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  1. Long-Term Follow-Up of Subjects in Gene Transfer Clinical ProtocolsVector Classes with Potential for Long-Term RisksCarolyn A. Wilson, Ph.D.Division of Cellular and Gene TherapiesCBER, 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

  7. 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?

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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)

  14. 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?