics you were talking about and Dr. Wara talking about, they a group patients for whom at least make them eligible to the gene transfer clinical trial, I guess question is it -- that we shouldn't do anyone other than those or should we be a little bit more positive about it to or considering it, not offering where considering it? There is always -- like your recommendation. I think "considered" is a better word and offers some slightly more positive long as it's couched within those inclusion/exclusion criteria.
I mention something else?
Other discussion then, yes, Dr.
I think you could modify the statement by saying limited to patients who have had failed or inadequate T-cell reconstitution from identical or haploidentical stem cell transplants because as Dr. O'Reilly pointed out and you have heard from Puck this morning, are people who have been transplanted who have become sky America -- sky meric but who -- chimeraic who have significant health problems and for whom gene therapy may be appropriate treatment.
In a case -- in that case... [ Feedback .
In the RAC recommends that... retroviral gene transfer for X-linked SCID should be considered for patients who have or inadequate T-cell reconstitution following identical or haploidentical stem cell transplantation and... who have intercurrent illness placing them at risk for cytoreduction or -- cytoreduction prior to -- for a secondary transplant. other words, good I do and we can wordsmith it a bit more later but, have made three changes. Right?
That that that's a very constructive and major, uhm, constructive change in the statement. And probably needs a vote of confidence the committee.
you want to should be considered or should only be considered?
That was the be considered. Because we're putting -- well, because the modifiers are later, yeah. thing, few words, for whom no suitable stem cell donor can be identified I think should come off if you listen to Dr. you know, even cord would be suitable. But again, rich says, would have to give pretransplant chemo if the patient is sick for that they couldn't -- so I think you just remove those few words.
And I did that. would be a very rare mull simultaneous.
Two (overlapping speakers). would be a very rare orphan from some really obscure place wouldn't have a potential donor present time. you to call a vote, then? [
I'm not comfortable with that language, be considered. too much a ringing endorsement my judgment. And -- and were it to read way would to know what those other modifiers were to come later. to me that our obligation be a bit cautious than that. I mean, we're to be the most risk averse persons on earth we have a primary to be more protective to be promotive so I think our fundamental mission is really defined by good guidance within the realm of we do and what we're fairly confident of. And to use the crystal or to make recommendations for shading into and setting priorities for what standard care is. I don't than that an attractive task for us personally. I think we know -- state necessary and conditions based on information we're highly confident of as we go did inAnd the extent that that's now modified, we have new information as we do, that we should incorporate that. And I think enough. we to say something further, ah long the line where something further along the line had other language think in our various iterations of drafts, and I remember that long day well, that we say something about that the risk-benefit a shifting dynamic, clinicians and may want to reconsider that as evidence emerges. And that have been some demonstrated therapeutic benefits well as some quite well-known demonstrated adverse consequences of a serious nature and that steams a much more -- that seem to me much more judicious kind of of the issue.
Uhm... I'm weighing both the arguments that have the for the of the word "considered" and then, uhm, Dr. concern about that. And of the things that, for me in today's very long sessions that had not been fully discussed is we now have a child that was treated with the gene transfer that was thought to be cured that got leukemia that was treated for leukemia and that had since died. I guess there is somebody who could sort of, uhm, suggest the scenario for what that child's would be like without having had the gene transfer might help me to understand what we're -- difference in language.
It be of those decisions I would presume. I just could offer I'm sorry...
don't know what the, uhm, clinical of the child was the time the gene transfer. But the child were well, and had a, uhm, half matched transplant, then theoretically could still be alive. But it sort of depends on, know don't know the clinical situation the the time.
Yeah. What we would be that the only infectious complications it we see that really become major risk factors for death going into transplant be either the herpes viruses or adeno-virus. So it's adeno, CMV, EBV, those are the ones. We have had in fact the first patient that we did had disseminated microbacterium avi. M. This is a clear population who had bcg Sys and are well. have had patients who have had a variety of other things, pneumocystic, RSV and otherwise whoSo my it is that it really -- very Beck da says, it really depends -- as says it really depends the status into the initial transplant. My own sense it is that it would be appropriate in the context of a patient who transplant who failed to engraft or failed to achieve immunological reconstitution consistent with reasonable health. That's the patient that is appropriate for the gene therapeutic evaluation. Because otherwise, how would that kid be, uhm -- would doing well.
Dr.
Can just to put this in perspective. best of the serious that Dr. has there is a percent mortality term ] of haploidentical transplantation in the series that I presented by the European colleagues when they counterbalance the risk/benefit of haploidentical term ] say the risk for mortality up to 20 percent. [ Heavy accent ] So a of that kind that would undergo haploidentical would have a risk of dying between 5 and 20 percent. In this series one patient died among 10 treated patients so that's a 10 percent. I would think that the things -- with different numbers, but they are comparable.
Dr. a short
Just short you looked at the kaplan Meyer curves you have in the computer now, you are also seeing though is that most of that mortality associated with the transplant issues are occurring in the same time frame as the mortality would be potentially expected you did not have adequate immune reconstitution. I showed you a very significant proportion the patients who died both in my series and Dr. series basically died the graft could take. So there is a radical in the timing of immunologic following the gene therapy mode, you would have to -- for exrarive base you are going -- comparative basis are going to have to compare them with kids to the transplant looking relatively well.
So in terms of Dr. Powers' recommendations for language, Diane, do you want to...
Dr. has typed up for us a revision which reads, in 2005, the NIH RAC recommends that......... should be limited, hearing Dr. Powers and miss Kwan's concern, go to "be to patients who have failed or inadequate T-cell reconstitution following identical or low -- haplo stem cell transplantation and or, 2, have intercurrent illnesses that would pose considerable risk as a consequence of cytoreduction prior to a second transplant." .
And we should prior to a further transplant because some of these infant and children have had several transplants before they come to attention.
Wouldn't than relevant for even a first transplant?
Well... I think If the of health is the child that such
No, because remember that Dr. Buckley presented us with large body of information that strongly suggests at least a subgroup of these patients do well with no cytoreduction and a ho -- and a haploidentical identical transplant so one would without, know, without fully developing an algorithm by the way I hope we will do in our plan manuscript, don't we want be that prescriptive this
would just that again. Again, in the X-linked SCID a rare child who would require cytoreduction prior to an information
You may wish at some later point to give full reconstitution but the fakt the is to get a tc graft in function it's a rare kid with X-linked is to fail.
A child presents with a minute naturing -- fulmin eighting viral prior to transplantation, how would consider that child? We would still with the transplant first. Without cytoreduction.
Then Dr. next issue is, should we add an additional sentence regarding ee involving -- evolving substantial... at least... -- evidence of Ben it present in the current gene transfer study but evolving evidence of substantial risk or should just leave that out?
It's a matter of difference to but I think it's -- probably goes without saying, the world we justBut if we the benefits we need to about the risks and need to speak the benefits because are both quite tangible. There may be some generalized value way in which the RAC recommendation is received and perceived the world by acknowledging that shifting dynamic and acknowledging that we do see a benefit and we see a serious risk. It just... it may be an -- to add something that I'm
You would be willing to craft a sentence for us? Thank
I would to add a comment. I too have some concerns this step of being a more -- think we need to be very cautious. The fact that Dr. Fisher has chosen apparently on his own to move to another vector system suggests to me there is some lack of confidence that he has in the of of this vector. And I would the fact that if we read our language from before, I think most people would anticipate that our response this time would be as conservative it was then even perhaps more so since this is a third case not associated with the first two. And so I'm -- we can't afford to step closer to this transition between gene transfer and gene therapy the basis of we heard today about the -- this success and other means which in years we weren't sure that the success was that level. So I think we to be cautious and I would -- I would not go to the point of saying that we are reaching home on gene therapy. It's too early use that if -- I could a term of experimental therapy in cases where this is the only alternative. I would be appropriate. But -- I think we need a little -- continue the caution we had expressed earlier. you bothered by the language that is up now?
not bothered the language that's up now. But I wanted to say that we were talking about being positive I think we need to be cautious identify of the issues because we need to answer some these other issues that suggest that would be very concerned in fact a third event occurred of such seriousness. .
I have suggestion and that would be, uhm, to put word experimental before retroviral up there. And that way, I think that people can take it they want. Thank you. good idea.
Doctor? I was kind of thinking along the same lines but we could go even a step further and specify the type of vector that have heard about the trials, you to do that. have in mind?
Lenti viruses are retroviruses so either we get generic about retroviruses we specify MLV-based as opposed to lenti virus. But... Dr. O'Reilly?
One word just before "to" I would change that from to and. rather than or. just to speak to the point about the vector, think that we really know whether it's the MLV vector or whether would be better with some other retrovirus vector so I it's best to leave it as retrovirus.
.
Okay, any other the language?
we should just let this stand as itI do. I'm quite persuaded technical term ] elegant statement of and probably need another further comment. The world knows it's dynamic and I think that's probably adequate. In the absence of any other more to statement, might be useful the committee to give its sense of the committee this is the appropriate statement this group and time from this group. Since I'm a the group it's not appropriate for me to instigate that so let that to Diane. (Laughter)
So going to read this into the official record, then. Short of no more wordsmithing.
Good.
"in the NIH RAC recommends that experimental retroviral transfer for X-linked be to patients who, one, have failed or have inadequate T-cell reconstitution following identical or haploidentical stem cell transplantation. And, 2, have intercurrent illnesses that would pose considerable risk as a consequence of cytoreduction prior a further transplant." .
Diane?
I guess I'm struggling with the "and" so have had one haploidentical have had another cytoreduction haploidentical, and they don't have ain'ter current illness are we forced to have a So what about that individual has a haploidentical, nonablation, has had -- has an illness, and then has second one, does not have an intercurrent illness they would not fit into that recommendation. Because they would not have an intercurrent that requires a third one. If they change word back to or... (Laughter) .
Depends on the the word "and." (Laughter).
Uhm
should be "or." To care of this. we will change "and" back to "or" very much, Dr.
Dr.
Well, you know, only issue is that if it's "or," it would say the patient does not need to have failed a primary transplant. what itYou can right a or Rye to gene therapy for SCID if it's or. He it could still be viewed with "or" that the could done without any at all. The comes in with intercurrent illnesses, that would pose a considerable risk outside a reduction this patient could go on to be transplanted -- could on gene rather be transplanted
They fall under the first category.
I we said if had illness
I had said that they had an -- if they had an intercurrent illness, they will get a nonablative transplant. would be the first choice. And that still fall into this.
Right. you have is or. That means that either/or could hold. And so what that basically would mean be that you -- people could read that and say that thin. Er current illness they don't need go to a transplant a priori. my problem. He [ term ] attorney. (Laughter). That was logic, actually. (Laughter). there is a lively about meaning of "or." (Laughter) ]
And?
Yes.
Okay. So leaving this at "and," would to take a vote. we will start with [Name to Captioner]. Aye or nay?
Aye. [ Roll taken[ Vote taken ] .
Thank you all. Okay. That's one issue. Can we go back to my... term ] .
Oh, Tom. . would like to propose adding another part of this recommendation for 2005. ... that the RAC recommend a study be done the feasibility of newborn screening for SCIDs either by lymphycyte count or by DNA-based assay for treks. PRECs. .
I think that's marvelous that shows my bias. I would recommend leaving the exact methodology
No problem.
Because I have a question about feasibility of PRECs in newborns as diagnostic. Dr. Buckley?
Well, Dr. Puck just published an article showing can do this on dried blood
Right.
So we know it's feasible. question is, I guess, uhm, logistical because people collect these dried spots they them an and put the mail and maybe 30 days later you might get the answer back. Whereas you did a white count the cord you would know immediately. And then you would able to transplant right afterBut might be better off just leaving the method out and that way, it might have more success.
I agree. Thank you.
Other comments or considerations?
Do we need second vote, Dr.If we add a sentence? We probably do. Dr. Lo?
are going about making recommendations for screening think it's a fairly well set of criteria one thinks in thinking about screening tests. And so we have to -- I I see a more about the false positives, false negatives, uhm, positive predicted
Just a study of the feasibility of... . And I is a is this really the purview of RAC?
Yeah.
The reason for suggesting it is have power but it a lot of recommendations... (Laughter).
Clout, that that might be a helpful kind of thing... (overlapping speakers).
United States public health task on screening which with setting national policy consider screening Indiscernible ] Me to no natures for k SCID. D X-SCID. Just by way a .
we want this a formal recommendation to go through the -- there is a national force on newborn screening. So the recommendation would need to go forth to that task force for and then would end up the of the barrel again. I like the of a recommendation of a study to consider feasibility but, bern initialing I hear what you're saying. Other thoughts? Miss Kwan?
there is this other mechanism, then
There
-- does that addition in this recommendation page is it a separate letter basically coming from the RAC directly to this body signalling that we spent considerable discussing this and feel that it's a would be invaluable to Car?
So Dr. just reminded me we are advisory and are not allowed to advise others who but we can ask the to draft letter to submit it to a advisory committee. Which sounds to to be this is... this is -- are don't understand. Sounds to me like the we should take to ask OBA to draft a letter of recommendation to to the newborn screening committee. We do so. We not have vote on that. . I have a request -- we certainly do that do have request that we finish the first task of revisiting the 2003 recommendations as they pertain to SCID, and you remember, sort of had two parts t a part that dealt with x linked SCID and had a part that dealt with ADA and other disorders. So just wanted to finish the revisiting the recommendations so we could tie up that loosened. [ Pause
The sentence doesn't need revising.
I said has to do with non-x-linked SCID and probably needs similar kind of restatement from this group. That's the language as it exists in 2003. Is there any need change that language? Is there any scientific reason to change the language in this paragraph? [
Or ethical?
The only I was wondering about was Dr. O'Reilly made point about it was the Jack mutation, right? It was of other classes of SCID mutations that he felt was in some senses analogous.
Jack is a form of X-linked SCID so when we talk about X-linked SCID instead of being for genetic mutation, we cover that issue that possibility.
So... [
I'm to we to the next paragraph and then vote on two together. next paragraph reads, "such studies may be justified contingent upon appropriate risk-benefit analysis, accompanied by implementation of appropriate informed consent and monitoring plans." Is there any sentiment to change the wording of this paragraph? Doctor?
excuse me. wanted to go to Jack 3. I apologize don't remember the chromosomal location of all genes but is Jack 3 X-linked? No, it's not.
That's true. It is a major signal transducer from gamma-c so it's on chromosome 19 so it's autosomal receptor. So we do need to change that. think because the adverse event that has occurred has been an X-linked SCID. With he don't any data or reason the present to extend it to JAK3 deficiency.
Miss Kwan?
Recommend this time RAC reaffirm the last two paragraphs. .
Once again we'll begin with a vote. [ Vote taken ] Thank you. 12KWR5*7 okay. So -- okay. So the next... Question -- back out to question 4. That has to do with other retroviral-based say what? Pause ] .
So the of the two 4s. (Laughter).
to do with other retroviral-based gene transfer studies. Is any reason to -- to, uhm, [ ]
this refer the February 2003 statement?
No, it takes the issue broader. I think not answer to some these questions are aimed at on record what I think has either been stated implicitly or explicitly through discussion today. And should be assessment the of the potential benefits of risk and protocol for other disease indications that employ retroviral vectors modified in of the of the events that have occurred both positive negative so how. And should target populations be in the assessment protocols involving conditioning regiments that might result in immunoincompetent states, for instance. This doesn't refer to a formal prestatement the RAC but a sense this committee's concern about other retrovirus-based studies. And whether one wants to a -- any statement about other based on what we have concluded that the RAC has recommended for SCID. Is there a in the committee for other retrovirus-based gene transfer protocols? There needs to -- there to be explicitly stated based the X-SCID model. Miss Kwan?
I apologize for being not scientific enough to or this is an appropriate question. But one of the things that was stated I think by Dr. Von Kalle was that you continue to store samples from these patients to see if an untoward event then they come back and do the lamI think more recently coming for June meetings is a protocol involving retroviral vectors in which I think promise is made the informed consent that that after the immediate of the sample everything would be destroyed. this a where we would want to make a different recommendation? .
I would suggest keeping the samples you found an adverse event downstream it will only after the fact that something bad you might able to glean information the hit that by back after so I would suggest to hold to the samples. going to recommendations for going the of the number 4s. Uhm, I think for just comparing indiscernible ] MLV-based vectors, MLV does have the unfavorable characteristic of favoring integration around transcription start sites and aldini's data that is that to you do gene trapping better. The are responsive to nearby promoters a result, more transcriptional talking back and forth. So I think it's unlikely lenti viruses would be worse and so something along the lines of encouraging more development of lengthie viral vectors might in order. It's not they will be better I think it's they will worse they might be better.
Any other comments on that issue? Pause ]
Yeah, yeah, that's right. But I think, uhm, just thinking terms of recommendations what would be useful going forward, just about comparative evaluation of lenti viruses Indiscernible ] Endorsed. Diane? I think that our 2003 recommendations up so well because are test, they the issues that were most appropriate, they are terse, and we didn't get much more broad or we talking about that's for internal use or otherwise, but I the that we didn't get bigger than we data for is helpful and in retrospect we looked smart back in
Because we didn't get bigger. So my would be to stick with what the specific things addressed and but interested in what might to say. want There is a discordance between dose response and an investigator -- I apologize I haven't read that paper that they are limiting themselves in dose and then the FDA whose comments include they would advise against the use of vector copy number or number of transduced cells to address the risks, that's really discordant in my mind and I think we should at least consider whether it's a recommendation or otherwise to actually encourage the investigation of the dose effect response although we probably can't conclude anything at this time. I'm not comfortable with saying don't look at it. And I'm also not comfortable saying we have a conclusion, but the investigators as was mentioned before, they have come to some conclusion and they know the data better than anybody. So... I would be interested in hearing what people have to say about making -- about encouraging the careful consideration of dose response with the increasing number of events. But you are just --
You are just going in with what you -- against what you just said.
I know, that being the one exception because that was presented to us today that was new. We have an MF3 now and there looks to be some separation in those groups albeit not statistical and not complete. And not concluding but encouraging that consideration.
But as you just stayed the investigators have already recognized this this is going forward and ro I don't think we necessarily is to look at the FDA recommendations and counterbalance each one of their points. I think again, our job is to considerate risk benefit ratios for subjects and, you know, I'm fully comfortable that the investigators are going to do the right science without us, you know, providing them a study section.
There is a way to do that suggest changes to appendix N that if this committee includes at least the question of the investigator's awareness of the dosage issue, in the Apepped disk N document that may be -- appendix N document may be useful way to incorporate this kind of thinking into -- yes?
For the record, [ Indiscernible ] I think one thing maybe you misunderstood. What Carolyn has said was, it wasn't what the FDA says not to consider copy number. We were talking about what the advisory committee suggested and what they said at the present moment especially if you take into consideration Dr. Fisher's trial, where he had a what we consider -- they consider higher dose initially for the first two subjects and then a lower dose because that was the recommendation, that he received, that maybe you don't want to take into consideration right now making a mandate that you have to set a copy number that doesn't mean that the FDA isn't asking sponsors to check copy number. There is a difference. What we are going to ask and what we are going to be asking for what the committee thinks -- since there is not a lot of data. You understand? The differences of course sponsors will look at this, of course the FDA will can for this information. But this alone may not be enough to make a decision on whether a trial goes forward or not. .
Her comments were preliminary in nature. They had not finalized those and they were transcripts. They had not transcribed the thing yet so I don't think it's anything officia that could even respond to to be quite Frank.
We are moving quickly towards 6:00. The energy in this room has taken a swift dive over the last 30 to 45 minutes so I -- need to propose two things. We wanted to make some firm recommendations about adding to appendix m. I'd like to defer that to the June meeting. And allow 45 minutes or so during the June meeting to consider appendix m in the context of the discussion today. Second, we do need to say something about 5 as it's listed. Is there any new information that we believe should be communicated to existing participants in on going protocols or prospective participants in new protocols, et cetera, based on what we heard today? Or should the informed consent document be changed substantially from what we recommended earlier? Dr. Deluca.
Just change the 2 to 3.
Correct. (Laughter).
Could I go out on a limb and say a positive thing about gene therapy? Phil said the only thing that's been new is one other case but we also have two more years of follow-up on a lot of those patients plus the British study where there is four, five new patients that were, quote, cured. So -- I -- I think... you know, you have a view that we have to be cautious but I also think we have to stand up for gene transfer when it seems to be working and there is a lot more data now. There is some negative but there is a lot that's positive so I think the numbers should be updated to account for all the positives and negatives.
Agreed.
Strongly agree.
What I want to say is that information about -- new information about both the benefits and risks of this gene therapy trial has been communicated to blah ba blah participants prospective participants and so forth.
We do not need to take a vote on a recommendation like that it's my understanding. I think it's quite clear that informed consent documents must reflect the current understanding of comparable gene transfer studies and so clearly, at a minimum, the number 2 has to be updated to 3 and at a maximum, the relative risk benefit of ongoing studies should be summarized at any informed consent document. [ Pause ]
On that note, is there anything else that anyone around the table or in the audience would like to add? And we'll return to this discussion in June. I want to thank everybody for participating today. It certainly I believe presented all of us with a fuller more complete understanding both of X-SCID and the options open for these patients and of the ongoing risk-benefit in the X-SCID gene transfer studies. So thank you all very much.