#  BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

Briefing Document For May 10^th^, 2002

Issues Pertaining To Inadvertent Germline Transmission Of Gene Transfer
Vectors

# 

# Introduction 

The unintended modification of genetic material in host germ cells by
gene transfer vectors during clinical trials remains a theoretical but
real concern. For the purposes of this briefing document, such an event
is referred to as Inadvertent Germline Transmission (IGLT). IGLT is of
particular concern in clinical trials in which gene transfer vectors
with the potential to integrate into the host chromosome are
administered by systemic routes. While germline transmission has not
been observed in clinical trial participants to date, findings obtained
from a recent study have renewed concern regarding IGLT.

Data from a phase I safety study involving administration of an
adeno-associated virus (AAV) vector carrying the factor IX gene into the
hepatic artery of subjects with hemophilia B were presented at two
recent meetings of the Recombinant DNA Advisory Committee (RAC). In this
study, vector sequences were detected in semen samples from the first
two subjects to which the vector was administered. In the context of
these findings, the FDA is seeking guidance on how to further develop
policies regarding IGLT, not just for this particular study, but in
general for all clinical trials involving systemic administration of
gene transfer vectors.

# 1. [BACKGROUND]{.underline}

# Pre-clinical animal studies

The data from animal studies regarding directed attempts to alter germ
cells with gene transfer vectors have provided equivocal results
regarding the ability of these agents to facilitate vertical gene
transmission to progeny. Using cationic lipid-encapsulated plasmid DNA
vectors encoding bacterial β-galactosidase, human growth hormone, or
green fluorescent protein, gene transfer to spermatozoa was observed,
both *in vitro* and following direct injection of vector into gonadal
tissues (testes, epididymes, or vas deferens; \[1-4\]). When injected
male mice were mated 2 to 3 days later with super-ovulated female
animals, vector sequence could be detected in the F0 progeny, in some
cases in as many as 50% of the fetuses \[5,6\]. Similarly, a study using
retroviral vectors injected into the hearts of spontaneously
hypertensive, newborn rats has demonstrated positive evidence of gene
transfer, as determined by PCR and Southern blot analysis, as well as
functional correction of the gene defect in the F1 and F2 offspring
\[7\].

Data on risks associated with germline 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 \[8\]). 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 \[9\].

However, similar attempts to create transgenic animals using adenoviral
vectors, either after *in vitro* exposure of mature sperm to the virus
or by direct injection into testicular tissues have failed to show
significant gene transfer to either spermatozoa or offspring \[10,11\].
In a study utilizing an adenoviral vector, murine spermatogenic cells
were exposed immediately prior to or during in vitro fertilization. When
examining exposed testes or pre-implantation embryos, no evidence of
germline transmission was observed. Additionally, treatment of either
male, female, or animals of both genders with high doses of adenoviral
vectors by intravenous injection (i.v.) failed to result in detectable
sequences in offspring, even when the gonadal tissues of the parental
animals were highly positive for vector sequence by PCR analyses \[12\].

Safety studies of AAV vectors, including the one used in the present
clinical trial, have failed to demonstrate germline transmission in
animal models \[13,14\]. When AAV vector encoding canine or human factor
IX, or a null AAV vector, were injected into either the muscle or
hepatic artery of four different species of animals (mice, rat, dog,
rabbit), vector dissemination to the testis was detected, although the
signal decreased over time. However, vector signal was localized to the
testis basement membrane and the interstitial spaces \[13\]. In contrast
to the results obtained from the current AAV FIX human trial, semen
obtained from the dogs exposed i.v. to AAV in this study had no
detectable vector sequences present by PCR analysis.

Taken together, these data suggest that the risks of IGLT are more than
theoretical, but are difficult to accurately assess in the available
animal models.

### Previous public discussions on IGLT

During the March 1999 RAC meeting, pre-clinical data on gonadal
biodistribution and the potential for IGLT were discussed
(http://www4.od.nih.gov/oba/rac/minutes/3-99RAC.htm). The consensus from
this meeting was that the risk of IGLT occurring in a gene transfer
clinical trial is low. Further discussion of IGLT became necessary when
data from a gene transfer trial conducted by Chiron Corporation revealed
the detection of vector sequences in the semen of a study subject. The
data from this study was presented at the BRMAC meeting held Nov 17,
2000. This trial involved i.v. administration of a murine
gammaretroviral vector carrying a factor VIII gene to subjects with
hemophilia A. At week nine post administration, DNA extracted from a
semen sample was positive for vector sequences as detected in 1/10
polymerase chain reaction (PCR) replicates. No positive signals were
detected in subsequent semen samples. Murine gammaretroviruses transduce
only dividing cells, thus mature sperm cells are unlikely targets for
transduction by these viruses. However, spermatogonial stem cells, which
are rapidly dividing, theoretically could be transduced by a retroviral
vector via hematogenous spread. Considering the physical barriers that a
systemically administered vector would need to cross, type A and B
spermatogonia would be accessible for transduction since these
progenitor germ cells are on the blood side of the Sertoli cell barrier
(for review see \[15\]). Since one cycle of spermatogenesis takes
approximately 64-74 days, the timing of the appearance of a transduced
progenitor's daughter cells in the semen is predictable. The protocol by
Chiron to test study subjects' semen samples took this timing into
account. The BRMAC committee agreed with the FDA instituting a clinical
hold when vector sequences are detected in semen samples from study
subjects. In order for the clinical hold to be lifted, the FDA approach
was to require that the sponsor demonstrate that three consecutive semen
samples, collected monthly, were negative for the presence of detectable
vector sequence. The committee also recommended that fractionation of
semen samples be performed as a follow up to a positive semen sample.
Using fractionation methods such as density separation, potential
contaminating transduced white blood cells can be removed from sperm
cell fractions.

At the RAC meetings of December 22, 2001 and March 7,2002 data were
presented from a trial being conducted by Avigen Inc.
(http://www4.od.nih.gov/oba/RAC/meeting.html). In this study, an AAV
vector carrying the factor IX gene was administered into the hepatic
artery of subjects with hemophilia B. As detected by PCR analysis of
unfractionated semen, vector sequences have been found in the semen of
the first two subjects treated. In the first subject treated, positive
PCR signals were detected up to ten weeks post administration, but no
signal was detected in multiple latter time-point samples. Only early
time-point samples have been analyzed on the second subject.
Conceivably, all subjects treated in this trial may have vector
sequences that are transiently detected in semen. In fact, vector
sequences may be detected in subjects' semen in all trials involving
systemic administration of an AAV vector. Additionally, with new
technologies allowing for higher titer vectors to be made, and with new
vector types being tested (such as lentiviral vectors), detection of
vector sequences subjects' semen might become more prevalent in future
clinical trials.

## Considerations for different vector classes

[]{.mark}

Due to the biology of the various gene transfer vectors being used,
specific consideration may be needed in terms of procedures for patient
follow- up. For example, unlike murine gammaretroviral vectors, AAV does
not have a strict requirement for dividing cells in order to stably
transduce cells. Thus, in addition to transduction of the spermatogonial
progenitor cells being a concern, transduction of mature sperm is also a
theoretical risk. In addition to AAV, lentiviral and adenoviral vectors
do not have a requirement for cell division in order to transduce cells.
The potential for host chromosome integration for these vectors range
from very likely to highly unlikely and must also be accounted for in
considering the appropriate follow-up procedures for subjects receiving
these vectors.

### 2. CURRENT FDA APPROACH

## FDA pre-clinical recommendations to assess potential for germline transmission.

During the conduct of pre-clinical safety studies of gene transfer
vectors, FDA recommends that the distribution of vector out of the
intended site be evaluated by PCR analysis of DNA isolated from a panel
of different tissues, including gonads (ovaries and testes). The FDA\'s
current recommendations for the design and conduct of biodistribution
studies are to include a minimum of 5 animals per gender, per dose
group, and per time point of sacrifice for analysis. If a single gender
is used in support of gender-specific indications (*e.g.* hemophilia in
males), then at least ten animals per group should be evaluated. The
route of administration of the vector should mimic the intended clinical
route as closely as possible. Studies should include a vehicle control
group, and preferably two or more dose levels of the test article to
both establish a no-effect level for biodistribution, as well as to
maximize exposure to the gene transfer agent. Tissue sampling should be
performed at terminal sacrifice, and should, at a minimum, include at
least one time point where peak gene expression occurs, as well as at
least one later time point. The later time points are to be determined
by the intended clinical use (*e.g.* if the clinical trial includes
plans for re-treatment with the vector, the later time point in the
animal study should approximate the day of re-administration). For
integrating vectors, the later samples should be obtained at least 60
days after injection, to determine the potential for signal persistence
through one complete cycle of spermatogenesis. To minimize the chances
of contamination of the samples by leukocytes present in peripheral
blood, every attempt should be made to harvest the tissues as cleanly as
possible, and to remove peripheral blood as much as possible at
necropsy.

**PCR Assay Conduct**

-   Assays should be able to detect a minimum of 10-100 copies of vector
    signal per mcg of DNA

-   A minimum of 3 mcg of DNA per tissue should be assayed

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-   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 a control for effects of tissue factors on
    detection of signal

## FDA current recommendations for patient follow up

In response to data from the AAV FIX trial, and subsequent discussions
at the Dec 2001 RAC, the FDA has modified its approach, and will only
institute clinical hold if the motile sperm from fractionated semen
tests positive for vector sequences.

## Current approach for follow-up of potential IGLT of gene transfer vector sequences to the sperm of clinical trial participants:

-   If semen tests positive for vector sequences, the FDA will allow the
    clinical trial to continue, but request that the sponsor begin
    fractionating semen at time points appropriate for the detection of
    a transduced progenitor in semen. Testing of fractionated semen
    should span at least one complete cycle of spermatogenesis.

-   If the motile sperm fraction tests positive for vector sequences,
    patient enrollment will be stopped and the clinical trial will be
    placed on hold.

    -   Subsequent fractionated samples from the same patient must test
        negative over 3 consecutive monthly intervals for enrollment to
        resume.

-   Use of barrier contraception is requested until 3 consecutive semen
    samples test negative.

The potential for assessing IGLT in women is especially difficult and,
to date, no clinical studies have proposed sampling of female
reproductive tissue.

# Informed consent

If during pre-clinical animal studies, vector is found in gonadal
tissue, this finding and the potential for gonadal dissemination should
be included in informed consent documents. If vector sequences are found
transiently in subject semen, sponsors will be asked to revise the
informed consent document in order to describe the detection of vector
DNA sequences within the semen of subjects tested.

**REFERENCES**

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    Dev., 1998. **50**: 406-409.

2.  Huguet, E. and P. Esponda. *Foreign DNA introduced into the vas
    deferense is gained by mammalian spermatozoa.* Mol. Reprod.
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3.  Sato, M., et al. *Sperm-mediated gene transfer by direct injection
    of foreign DNA into mouse testis*. Transgenics, 1999. **2**:357-369.

4.  Yamakazi, Y., et al. *In vivo gene transfer to mouse spermatogenic
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5.  Sato, M., et al. *Testis-mediated gene transfer (TMGT) in mice:
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    generations.* Transgenics,1999. **3**: p. 11-22.

6.  Sato, M., et al. *Direct injection of foreign DNA into mouse testis
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7.  Reaves, P.Y., et al., *Permanent cardiovascular protection from
    hypertension by the AT1 receptor antisense gene therapy in
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8.  Jaenisch, R., et al., *Retroviruses and insertional mutagenesis.*
    Cold Spring Harb. Symp. Quant. Biol., 1985. **50**: p. 439-445.

9.  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 Dev., 1987. **1**: p.
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10. Hall, S.J., et al., *Direct exposure of mouse spermatogenic cells to
    high doses of adenovirus gene therapy vector does not result in germ
    cell transduction.* Hum. Gene Ther., 2000. 11:1705-1712.

11. Peters, A.H., Absence of germline infection in male mice following
    intraventrical injection of adenovirus. Mol. Gene Ther., 2001.
    **4**: p. 603-613.

12. Ye, X. et al. *Evaluating the potential of germ line transmission
    after intravenous administration of recombinant adenovirus in the
    C3H mouse.* Hum. Gene Ther., 1998. **9**: 2135-2142.

13. Arruda, V.R., et al., *Lack of germline transmission of vector
    sequences following systemic administration of recombinant AAV-2
    Vectors in males.* Mol. Ther., 2001. **4**: p. 586-592.

14. Favre, D., et al., *Immediate and long-term safety of recombinant
    Adeno-associated virus injected into the nonhuman primate muscle.*
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15. Gordon J.W. *Germline alteration by gene therapy: assessing and
    reducing the risks.* Mol. Med. Today. 4: p. 468-470.