Confirmation Number:370957 Event Started: 6/15/2005

Good morning, everybody, I have the privilege of welcoming everyone to this 100ct meeting of the Recombinant Advisory Committee. Hundred meetings. Any rate, hopefully this one will be as prodic tb as those that have gone in the past. Again, I welcome everyone to participate actively including those here from the public in our deliberations and discussions we will begin the minutes of the from March 16th, 2005.

Hello, the read the minutes from the last RAC meeting and there were issues that were clarified and I think they are fine now.

Dr. Barkley, any comments?

I read them carefully as well and again it -- I was impressed with nare quality and clarity and it's education to read them again. I adopted

Always an education to reread what we discussed. Any other points from other members of the RAC? Would someone like to move them to adopt the minutes. I move to adopt the minutes.

And I second it. All have to go through -- so our first vote.

Dr.

-- pours? --

I'm just looking for where I am. -- thank you all very much we will move forward to our next discussion while waiting for Dr. -- he is here? Dr. Kington, welcome and we were glad you can join us this morning. We have a place for you here at the front table. Dr. Kington is the deputy director of the national institutes of health and is here to present the certificate for appreciation and say a few words.

Thank you. Pleasure to be here. As deputy I get to do a lot of things. of the things I get to do a lot of is saying a few words and that can be on my epithet, he said a few words. Really pleasure to represent doctors and the Singhtary this morning. -- secretary this morning. This committee actually is an extraordinarily important committee and we wanted to take this special occasion to thank those of you who served this committee as sort of advisory role that actually has extraordinary impact. Actually, that -- don't have an impact but you definitely have an impact. This was a critically important advisory role and played different parts of providing perspectives to help the agency and the department deal with the significant safety dimensions and the clinical applications related to recomeinant DNA research. I learned by the American association of association of advancement of science for this committee for its main correct.

The freedom and responsibility award which is remarkable given it was selected for individuals and to give this a committee in all of you who served on committees I think is an indication of how important your role has been. I hear a lot of good things from doctor Patterson about the various advice that you give and it's a pleasure to get -- for those who will be -- I can assure you that your impetus has been valued and we will still continue to call upon you. You are not out of the loop yet. We have a remarkable tendency to call people over and over and over again to in our committee. We have over 20,000 committee assigned, scientists from all parts of the scientific community who advise us on various commities and advisories committee and study sections and it's a strength of agency and the department to allow -- and clinical perspectives and patient's perspective and advocacy perspectives to do the right thing and this is one part of our recognition of those services. You have to attorney give me if I mispronounce your names p. There are no nonics here. Dr. DeLuca. Dr. DeMint. Thank you. This is going to give me trouble.

Doctor Kington again thank you very much for taking the time to join us this morning.

Pleasure our next agenda item is to re-- our next agenda item is to have Dr. Patterson discuss the review with us the rules for conflict of interest.

Thank you. As you know this is a standing part of our agenda but to read into the record our rules for conduct -- conflict of interest review. Be a member of this committee makes you a special government employee and be there subject to rules of conduct that apply to government employees. The rules and regulations are explained in the report titled standards of ethical conduct for employees of the executive branch. You may remember each of you received a copy of this document when you a were appointed to committee. In addition to following the rules, we like to review the steps that we take and ask you to take to ensure any conflicts of interest are both identified and addressed. As you know, the before meeting you provide us with information about your personal, professional and financial interest and we use this information for basing assessing if you have real, potential or apparent conflicts of interest that could compromise your ability to being objective to give advice during committee meetings. While we waive conflicts of interest for general matters because we weave your ability to be objective that won't affect your interest in such matters we rely to a great degree on you to be attentive in our meetings to the possibility that an issue will arise that could affect your interest in a specific way. So just want to underscore that point that during the discussion of both issues as well as specific protocols it's important to be is continually mindful of the dynamic nature of the review and that issues might come up that you weren't aware of during the initial conflict of the screening and be mindful to alert us to any conflict even in the course of the meeting. If this happens we ask you to recuse yourself from the discussion and leave the room and not to leave the table or sit in the audience. And you are required to recuse yourself from the preliminary protocol review process when you have a real or apparent conflict of interest. Involving a particular protocol. If you have any questions about the rules of conduct or conflict of interest, our committee management officers will be happy to address them. Thank you.

The next agenda item is to rediscuss the RAC recommendations for conduct of ongoing and future gene transfer studies in X link severe combined -- deficiency. We began this discussion shortly after the initial two children in the French X-SCID study developed a proliferative disorder subsequently found to be a form of acute limb foesetic leukemia. Does anyone know a third infant, child was diagnosed with that same disorder post gene transfer early this winter. And one of the initial children died with their leukemia. The other back drop to this discussion is that 11 children received gene transfer in Paris. Ten had some of the 11 had some immediate, meaning within months, and continued evidence of increased well being and reconstitution of their underlying SCIDs. So we and the rest of the scientific community are left a dilemma which not a disorder X-SCID which is uniformly fatal if not treated. When gene transfer is found to be relatively open conscious as it was and is in this cohort of well defined children, how much risk should one assume in the conduct of these studies? We met as a group several times to examine the scientific information, which has emerged and has been very on a personal note, very carefully done since the initial reports of leukemia in this cohort. And I believe all of us would agree that the science which has been carry out by multiple investigators really rests on the careful accumulation of samples from this cohort of children, both before their gene transfer and subsequently. And if it weren't for the careful sample collection the studies that had been done to examine why the leukemia has occurred would not have been possible. We most recently examined the information in March of this year, and readvised our recommendations, although -- reviced our representation -- revised our recommendations although rapidly. And -- it's the slides. We revised those recommendations between March and today. New information has emerged, although nothing directly related to the gene transfer. And we are presenting for you this morning a reconsideration of the recommendations in March. And that's what we will discuss over the next ten minutes or 20 minutes or so. And these are the questions for and the review again -- I'm moving. And to give credit to the appropriate people when I arrived this morning I wasn't certain that there were any overheads or slides prepared. That was exactly 30 minutes ago. And within the last 30 minutes our wonderful staff has put together overheads to guide us through this discussion. So the questions for consideration are the same as those that we worked with in March. And I think everyone can read them. I already commented on the first. I suppose I should read it into the record. Should the assessment of the balance of the potential benefits and risks in X-SCID protocols be modified once again? In 2003, we made a series of recommendations, the most important one was, quote, retro viral gene transfer for ex-link skids for patients who have failed -- stem cell transferation or no suitable stem cell donor can be identified. End quote. And rather than discussing that at this juncture, I think we will move forward fairly rapidly to the statements that we put together in March. Then ask simply is this still how we wish to proceed? Point four, extends that potential point three, extends the potential qualifications to other SCID protocols beyond X-SCID and includes the ADA deficient SCID protocols and possibly chronic grand uleo TOU -- disease at least reaffirmed for everyone is not a form of X-SCID. It's not a deficiency or an abnormality in lymphocytes but rather in luterofalls Mono sites. It's a different form of immuno+ deficiency affecting a different group of cells but still monogenic in nature. Four, even moving beyond immuno+ deficiency, are there other disease indications using retro viral vectors that we should rethink in light of the X-SCID information. And finally, what new information should we ask be included in the informed consent document to reflect the X-SCID experience. And should appendix MM be modified? We asked earlier how might the risk of leukemia be reduced in gene transfer studies using retro viral vectors. We discussed that at length and that's really not the purpose of this morning's discussion. And then next something that is an underpinning for all of our discussions, what is the ethical status of a research intervention that provides affective therapy as a does in X-SCID but carries a severe and recognizable risk. And it looks like those are the slides that the staff put up for me. So if you will give me a moment, I will get my handout. In your pacts, you -- packet, you all received a copy entitled follow up from March '05 safety X-SCID symposium, if you all please open that and go to the last section which is the draft of the conclusions and recommendations that we made in March. We reviewed a substantial portion of what is summarized here for us, and probably can agree to all of the bullet points that are above the formal recommendation. And those bullet points include first the majority of children in the X-SCID gene transfer study had major clinical improvement to date. Are there any comments or changes in that sentence? All right, second, of the nine children in this study who had successful engraphment of their gam eas transduce cells, three developed leukemia three years after treatment and required chemotherapy. One participant subsequently died. The overall frequents of this adverse -- frequency in this trial cannot be -- that's straight presentation of fact. Next, the gene transfer was a clause of the leukemias. Next, the occurrence of leukemia in this protocol is not a random event and constitutes a series of at risk in this study. Comments on wording. All right, next, some subjects in gene transfer studies for nonX-link skid, AGA skid and chronic grand lmitous disease experienced mild to moderate clinical improvement. Now, apparently at the recent ASGT meeting and I did not attend that meeting, there was a report of significant clinical improvement in two adult subjects in contrast to the infants who were enrolled in the gene transfer study and X-SCID. So two adult subjects. After gene transfer the retro virus expressing GT of 91 fox which is form of chronic grand lmitous disease. My question have a personal question would like the group to assist me with, is that I have not personally reviewed that data nor have I seen it presented or written and I am a little bit concerned about including that specific a scripter in a -- des scripter in a RAC recommendation and I'm wondering if we should just go back to the X-SCID paragraph that we have and not include chronic granuleo us to disease but I would -- granuleomate us it disease.

Regardless if it has been published.

I think -- I agree. I think it's very specific because it actually mentions the precise gene and as everybody here probably knows there are multiple causes of chronic grand lmitous disease in terms of genetic abnormalities and I think this is specific date thaw we haven't reviewed. Provocative, interesting. All right.

I don't recall us discussing the integration of the participants with -- inaudible --

I'm sorry where are you?

I'm sorry, I missed that in the discussion. Should we go back to that paragraph and then move forward to the end? All right. There is new language in the intro ducktory paragraph that -- introductory that is open, the entire paragraph is open to discussion. Online seven -- on line seven, three children inserted two children has been inserted and is that certainly correct. On line 10 the description of the integration sites is expanded and is that, Dr. DeLuka, that's a section you would like to go over?

I wondering where that came from, the published information. The last meeting we didn't know where -- inaudible.

I haven't seen that information published either.

I tend to ask the investigator from the French of the X-SCID link report recent finding of that integration -- participant. But this data will present it as a meeting but I don't believe it is published.

That's correct, it's not published.

It's not published? So we are in the same I would say predicament or position with this data as we are with the chronic granulomitous data provocative but not published or reviewed by any of our members.

Diane, I make a suggestion because it is clearly data that's been made publicly available, although not yet peer reviewed and published, perhaps, and furthermore it was not presented at the March meeting and so I wonder if it would be reasonable to go back to the initial language but add a footnote to that language stating that additional information has been recently presented at the ASGT and describe what was presented there. It might be a way to handle it. Well, if we did that, I would propose actually referencing it to a specific abstract or presentation of the ASGT so that people could get back to the primary information. I'm a little uncomfortable moving in to RAC language what most of us would agree is unpublished information. But I would like some further discussion. Yes?

I actually think in some ways that this particular point is different to the point you raised earlier. The point that you raised earlier relates to a whole area of information that we haven't had an opportunity to review. While I look at this information as more of additional data that has come up since we were able to have a full discussion. So I actually like the footnoting or some option such as that so that it's an information point as opposed to part of the recommendation. In the other area, the chronic granulomitous disease I think it should be left out all together.

There is a qualitative difference between the kinds of information we are discussing.

I think you have to take into consideration this data was released really before the ASGT meeting and is new information. It's not clear what this information means and I'm not saying isn't Val vau led because it's been sequenced but you have to take this into consideration, this was just released a few weeks ago.

It's an interesting discussion issue because the question before us is, what do we do with information that is very new-- was anybody of the RAC members, was anybody in the session at which this was presented at ASGT? None of us have had the opportunity to even interact with the investigators and we haven't seen the data?

You could be a little less specific if you wanted to and you say use the word to appear involve LMN2 and other genes and I think it's important that LNO2 was involved because it links it to the other two.

I believe at the last RAC meeting when we were discussing this -- in March I thought I heard one of the discussants remark that at the time they didn't think LNO2 was involved, okay? So now it is involved and I like the doctor's comment, appears to involve LNO2 and possibly other ONCO genes and they probably couldn't tell and there may not a way to tell which of these integration events is causative of the leukemia. So I don't know -- that's relevant to list them like that.

Dr.

Although it's certainly anecdotal, I think people in the retro virus community began talking that there was an LNO2 insertion, apparently, although I haven't seen the data myself as early as early April so actually quite soon after the meeting. But I do like the suggestion appears to involve and perhaps others.

And if the wording were that, which I really like your suggestion, then we could even list the others and others including "appears to involve" and "others including." Other thoughts? MissKwan.

The issue here is RAC acts as an information disbursle to the public because we were the only group that all of these discussions in public all the time. To let people know that additional information is around but that they should go looking for it themselves because we can't stand behind the validity of it.

have a concern, though, if indeed these data are correct and if it were to be determined that these other integration sites were the cause of the leukemia itself, it would suggest to me that this is information that we should look out very carefully because it might in fact increase the level of risk we see using this vector and it may augment some of the -- of our recommendations. I think it requires very serious conversation with those who generated those data and perhaps reassessment of some of the statements we made already.

Certainly agree with that. But in terms of finalizing a statement about X-SCID, the -- and gene transfer, the issue is how much of this information should we include. And I am comfortable including "appears to involve." I'm comfortable loosening the language and then leaving the information in the body rather than resorting to footnotes. Dr. Lo.

This is clearly an important and moving target. This will be the data or from the French are published and future events dictate.

Dr. Patterson.

Just hear about what we are hearing right now the phrase is "integration sites in the cell third participant appears to involve BMI1LY1LMO2 and" and I think a we have heard here is this information would be put as a footnote with a specific reference to the abstract and presentation that ASGT and would while it's currently says it appears to involve, we further saw in that that a third participant appears to involve LMO2 and possibly other onco genes and list those. And I think to Bernie's point I think that it's true that each day goes by hopefully we will have more information and we could sit here constantly and revise these at some point we have to fish or cut bait and go out with the conclusion based on the information as it is today but it's important to revisit and I might propose that we invite the investigators to come and present this data at an upcoming RAC meeting so you have a chance to hear the data first hand as questions and discuss it and revise your recommendations as need be. Does that accurately reflect sentiments?

Dr.

Might I suggest that in that -- where you convey that information we make the statement that the RAC will be inviting the scientists to discuss this at the next RAC meeting so that they know it's a continuum.

Let's move on back then to the issues under the clinical bullets. And the last bullet currently states some subjects of gene transfer studies for nonX-SCID, eg, chronic granulomitous disease experience mild to moderate clinical improvement. And the question in my mind about this bullet is that the nonX-linkSCID and chronic granulomitous disease, and chronic granulomitous disease is not a form of nonX-SCID first. Second, I don't recall our group reviewing at all since the initial discussion in the fall immediately following the first two X-SCID SAEs, I don't recall our group sighing the chronic granulomitous disease data. So I actually would suggest removing any comments about chronic granulomitous disease from this document and narrowing the document when we mentioned disease to just X-SCID and nonX-SCID. Is there any discussion of that approach? And then we come to the recommendations. The first sentence of the recommendation has been modified. Pending further data or extend with theeing circumstances reviewed on a case by case basis, retro viral gene transfer studies for X-link SCID limited to -- identical stem cell -- period. So so exclude or for whom know suitable stem cell donor can be identified. And the reason for doing that at least that we discussed in March, is that it is I suppose remotely conceivable that we would not be able to identify a stem cell donor but it's very remote that we couldn't find either a you have a comment?

I would say there is a chance that the -- may not be -- and may not -- criteria. Inaudible.

You're correct. Without going through all of the specific criteria, for HAPLO donors that they do exist

Particularly -- inaudible.

So are you proposing that we leave in the statement for whom no suitable stem cell donor can be identified? To make certain that every child is potentially eligible. Dr.

I think that will take out the rest of that paragraph and it adds nothing to what is already known about standard practices and procedures and informed consent and monitoring. It makes it clear anyway in that it's case by case. I prefer us not characterize experimental investigations. I wouldn't use standard of care language anyway as compared to standard treatment. At least cases extinguish those two -- Standard of care has liability for some people, lexicon not all -- I would just rid and I don't this it adds to our vision or any special advice we have to offer the world. Then would you leave that comment about the stem cell donor -- inaudible. I had a similar memory from that discussion but what it changed.

The group has been struggling and that's what the emphasis is from the doctor attending the ASGT meeting, the group has been struggling with the continued debate amongst those who provide HAPLs to babies with X-SCID and nonX-SCID. The continued debate about the relative efficacy of HAPLOs in that study, identical marrow transplants in that setting. You will recall we had an extension discussion during the March meeting about the need to ablate and prepare the marrow in babies with X-SCID versus not doing that and we had two very senior established investigators present to us, Dr. Buckley and Dr. O'Reilly who came to us with very different opinions, both with longstanding and extremely successful transplant programs. One using ablateo therapy and the other not. We were left with a dilemma. We can handle that by not matching it at all, which I think Dr. Powers is a you are saying, there is no established -- the discussion between those two experts and the different but related discussion at the recent meetings about of HAPLOs means that there is no standard approach. That this is -- and I wouldn't call it experimental. It lies somewhere in between. It's a judgment about how to manage and care for a baby with X-SCID.

I think my main point is we ought not be the business of characterizing which of these are experimental and we shouldn't be experimental or standard of care. These are all case by case gum of unproven things. What I don't know is whether as Dr. Says we should perhaps reinclude all for whom those suitable stem cell donor can be identified because it did sound as if the last meetings -- that there are instances which that seems to be an appropriate avenue and I wouldn't want us to foreclose that possibility by leaving that part of the sentence out nor would I want us to foreclose haul these continuing debates by adding any further material. I mean we probably shouldn't rush to some premature closure.

I think we are all in agreement. As I look at the wording this morning, the clause we were talking about or from no suitable stem cell donor could be identified really relates to inclusion and exclusion criteria and we were narrowing them too much if we delete that clause and the remainder of the paragraph deals with how we should treat babies with X-SCID and not our purview. I like your proposal very much. Let's clean this up, simplify it, reinclude that the clause that the doctor commented on and not use the rest of the paragraph. Which returns us, I must remind people almost exactly to our earlier statement, which was very clean in my mind that the final bullet is straightforward. We are not recommending any modifications to it at all. Any further discussion? Dr. Patterson, do we need a vote?

Sure do.

Okay.

I thank you all for thinking about this over the last two years and it will continue to -- the statement will continue to be modified as others have commented on as new information becomes available to us. And we on staff will make certain that as important information moves into the field we invite the investigators here in a timely manner to present that information. So thank you. We will -- yes?

I would like to raise a question related to the discussion we had last time and that was the presentation I thought rather compelling evidence that half identical transplants worked quite well in early infancy and not infected children and there it was a iffy proposition done later in patients that were already infected. And at that time we discussed appropriately then any issues relates to screening for X-link SCID to be part of our recommendations but was going to think about ways to weigh into that discussion going on in various -- inaudible. I can send you all out by E-mail to the entire committee we did actually look into that and the first of all there is a separate committee, a second father-in-law -- secretarile advisory committee to add to the screening that are currently in place for newborn screening. And the test for SCID has been on the docket or on the table and under discussion until recently how a reliable easily reproducible highly sensitive test with good reliability has not been available. Primarily bio-chemical. The more recent test developed by including the team here in NIH for doing PCR based diagnostics has recently come forward and under evaluation. This issue is back on the table again and under consideration. It is recognizing the efficacy in many instances of early transplant in these infants. The issue is one that has come up frequently but it's been the quality and the reliability of the test that is at issue.

would add to that that we are all struggling with including newborn diagnostic testing for X-SCID in our newborn panels. And certainly I can speak for California, we were looking carefully at using the PCR based methodology which does come from NIH primarily and including testing for X-SCID in our newborn panel. Other comments or questions? I would like to move forward to our first protocol for this morning. The discussion of human gene transfer protocol 6 13RBGS Phase 1 dose escalation study of intratermeral herpes simplex virus mutant rRp450 in patients with retracktory sarcoma or neuroblastoma. I been asked after I introduced the protocol to announce the recuseles of Dr. Hess lap has already left the room. She is a member of DSMB for this study being conducted at Cincinnati children's hospital. I understand that Dr. Timothy Cripe, M.D., Ph.D. will lead the discussion this morning. Thank you, Dr. Cripe.

Thank you Dr. Wara, members of the RAC. Pleasure to be here today. I appreciate your thoughtful review of our protocol today. Look forward to in-depth discussion today. I will be discussing our proposed proceedical which is a face I dose -- rRp450 in patients with retracktory sarcoma or neuroblastoma. I would like to begin by talking about the tumor targets we are looking at for this trial. Proceed then to discuss briefly on collatic and use for potential cancer therapy and describe vector we are planning to use rRp450, the structure, efficacy data and safety data and describe and outline the clinical trial. Beginning with the first topic, the tumor targets we are looking at for this clinical study are relapse soft Tish knew sarcoma in pediatric is ram dough -- sarcoma which occurs in children as young as adolescent and young adults particularly those relapse. -- which is the most common soft tissue sarcoma if adults and variety of other sarcomas that we tested in preclinical studies that maybe eligible for this trial including for instance malignant tumors which are common in patients with neuro fibromaitoussis. In addition this clinical study would include patients with relapsed bone sarcomas and osteo sarcoma or one of the members of the sarcoma sarcoma of tumor's the final target is relapse neuro belstima. What they have common is their dismal prognosis when they relapse as illustrated by the neck several slides. This is a survival curve of patients after their diagnosed of the sarcoma and in this particular graph it's for those that are in the -- area. And head and neck that as you can see if most patients do not survive this, very few do and it depends a little bit on the characteristics of their relapse regional versus CNS, et cetera. Similar data are found for the other tumor targets that we included in this study. Here are data for relapsed osteo sarcoma where most patients die of their disease the first year, year a half following relapse. Survival after relapse Ewing sarcoma is no better with the curve dropping close to zero the first year for those who have received paltive therapy with mild prolongation of survival with therapeutic treatment meaning somewhat usually experimental agents on clinical studies. And finally survival after diagnosis of neuroblastoma and in this case it's not even relapse but high risk neuroblastoma and those with certain age groups the survival again is dismal. So these collectively these diseases represent significant unmathematical need moving on to the discussion of -- attractiveness as potential cancer therapeutic are many fold including their ability to kill cells directly. Often turned okayo liesis. And bypassing therapy that often mediated by p a 3 mutations and ability to self-propagate within a tumor and overcoming the gene transfer inefficiency that is commonly found with recommendation incompetent vectors and enhanced delivery of therapeutic genes. The -- are illustrated on this slide with the genomic structure across the top and consists of a long region and unique short region both of which are flanked by repeats. The NV1020 vector that is reviewed in the first 100 RAC in the past, contains a deletion or replacement of genes in the internal repeat region with replacement of likeo protein sequences from agency which was to using it as a vaccine for both types 1 and 2. This deletion and replacement results in deletion of the UL55 and 6 genes. There is a deletion of the UL24 gene. Mechanism of tumor cell Shrektivity for this vector is not characterized. The ability of the virus to replicate because it is impaired partly it's thought because of the deletion of the -- repeats in the middle allowing the virus to not undergo its proper recommendation but it does have intact the gamma 131- a gene which is touted as a neuro veer license gene which is important to be deleted for safety. One of those copies in the gene and other repeat on the geno. And TK deletion. But it has been replaced under control of the early promoter alalpha 4. Complete absence of the and coded gene gamma 131.5 demonstrated by the deletions of the long repeats. And it also has insertionle mute genesis in the ICP6 gene for the gene and coding ICP6 which encode the large sub unit private -- re-- and in that vector there is the beta Gary Locketic gene. Final vector that's tested in humans fairly extensively is 17-16 most of this work has been done in Europe. It contains simple simply deletions of the gamma 131.5 genes. These vectors have been used in I'ms on doses used in this slide. The 1020 gene is used in published in abstract form by human -- with intraPatic arterial injection for patiences with metsetic colon cancer to deliver up to the plaque forming units without significant adverse events. has been used in intracerebral or tumoral or for brain cancer. Up to three times ten the ninth by forming units and 1716 has been used in patients with brain cancer with tumor injection and head and neck squamous cell carcinoma with tumor injection and doses up to one time tens to the sixth. Vector we would like to use in this trial is rRp450. This contains insertionle deletion mutation in the UL39 gene coding ICP36. In place of that gene appears the CDNA encoding -- which is p450 enzyme pro drug enzyme that activates -- it's under control of the early ICP6 promoter from the virus. This gene retains or this virus retains the native -- and it's sensitivity to a -- The HSV1, 139 or I-36 protein, coding the large -- converts -- as illustrated on the slide. This critical for virus recommendation to generate sufficient pools of -- for virus recommendation and also critical for reactivation of the lateen virus. Tumor cells are known to express high levels. As illustrated in this from our laboratory with this panel of human malignant tumor using also the monkey kidney as a control and these are compared on the farther left in norm human cells which do not express high levels. So this differential expression of this enzyme is the major basis we believe for the selective recommendation rRp450 in actively dividing tumor cells. We published the number of papers showing the efficacy of the vectors for the target many of the target tumor times we like to include in this clinical study both cell lines and human xeno graph data to show the cell line date u a is ile strighted -- illustrated on this -- indicates a ten fold increase in susceptibility or sensitivity to the virus in an MTT assay. We looked at random sarcoma and Ewing sarcoma one cell item and itema and large panel of cells derived from neuroblastoma and new from it was not included in our appendix and maaing ininant tumor cells. With the vectors in clinical trials we found a broad range of sensitivity to these vectors both. G207 was weaker in general and the Ewing sarcoma cell line was not sensitive to either of these. Where as the neuroblastoma and malignant tumors were quite sensitive to infection and -- by the vectors. RRp450 showed somewhat increased sensitivity or siteo toxicity for many of the cell lines particular note was the Ewing sarcoma cell lines which appeared sensitive to infection with rRp450 increased above that of those vectors that are used in clinical trials. In addition to even those cell lines that showed quite a bit of sensitivity, neuroblastoma and the cell line they also showed increased killing with the rRp450 vector. This data suggest that rRp450 may be more ef conscious than those vectors in clinical trial and parentthetcally some the criticism of these vectors has been that although they have been shown to be quite safe in human study, the efficacy has not been robust. Using xeno graph tumor models with human -- from human tumors placed in mice we shown remarkable antitumor effect with a single injection of some the different viruses as shown here with NV1020. When tumors were fairly large between 250 and 750 millimeters cubed and in this study that single injection gave us a 50% complete response rate four out of eight tumors shrunk in the eight weeks disappeared and remained absent in the animals until the end of the study. The other in another two we had a partial response meeting that the tumors to less than 50% of their original size although they grew back to progressive disease. And one of them appeared to be have stable diseaser to awhile and then grew back as well. We had an overall response rate of 75% and we have similar data that we now published in models of neuroblastoma as well. Using the rRp450 vector and in this study at a lower dose 6 times to the fifth, we also had a significant antitumor effect with a 50% complete response rate, five of these ten tumors shrunk in the first three to four weeks and disappeared. of them show prod gr'sive disease and another several had stable disease at the time of the end of the study. Considering a complete response and a partial response to the response rate here was 90% or nine out of ten and base ten. Most of the concern with this vector has been its potential toxicity and therefore we embarked on a number of safety studies recently I would like to review. began those with looking at the attain wings of our rRp450 recommendation in normal human cell lines. This data repeated several times from normal human hepet site showing hepetsets showing ROP4350 or as a control. The apparent strain from rRp450 was derived and the cost strain. Here examples are shown for multiplicity for infections. 01 .1 and the culture was measured at one hour, 24 hours, 48 hours and 72 hours. In the rRp450 cultures, infected cultures we did not detech virus recommendation over time the amount of recoverable replicating virus as determined by plaque assays fell off with half is that seen when add virus to immediate alone without cells. In contrast the cost vector replicated robustly increasing three to four orders of magnitude over those 72 hours. So these data suggests the rRp450 is quite attain with theed and normal human pepetsets. We know in cells that divide this culture andup hepetsets are not healthy and do not take well in terms of dividing in culture but cells that divide are predicted to support virus recommendations than those that are were live are theed cells to have enough regulation reducatis. In cultures of prolive are theeing which came from donors we do detect robust virus recommendations both of rRp450 as well as -- and in this experiment we included the NV1020 vector in human trials and showed it replicated to a similar degree in these cultures. Interestingly and has predicted when the cells were differentiated with calcium we no longer saw probust recommendation of the rRp450 vector. We did see continued recommendation of the wild cost cost vector on the far right and interestingly enough we saw fair amount of recommendation of the NB1020 vector. So the data actually suggest rRp450 was more attain with theed and attain with theed if not more in NV1020. Terms of our safety studies we performed a variety of studies in mice. And we took basically what was our highest available or given volumes and tighters and injected mice with to the 8th plaque forming units of virus. We use virus alone plus Foss md. Fosso md was added 24 hours after the virus injection to allow for transgene expression from the virus. The clinical studies after both intraVenus and tumoral virus injection were performed on day three and late time based orecommendations by the FDA and we found no clinically significant effects on animal waits. Complete blood counts electrolytes renal function or liver function in the detail graphs of those are in appendix M. We tested virus with the additional psycho Foss and did not see added toxicity due to virus that we in addition to that toxicity with -- as predicted it did drop blood counts those recovered. We also performed survival studies with both intravees if and intracerebral virus injections. The data on this slide with the well type virus are new but used as a comparison to the data with the rRp450. So when we gave animals this virus intravenously or intracerebraly it was toxic and had tended fifth dose of plaque forming units nine of ten animals were alive but all of them had neurologic toxicity, paralysis and nine of those recovered. So there did appear to be morbidity at that level and doses higher than that, none of the animals survived and these animals in these groups died within three and seven days following intraVenus injection. For the intracerebral injection, two of the ten animals died. Ten to the fourth and all of the animals at ten to the fifth. These deaths occurred within predominantly in the first week following virus injection. These data suggest that this model is suitable for assessing toxicity. At least to this degree. In contrast when we used rRp450 at the highest dose tested with well type 10 to the eight -- all of the 20 animals injected with intraVenus virus have survived and the experiment is out to 150 days. Intracerebral injections two of the mice die shortly after injection and was thought to be due to the procedure and or the anesthesia but 18 of them have survived and now out approximately a month or so. So the data suggests there is quite several orders of magnitude difference in VIVO toxicity of rRp450 compared to HSV. Similarly in those studies we have added extra -- Foss md 24 hours after injection of the virus. Here are the data from the previous slide recording rRp450 in animals given psycho Foss md alone we routinely lose about 10% of those animals due to toxicity from psycho Foss md presumed low blood counts susceptibility to infection and when we add in the virus to psycho Foss md we also lose about 10% no different from psycho Foss md alone. These data actually suggests that the combination is no more toxic than psycho Foss md alone. So finally I like to discuss our proposal for the clinical trial. Our aims are to establish the maximum tolerated dose of rRp450 administered as a intertumoral injection within the con fineses of the dose escalation scheme we won't continue to escalate until we get maximum tolerated dose but rather escalate into the highest dose that we are able to give based on the tighter and rather try to push it even further. Secondly to determine the dose limbing toxicity of using intratumoral of rRp450 in humans. Our secondary aims are to measure antiviral immune response to measure the systemic Byrumia and cee fine the antitumor activity of rRp450 within the confines of a Phase 1 study. Pairthen catted as well this study will serve as platform for future studies with the combination of psycho Foss md order to enhance efficacy. Our criteria are patients that are older than age two and younger than 30 so they may be treated at a single institution. Cincinnati children ps hospital. Hiss logic diagnosis of the sarcoma neuroblastoma that is relapse to therapy where there is accessible measurable disease accessible meaning it has been determined by the interventionle radiologist and the life expectancy of the subjects will be required to estimate at greater than eight weeks. Our exclews criteria are those that we use in standard pediatrics face I driveteria no acute toxic affects are active and ongoing from their prior therapy and sufficient time from prior therapy to recover from those such as mild suppressive chemotherapy and othery ino plastic biologic therapy and radiation autologous bone marrow transplant and no patients would be allowed if they received the transplant prior to gene therapy, pregnant or breast-feeding. Our treatment plan is to administer the democratic CT guided intratumoral injection. Virus we propose to enroll three young adults first in the age range of 22 to 30 and subsconly for each dose stratta is then enrolled pediatric patients. We will give up tower injections three weeks apart and initially plan for six dose stratta beginning at one time down -- and escalating out to three times ten to the ninth plaque forming units. Our dose determination was done because there is no maximum tolerated dose determined in humans yet. Each of the studies I cited gone up to the previously determined maximum dose. Again, the maximum dose with NV1020 which appears to be similar in terms of its attain wings if not less attain with theed with rRp450 was 1.3 times ten to the sendth. NCI and -- has allowed us and pediatrics in general to begin phase I dosing at pediatrics at 81% of dote es which is one times ten to the 7 pght. Procure a gram basis the exposure will be at this initial dose at 10,000 foal less than the maximum dose tested in mice which was ten to the eighth|1 th |0. Regarding good manufacturing production of the vector, I learned on Monday the national gene vector laboratory has provisionally agreed to support this project and need administrative issues though I not received the official notification and if that's the case, then we would plan po produce this vector in the human gene therapy -- university of Pittsburgh. In fact, through Dave we had a number of successful premanufacturing runs with rRp450. The method that he uses are purification methods though we have not been privy to the details of those methods. He has achieved 8.5 plaque forming units per mill. And with these premanufacturing runs which feed quite sufficient for the kinds of dose pros posed in our study. So just to conclude my formal remarks, the intended participants have incurrable cancers and therefore there is quite unmathematical need. Cancer model of these diseases are susceptible to -- the efficacy of ROP450 is attractive because of the increased siteo toxicity and use it as a platform for future combination with and rRp450 from our study appears safe in that it's attain with theed by fold versus wild type -- cells. It is at least attain with theed as MV1020 which is in human trials. High doses tolerated by mice. And there is no added when combined with psycho Foss md. Therefore it is our belief that rRp450 warrants safety testing in subjects with retracktory potential -- cancers. I look forward to our lively discussion about this proposal. Thank you very much. We will begin this morning by asking the individual reviewers to review their initial concerns publicly for the record and then to state whether that concern is resolved or continuous. Those concerns that continue will generate some discussion with you. If you like to stay there, that's probably -- you may either choose to stay there

I'm happy to main.

Why don't you stay right there. Dr. DeLuca, would you like to begin?

Sure. I would like to thank Dr. Cripe for the presentation today. His responses he has answered some of my questions, quite a few things. So what I would like to do is to go through each point and then perhaps -- a point we didn't have a discussion. I think we already been through what the -- protocol. The stated objectives I would like to add something here, the state of the object Science Centertives of the protocol were to opts objectives of the protocol. I didn't so there is one thing I need clarification now pending your talk is the -- do one of the objectives of the protocol depend on the expression of the rRp450 gene?

Do one of the objectives? This first study do not depend on the expression.

That's clear. So most of my concerns had to do with safety and toxicity of the virus and I will go through those. I had some questions about the slant put on the tropism of the virus. This virus and there is actually a very good paper out there from 1988 from Goldstein and Weller for people who first made this virus who showed that this virus -- is defective for growth in dividing cells. Normal human di employed di ployed. The other important thing which we will get to in a minute is it's temperature sensitive for growth no one knowize. It elevated temperatures the virus will not grow even in dividing cells. So given that and the results of another paper from Don Cohen's lab in 199 where he first looked at the pathogensis of this model and showed that the virus was attain with theed in the growth in the -- and for the establishment and reakes have of latiness that his conclusions were that because the mouse has an elevated temperature, one has to use -- these are -- I could quote this thing but I would have to get online to do that and we referent allowed to, apparently. -- we aren't allowed to, apparently. The main conclusion is that you could not extrapolate from mouse to humans with respect to the pathogeninousty of this virus because of the elevated temperature of theThis to something that the and I had was that a discussion about the need for some other animal model or for primate data which was done with the other two herpes, the other three herpes trials, the one with the gamma 4.5 mutant and one with the big deletion and the one with the ICP6 gamma 34.5. So I would like to have some discussion on why -- I'm not sure I understand your response why you think you don't need a primate model or some other animal model to test for safety.

Sure. I think in my discussion with members of the FDA their feeling was that the need for various animal models is dependent what they take into consideration is risk benefit analysis of the proposed clinical trial and proposed route of the administration, et cetera, and their feeling was that even primate studies would not necessarily accurately reflect the situation in humans and that ultimately if one needs to test safety of this vector it needs to be done in humans and therefore their criteria and level of concern would be predicated or dictated by in part by the risk category of the potential enRollies of the clinical trial and they felt that the showing safety in mouse models might quite well be sufficient, obviously they couldn't give a final verdict until they reviewed all the data. And the fact that the data from the 1980s showed elevation of the temperature attain with thees to some degree the virus -- we have taken the temperature of our mice in our studies. I know that the average mouse is slightly warmer than the average human. Approximately 38 degrees. And it's true there may be some attain wings of the virus at that temperature. I don't know our data about continuation of -- herpes or any other type of mutants as well. I guess our bottom line was the -- it wasn't clear that it seemed clear that this virus is with theed compared to wild type in these mouse studies and indications from the FDA is that may be sufficient. So in the interest of being able to move the field forward, in a relatively timely fashion, and actually to make it doable from resources standpoint, we have not undertaken those studies.

Well then let me skip forward to my sixth point. Tan little bit on that. If we look at the other viruses that are in clinical trials they have all been tested in in primate models, not human primate models. The two I want to talk about are the G-207 and 1716. Those are good ones to think about because G-207 has the same mutation that you have here. That has been up to three times ten to the ninth and very attain with theed. Now taking the single mutation, 1716, the one that was done over in Europe, they only gone up as high as ten to the ten to the six in virus and that's because that virus is -- that virus is easy to hurt monkeys with. And you have a starting dose of 10 to the sendth here. Due to the lack of testing -- ten to the seventh here. Due to the lack of testing shouldn't you consider lowering the dose especially since you are proposing to inoculate children.

I think it is reasonable question and my written response we had basically been worried about losing efficacy by going to -- I guess we will be happy to revisit that question.

What's the primary objective of the protocol and the primary objective of the protocol is safety.

Correct. However, it's against the code of federal regulation to do clinical trials on children without the possibility of some benefit and therefore we felt that this was a dose that did well in our mouse studies in terms of efficacy and therefore had the likelihood of potentially having benefit in humans. But again, we are happy to consider a lower dose if the lack of primate data dictated that consideration.

My third question is, has a marker rescue version of this virus been generated to assure the attain wings is solely due to the coding sequence? The reason for doing that is because this protocol will be precedent setting and that's why it's reviewed here. The a virus with a single deletion in it has never been used in people before. We want to know whether the attain wings is due to that deletion just in case other similar protocols come forward in the future. If this does have additional mutations in it which are likely that often happens with herpes, then it may be attain with theed for another reason. Now I know you said that in the 1020, that rescue weren't generated for that but they were for the G-207. So something it to consider. And the other thing would be give an the novelty of this vector for use in humans and the fact that the p450 gene is not employed in this application, it be better if you just had an ICP6 mutant that didn't, prex p450 and -- p450 and the virus will be established latiness and it can replicate and have expressing this p450 gene and it adds another variable that really isn't used in this trial. It doesn't -- it seems like it's an add-own. Almost asks for expression data which you don't include in this. There is a transgene here and there is not expression data given for this transgene in a clinical -- in a kind of model. It would be clear and better science if it was just ICP6 deletion unit.

Now, in my view it speaks somewhat to the practicality of the matter in formers of what we like to do is use this as a platform to use fuseture studies and as you know if we were that want to expand to a p450 transgene expressing vector it would require enormous amount of resources, time, effort, money to then redo all the studies that we are proposing and all of the preclinical data with the separate vector. of that data will have to be reduplicated again with vector now that doesn't contain that virus where as if we show safety with this vector that we will look forward to look at transgene expression and potentially expand that array of studies that could be conducted with this vector in the future. I think it's somewhat of an investment in the future and include that now.

In appendix M in this trial we usually call for or requires an expression data transgene for this particular trial in some kind of model duration of expression of what cells are the transgene expressed. Are data like that available?

Data like that are available. Most of that is done by Kent as general who has looked at transgene expression, functionally. Mainly there is not great assays for functionle transgene expression other than taking superor conditioned media from virus infected cells and using that to show toxicity of other cells from it. Akin to one might see with the addition of the chemotherapy to those cells with appropriate controls, of course. I'm not aware of any inviiveo data.

How about the western block? Flies the IV6 promoter?

Yeah, data there. We haven't been focussing on the use of that transgene, I don't have those data at my laboratory.

Do you think this will listen to the rat -- than listen to the immune response?

It may well but there are a lot of foreign genes expressed because all the virus genes are foreign. Usually with at least in mouse models of the infection there are immuno+ dominant peptide that most of the antitumor activities again in many cases depending on the mouse train is from lack of protein B.

Most of these people have antibodies to herpes.

Many will and if we go down to the lower age rates are talking some of them may not. But I don't think that the contribution of the rat gene to the immuno+logic reaction will be significant relative or within the contact of all the other viral genes that are being expressed.

And my last point is to what extent does the virus express lay-in see used in a modwell cancer? I know in the standard herpes lateensy model the establishment is very low. Now you are putting in the cells where presumably theoretically this virus will replicate well and generate a large pool of virus and then can go and feed peripheral neurons. So the question is in these mice that get these, that's your treat, cure, are there ganglia loaded up with the herpes rRp450 gene.

We have not looked at that. I worked closely with Nancy who is a expert in herpes lateensy and it's not known and it's clear administratoring high loads of virus by other routes than what I acquired may result in cells containing lateen genomes that otherwise would not have and that's an unknown and something that its yet to be looked at. don't have any data about lateensy in our animals but we have tissue sections in our animals that could be looking for a lateen virus. We aren't concerns because lateensy is a welcome risk in this high risk population in patients that will succumb to their disease.

And lastly, did you actually test for -- sensitivity.

We did. I can show you a graph if you like to see it. That was tested in the contact of psycho Foss md and against ant cyclever with a concept the fear that the psycho Foss md might inhunt virus recommendation and so actually in that experiment was used as a control and that it inhibited rRp450 recommendation four or five orders of magnitude where as the psycho Foss md did not.

You had minor points and I think were were -- they were taken care of and that's all have I.

Dr. DeLuca can you review for us your remaining concerns?

My remaining concern, the reliance on the mouse safety data. The marker rescue virus and the use of the rRp450 gene. And the high starting dose rate.

Thank you. Dr., let's move to you.

Dr. Cripe, first of all an apology in order to talk to the microphone have to turn my back it to you. But thank you for your responses to the concerns that I raised. My major concern again is the safety issue and the lack of not human primate data that Dr. DeLuca has reviewed. And that has been discussed. So I still concerned about that, but I really would defer my concerns as well to Dr. DeLuca who is far more expert on herpes. A second concern was that of about immune compromise in patients who have already undergon significant chemotherapy. That has been addrested, although I must say I still am concerned by the comment that immuno+ deficiency related to chemotherapy is not expected to increase virus related toxicity. My third concern was about the insertion of the siteo chrome p50 gene when it's not going to be used the same as Dr. DeLuca has raised and I guess I'm satisfied with the response in part that this is a platform for future study. I raise the question that about comparing the biologies of the tumors to be treated in adults and children. I think the idea of treating adults first before the children in each given doses is highly appropriate. However, with that result in problems related to the different biology of the tumors this has been addressed satisfactorily. In the original protocol that three different potential sources of the transfer vector were discussed and been selected and that has been addressed by the presentation today. And my final concern then was about the informed consent and the expectation of benefit that was raised that also has been addressed by the regulations regarding studies in children. I have a couple of questions related to the presentation. And one just by the way since this virus the attain with theed virus can grow divided cells which include the get, does herpes infect gut cells and do you see shedding of herpes in the stool if so is that something that should be looked for?

Most of the time -- I'm assuming you are asking in certain native certain infections one see shedding of the stools. Most of the time the infections are local, mucosal and -- checked by the immune system. I actually had patients with severe immuno+ deficiency which acquired herpes simplex virus die of HSV disease which disseminated openthal infection in those one rare case one be able to virus in the stool. I'm not aware of the study. Usually it's a local disease.

And then I wondered if you could go back to the two slights you -- slides you showed about the response to the intratumoral injection into the mice. It looks to me as if three of the mice if I'm looking at the numbers right had a complete response. Three others that had a partial response that not sustained and then one started to see further recommendation or further growth of the tumor and one that clearly had an early response but then failed. So where does the 90%

Five of them ticked down in the slide here.

Five, six -- there is ten in the study. Five have -- each of these is a different mouse and they are different symbols but it gets a little difficult to discern from one another as they are clustered in here. There are five.

So there is a 50% -- this from a single injection?

Single injection. Yes. 50% complete response rate and another four that had a partial response rate. In terms of categorizing their best response, these four or these nine out of ten is what I would say has the response rate. Two or three of these that had partial responses eventually regrew at least to the point where we would categorize them a stable disease which means they are 50% of their original size and one had progressive disease which means it grown beyond 50% of its original size. And the other one remained down here as a portion response less than 50% so that's -- it's right there.

And the next slide please.

This is within the 50. Similar data are here. Four of these had complete response and remained on this line at the end of the experiment. Two of those the trunks were less than 50% of their size and therefore categorize as partial response and went on to regrow. One of those that its best response was within 50% of its size. And stable disease went on to have progressive disease and one had earlier progressive disease. So again the initial response would be six out of the eight or 75%.

And in these mouse study dozen you have data on repeated injections as you are planning to do with --

We have data on repeated injections. I didn't show that graph. We have given mice published in our paper courier now. There were -- we gave mice ten different injections, three times weekly, Monday, Wednesday, Friday for ten doses and that was an attempt to be able to shrink larger tumors and I might parentthetcally add that 90% if not more of the study if not more out there tumors there are 50 to 100 millimeters cubed. Very few had large tumors. We decided we wanted to have efficacy in large tumors in mice. So these are quite large in mice. We taken it up to 1100 millimeters cubed and those president experiments where we did ten repeated injections and did get some efficacy in the larger tumors as well.

Are there any studies where the injections let's say three or four weeks apart were done? Something more similar to the clinical protocol?

No, we have not done those because I guess the closest thing we have done is published in our paper which is on neuroblastoma where we have injected tumors as they have grown here. Most of the time they are shrinking and no need to inject them. And those that have regrown we have taketen upon ourselves to do reinjection at points where it's clear they have progress fen disease and in those cases we seen complete responses in many cases actually. There is no data -- no evidence that these cells are actually acquire resistance to HSV. believe it's a matter of dosage and the cells are dividing faster than the virus is by giving it a boost we overcome that. It would be helpful to have seen that. Thank you.

Doctor.

I believe that when I left the room it was stated that federal regulations for research involving children require that you can't involve children unless there is prospective of direct benefit and that's not true. I wanted to point out that there are four categories of research that are allowed for children, one is research not involving greater than minimal risk. The second one is research involving greater than minimal risk but presenting the prospic of direct benefit to the individual subjects. The one that requires that there is direct benefit to the subjects. The other one, third one is research involving greater than minimal risk and no prospect to den -- generalizable knowledge about the subject's disorder or condition and the fourth one which is only approvable by a second father-in-law panel -- secretarile pattern that presents an opportunity to alleviate a serious problem affecting the health and welfare of children.

Clarification, the first or four of those don't apply in this case. Clearly the first one which is can't be greater than minimal risk in healthy children applies. The second one is the one I cited and the third one there is generalizable knowledge about their disease which I don't see this study will yield. I still think that it's not fitting with the code of regulations as written that we would conduct this trial if there weren't any potential benefits. It's not to say that our consent form stating there is a benefit in fact we say specifically that you may not derive any benefit which is standard language for our phase I studies.

So, there is ongoing confusion in the pediatric community and among IRBs about this precise issue that has just been discussed. In other words, in what groups of phase I studies should we move forward in children without prospect of direct benefit to the participants. I think that's a subject that's beyond resolution in this forum, but agree with its having been raised in the contact of this particular study.

Yeah, I haven't really looked closely enough at the protocol to be able to say whether or not this fits into any of these individual categories. However, just from what I know it seems to me that this research would be likely to yield generalizable knowledge about this disease. The times when you get into trouble with that category is when you involving normal subjects, normal children and I don't believe you are planning on doing that now. Those normal children don't have a disease or a disorder or condition so that's when people usually get hung up on that third category. Or that you could argue perhaps that in this case it has to be just a minor increase over minimal risk and it may not fit that part of it. I can see that possibility.

Yes, the experience we had with IRB suggest that category three wouldn't have gone very well.

So the conflict here is what we have been whispering back and forth about and that is the application of the guidance to identification of the starting dose. In other words, the balance between risk benefit to a particular child subject. And Ms. Kwan on that note I will turn to you.

Actually, I would like to before I begin my independent comments to sort of piggyback on the conversation because in listening to doctor-recite the categories, I'm sensing that it might be a kind of a bizarre application of the wording that is being used. I think to me what that says is you are not supposed to expose kids to a heightened risk if there is no potential for benefit. And that makes sense. But I don't think the answer or the response to that is to therefore up the dosage. I think the intention is to decrease the risk rather than to up the ante, so to speak. So that's a lay person's interpretation of what those guidelines probably should be meaning. And I think this isn't first time that this has come up. I think even last time something similar came up. On to my specific review. I was particularly concerned because the population of potential participants are children who have almost no alternatives. And for children as young as two years old I think it's very emotional issue for the parents to be making these decisions. And that makes it all the more important that the explanation given to the parent is absolutely clear because I think the emotions is do anything to save my child. But I think what we really need to convey is if there is not a reasonable chance of helping the child, is it reasonable to expect the child to be exposed to higher risks of greater discomfort in the process of dying essentially. And I think that really needs to be scrupulously handled. So I was very concerned that the language both in the nonscientific abstract and the informed consent seem to me to be very complex, much too technical, and not really addressing some of the issues that I think that the parents of these children should really be confronted with in making these decisions. A number of the specific requests I made have been addressed. The lay abstract has been modified and so have a number of the statements in the informed consent document. But I would just forewarn people I think a lot of the times people are using some of these automated readability indexes to determine whether or not they fit into this eighth grade or below understanding and those are mainly based on statistics such as how many syllables are used in the words you and that often doesn't come really recognize what's practically true of people Reading the documents. So even though there have been modifications, I find that it's still really quite complicated and that may be a reflection of the fact that the issues involved in this protocol are complicated. And I think we did have a symposium at a previous RAC meeting where our outside experts had really strongly said it's not so much what's in the written documents, it's how you handle the discussion. So I would add to my comments that a lot of this can't really be assessed at a RAC meeting but that the investigators really need to be alert to the fact that they have to be extremely sensitive to how this is explained to very emotional parents at this point. Some specifics there was a difference between the age groups that were allowed and I believe that the investigators have now decided that the age range will be from two years old to 30 years old. There were some documents that showed children as young as one year would be enrolled and that's been corrected. I asked that in parts of the document that talked about herpes simplex virus that some recognizable examples be given in and that has been inserted. I asked in general that terms such as patient therapy and treatment be replaced. And that has been done. I also asked that some language would -- which said if you have an adverse result and there are unanticipated infections that you would be treated and my comment was that the language suggested that you would be treated successfully so that there a little change in that so that they recognize that even though there would be treatment, it's possible that the treatment would not be affective. And finally just some again language in the informed consent that said there are other risks and this will be explained and the response was that the explanation would be on a case by case basis in discussion. And that seems to be satisfactory. Just in terms of the conversation that has gone on in this review, I just really think that overall the risks of causing tremendous discomfort or prolongation of discomfort for the participants should really be carefully discussed with parents. If in fact so much is still unknown and those unknowns may get very marginal that there will be any kind of positive effect.

Thank you very much. Any response to?

I appreciate Ms. Kwan's comments I think they are appropriate and thank you.

Dr.?

Thank you.

All right.

Any further discussions from RAC members?

Hi, I'm sorry. I reviewed this protocol in detail so you may have the date -- if you ever done these injections into mouse tumors in fully immuno+ competent mice and also in the situation where those mice have already got some sort of latent herpes infection?

We have done the former and not the latter. We injected in our random -- mouse model in a genetically susceptible mouse for a knockout hepetset growth factor these mice get early onset -- sarcomas and we are working with that model to do exactly what you said. That model actually is quite aggressive. These tumors grow quite rapidly and we have not seen any adverse events from those injections but we haven't got very good efficacy and I think the tumors are just too aggressive for the virus to do its thing. We have not preimmunized or exposed animals to HSV prior to these.

So the injections where you don't cure -- where you can't cure the mouse tumor, how long does that normally take to kill the mouse?

It usually the mice need to be euthanized or the rule at our institution is when the tumors reach 10% of the animal body weight and those models it takes seven to ten days, at the outset maybe 14. That's as far out we were following those mice following.

And you haven't been able to do --

In the repeated injections we can get a little delayed growth and get them out to three weeks or so. And we have done that and again not seen adverse events but not many watch -- I guess my question really relates partly to efficacy obviously but also partly to the issues of toxicity. With these studies which you shown I would predict there would be complex toxicity which might be associated with immune response both to the virus and tumors and any other latent virus existed in the patients. I raise that as another sort of toxicity issue.

I think those issues have certainly been discussed in the contact of the other clinical -- con text in other clinical trials regardle of which mutant interestingly in the G-207 model the antitumor effect is only seen when there is a immuno+ competent animal and they lose that effect in the -- and been shown that the antitumor effect is actually more cytotoxic T-limpset to the tumor specific antigen rather than the virus recommendation.

Is it not possible to use some other model not necessarily -- sarcoma to get some of those data with your virus in immuno+ competent model?

We could test other -- in fact that has been done at Mass general he has used mouse colon cancer models. But colon cancer models are immuno+ competent animal. His model injection the tumor sells and follows the virus injection and either in the spleens they get metsetic lesions in the liver and it's delivered by injecting it into the spleen and they have done extensive studies in that record with rRp450 and haven't seen any adverse Evans and has pretty good efficacy in the mouse colon cancer models.

Thank you.

I would like to make some comments coming from the perspective of someone who has done and is doing human gene therapy for cancer patients. There is always a huge battle within everyone's mind between practicality and, quote, good science. The kind of thing you can do in mice. And I think it's important to discuss that and I was Dr. DeLuca's comments I think very important in that and I have suggestions where you may try to balance those two. Obviously if you are forced too far to the perfect science, the field will never advance and never be able to get into human trials. On the other hand we need some level of safety. It seems to me from just a practical point I accept your explanation about keeping the transsheet in. To rye try to do all the ting in phase I and have to repeat the whole thing with the second virus kill the program which is impossible to do that. That's another problem. We are using old virus by the time we get to humans. It's important to think ahead as you did. My personal opinion is that I don't think it would add significant risk. The immediate response you generate might be good in terms of also being something that would help get rid of the tumor. On the other hand, you aren't or you are not proposing doing primate data which I think is risky since this is a different virus. So I agree that I think the start -- if you aren't going to do primate data that's very expensive to do. The starting dose needs to be lower. Especially based on the data from the European studies where they saw toxicity. If you worried about this no possible benefit in children which I'm not sure is really an issue, then start with the with the lower doses until you feel more comfortable. So my suggestion then would be to try to go down it a low dose, 10 to the six or maybe lower for the first few patients. If you feel you must do that in your adult patients and move forward especially see if you aren't doing with the primate studies. I think these patients are in desperate need. There is nothing to offer. There are three trials with herpes. This is less risky Not injecting it into people's brains. The virus does seem to be more active. May be more toxicity and will have to find that out. I believe it's an important trial to move forward with. If you are not going to do the primate data and the FDA will guide you on that, then I would suggest starting at lower doses.

Can I add?

Yes.

I think the lower dose is a good idea and really does address the issue raised by Dr. DeLuca. And you are not going to get in trouble clearly with the provision that says you need efficacy because you aren't throwing away your high dose which is where you expect to see efficacy. That's not an issue.

I would be happy to do that.

I was going to ask you to given the clarification of the federal R regs that we heard from Dr. Borrow to review the rational from the ten to the searchth starting dose the concern of the group are clear. How would you respond so that we can structure our formal response to you?

Sure. I guess we would be happy to start at a lower dose and actually reviewing and looking at the data on the slide in the experiment when we did use rRp450 which we take more efficacyious and at the time we had limited and only given ten to the fifth or six times ten to the fifth and got significant efficacy. I think in retrospect there is quite good reason to believe that even a lower dose may have potential for benefits.

Thank you. I think we -- doctor?

I would agree with Dr -- about development of a program that involves virus that has components that would be necessary for the first study. Are there preclinical data perhaps in the model that Dr -- asked you about where the virus alone wasn't sufficient with getting psycho Foss md over the top in the immuno+ compromised.

Yes, at the end I'm not sure if I deleted -- inaudible.

I think everything after the black box. In reserve. I will describe it to you but pictures are really better. I will show you some cell data and these data from a group. Doleen cancer model. A panel of five cell lines. In the first lane you can see that's -- the lights there. These are the control survival of the cells by definition at 100%. Psycho Foss md alone the dose used had a minimal effect on the survival of the cells. Psycho veer loan had a minimal effect on the cells. Virus alone reduced cell survival down to the 30 to 40% range and psycho Foss md plus virus reduced it further. Again psycho veer plus virus inhibited the virus effect on the cell line. In addition, they did -- this is the modal that I alluded to. The hepatic metastasis colon cancer model with -- and this is looking at theism of metastasize in the liver, creating 100. Psychos if md, virus alone with PBS control and metastasize were down to 10 to 15 and virus -- virus with psycho Foss md was down to five. The data suggests that the additional of psycho md even with the setting will add to the efficacy.

Do those results depend on the expression of the rRp450 gene? -- p450 gene.

I don't believe they measured that in that paper. But I would have to defer that to data.

Did those results depend on intact immune system so had affected the immune system and took the immune system away did you get the same?

Usually see the same results in the human Ken graph models as well as -- xeno graph models. I would like to see the literature on that.

Am the met tesic model?

Uh-huh. I know they also did pass imings in in administration of antibodies which did not inhibit the result.

Dr.?

One last thing. I do think the latenty questions are important and those are easy experiments to do I would recommend that you do them model like this or your nude mouse model and go back and answer those questions what type of lateensy in the ganglia of things.

Discussion from the public? Yes, sir, could you please give us your name.

Luis -- university of California in Irvin. I want to introduce a concept here which RAC doesn't seem to be thinking of as you move forward with your approval of applied human viruses to people, you need to start worrying about some especially rare but potentially significant events of creating a virus from recome byings in which us unlike we start off. We had experiences with trying to eliminate polio of continue with theed strains picking up sequences from unexpected sources. I think RAC needs to worry about that a little bit with respect to humans harboring a lot of other potential sources of recombination -- recombination.

Thank very much. Thank you very much for your clear presentation and interaction with the RAC.

thank you for the opportunity.

I have taken a stab at this. So let me read what appear to be the remaining concerns. Number one, concerns remain that the mouse model utilized for the preclinical data indicating safety may not be fully valid. I'm trying to make the language appropriately loose. May not be fully fully valid because of the elevated temperature of the mouse attain with theeing recommendation. The immuno+ virus and thus potentially underreporting toxicity in man. Preclinical studies in a nonclinical primate model should be -- I put under take and should be considered is probably more appropriate. So should be considered. Number two, collusion of the p450 gene may not be fully justified in this viral transgene as the virus will life likely establish long lateensy and expression of rRp450 is not -- p450 is not an integral performance of this protocol. We had a lot of of that during the last half hour the discussion really has I think tried to balance using a particular study as a platform for future studies versus the construct of the exact protocol we were reviewing. I am in favor of deleting what I have just read from our concerns and want to know if there is anyone who objects to that.

If you want to, you can say however these concerns are balanced by issues related to practicality and using this as a platform in the future. Both sides of the story there.

Inaudible.

We could focus on the p4 50RBGS we have how many genes in herpes, why are we focussing on the p450 specifically. I think this is really a -- it should be a focus of the study where we actually induce or use it possibly but not necessarily -- just another gene that we have in there.

Dr. DeLuca, want to comment on that?

Sure. I was concerned because it's not normally expressed. Recomeinant transgene. The RAC gene -- there will be a response to herpes before the injection -- inaudible.

Again, concepts of the herpes genes producing immune response. I'm having trouble trying to find a safety issue in that. That's my point.

Although I raised that question, too, I guess I would go along with the issue. We tend to focus on not having genes in being transferred that aren't essential. I think that's been recently justified here.

I just deleted it. Thank you. Now the new number two, given the ongoing concerns with regarding the relevance of the mouse model and observation that ICP6 mutant replicate in the brains of young mice consideration should be given to decreasing the starting dose to less than 10 to the 7th and decrease risk of this vulnerable patient population. Dr.?

Just one thing that we can discuss and I guess mostly I'm wondering what everybody also, what about the notion of selecting -- positives, will that decrease safety because with the young kids you have a potentially --

That's a possibility. Actually, that's what I guess the RAC decided when they reviewed the NV1020 protocol. Was to go forward with seera positives first. That was with young people. And this much then go forward with seera negatives. And I don't -- I don't think that it would have been -- the outcome would have been different if they just when at the same time. The way things worked out. It is a possibility.

But selecting for zero positives when the subject population is so young is a strategy that is likely to exclude almost all of the desired subjects.

It would certainly potentially exclude children below the age of five, possibly.

Yes. And the tumors that are mentioned in this protocol are those which frequently affect very young children. So I think we are getting into a piece of the protocol that really might end up totally reducing it for marginal benefit.

Perhaps reducing the starting dose takes care of it.

I think so.

That's fine. I want to make sure that --

Thank you. Other -- all right and number three, given the on going concerns regarding this about -- begs to Ms. Kwan's comments given the ongoing concerns given potential risk to children the informed consent document should be reviewed for language clarity in order to assure the consent for this vulnerable possibling will be obtained following the complete understanding of potential risks including discomfort. I would like motion for approval.

Couple of others.

Please.

People the animal model -- to examine the extent which the virus established laytensy where the virus established lateensy purposes in okay.

And what animal model, Dr. DeLuca, other --

Think they only used one.

You mean species or particular tumor models? Only one it works.

Well, you used a mouse model where you put -- not model but tumors. Right.

So it's important that that be -- that lateensy be established and I have that as part of point two and I deleted it. What impact will that information have because it's like lie that latency does occur. What impact will that have on the design of this study?

Actually, one of the rationales for doing this is the virus establishes latency very poorly in a mouse model. Now there is a situation where they are providing a whole bowl of susceptible cells.

It would affect risk benefit analysis if you were establishing latency or not. I think it's important to know that.

Okay.

I have writ in number four, determining extent to which the virus established latency in the peripheral nervous system in the mouse --

In the model cancer. If you are not providing those susceptible cells you are not darn.

In the cancer model. To fully assess or fully develop the risk benefit analysis.

Just to clarify that that means if we find that there is 1% or five percent or 100% latency we will move forward but put in the consent document for the data.

That's my understanding. That is why I asked the question. It's important to understand the degree of latency established but not a protocol stopper is the way I --

Is that fair?

Correct other issues for discussion?

In the statement of starting with the lower dose in the first discussion of ten to the fifth and ten to the sixth will we recommend a specific dose.

I did and my second point consideration should be given to decreasing the starting dose to less than ten to the seventh. We could recommend a specific dose. Unless someone can help me with this, I don't personally have the information from the protocol as written to make that kind of recommendation.

And should that recommendation be done in the contact of lack of primate studies or --

Remember our first recommendation states preclinical studies in a nonhuman primate model should be considered and our second recommendation then is, given the ongoing concerns regarding the relevance of the mouse model, consideration should be given to decreasing the dose to less than. So we were nowhere saying that a nonhuman primate preclinical study needs to be done. We are saying consideration should be given to the information which we have available suggests that the starting dose should be less than and we are not prescribing a dose.

One other question about that. One of the reasons we avoided primate studies we don't have a tumor model to emulate the clinical trial for the primate model. What kinds of studies and what would be sufficient.

We can't define that for you. You have to to the fda. Anybody doing cancer therapy doesn't have a tumor model for the primate. There are preclinical studies you could do. Usually say worst case scenario direct intracranial injection, iv injection if you fled do that and that will be determined between you and the fda primarily you can discuss that. But that's the usual -- there are no tumor models in primates. We usually go with the worst case scenario.

All right, may have I -- may I have a motion for approval, please. Dr. DeLuca.

I want to see what people think about the other recommendation that a rescue -- to assure the attain wings solely due to the mutation since this will be precedent setting. And people do and they don't do that and I know if you wanted to publish it in the journal of -- you wouldn't be able to simply for simple esoteric study there if you didn't do that experiment. The reason I think that it could be important is other mutation dozen occur especially when you generate viruses by transvex. And if the attain wings of this virus is in part to another mutation it doesn't give a feel to a clear picture of what the toxicity of the virus is.

I agree. I think that should be done and it's very important issue for a brand-new virus.

It's a fairly simple experiment you can do. You don't have to do a lot of go through and repeat everything.

So given the likelihood that the gene money fieed virus will be utilized in future studies, it is important to assure that the attain wings is solely due to the deletion of L.A.-de-da inserted?

They are going to use this particular virus regardless what the outcome of the study is, the virus going in with and so that would put a couch in the language of please give consideration of this because this will obviously help the failure points and --

Other people may come put in protocols utilizing this or similar approach and use this as precedent. They will have to do this work and find out the virus replicates better.

I guess the question would be to what extent does it rescue it if it doesn't totally rescue it and to what extent does it inhibit moving forward with this protocol even if it's shown to be a safe virus?

I would say none at all.

We don't know all the mechanism of how preed Nissan works -- preedson works.

Then I guess the RAC should consider publicly reviewing every protocol and that's what we would be heading if we don't make sure that the precedence that are establishing are based on sound finds.

We aren't asking to define the mechanism of how the virus works. Only to prove that's the gene that is responsible for the attain wings.

Okay. Just give me a second. People want me to reread what I written? Dr. Powers says no. I will take no. I'm going to go over this. We have changed it so morn much that I think it's important before we vote. One, concerns remain that the mouse model utilized for the preclinical data indicating safety not be fully valid because of the elevated temperature of the mouse. Atten with theeing recommendation of the mutant virus and underreporting toxicity of man and nonhuman primate model should be considered. Two, given the ongoing concerns regarding the relevance of the mouse model and the observation that icp6 mutants replicate in the brains of young mice consideration should be given to decreasing the starting dose to less than 10 to the seventh in order to decrease risk to less vulnerable patient population. Three, given the likelihood the gene modified virus will be used in the future crrtion should be given to the generation of rescue version of rRp450 to be given that is generated to assure that the attain wings is solely due to the deletion of the icp6 coding sequence. Four, given the ongoing concerns regarding potential risk to children and that's the informed consent piece we haven't changed that. And five, to determine the it which the virus establishes latency in the peripheral nervous system in a mouse in a cancer model to fully develop the risk benefit analysis. I will have to beginning wording of that and I will do that. Given those five comments, could I have a motion for apofl?

Second.

Thank you, both. Let's take a vote then. Thank you all. We will take a break. And we will return in 30 minutes. RP RP

Our next -- wait, conflict of interestwe have one investigator, Dr. He is lip in conflictwith the next study we're reviewing, 699. again, he is a member of a DSMB.First study being conducted at Cincinnati Children'sHospital.She is also a consultant to St. Jude children's researchhospital and therefore is excused for the discussion ofthis study.Protocol is entitled a pilot study of Temozolomide andBenzylguanine for the treatment of high rate gliomagenetically modified for chemoprotection.And two investigators will be presenting.We're going to start with Dr. Lars Wagner. Good morning.I wanted to thank Dr. WARA and members of the RAC forallowing us to participate and for your reviews.This will be a two-part study.We'll be discussing some of the clinical aspects of thetrial and then the study's sponsor Dr. Von Kalle will bediscussing some of the safety issues such as insertionalmutagenesis.I wanted to start by providing some clinical backgroundregarding the disease we'll be treating.Brain tumors are the leading cause of death in childrenand high gried gliomas are clearly associated with apoor prognosis such that the five year progression forsurvival for kids with this disease is about 5% and themedian overall survival ranges from -- it's more he thatwill in adults where the five year survival is about 5%.So this is clearly a disease type and a patientpopulation that is in need of more effective therapies.Historically, patients with high grade glioma have beentreated with a multimodality approach that involves thesurgical removal of the tumor.This is important but not expected to be curativebecause of extensive microscopic infiltration of theFocal radiation is then provided in this maneuverprolongs disease free survival but doesn't really impacttoo much on overall outcome and so clinicians have oftenoffered additional chemo therapy following radiationprimarily in the past using nitro agents.This may modestly improve survival perhaps 5 to 10percentage points but in general is not curative andtreatment with myotro surrrhea -- requiring delays indose reductions and nonlife threatening pulmoto logicbut really the main problem is tumor cell resistance tothe chemo therapy agents that we've used.One of the big developments in the field ofneuro-oncology the past decades has been the use of has a nonto profile and soin a large phase 3 trial that was recently reported theuse of Temozolomide used after surgery concurrent withand then following radiation significantly improvedsurvival for adults glioblastoma compared withtreatment of radiation alone.And so this type of treatment with Temozolomide duringand after radiation is now kind of evolving as the newstandard of care at least for adults with glioblastomabased on the limited to bes sisty and the use of oralThis approach is currently being evaluated in pediatricpatients through an oncology group trial but similarresults are expected and while this is an encouragingdevelopment in the field, still with a two-year overallsurvival rate of 26% in adults indicates Temozolomide isreally going to expected to be curative for themajority of newly diagnosed patients.And the primary problem of course is tumor cellresistance.We know that MGM T as the DNA repair protein that's theprimary repair system and MGMT is set to remove addictsby either Temozolomide or conventional agents.And this is a scheme attic representation in which thechemo therapy agent here nitrous yourrrhea deposits thegroup on the O 6 position of DNA and then that can beremoved in a one to one reaction by MGMT.And MGMT really appears as a relevant therapeutic target it's expressed in about 90% of Peted pediatricgliomas and about 90% of adult gliomas and theimpression to Temozolomide in patients withhigh gried gliomas and so the more the MGMT expressionthe less response is scene.And now there's Benzylguanine which effectivelyincreases the sensitivity of tumor cells toTemozolomide.So that preclinical been used to lead onto clinical trials, phase one trials of Temozolomide and 6 Benzylguanine both in kids and adults.And unfortunately, we've seen some exacerbation ofhematologic to bes sisty and this is thought to be due inactivation of low but still protective levels ofthe hematic skrem cells and this is required a dosereduction of over 50% for Temozolomide in phase 1 trialssuch that the usual dose has to be reduced to a maximumtolerated dose of 75 milligrams per meters squared perday.In a similar in adults showed a requirementofMaybe the optization of this Temozolomide BG approach require a method to help circumvebt the hematologicto bes sisty.And one of the developments that's helped push thisforward has been the creation of the mutant form aproline has been substituted for LYSONEcreate add form of the enzyme which is resistant to bothTemozolomide and O 6 BG and so this kre creates a way toof to overcome toxicty and if you get repeateddoses of this combination after transduction then youcan kill the nontransduced hematic transduced cells andthere by increasing the relative percentage of protectedcells and the so called enrichment effect actuallycausing less myolow suppression with further therapy andthis is in sharp contrast with what we see withconventional where the myolow suppression gets worsewith each course.So this overcomes initial transduction efficiency bytransducing out the selected cell population withselected treatment.This may allow for increasing doses of Temozolomidewhich could lead to superior antitumor effects at leastin a clinical model.So to briefly summarize about what we know about thepreclinical evidence of this type of approach using theMGMT P 140 K mutant, we've seen that protection frrchemo therapy to beicty is seen both with mouse andhuman stem cells and this leads to improvement in the counts with continued therapy and durablechemoprotection is observed even when doing serialtransplants into another mouse that the mice are able totolerate higher dose intensity and this translates intoa signe graft model into antitu mar activity so nicelyproving the principle at least preclinically.And this leads to multiprotection enrichment in a modelin which the percent of marked cells went from 15% allthe way up to 90%.This also in the Al yes nays setting increased from 95%and highlights the potential approach of this Al geneticsetting.You take the transgene and put it into the stem cells,up matly administering it to the patient and here wehave just a sampling of what's in the stem cellcompartment with a few transduced cells in thebackground of many nontransduced cells but withtreatment of Temozolomide and BG then you can kill offthe nonprotected cell tells and then causing enrichmentand expacks of the chemo protected cells and this canlead to multilineage enhancement here.So our clinical trial is set up in the following way.We chose to do a pilot study of ten patients and wechose ten patients because this seemed to be the minimumnumber of patients that we hoped would accomplish allthe study objectives here and this number of patients issimilar to what has been seen in other pilot studies.And so the main study objectives is really to the feasibility and the safety of genetra*bs fer and the proposed chemo therapy regimen andthen also to assess the efficiency of gene transfer andthe durability of the MGMT expression to ashes thedegree of chemo resistance and ability to dointrapatient dosing of Temozolomide and so this will beone of the hallmarks of success in this clinical trialis the ability to -- whether this can meaningfullytranslate to a dose increase in the Temozolomide.And so we'll be carefully dose escalating patients in anintrapatient fashion using extent of the transunitexpression as one of the criteria for dose escalationand then responses in the confines of a very small pilotstudy.I'm just noting these responses, but of course thisnot going to be a demonstration of the efficacy of thisapproach.The population we tried to be very careful aboutwho we would include and exclude.We chose children five years of age and older becauseyounger children with brain tumors are often treatedwith different radio therapy approaches and we wanted auniform population with which to provide treatment to.We chose new patients instead of relapse patientsbecause we wanted patients to have an expected survivalat least for the length of the entire study and then type patients adequate organ function. excluded patients that may have either too good of aprognosis or too poor of a prognosis.So may not be appropriate for this experimental type ofapproach.Similarly shs patients with this type of histology eventhough they're high grade gliomas do better withconventional therapy and would not be appropriate forenrollment on this trial.Patients with extracranial disease may not have theexpected life expectancy to last for the duration ofthis trial.The study is designed into three blocks, the first ofwhich we'll term the standard of care block and again,this experimental trial is really piggybacked on to whatis currently available treatment for patients with newlydiagnosed high grade glioma so this will includeresection followed by radiation and kind of a standarddose of concurrent Temozolomide.The second part will be the gene transfer part wherepatients receive two days' wo*t of low dose bu sul feignto create space and allow for transfer.This is not a myolow aplaytive regimen.This is to create space and this is a drug and thesedoses that has been used in other gene therapy trialsfor this purpose.And the third part will be the more experimentalpart where we'll try to achieve dose escalation and so will receive chemo therapy with six monthlycourses with intrapatient dose escalation as toleratedif they meet certain criteria.The study scheme looks like this.Patients undergo surgery.We collect stem cells by aforree sis.In the lab these undergo cell collection andtransduction and administration to the patient at theappropriate time.Meanwhile after stem cell collection patients undergoradiation.We have MRIs KIND OF BUILT IN to make sure thesepatients are not clinically progressing and of course not receive further therapy.Block 2 is low dose bul sul fan.We expect recount recovery to be about tloo to fourweeks and proceed on to block three which is the sixcourses with attempts at dose escalation in courses 2through 6.So this intrapatient dose escalation will be dependenton the absence of dose limiting toxicty as defined inthe protocol in previous courses as well as some evidence of presence of the transgene andperipheral neutrophils and so we describe the definitionof a marked A and C or absolute neutrophil count and wecan get some estimate that these patients may indeedhave at least some degree of chemoprotection.And then if they meet the criteria we would increase thedose.This was the was reached in a mediate tricktrial without any additional stem cell support so we think based on the previous phase 1 trial thatthis dose combined with the O 6 would be a safestarting point and we'll try to increase by 30 to 35%with subsequent dose levels.If a patient was able to make it all the way through bycourse 6 they would receive almost four times as much as they would have been able to get justbased on the conventional phase 1 trial.So the study end points involve safety and of coursewe'll be assessing toxicty involving standard NIHcriteria and then throughout the protocol and thetreatment will be monitoring blood and bone marrow fromsoxicty and we'll describe that a little more later. be looking at the extent of gene transfer of realtime PCR.The efficacy of gene transfer is assessed by MGMT and then from a functional standpoint theability to actually dose escalate and give higher dosesof Temozolomide in block three and then looking at theantitumor activity with physical exams and MRIs so thistherapy is not without potential toxicty and Dr. Kallewill be reviewing this in a little more detail but thereare efforts in protocol to help address theseconcerns, so looking for secondary leukemia frominsertional mutagenesis and built in are plannedevaluations of the blood and bone marrow forconventionalmorph logical and then clialty and the useof the land PCR for determination of monoclonalinsertion sites will be also evaluating for developmentof replication retrovirus and severe acute infusionrelated toxicty from the product.And then another potential concern is delayedengraftment.Patients will have in the freezer a backup of stem cells that can be given in thissituation and for the first three of theseconsiderations there is a stock in place if any of thesefirst three toxicties occur and they'll be evaluatedbefore the study can continue.And just to highlight some potential clinicalapplications.What we're trying to do in this trial and this could bealso applicable for a disease like metastatic melanoma,modifying the Al yes nate -- leukemia graft is alsoanother avenue of integration and then transfer bysingle disorders and this had been demonstrated bythe preclinical model.Soy eel turn it over to Dr. Kalle who will be talkingabout the laboratory monitoring and some of the othersafety issues.

Can I ask one question on the trial?Are giving one dose of the stem cells?Are you escalating that and how did you choose that dosein your clinical protocol?

So there will be just a single administration of thestem cells.It is somewhat dependent on the amount of cells that arecollected.There will be -- for every patient a backup kept as anunmanipulated frozen Al kwaun in the case of delayedtransfer.We're setting some limitations on the range of stemcells that will be transduced and administered into the but going to be hard to prescribe a -- acertain dose for patients because that's going to besomewhat dependent on the number of stem cells

I just quickly wanted to go and summarize some of thepreclinical as well as followup experience that we havewith regards to the gene transfer of this transgene.This is very quickly a summary of some of the mouse data with MGMT as one of the examples where youcan see primary recipient after two cycles of selectionand you can see the gene marking actually stays present.Our group in Seattle as has already been mentioned hasdone the same in the canine model where you can see thatcourse rs of selection raised the number of marked cellsin the frifly all the way up to almost 100% in thismodel and some information that you can't see on thisslide is also that selections at intensities thatproduced neutropenia down here have no longer producedneutropenia or some cytopenia at the later stages ofselections.Here's the date that Lars already mentioned with thedonor timerism that adult stem cell behavior could bemodified in that the recovery was returned to greaterthan 95% donor just with the selection process alonewithout any T-cells which would be a majortherapeutic accomplishment in hall yes nays Joantransfer.I wanted to use the opportunity as I've been asked tosummarize some of the insertion sites that have been inthis trial.Of course it's in principle a dose finding to these genetherapy vectors and gives information about stem cellinsertion site distribution.The cell type dependence of such insertions.Genomic side effects if there are any and behavior ofparticular vector types and I've already talked to thisgroup in March about this so I will quickly go over thisas I've shown before.We have developed method dolling that can identifydifferent insertion sites even in a complex group andallows us to have sequencing information about thesequence and have a more moleccue lar mark for eachinvolved clone.You're also aware of the clinical trials both fromDr. Fishers and the group in Paris as well as fromDr. Thrash's group in London where again the C functionis replaced by a retrovirus vector and I had shown dataearlier in that trial we can see a very pollicclone recovery in the CD 3 component.But we can basically identify by sequencing these thatsome the cells that have been corrected are actuallyproduced both granule sites.Macrophages and lymphosites meaning that it couldqualifyChronology in the cases the first to leukemia sideeffects in the fr*efrng trial and you have heard and hada discussion this morning about the third case, thatfrom the moleccue lar mechanism is actually fairlysimilar and we have gone on to do a more in-depth of post transplantation home porsis and looked at the insertion sites.It has been published and we have also found evidence inthe clinical model and again, I already talked aboutthat in March that the distribution isn't completelyhome general yous and the cross of chromosomes isto the genes than the genetic nfks as such.There's also a bias after retrovirus insertion so theinsertions actually sit closer to the start site.If we look by gene ontology analysis of where theseinsertions occur you can basically see the genes withkinase activities transforrace activities and someothers in these trials have a statisticallysignificantly increased likelihood of becoming hits,probably mainly because such genes are expressed in thetarget cell population at the time of retrovirus entry.So summary of what we that 60 -- about 60%of insertions occur within or close to genes and ofthese 20, 50% of the first 20 sit around the front ofthe gene.This clustering around some common insertion sites and about that in a minute and there are before and after and some of theseinsertions have some influence on the biology of the cell clones and then there's also differencesbetween trials in that severe adverse events have onlybe observed in one of the trials far and it will beinteresting see what -- what happens with the othertrials.But also we have to keep in mind that in summary, allthe immune know dep efficiency trials, the ADA trial andthe German trial, so far have treated 27 patients.These are the French, British, Italian combined.26 of the 27 are alive and the vast majority of themThe successful correction has been described in theliterature in 24 out of 27 from these patients. so far had three SAEs reported and two of the threepatients affects by this have survived the SAEs and arestill using the immunity of the successfully correctedcells basically for their day-to-day immunity, andagain, there seem to be some very significantdifferences between the different types of trials, theADA and the SCID wanted to close with summarizingsome of the more recent experience that we have had withthe -- our colleagues who have conducted these KTDtrials.These are the trials of the German and Swiss groups.The principal investigator in frank further, we havefollowup of over one year and close to one year in thefirst two patients.This trial is remarkable because more than 20% of myolowpor ree sis has been corrected in both patients byretrovirus vector so this not -- basically anisolation to mile porosis.You also see that there is a more regular pattern ofthe LAM analysis toward the second half of theobservation period.We have looked into this more closely.You can see here as a red line the marking level or thepresence of transgene in these patients that hasincreased in long-term he ma toe paresis that it has upto about 50% peripheral blood cells and we couldidentify that certain insertion sites basically -- wenow think selected by growth are doing the course ofthis process before this whole process plateaus out andwe have followup of more than a half a year with and it seems to be a stable process.The second patient shows a very similar picture ofstarting out around 15% and plateauing at around 50% ofcorrection in sites.One of the most frequent hits, we basically found threehits, two of which we're working on the moll cueevidence.You see the insertions on the first and the second also.This is a trial where very many corrected cells havebeen reinfused and 1.5 to 2 insertions had occurred percorrected cell and you can see that there is very strongclustering of insertions around certain locations evenwithin the gene that allows the activation of this geneand then facilitates the expansion of these affectedclones.We can then show by long-term followup that thatbasically -- the plateauing is also happening at thelevel of each single clone.You can see that there's basically, if you so wish anexmro ra*r ration of the -- and that then stabilizes outover the course of time.You have to keep in mind that none of these patientshave a serious event or anything similar by thedefinition of the trial and that the leukosite numbersin these numbers not elevated and show no signs ofincreasing.I wanted to put in as a reminder also here that the bothanalogous and ADA trials that we've been following from on has a contribution pattern that is biologicallystable over the course of time.So in summary, on random insertion had some veryobviously positive results in these patients in thetrial we've seen unprecedented levels of correction evenprior to the expansion of the cell -- of the cells and Ididn't have the time to go into this, but both patientshave substantially cleared their preexisting infectionso there seems to be a strong therapeutic benefit fromthe trial.These are likely single and double copy insertions oftheThe cells seem to be continued to be regulated in growthand some of the clones tend to disappear over time so wethink that many of these may actually be burning outover time.We do not know what the expression pattern of thesegenes in long-term he ma to forree sis is so we do notknow what the normal physiology of these genes or somechanges that have to do more with regulation of apoptosis and regulation of operation.We have likely identified regulators and none of thesecell types also not in animal models where this findingcan be closely reproduced.This also shows that prospective screening by the methoddolling we apply is possible and so if we look at therisk benefits of these clinical applications as I saidearlier, number of modified stem cells over thecourse of time that these trials have managed to put infor a single patient have reached very high numbers.We have started to see therapeutic efficacy, not --mostly based on the increased efficacy and of coursethis is the therapeutic window that will also definewhen we will see potential side effects of thesemeasures.So I would like to conclude that stem cells can begenetically modified and effectively selected by what weare proposing.The side effects are clearly based on gene activationsand so far there's one major mechanism that has beenidentified.Determination of stem cell contributions at the clonelevel offers insights and gene transfer continues tooffer unique theer putic opportunities.With that would like to close and thank thecollaborators in Cincinnati who have made the definitionof this possible and we'll be happy to take yourquestions.

Thank you very much not only for presenting for thisparticular study but for updating us once again.We should have had you speak before our first discussionthis morning.I'd like you both, Dr. Wagner, perhaps you could go tothe podium as well, and we're going to open discussion.We'll do that by moving from one to the other of theformal reviewers for the protocol and asking you tointeract directly with them and then we'll open yourprotocol for broader discussion.So our first -- I have owlOur first discusser, Dr. Rosenberg.Is that all right?Great.

Thank you very much for your presentations and alsofor your thoughtful responses to our reviews.Most of my particular questions, many of which have beenanswered, so many of my questions have been answered andmost of my questions know kused on the retroviralaspects of the protocol.So it will be those that I'll cover.And I'll go through them in part for the record, I dohave a couple of remaining questions.Several of my questions related to the efficacy withwhich transduction was going to be improved inyour -- in your protocol, and you certainly haveresponded that you estimate 20 to 40% of the cells to becarrying an insertion and probably about 1 insertionwere cell.Although I didn't directly ask it in my questions, I'mwondering if you could give some feeling for how manycells or the range, I realize you don't knowspecifically will be infused into each patient and myreason for asking is what's the chance that eachindividual will receive at least one cell that has anintegration more or less gene?

The amount that we're -- that we're -- isbased on -- and then based on the outcome of thosecultures that usually are between 50% and 150% of thatnumber be reinfused.That, of course, means that there are basically millionsof cells per patient that can potentially carryinsertions and so as we have described earlier, thestatistical likelihood of integrations in the vicinityof active genes is basically given in these patientslike for all of the other trials.

Right. just wanted to make sure that was -- that was clear.One thing that you included, but if you couldjust comment on that now, you are using cyber neck tincomponent to enhance your infection and I realize you'veadded references to that, but since that didn't come upin the discussion, if you could briefly comment on howthat actually works.

Yes.This actually a recum bant and fiber neck tincomponent that allows to coat the plastic of the culturevessel and basically contains binding sites both for theretrovirus envelopes as well as for the target cellpopulation.Now current understanding produces a vicinity betweenthe two.It also seems to be the case that long-term survival ofrepopulating cell is better. is something that has been described as early as1995 and '96 and has been a standard for transductionprotocols that in itself is not a test question forthis trial.

My next series of questions relate really to thenature of the CD 34 positive cells that are to be usedand I wonder if you could comment a bit more clearly, Iknow we -- everyone knows perhaps less than might beideal concerning the nature of the populations that areused and how those populations may vary from patient topatient or trial to trial, and you've certainly pointthat out in your response, but I'm wondering if youcould comment a bit more on steps that you will betaking to try to increase information to the field as tothe specific types of cells that are infused into thepatient beyond what we Okay.This is an interesting question.The validity of the -- especially CD 34 positivesubpopulations for long-term recovery has not beenclearly established.Of course lineage negative cells are supposed to be theones that carry short and long-term repopulationalthough some lineage markers may be on short termrepopulating cells so our analysis on which cells haveactually been genetically modified will focus more onthe post transplantation and on the analysis of thedifferent markings that we have seen or demonstrated inthe SCID trial we can identify the relationship betweenIt will be very difficult because basically the analysisof a given cell in the pretransplantation material willuse up that cell for the purpose of transplantation.It will be very difficult to do studies that try topredict behavior from the pretransplantation immunotype.

I think several of us asked questions concerning --oh, wait.I left one thing out and I -- we did ask about thepotential autogenic potential of the transgenes to beused and you comment that -- that there's no knownpotential for that and there were also questions raisedwhich I believe you've addressed concerning theconcerning the length of time it would take to obtaindata from your patients and how that data might be usedto the trial.And I believe you've answered those questions.My question related to the use of the particularenvelope gene that you had chosen if your virus and Irealize it certainly is a highly efficacious one whichyou use to point out.I'm wondering if there's anything that you can saybeyond the efficacy that might lead us to wonder ifdifferent populations or tell us anything about whetherdifferent populations of progenitors may be infectedwhen this envelope is used as opposed to other envelopesthat are used with other retroviral vectors,particularlily I'm thinking of the difference betweenthe trial -- the fisher trial and the Thrasher trialwhich I believe use different envelopes.

Yes.And that is an interesting point, of course, and a very question.The differences between the slasher and the fisher trialwere that the Paris trial was used in an tlo toepicenvelope where as Thrasher's trial was using thewhich the same envelope that we're using.I think the agreement is that the efficacy especially inthe immature long-term repopulating -- so we wouldexpect to see a more efficient gene transfer into, ifyou so wish, he ma to poetic cells.

Thank you very much.

Dr. Rosenberg, do you have any remaining concerns?

I do not think so. Because I heard a discussion suggesting that all of carefully written concerns have been addressed.

I believe that that is true.I think from all of this, the one thing that -- and itdoesn't speak specifically to what we've heard aboutthis, really relates to ways to understand better whichinfected cells really contribute or have the potentialto contribute to problems in particular patients.However, believe those concerns are addressed withrespect to this specific trial.

Thank you.Dr. Nem ro?

So I -- I really was impressoed by the thorareness ofthe trial and the multiple stopping points that had beenbuilt into protect patients and I thought it was a verynovel but a complex trial that merited furtherdiscussion and I had many of the same questions thatDr. Rosenberg raised.And I'd like to maybe follow up on a couple of those anda couple ones as well.So it's mainly clarifications rather than concerns fortheSo I wondered during the collection of the CD 34positive cells, there's also likely to contaminationwith CD 34 negative cell populations.Will you be looking at which, you know, the -- thepresence of those cells and also is there a concern thatthere may be contaminating glee mall sales or cancercells that would also be potentially transduced andcreate a problem for treatment?

I think Lars Wagner can answer that question becausehe has just undertaken a review of the literature withrespect to the presence of such cells in the bonemarrow.

Yeah.That is a theoretical concern but should be exceedinglyrare.There have been multiple studies, the use of autologousstem cells before and this has not been found to be aclinically significant problem.There's never been a reported case of brain tumor cellsbeing contaminating the stem cell product and thenhaving complications from that.The incidence of extracranial me ta*s cease at the timeof diagnosis is exceedingly rare with high grade gliomaso it's probably less than 1% of patients will haveextracranial metastasis so it's probably more uncommon the bone marrow would be involved.In a review of the literature, there have been 13 casesof bone marrow involvement with glee owe blastoma, onlyone of which was at the time of diagnosis and about 85%of these cases would not have been eligible for thestudy because of hematologic problems so hopefully thiswill be an exceedingly rare problem that we will notencounter.

Thank you much.I -- one of the other questions -- clarification was toto indicate the decision of using bio safety level 1versus bio safety level 2 containment in theinvestigator was -- in his written response was able toclarify the decision to use bio safety level 1 and I'm that.One of the things itches interested in was thatwe've been discussing the -- the problem with MLVvectors and the SCID trials in the last several monthsand because of the experience of the investigator, I'mwondering was any thought given to different retroviralvectors in designing these kinds of trials that perhapsmight have less or either built in regulatory sequencesor perhaps less propensity to insert in the five primeregion or near the five prime regions of -- of indodgenows

That is of course an ongoing thought process and aswe go forward with these trials.However, I think we're still at the stage where we'retrying to understand what the actual distribution is andwhat the influence on the biological fate of these cellsexactly is.So in terms of the feasibility of putting these thingsand also the safety of putting these things forward intoclinical trials, I think a lot of research, preclinicalresearch has still to be conducted.We have to find out whether the newly envisioned vectorsare actually as effective and as safe even as the olderones.And so I think what we're -- we're intending to use hereis if you so wish, a state of the art retrovirus vectorand incorporating our current knowledge.

Thank you.So another question that I had really was more of aclarification.In the written response, you indicated that 20 to 40% ofthe CD 34 population is transduced.But in another look, the appendix M in the proposal,there's an indication that there's 35 to 64% that areactually transduced with a vector having the MGMT gene.So I'm wondering which of those is the more accurateassessment of the transduction?

Okay.The that we stated with regards to what we'reexpecting from the trial is incorporating our experiencewith the scaleout to the clinical dimension of the data is what has been done on a smaller and as you be aware, we have an active genecorrection trial for anemia and have some experiencewith the upscaling now of similar types of vectors tothe procedure and so those are the numbers that we'reexpecting to see in CD 34 cells.

Okay.So one of the other questions came up also in thewritten response and you indicated that there a --one of the significant differences between the Britishand French trials in the transduction was using serumfree transduction.I wondered if you could clarify what that meant.

Okay.

What that's related to.

Yes.The media that are actually used to culture the cellsexVIVO contains animal ser ra in one case and contain noanimal products in the case of serum pregene transfer.The serum containing media are tlauth to induce moreproliferation and also probably more differentiation inthe cell populations and the -- the thinking is that theserum free conditions maintain a more immature state anda less proliferative state of the target cellpopulation.

And that's related to the fiber knack tin that's inthe serum probably?

No, these are probably growth factor come poebts andother components.The fiber neck tin I wouldn't think because the fiberneck tin is also a component of the serum free cultures.

Okay.Let's see.So I had several other questions that were addressed inthe written response, and one of which was to look forrearrangement of the vector in CD 34 positive cells andthe invest sga torr indicated that was technicallydifficult to do, but it has been looked at in the healcells.The other question I had was whether they have looked atgerm line transmission in