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

1. CTGTAC March 4, 2005, Topic II Update: Retrovirus Vector-Mediated Insertional TumorigenesisCarolyn 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. Psi (Internal Promotor) Transgene LTR LTR LTR LTR LTR U3 R U5 U3 Read-through Transcription Dysregulated Gene Expression Distal Gene Activation Tumorigenesis Gammaretroviral-Mediated Endogenous Gene Activation 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-X1Hacein-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. 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 106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. RecommendationsActions BRMAC Meeting February, 2003

10. Recommendations for X-SCID, IL-7 and JAK-3 deficiencies • 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 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. 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 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 HSCConsensus, 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. 3 SCID (new hold) 6 no response 1 continue hold Administrative and IND Status of 28 INDs Placed on Hold, January, 2003 Number of INDs

19. 15 INDs Received Since January, 2003Using Retroviral Vector 4 7 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 Research • June, 2004 • Cosponsors: FDA, ASGT, BIO, PhRMA • Workshop Summary, 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) *Based on Wu, et al, 2003, Science 300:1749

25. Results of Simulation Modeling Number Vector Integrations in LMO-2 per 106 cells Probability that in a treatment of 106 cells, one or more cells with vector integration in LMO-2 engrafts

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

27. Update P10* • Treated at 8 months of age • 11 x 106 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 protein • Complete remission with first line treatment, steroids * Data By Permission, Jean-Hughes Trouvin, AFSSAPS, and Alain Fischer

28. Comparison of Patients with Leukemias *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 *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 Buckley, RH, 2004, Annu. Rev. Immunol. 22:625 • 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

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 • Gaspar, et al, 2004, The Lancet 364:2181 • Weinberg (by permission), no children treated • 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: • InADA-SCID relative to X-SCID. • In other clinical indications.

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