Notes

Outline

iNOS Gene Therapy to Prevent AV Graft Intimal Hyperplasia Principal Investigator:  Edith Tzeng, M.D.

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Vascular Functions of Nitric Oxide Vasodilator Antiplatelets Antiinflammatory Antiproliferative for smooth muscle cells Antiapoptotic for endothelial cells Proproliferative for endothelial cells

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NOS-Based Gene Therapy Harness the vasoprotective properties of NO to prevent intimal hyperplasia following vascular injury Limit the delivery of NO to the region of vascular trauma - minimize systemic side-effects

Enzymatic sources of NO Endothelial NO synthase (eNOS) produces small bursts of NO vascular homeostasis Neuronal NO synthase (nNOS) produces small bursts of NO signaling Inducible NO synthase (iNOS) in response to inflammation, sepsis produces large quantities of NO

Recombinant Adenoviral Vectors: first-generation adenovirus

iNOS Gene Transfer Inhibits Intimal Hyperplasia in Injured Rat Carotid Arteries

Persistent Inhibition of Intimal Hyperplasia by iNOS Gene Transfer

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Gene Transfer Efficiency in Vein Grafts

Nitrite Accumulation in Organ Culture

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Preclinical Toxicity Studies Weight Adjusted Dosing in Rats

Distribution of Systemic AdiNOS Administration 3 days - PCR (+) for iNOS in liver and spleen (in one rat, also see in aorta, muscle) 14 days - PCR (-) in all tissues, including testes histologic examination pending

Clinical Grade AdiNOS Sequencing of entire construct nearly complete RCA testing of master viral bank of AdiNOS – negative at 1 x 109 pfu (AppTec Laboratory Services) Testing for adventitious agents in master viral bank underway Awaiting results to proceed with production of clinical grade AdiNOS

Advantages of iNOS Gene Transfer Diffusible product (NO) so high gene transfer efficiency may not be necessary Greater NO synthetic capacity than eNOS Rapid uptake and inactivation of NO by hemoglobin – reduces systemic toxicity Multiple vasoprotective actions

Forearm AV Graft for Hemodialysis

Rationale for AV Graft Model ~70,000 new HD patients annually and ~200,000 existing HD patients (12/31/99) ~60% of all permanent HD access = AV grafts Annual AV graft placement ~100,000 Ideal clinical model of intimal hyperplasia Intimal hyperplasia within a normal vessel Ease and safety of virus administration Easy surveillance (doppler, flow measurements on dialysis, physical exam) Low risk to patient if therapy fails

Inclusion Criteria All ethnic groups Gender:  male or female Lower age limit:  18 years Upper age limit:  none ESRD requiring new AV graft for HD access Females - post-menopausal, oophorectomy or hysterectomy, or on oral contraceptives Males - barrier contraceptives and sexual partners must use oral contraceptives Reasonable surgical candidates Imminent need for HD or already on HD No anticoagulation within 30 days Laboratory values:  WBC>3.0x109/L, platelet > 100,000/mm3, nl LFTs

Exclusion Criteria Fail to meet inclusion criteria Undergoing placement of primary AV fistula Undergoing revision of previous AV graft Taking steroids for disease processes (ie. transplant, COPD, arthritidies, autoimmune processes…) Hypercoagulable state On the active transplant waiting list Confirmed pregnancy or < 3 months post-partum Documented central vein stenosis on side of AV graft Participating in other investigational studies Life-expectancy < 12 months Unable to give informed consent or comply with followup

Enrollment Patients referred to vascular surgery for AV graft by nephrologist Patients being referred for peritoneal dialysis or central venous catheters will not be considered Only patients who will be dialyzed at UPMC affiliated hemodialysis facilities will be enrolled Meets inclusion/exclusion criteria Signs informed consent Suitable arms veins during operative exploration

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Dose Escalation Scheme

Post-Procedure/Discharge After AV graft placement, patient will be taken to recovery room or back to same day surgery unit Patient will be monitored (HR, BP, RR, temp) every 15 minutes for one hr If vital signs remain stable, patient will be discharged home Follow-up schedule will be discussed prior to discharge

Schedule of Patient Evaluation

Study End-points Primary Dose escalation - safety Evidence of systemic viral dissemination Adverse events (systemic inflammation, local inflammation, bleeding, aneurysm formation…) Secondary Evaluation of graft function Time to first graft revision or graft failure Evidence of venous anastomotic stenosis by duplex of fistulography

AV Graft Function Physical exam – loss of thrill or pulse Maximum venous pressures on HD at flow rate of 400 ml/min Weekly measurements > 230 mm Hg on 3 occasions Duplex imaging Narrowing of venous anastomosis Flow rate < 800 ml/min 20% reduction in flow rate Fistulogram > 50% narrowing requires surgical revision