# Presentation: 539366

## 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

## 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

## 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)

**Notes:**
Potential pathogenic consequences of viral infection
The example of RCR with retroviral vectors will be discussed in more detail in the next talk by Dr. Bishop
Therefore, I will focus my comments to the latter two risks with the aim of providing for your consideration the scientific rationale for performing long-term follow-up of patients in gene transfer clinical trials.

## 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

**Notes:**
Because our genome is diploid, many of these effects will still be silent in terms of any altered phenotype - especially those insertions that disrupt gene expression  

However, in those cases where a gene is normally silent or tightly regulated, but now becomes constitutively expressed at high levels, a diploid effect will not help.  If the gene also happens to be one that may cause unregulated cell division due to unregulated gene expression, the concern is that one outcome of integration is that of tumor formation.

## 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

**Notes:**
Or 1/625 integration events may be in a locus encoding an oncogene.  
But this does not mean that tumors will necessarily result.  Tumorigenesis is a multi-step process.  Replicating retroviruses that cause tumors do so after long latencies, because several steps are required.  After provirus insertion, additional events must occur.  For example, additional recombinational events with endogenous retroviruses are often a requisite to tumor formation in murine models.

Therefore, the risk of tumorigenesis from genomic integration of  gene transfer vector sequences is difficult to predict and can really only be determined from empiric observation in the clinical trials.

## 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**

## Slide 7

## Slide 8

**Notes:**
Clearly, risk of insertional mutagenesis is greatest with retroviral vectors and AAV.  However, plasmid DNA and adenovirus vectors do integrate at low frequencies, and different mode of transfer or in the case of adeno, vector design, may result in increased frequencies.  Therefore, one of the questions before the committee is the following:

## 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?

## 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

**Notes:**
The example of RCR with retroviral vectors will be discussed in more detail in the next talk by Dr. Bishop
Therefore, I will focus my comments to the latter two risks with the aim of providing for your consideration the scientific rationale for performing long-term follow-up of patients in gene transfer clinical trials.

## 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

**Notes:**
Genetic disorders, such as metabolic diseases, or other single gene defects

## 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

## 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

**Notes:**
Sato, et al, injection of liposome encapsulated plasmid DNA into testes resulted in transfection of testicular spermatozoa, as evidenced by transgene detection in F0 progeny at frequencies from 4.3-91%

Of particular interest, a report in rats that received intra-cardiac injection of a retroviral vector had not only the altered genotypes, but even the corrected phenotype in F1 offspring of animals that received the injected vector

## 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.

## 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)

## 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?