BRMAC briefing document for Day 2 November 17, 2000

Follow-up of Subjects in Gene Transfer Clinical Trials

[Session IV. Issues in germ line transmission]{.underline}

Inadvertent germ line transmission remains a theoretical but real
concern for gene transfer clinical trials. The risk is particularly
applicable in clinical trials where integrating gene transfer vectors
are administered by systemic routes. While germ line transmission has
not been observed in clinical trials to date, there are preclinical data
suggesting this is more than a theoretical risk. For example, data from
one study in rats wherein a retroviral vector was injected intracardiac
showed evidence of gene transfer not only in the treated rats but also
in their F1 and F2 offspring as well, demonstrating that germ cells were
transduced by the retroviral vector \[1\]. In other cases, germ line
gene transfer may only occur with more directed attempts. For example,
direct injection of liposome-encapsulated plasmid into mouse testis
followed by breeding was successful in transferring the transgene to
offspring \[2\]. The relevance of these kinds of reports is less
obvious, but they support the conclusion that the risk is real.

Data on the risks associated with germ line gene transfer are available
from studies performed with retroviruses, where these viruses were in
fact used as a tool to derive mouse strains with recessive lethal
mutations induced by provirus mutations (for review, see \[3\]). To
assess the mutagenic effect of retrovirus insertion, heterozygous
animals were intercrossed in order to obtain homozygous animals at each
locus. Combining results from a number of studies, two out of 48 crosses
produced recessive lethal mutations \[4\].

**Discussions with the Recombinant DNA Advisory Committee**

On March 12, 1999, the Recombinant DNA Advisory Committee (RAC) met to
discuss gonadal biodistribution studies and potential risk of
inadvertent germ line transfer (see
http://www4.od.nih.gov/oba/3-99RAC.htm). In summary, although the risk
of inadvertent germ line transmission was considered low, the RAC
recommended platform studies for certain classes of viral vectors to
support human studies**.**

FDA\'s current recommendations for biodistribution studies:

**Animal Treatment**

-   Route of administration should mimic intended clinical use as
    closely as possible

-   Inclusion of vehicle control, at least two doses of test article (to
    establish NOEL for biodistribution, and to attempt to maximize
    exposure)

-   Sacrifice/sampling times should include at least one time point
    where gene expression is at peak, and one later time point

-   If integrating vector, should include time point of at least 60
    days, to determine potential for signal persistence through one
    complete cycle of spermatogenesis

-   If planning on repeat administrations for the clinical trial, later
    time point should be at approximately the time of vector
    re-administration planned in humans

-   Whenever feasible, peripheral blood should be removed from tissue
    samples as much as possible at necropsy

**PCR Assay Conduct**

-   Assay should detect a minimum of 100 copies of vector signal per mcg
    of DNA

-   Minimum of 3 mcg of DNA per tissue should be assayed

-   Two mcg of DNA per tissue sample to be assayed directly, with no
    further treatment

-   One mcg of DNA per tissue to be "spiked" with positive control for
    vector amplification, as control for effects of tissue factors on
    detection of signal

**Patient follow up**

FDA has not developed specific recommendations regarding semen analysis
or other tissue analysis to assess germ line dissemination. Most
retroviral vectors are administered *ex vivo*. The requirements for
patient testing (which does not include semen analysis) are outlined in
section III. In the one ongoing clinical study of direct IV injection of
a retroviral vector, semen samples are obtained at time 0, week
2,4,6,17,29, and 52 weeks. Study participants are to use barrier
contraception for one year after vector administration. Recently a
patient sample was positive by PCR, and the study was put on clinical
hold until 3 consecutive semen samples were negative. In another ongoing
study of 3 direct (IM) administrations of an AAV vector, semen analysis
is performed at time 0, 1,2, and 3 months. As above, any positive sample
will lead to a clinical hold until 3 consecutive samples are negative.
The potential for gonadal dissemination is included in informed consent
documents.

**References:**

1\. Reaves, P.Y., et al., *Permanent cardiovascular protection from
hypertension by the AT1 receptor antisense gene therapy in hypertensive
rat offspring.* Circulation Research, 1999. **85**: p. e44-e50.

2\. Sato, M., et al., *Testis-mediated gene transfer (TMGT) in mice:
successful transmission of introduced DNA from F0 to F2 generations.*
1999.

3\. Jaenisch, R., et al., *Retroviruses and insertional mutagenesis.*
Cold Spring Harbor Symposia on Quantitative Biology., 1985. **50**: p.
439-445.

4\. Soriano, P., T. Gridley, and R. Jaenisch, *Retroviruses and
insertional mutagenesis in mice: proviral integration at the Mov34 locus
leads to early embryonic death.* Genes and Development, 1987. **1**: p.
366-375.

**DRAFT Questions to the committee**

1.  If semen positivity is identified in patients in a clinical trial,
    our current approach is to place a clinical hold on the study until
    it is determined that semen positivity is transient, at which time,
    the trial may be resumed. Please discuss whether this approach is
    appropriate.

2.  In cases where a positive PCR signal is observed in patient semen
    samples, please discuss methods to determine cell source for the
    positive signal (e.g., in situ hybridization, fractionation of semen
    into sperm and lymphocytes, etc.).