1. CTGTAC March 4, 2005, Topic II Update: Retrovirus Vector-Mediated Insertional Tumorigenesis Carolyn A. Wilson, Ph.D. DCGT/OCTGT

2. Update Review, Retroviral Insertional Mutagenesis Review and Brief Update: X-SCID Gene Therapy Clinical Trial in France –FDA Responses (then and now) BRMAC, February, 2003 –Recommendations and Actions Detailed Update: X-SCID Gene Therapy Clinical Trial in France Today’s Presentations and Questions

3. Gammaretroviral-Mediated Endogenous Gene Activation Distal Gene Activation Read-through Transcription Psi (Internal Promotor) Transgene LTR U3 R U5U3 Dysregulated Gene Expression Tumorigenesis Gene Disruption

4. Events Leading to BRMAC, 2003 11 children with X-linked SCID treated in France with autologous CD34+ cells modified ex vivo by retroviral vector encoding gamma-c (  c) cDNA. –10/11, Clinical and laboratory evidence of engraftment and benefit –2/10 children with engraftment developed leukemia Cavazzana-Calvo, M., et al. 2000. Science 288:669-72. Hacein-Bey-Abina, et al. 2002. New England Journal of Medicine 346:1185-1193. Hacein-Bey-Abina, S., et al. 2003. N Engl J Med 348:255-6; Science 302:415.

5. LMO-2 Associated Clonal T-Cell Proliferation in Two Patients After Gene Therapy for SCID-X1 Hacein-Bey-Abina, S., et al, 2003, Science 302:415 Retroviral Vector: 1) Integrated into LMO-2 2) Resulted in Transcriptional Activation of LMO-2 locus

6. CategoryRevise Informed Consent Monitor Clonality Clinical Hold SCID, All Active Hematopoietic Stem Cells Yes Inactive HSCYes Require to resume trial All other retroviral vector clinical trials Yes No, Recommend January 13, 2003 Letter to Retroviral Vector Sponsors All sponsors were asked to provide risk/benefit analysis. CBER evaluated each response.

7. New developments in X-SCID Gene Therapy Clinical Trial in France Resumed treating patients, May 2004 with following changes (1 patient treated to date): –Maximum cell dose of 10 x 10 6  c(+) CD34 + /kg –Absence of family history of childhood cancer –Absence of cytogenetic abnormality –Presence of at least one infectious episode (i.e., pneumopathy, herpes infection, BCG infection, etc) –>6 months of age P4: Relapse leukemia, died October, 2004 P5, Status: Complete remission, after completion of chemotherapy P10: presented with T cell lymphoproliferation

8. FDA Response Three INDs placed on Clinical Hold –X-SCID (2); ADA-SCID (1) –Revise Informed Consent –Notify IRB All sponsors that use retroviral vector: –Informed of new events Notified IRBs

9. Recommendations Actions BRMAC Meeting February, 2003

10. Recommendations for X-SCID, IL-7 and JAK-3 deficiencies Unanimous Vote in Favor:Unanimous Vote in Favor: –Only allow use of gene therapy in children with these conditions when there are no alternative therapies available

11. Protocol Changes for X-SCID US INDs, Consistent with February 2003, BRMAC MalechWeinberg Previous BMT with persistent T and B lymphocyte impairments Compromised quality of life Older, 2-20 years old Case by case review by IRB, FDA Absence of eligible HLA-matched sibling High risk for haplo- identical transplant (e.g., infections) Cell dose limit of 2 x 10 7 cells/kg total (~5 x 10 6  c+) Shown with permission from Drs. Malech and Weinberg

12. ADA-SCID : Spectrum of Viewpoints No fundamental biological difference in ADA- SCID to render outcome different than X- SCID Transgene (gamma-c) not having effect Cell dose lower in ADA-SCID X-SCID transgene product, gamma-c, has potential to be oncogenic via constitutive activation ADA-SCID transgene product simply metabolic –“apples and oranges”, more potential for gamma-c to be “second hit”

13. No consensus Recommendations for ADA-SCID – No consensus Remove Clinical Hold Use Gene Therapy only in children who fail PEG-ADA or fail BMT –Only for those with no alternative therapy Children on PEG-ADA have disadvantage for GT, lose in vivo selection –Remove PEG-ADA to provide selective advantage

14. Protocol Changes for ADA-SCID US IND, Consistent with February 2003, BRMAC Kohn (ADA-SCID) Treat children >6 months old Children will not receive PEG-ADA starting 2 weeks prior to treatment and following transplant (or were never receiving) Busulfan treatment 5 days prior to transplant Shown with permission from Dr. Kohn

15. Recommendations: All other Protocols Using Ex vivo Retroviral Vector Transduction of HSC Significant risk with any HSC therapy when adequate numbers of transduced cells are infused. –Important consideration in risk/benefit analysis –Ask sponsors to analyze probability for their trial Risk not the same with other trials as with X- SCID Suitable when no alternative effective therapy; failure of standard therapy

16. All other Retroviral Vectors: Transducing HSC Consensus, Vote Reevaluate trials on hold –Risk/benefit of GT vs. existing alternative therapies –Case-by-case –Appropriate informed consent documents 18 yes 1 no –Encouraged FDA to request an analysis from sponsors of the probability of a similar adverse event occurring in their individual trial.

17. FDA Implementation Evaluation of each sponsor’s responses to January, 2003 letter, all indications –Informed consent revisions –Threshold for clonality analysis –Risk/benefit analysis

18. Number of INDs Administrative and IND Status of 28 INDs Placed on Hold, January, 2003 3 SCID (new hold) 6 no response 1 continue hold

19. 15 INDs Received Since January, 2003 Using Retroviral Vector 7 4 4

20. Other BRMAC Recommendations: VECTOR SAFETY Advise investigators to develop vectors with improved safety. Important issues identified –Suicide vectors –Insulator elements –Cell dose –Number of integration sites

21. FDA Actions: VECTOR SAFETY National Toxicology Program, Collaborative Study: FDA, NIH, Chris Baum, David Emery Systematic approach to study effect of various parameters on risk of vector-mediated tumorigenesis: –Delete U3 from LTR (SIN) –Insert Insulator element in LTR –Modulate MOI used for vector transduction –Include both gammaretrovirus and lentivirus vectors –Mouse model established by Chris Baum –Analyze in study of sufficient size to give 90% CI in negative result

22. Other Related FDA Action Workshop on Long-term Follow-up of Participants in Human Gene Transfer ResearchWorkshop on Long-term Follow-up of Participants in Human Gene Transfer Research –June, 2004 –Cosponsors: FDA, ASGT, BIO, PhRMA –Workshop Summary, www.asgt.org www.asgt.org

23. Other BRMAC Recommendations FDA shouldn’t extrapolate an AE from one trial to others with same vector, target cell. –FDA did not place non-SCID INDs on hold after news of third child with leukemia in French X-SCID trial Develop committee to determine number of integrants that would provide reduced risk –FDA committee to develop statistical approach and risk assessment Carolyn Wilson and Dan Takefman, DCGT Steve Anderson, Associate Director for Risk Assessment, Office of Biostatistics and Epidemiology

24. Computer Model Assumptions: Estimation of Number of Vector Integrations in LMO-2 ( 2 loci) CharacteristicSize (kb)Probability Random Insertion Probability Gene Biased Insertion* Upstream LMO-2 -5-10 kb -1-5 kb - 1 kb 10.0 kb 6.66 x 10 -7 3.55 x 10 -6 5.3 x 10 -6 LMO-2 Gene 0-1 kb 1-5 kb 5-10.8 kb 10.8 kb 6.66 x 10 -7 5.3 x 10 -6 3.55 x 10 -6 1.08 x 10 -6 Downstream LMO-2 11-16 kb 16-21 kb 10.0 kb 6.66 x 10 -7 1.08 x 10 -6 6.66 x 10 -7 *Based on Wu, et al, 2003, Science 300:1749

25. Results of Simulation Modeling Vector Copies/CellRandom ModelGene Biased Model 12050 5100250 10200500 Number Vector Integrations in LMO-2 per 10 6 cells Probability that in a treatment of 10 6 cells, one or more cells with vector integration in LMO-2 engrafts Vector Copies/CellRandom ModelGene Biased Model 11/50001/2000 51/10001/400 101/5001/200

26. Update on P10 in X-SCID Gene Therapy Clinical Trial in France

27. Update P10* Treated at 8 months of age –11 x 10 6 CD34 +  c(+) cells/kg No clonal expansion seen at previous time point tested (2 months earlier) Approximately 33 months post-transplant, presented with T cell lymphoproliferation –CD4 and CD8 positive –Gamma-c positive –Vector clonality and integration site under investigation No LMO-2 integrants were detected in earlier time points; No high expression of LMO-2 proteinNo LMO-2 integrants were detected in earlier time points; No high expression of LMO-2 protein Complete remission with first line treatment, steroids * * Data By Permission, Jean-Hughes Trouvin, AFSSAPS, and Alain Fischer

28. Comparison of Patients with Leukemias CharacteristicP4*P5*P10 # Age at time of treatment 1 month3 months9 months Dose  c +, CD34 + cells/kg 18 x 10 6 20 x 10 6 11 x 10 6 Time, Type T cell proliferation 30 mos,  +, 1 TCR 34 mos  +, 3 TCR 33 mos TCRV  5 Retroviral Vector Integration In 1 st Intron, LMO-2 In 5’ UTR, LMO-2 3 vector integrants Sites TBD *Hacein-Bey-Abina, S., et al, 2003, Science 302:415 # Data By Permission, Jean-Hughes Trouvin, Afssaps, and Alain Fischer

29. Status of Other Patients Patient Age Treated (m) Dose  c+ CD34+/kg Follow-up (yrs) Status P1113 x 10 6 ~5.9Healthy P275 x 10 6 5.8Healthy P385 x 10 6 GT failed*BMT,healthy P661 x 10 6 3.8Healthy P7114 x 10 6 3.7Healthy P8622 x 10 6 3.1Healthy P971 x 10 6 3.1↓T cell ; BMT, healthy P1185-7 x 10 6 1 monthHealthy P1215.5 yrs22 x 10 6 NANo T cell reconst. * BCG vaccination, slenomegaly;  c+ cells in spleen # Data By Permission, Jean-Hughes Trouvin, Afssaps, and Alain Fischer

30. Regulatory Status of X-SCID Clinical Trial in France On clinical hold, request of investigators “ …a risk of insertional mutagenesis is clearly identified today with the combination type of vector/nature of the transgene. This risk warrants the necessity of reconsideration on the protocol and method used for the SCID-X1 gene transfer….the use of safer designed vectors is to be envisaged in an attempt to reducing the insertional mutagenesis risk. ” -AFSSAPS

31. Likelihood of Leukemia in SCID Patients After Bone Marrow Transplant?

32. Summary Results from Immune Reconstitution of 132 SCID Patients 117 Haploidentical; 15 HLA-identical 102 survivors (62 on IVIG) –96 >1 year –68 >5 years –37 >10 years 30 deaths –24 from viral infections –2 from Candida sepsis –1 each, mitochondrial defect, nephrotic syndrome (subsequent to chemotherapy for mis-diagnosed malignancy); pulmonary hypertension No hematologic malignancies Buckley, RH, 2004, Annu. Rev. Immunol. 22:625

33. Role of  c Transgene Product? AGAINST: Hacein-Bey-Abina, S., et al, 2003 –No over-expression of  c in leukemic cells of P4, P5 –No sequence changes in  c transgene and entire retroviral vector in leukemic cells of P4, P5 –No constitutive activation of JAK-3 No observed hematologic malignancies in other gene therapy clinical trials in X-SCID FOR: Dave, U., et al, 2004, Science 303:333 –Identification of leukemia with insertion in both  c and LMO-2

34. Other Gene Therapy Clinical Trials in X-SCID 1.Gaspar, et al, 2004, The Lancet 364:2181 2.Weinberg (by permission), no children treated 3.Malech (by permission), 2 children treated

35. Unique Characteristics of French X- SCID Gene Therapy Clinical Trial Media Additives During Ex vivo culture and transduction: –Fetal calf serum –Higher concentration IL-3 –PEG-MGDF vs. TPO Transduction Conditions –Shortest prestimulation period –Use of amphotropic MLV envelope on retroviral vector pseudotype Longest time to follow-up (5 years) Significance??

36. Topic II: Agenda and Questions

37. Topic II: Speakers Relevant Data from Animal Models: Dr. Cynthia Dunbar Dr. Utpal Dave Dr. Christopher Baum Update from Human Experience, ADA-SCID Dr. Donald Kohn

38. Questions for Committee

39. Question 1 What incidence of leukemia in clinical trials using retroviral vector-mediated gene therapy for treatment of X-SCID meet the following? “Human subjects are or would be exposed to an unreasonable and significant risk of illness or injury.”

40. Question 2 How to reduce the risk to subjects? Dose Vector Design

41. Question 3 Risk/benefit considerations: a.In ADA-SCID relative to X-SCID. b.In other clinical indications.

42. Question 4 Limit on vector copy number per cell for cells transduced ex vivo with lentivirus vector?