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

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

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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)

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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

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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

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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

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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?

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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

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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

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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 2891152, 2000

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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

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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 2357
• 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
  85e44-e50.

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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)

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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?