1. Long-Term Follow-Up of Subjects in Gene Transfer Clinical Protocols Vector Classes with Potential for Long-Term Risks Carolyn A. Wilson, Ph.D. Division of Cellular and Gene Therapies CBER, FDA

2. Overview Scientific basis for long-term risks of gene transfer –long-term patient follow-up –short-term patient follow-up Properties of gene transfer vectors Gene transfer vectors and methods that share these properties

3. Properties of Gene Transfer Vectors with Potential for Long-term Risks Integration into host genomic DNA in somatic cells Integration into host genomic DNA in germ cells Contamination with replication-competent virus (integrating viruses)

4. Integration into Host Genomic DNA in Somatic Cells: Biological Effects Expression of the transgene product Chromosomal rearrangement, e.g., translocations Activation of gene expression via strong viral promoter or enhancer (up to 100 kbp distally) Disruption of transcriptional or translational control regions Dysregulated gene expression

5. What Is the Likelihood that Gene Transfer Vector Integration Would be Tumorigenic? ~80,000 genes (3-5% of genome is coding or transcriptional control regions) ~130 loci identified as “oncogenes” or proto- oncogenes (0.16% of total)* Tumor formation is typically multi-step process Insertional mutagenesis being potentially the first *The Cancer Genome Anatomy Project, Tumor Suppressor and Oncogene Directory

6. Rationale for Long-term Follow-up to Identify Risk of Vector DNA Integration Studies with murine retroviruses demonstrate –High level of virus replication is required for tumorigenesis –Multiple steps are involved in tumorigenesis past provirus insertion –Long latencies prior to tumor formation Data suggest risk of tumorigenesis from gene transfer vector integration is low, effects are long-term

9. Long-term follow-up -- Unresolved issues Gene transfer vectors –range of integration frequencies –variable integration frequencies What properties trigger the need for long-term follow-up? –Characteristics of gene transfer method? –Minimum frequency of integration events?

10. Properties of Gene Transfer Vectors with Potential for Long-term Risks Integration into host genomic DNA in somatic cells Integration into host genomic DNA in germ cells –Effects are long-term –Patient follow-up is typically short-term Contamination with replication-competent virus

11. Integration into host genomic DNA in germ cells: Risks Biological Effects: –None –Genetic disorders –Birth defects –Lethality to developing fetus Societal Issues: –Deliberate germ line alteration deemed unacceptable –Unknown public acceptance of inadvertent germ line alteration

12. What Is the Likelihood that Gene Transfer Vector Integration in Germ Cell DNA Would Be Deleterious? Zebrafish and Mice –Retroviruses used as insertional mutagens to study development: Effects require homozygosity Transposable Elements in the Human Genome* –33 retrotransposition events identified result in human disease (hemophilia A and B, ß-thalassemia, muscular dystrophy) –Background rate of retrotransposition: 1/50-100 germ cells *Kazazian, H., Science 289:1152, 2000

13. Potential for Integration into Genome of Germ Cells Highly dependent upon route of administration –Ex vivo gene transfer, little to no risk –Localized injections, low risk –Localized injections to gonadal regions, risk –Systemic injections, risk

14. Potential for Integration into Genome of Germ Cells Localized injections to gonadal regions –Sato, M., et al, 1999, Sperm-mediated gene transfer by direct injection of foreign DNA into mouse testis. Transgenics 2:357 Systemic injections –Reaves, P.Y., et al, 1999, Permanent cardiovascular protection from hypertension by the AT1 receptor antisense gene therapy in hypertensive rat offspring. Circulation Research 85:e44-e50.

15. Summary Factors influencing long-term risks –Integration of gene transfer vector –Dose of gene transfer vector –Presence of replicating integrating virus –Route of administration (for germ cell integration) –Immune status of recipient Long-term adverse events predicted –Malignancies (somatic cell integration) –Genetic disorders, birth defects, embryonic lethalities (germ cell integration)

16. Will long-term follow-up of patients provide scientific data to assess the long-term risks of gene transfer research? If so, how can this best be achieved?