Briefing document\
BB‑IND 9398

bIOLOGICAL rESPONSE mODIFIERS aDVISORY cOMMITTEE\
mAY 9‑10, 2002

![](media/image1.wmf){width="1.325in" height="0.7013888888888888in"}1301
Harbor Bay Parkway\
Alameda, California 94502

April 19, 2002

Table of Contents

[i. Background And Rationale 3](#i.-background-and-rationale)

[I.A. Background 3](#background)

[I.A.1 Hemophilia B 3](#hemophilia-b)

[I.A.2 Factor IX Gene and Protein 5](#factor-ix-gene-and-protein)

[I.A.3 Biology of Adeno-Associated Virus (AAV) Vectors
6](#biology-of-adeno-associated-virus-aav-vectors)

[I.A.4 Description of the AAV‑hFIX Vector
6](#description-of-the-aavhfix-vector)

[I.B. Manufacture 7](#manufacture)

[II. clinical update 7](#clinical-update)

[II.A. Subject 1 8](#subject-1)

[II.B. Subject 2 10](#subject-2)

[III. Preclinical Update 12](#preclinical-update)

[III.A. Pre-Clinical Efficacy Studies
12](#pre-clinical-efficacy-studies)

[III.B. Molecular characterization of vector DNA in liver
......................................................................................
12](#molecular-characterization-of-vector-dna-in-liver)

[III.C. Safety and Biodistribution Studies
13](#safety-and-biodistribution-studies)

[III.D. Immune Responses Against the Expressed Coagulation Factor
19](#immune-responses-against-the-expressed-coagulation-factor)

[III.E. Summary of Planned Studies 20](#summary-of-planned-studies)

[IV. References 21](#references)

# i. Background And Rationale {#i.-background-and-rationale .unnumbered}

## Background

### Hemophilia B

Hemophilia B is the bleeding diathesis that results from a deficiency of
blood coagulation factor IX. The disease is X-linked and affects
approximately 1 in 30,000 males. Current treatment of the disease is
based on intravenous infusion of clotting factor concentrates, either
recombinant or plasma-derived, in response to bleeding episodes. The
major morbidity of the disease is arthropathy that results from
recurrent spontaneous bleeds into the joints. A major cause of mortality
is bleeding into critical closed spaces (e.g., intracranial,
retropharyngeal, or retroperitoneal bleeds). The introduction of
factor IX concentrates and of home infusion protocols in the 1960s and
1970s represented a major advance in the treatment of hemophilia.
However, widespread use of contaminated plasma-derived clotting factor
concentrates resulted in a high rate of human immunodeficiency virus
(HIV) infection among individuals with severe disease. Seroconversion
studies document that most patients with hemophilia were infected
between 1978 and 1984 (1). In the US, most patients with severe
hemophilia who are over the age of 20 are HIV‑positive, and many have
already died from complications related to the acquired immune
deficiency syndrome (AIDS). The development of recombinant factor IX
protein in the 1990s ensured an additional level of safety, but these
products have not circumvented all of the problems of protein-based
therapies.

Most individuals with hemophilia B have severe disease, with factor IX
levels of \< 1%. A generation of clinical research in patients treated
with clotting factor concentrates has documented that minimal elevations
in the levels of circulating clotting factor are sufficient to prevent
much of the morbidity of the disease. The most comprehensive data are
contained in the Swedish prophylaxis studies summarized in Lofqvist
*et al.* (2). Since 1958, most hemophilia patients in Sweden have been
maintained on a regimen in which factor is infused on a regular basis
rather than in response to bleeds. The doses used have been gradually
increased through the years; the current regimen involves infusion of
25--40 U/kg two--three times weekly. The goal of therapy is to maintain
trough factor levels \> 1% of normal. Data from the most recently
published summary showed orthopedic and radiographic joint scores of 0
(normal joints) for all patients under the age of 15; radiographic joint
scores in the range of 5--20 would be more typical for individuals in
this age group treated only in response to bleeds (3). Thus, the
favorable effect on joint disease of keeping levels even marginally
higher than baseline is clear.

Before the AIDS era, central nervous system hemorrhage was the most
common cause of premature death in patients with hemophilia (4). In
several retrospective analyses and in one prospective study, the
prevalence of CNS bleeding ranged between 2.6 and 13.8%, with mortality
rates between 20 and 50% (5-7) and morbidity rates (including seizure
disorder, motor impairment, or mental retardation) of 40--50% in
survivors (5). These bleeds can be present with few or subtle
premonitory symptoms, and the bleed can impair the patient's ability to
treat himself. The literature documents that these bleeds occur
predominantly in patients with severe disease (\< 1% factor level) (5).
Thus, it is reasonable to suppose that raising the levels of factor even
slightly ( 2% of normal) would improve the chances for avoiding this
life-threatening complication of the disease. The incidence of CNS
hemorrhage can also be reduced by the use of prophylactic factor
infusions (4).

Prophylactic therapy for hemophilia has not achieved widespread
acceptance in the US for several reasons. The expense is prohibitive (an
average adult patient with severe disease who treats [only]{.underline}
in response to bleeds spends \$50,000--\$100,000 on concentrate each
year). In addition, in children treated prophylactically who require
indwelling intravenous catheters for infusion there is an attendant risk
of catheter-related infections (8). In addition to the cost and the
inconvenience associated with the need for intravenous infusion, the use
of plasma-derived concentrates carries the additional risks of
blood-borne disease transmission associated with all blood products.
Current viral inactivation procedures have greatly reduced, but not
eliminated, this risk (9).

Gene therapy, a recently proposed alternative approach, would
potentially allow patients to realize the benefits of prophylaxis,
including reduced incidence of CNS bleeding and of hemophilic
arthropathy, without the need for indwelling intravenous catheters or
frequent factor infusion.

Several features make hemophilia B a good model for gene therapy. The
first advantage of hemophilia B as a model for gene therapy is that
precise regulation of transgene expression is not required. Therapeutic
range is remarkably wide, from  1% to 150% of normal. It is clear, based
on data from infusion of concentrates into patients with hemophilia B,
that levels as high as 150% are not associated with ill-effects since
the protein circulates as a zymogen (inactive precursor). Second, as
stated, even low levels of between 1--5% of normal provide prophylaxis
against chronic arthropathy and CNS bleeding. Patients with levels of 5%
have much milder disease and only rarely experience spontaneous bleeding
episodes (although they exhibit abnormal bleeding in response to
hemostatic challenges such as surgery or trauma). A third advantage of
hemophilia B is the availability of large and small animal models for
the human disease. Clearly, animal models are a major asset in efforts
to establish an experimental basis for gene therapy. In the case of
hemophilia, there are well characterized naturally occurring canine
models of the disease and genetically engineered hemophilic mice
(10-13). Finally, it should be noted that determination of therapeutic
efficacy is straightforward and unequivocal in the case of hemophilia B,
since plasma levels of factor IX are easy to measure and correlate well
with clinical severity of disease.

### Factor IX Gene and Protein

The human factor IX gene is located on the X chromosome at Xq26 through
Xq27 (14). It spans 35 kb and consists of eight exons separated by seven
introns (15). The exons correlate approximately with the functional
domains of the protein. Thus, exon 1 encodes the signal sequence, exon 2
the propeptide and ‑carboxyglutamic acid (Gla) domain, exon 3 a short
connecting peptide, exons 4 and 5 the first and second epidermal growth
factor (EGF)-like domains respectively, exon 6 the activation peptide,
and exons 7 and 8 the catalytic domain and the long 3\' untranslated
sequence. The human cDNA is 3 kb in length, encoding a mature protein of
415 amino acids. The factor IX genes of all species studied to date
(including the human gene) contain a long (\~ 1.4 kb) 3\' untranslated
sequence. The precise function of this sequence is unknown, and it has
not been included in most recombinant vectors expressing human
factor IX.

The biosynthesis of factor IX is complex because of a number of
post‑translational modifications that are required in order for the
protein to have full biological activity (16). These include proteolytic
removal of the signal sequence and the propeptide; ‑carboxylation of 12
glutamic acid residues at the N-terminus of the protein in a
vitamin K-dependent reaction; and glycosylation at specific residues in
the light chain (the N‑terminal portion of the molecule) and the
activation peptide. The enzyme ‑glutamylcarboxylase, which catalyzes the
conversion of specific glutamic acid residues to -carboxyglutamic acid,
is found in high levels in hepatocytes, but is also present in a variety
of other tissues including muscle (17). Biologically active factor IX
has been synthesized in a wide range of cells including CHO cells,
endothelial cells, myoblasts, and fibroblasts (18-20).

### Biology of Adeno-Associated Virus (AAV) Vectors

Adeno-associated virus is a non-enveloped, replication-defective
parvovirus that has not been associated with human disease. AAV vectors
are derived from the parent virus by removing all of the viral elements
except for the inverted terminal repeats (ITR) and inserting the gene or
genes of interest and their associated regulatory elements. The
long-term safety of these vectors in humans is unknown; however, initial
results of two clinical trials using AAV vectors to deliver the gene for
the cystic fibrosis transmembrane regulator (CFTR) directly to the
respiratory tract have not resulted in adverse events.

AAV vectors do not require actively dividing target cells to achieve
efficient transduction, as demonstrated in post-mitotic cells of brain,
liver, and muscle *in vivo* (21-26). Also, because all of the viral
genes have been removed, there is no immune response directed against
the transduced cell due to viral gene expression. This accounts, at
least in part, for prolonged (months to years) transgene expression
observed *in vivo* following a single administration of an AAV vector.
Several groups have now established that AAV efficiently transduces
liver cells following a single administration via the portal vein
resulting in long-term, (\> 2 years), dose-dependent transgene
expression (25-27).

### Description of the AAV‑hFIX Vector

The AAV‑hFIX vector was derived from the naturally occurring virus
adeno-associated virus (AAV) that has not been associated with human
disease and is ubiquitous in the environment. The virus consists of a
single-stranded DNA genome encapsidated in a protein coat. The AAV
genome consists of three elements: the *rep* gene, the *cap* gene and
the inverted terminal repeats (ITRs). The *rep* gene directs production
of the proteins that enable the virus to replicate its genome. The *cap*
gene directs production of the protein coat. These two viral genes are
flanked by the ITRs, identical segments of DNA required for replication
and encapsidation.

The AAV-hFIX16 vector is derived from wild-type AAV using recombinant
DNA techniques and contains a single-stranded DNA genome of 4492
nucleotides. All of the viral genes have been removed and replaced with
the following: 1) an expression cassette that contains the human
1-antitrypsin promoter coupled to the human apolipoprotein E enhancer
(28) and hepatocyte control region (29) 2) exon 1 from the human
factor IX gene; 3) a portion of the human factor IX intron 1; 4) exons
2-8 of the human factor IX gene; and 5) the bovine growth hormone
polyadenylation signal sequence. Small intervening non‑functional DNA
sequences are derived in the process of assembling the genetic elements
through recombinant DNA techniques. The expression cassette is flanked
by the 145 nucleotide inverted terminal repeats derived from AAV type 2.

AAV-hFIX16 vector is produced by transfecting Human Embryo Kidney (HEK)
293 cells with plasmid DNA encoding the genes required for vector
amplification and packaging. Vector particles are released from
harvested cells by freeze/thaw, purified by density gradient
centrifugation, and concentrated / diafiltered by tangential flow
filtration. Each lot of AAV-hFIX16 is tested for identity,
concentration, potency, purity, pH, sterility, and appearance.

## Manufacture

AAV-hFIX16 vector is manufactured in compliance with FDA Current Good
Manufacturing Practice (cGMP) Regulations by Avigen, Inc, 1201 Harbor
Bay Parkway Suite 1000, Alameda, CA 94502.

# clinical update

Based on previously published preclinical efficacy and safety studies in
animals, a human clinical trial has been initiated using a rAAV vector
to deliver the gene for human factor IX into the liver. The AAV vector
containing a gene for human factor IX (AAV-hFIX) is administered into
the hepatic artery via arteriography. The study design is open-label,
non-randomized, single administration with inter-subject group dose
escalations. Up to 10 subjects with severe hemophilia B will be enrolled
and assigned to groups of two to four. Subjects within each group will
receive the same dose of AAV-hFIX16 vector as indicated in Table 1.

**Table 1**

![](media/image2.png){width="5.0in" height="2.790277777777778in"}

> ![](media/image3.png){width="5.0in" height="2.875in"}**Table 2**

Following vector administration, subjects are evaluated for local or
systemic toxicity by clinical and laboratory assessment during the
initial hospitalization, the first week following treatment, and then
periodically for the first year. Complete blood counts, coagulation
studies, and serum chemistries are regularly monitored. In addition,
Factor IX inhibitor titers and vector shedding (urine, blood, saliva,
and stool) are evaluated. To evaluate the potential for vertical
transmission of AAV vector, semen samples are obtained at baseline,
during week 1, at months 1--4, and then monthly until 3 serial samples
are negative for vector DNA by PCR. The semen samples at two, three and
four months (days 60, 90, and 120) are subjected to a fractionation
procedure that will allow testing of the motile sperm fraction for
vector sequence. Currently, dose escalation cannot take place unless all
subjects in the previous cohort have been shown to be free of vector
sequence in the motile sperm fraction at day 60. Subjects are counseled
to consider banking sperm prior to administration of AAV-hFIX16, and to
practice barrier (condom) contraception until three consecutive monthly
semen samples are negative.

## Subject 1

To date, two subjects have been enrolled and treated in this study. The
first subject had no serious adverse clinical or laboratory events
(Tables 2,3) as now observed to Week 24. Vector shedding in various body
fluids were determined by a PCR assay with a sensitivity of 10 copies
per microgram of total genomic DNA. All semen samples were analyzed in
triplicate. Vector sequences were detected in serum out to 14 days, and
detected in semen out to 6-10 weeks but have subsequently been negative
(Table 4). Fractionation of the semen sample from week 3 indicated the
motile sperm component was found to be negative for rAAV vector
sequences while the seminal fluid was positive (Table 5).

#### Table 3 {#table-3 .unnumbered}

![](media/image4.png){width="5.25in" height="2.75in"}

##### Table 4 {#table-4 .unnumbered}

![](media/image5.png){width="5.25in" height="2.772222222222222in"}

#####  {#section .unnumbered}

#####   {#section-1 .unnumbered}

##### Table 5 {#table-5 .unnumbered}

![](media/image6.png){width="5.125in" height="2.875in"}

## Subject 2

A second subject with severe factor IX deficiency was enrolled into the
study approximately 8 weeks ago. The procedure was tolerated without
adverse clinical or laboratory events (Tables 6,7). PCR analysis of
total semen has determined positive vector sequences in the week 1, 4,
and 8 samples. Fractionation of the week 8 specimen could not be
performed using the Andrology Laboratory's standard fractionation
procedure, due to the low volume of semen obtained (Table 8). PCR
analysis of total semen samples to date show a pattern similar to that
seen with the first subject, ie. gradually diminishing intensity of
positive signals (Table 9).

######## Table 6 {#table-6 .unnumbered}

![](media/image7.png){width="5.375in" height="2.5in"}

##### Table 7 {#table-7 .unnumbered}

![](media/image8.png){width="5.25in" height="3.486111111111111in"}

##### Table 8 {#table-8 .unnumbered}

![](media/image9.png){width="5.25in" height="3.5in"}

########  {#section-2 .unnumbered}

![](media/image10.png){width="5.25in" height="3.0in"}

# Preclinical Update

## Pre-Clinical Efficacy Studies

Numerous studies have been performed by a number of investigators
(25-27,30-33) to demonstrate the efficacy of delivering an AAV-FIX
vector to the livers of normal and hemophilic mice and to hemophilic
dogs. Normal mice injected with the AAV-hFIX vector being used in the
clinical trial at doses ranging from 1.0 x 10^11^-7.0 x 10^13^ vg/kg
have produced circulating FIX levels of between 50 and 25,000 ng/ml,
representing 1-500% of normal. Hemophilic mice injected with a different
AAV-hFIX vector at doses of 8x10^12^ - 2.2 x 10^13^ vg/kg resulted in
similar plasma FIX levels of 1000-20,000 ng/ml, resulting in the
amelioration of the bleeding diathesis in these animal (33). Efficacy
has also been demonstrated in hemophilic B dogs following injection of
an AAV-cFIX vector. Our data demonstrate that doses of approximately
10^12^ vg/kg resulted in sustained Factor IX levels of 4-12% (30). In
addition, Wang et al (32) have seen levels of 218 ng/ml following
administration of 4.6x10^12^ vg/kg of an AAV-cFIX vector containing a
different expression cassette.

## Molecular characterization of vector DNA in liver 

AAV gene transfer to the mouse liver has been studied at the molecular
level by a number of investigators. Following injection into either the
liver or the muscle, expression rises gradually over a period of 2--8
weeks. Using DNA in-situ hybridization it has been demonstrated that
most of the hepatic nuclei take up the vector following portal vein
administraton of 3 x 10^10^ to 2 x 10^11^ vg per mouse, but only about 5
percent of the hepatocytes are stably transduced (34). Vector DNA is
converted from the single-stranded input form into a double-stranded
genome in the stably transduced cells. The molecular structures of the
double-stranded genomes represent a complex mixture of circular and
linear DNA forms. Some of the double-stranded DNA forms are present as
concatemers, with equal numbers of head to head, tail to tail, and head
to tail structures (35) An ongoing area of investigation has been the
attempt to determine whether these high molecular weight forms are
integrated into the host cell genome or persist as episomal forms. Miao
et al. (36) have recently provided two types of evidence suggesting that
the vector concatemers and/or integrated DNA forms were present
following injection into the portal circulation of the mouse. They used
fluorescence in-situ hybridization (FISH) analysis performed on
metaphase spreads from mouse hepatocytes to demonstrate that a
hybridization signal arising from an rAAV probe was found on sister
chromatids in metaphase chromosomes. Finally, Nakai et al. (35),
isolated rAAV-mouse chromosomal junction fragments from transduced mouse
liver. Over 15 of these fragments have been molecularly
characterized**.** These early studies using methods that established
integration, were not able to quantify the amount of integrated AAV DNA
in liver. More recently an in vivo liver regeneration assay was used to
demonstrate that in most cases, chromosomal integration of rAAV vector
genomes was relatively inefficient (approximately 5%). These studies
demonstrated that the majority of gene expression was derived from
episomal forms of the vector (37).

## Safety and Biodistribution Studies

Safety of rAAV delivery to the liver has been evaluated in five animal
species: mice, rats, dogs, non-human primates, and most recently rabbits
(see Table 1). A GLP toxicology and biodistribution study was performed
in mice and rats, a non-GLP study in normal dogs evaluated toxicology
and gonadal/semen dissemination of vector, and a GLP toxicology study
was performed in non-human primates that also included analysis of
dissemination of vector to liver and gonadal tissue (the biodistribution
analysis was done in a non-GLP setting). Recently we have demonstrated
that rabbits are the most useful species to assess vector distribution
to semen and to monitor the kinetics of its clearance.

![](media/image11.png){width="5.625in"
height="3.6555555555555554in"}**Table 1 Summary of Safety Studies**

There was no evidence for vector related toxicity in rats, dogs or
non-human primates at doses of rAAV up to 10^13^ vg/kg. In this document
we will focus only on those aspects of the studies listed which address
issues related to potential for inadvertent germ line transmission.

Biodistribution of rAAV vector or in some cases just gonadal
dissemination of vector was evaluated in each of the safety studies
described above.

A GLP study using mice was performed to evaluate the distribution of
vector to tissues, including gonads. Animals were injected intravenously
with AAV-murine F.IX at 1.7 x 10^12^ vg/kg and sacrificed at day 31 or
day 91 post-injection. Using genomic DNA obtained from gonadal tissue, a
PCR assay revealed vector sequences in all injected animals (n=4) after
31 days. However, at day 91 vector sequences were seen in only 3/4
animals, and the PCR signal was weak in one of these three compared to
the intensity in the first cohort of animals (Table 2).

![](media/image12.png){width="6.0in" height="4.25in"}**Table 2**

The GLP rat study included quantitative PCR analysis of seven tissues
and blood at two time points (days 50 and 92) following vector
administration. Three doses of AAV-hFIX ranging from 1x10^11^-1x10^13^
vector genomes per kg were used, the highest dose being 50 fold higher
than the starting clinical dose. Vector was observed in the testes in
animals from all groups, 50 days post injection (Table 2). However, a
dose response was observed as more animals in the high dose group were
positive. By 92 days post-injection no animals with positive testes were
observed in the two lower dose cohorts, indicating clearance of the
vector over time. Relative to the target tissue, liver, gonadal tissue
had 1000 fold lower levels of AAV vector sequences.

A less comprehensive non-GLP study in dogs evaluated biodistribution of
the AAV-Null vector in three tissues (liver, spleen, testes) and semen.
In this study, the vector was administered to the hepatic artery using a
catheter under fluorographic guidance, similar to the procedure that is
being used in subjects. Southern blot analyses detected vector sequences
in all four lobes of the liver (0.05-0.27 copies/diploid genome), but
not in spleen. Using a PCR assay with a sensitivity of 100 copies per
microgram of genomic DNA, vector sequences were not detected in testes
90 days post-injection (Table 2) nor in semen samples collected 0, 7,
30, 60, and 90 days post vector infusion (38). These findings were used
to support delivery of AAV to the hepatic artery using the
interventional radiographic procedure, while minimizing the probability
of vector dissemination to non-target tissues, such as spleen and
gonads.

The GLP non-human primate toxicology study included a non-GLP component
to assess vector dissemination to liver and gonadal tissue, however
these studies are not yet completed. Following initiation of the study
we attempted to collect semen from the animals. After several failed
attempts, samples were collected on days 82-89. PCR analysis of DNA
extracted from both excipient treated and AAV injected animals was
positive. The source of the contamination could not be determined;
therefore, we made a decision to sacrifice the animals to collect the
histopathology data, which was the primary goal of the study. We
subsequently evaluated the testicular tissue using immunofluorescence
for the presence of heparin sulphate proteoglycan (HSPG), which has been
reported to be the primary receptor for AAV (39). This analysis clearly
demonstrates the presence of HSPG in the interstitial space surrounding
the seminiferous tubules, but is completely absent from spermatogenic
cells within the tubules, suggesting that AAV is unable to infect
spermatogenic cells (Figure 1).

**Figure 1**

![](media/image13.png){width="5.25in" height="3.375in"}

Following the discovery of vector sequences in the semen of the initial
subjects enrolled in the hepatic artery trial, we evaluated rabbits as a
model to study shedding of vector into semen. Rabbits were injected
intravenously with AAV-FIX at doses ranging from 1 x 10^11^ to 1 x
10^13^ vg/kg. Semen was collected at time points ranging from 7 to 87
days following injection. DNA was extracted and analyzed by a sensitive
PCR assay that can detect as few as 10 copies of vector DNA in 1 g DNA.
For some experiments, semen was fractionated by density centrifugation
and DNA was extracted from the motile sperm fraction and the non-motile
sperm fraction. Initial efforts to fractionate rabbit semen using
protocols developed for fractionation of human semen showed heavy
contamination of the motile sperm fraction with other cells and with
non-motile sperm. Revision of the protocol to include centrifugation at
higher speeds resulted in a pure motile sperm fraction.

Results show that in rabbits injected at a dose of 1 x 10^11^ vg/kg, DNA
extracted from total semen was consistently negative for vector
sequences (Table 3). At a 10-fold higher dose, total semen was positive
for vector sequences through day 22 and negative thereafter. At 1 x
10^13^ vg/kg, semen DNA was positive for 44 days and thereafter
negative. For the analysis of motile sperm, the initial time point in
the mid dose group was positive, with all samples negative thereafter.
This likely reflects an artifact, since the samples at this time point
were fractionated using the protocol developed for human semen.

> **Table 3**

![](media/image14.png)

In earlier work, we have shown that AAV vector injected either
intramuscularly or into the intravascular space spreads by hematogenous
dissemination to the gonads (38, see attached paper). FISH analysis of
rabbit testis in these previous studies suggested that vector tends to
adhere to the blood vessel walls and to testicular basement membrane,
structures that are rich in heparin sulfate proteoglycan, which binds
AAV-2 tightly (39). However, FISH analysis was positive only at 7 days,
never at later time points. In addition, attempts to transduce murine
spermatogonia directly with AAV-2 in culture were negative (38). The
results reported here are consistent with these earlier findings, and
suggest that vector that spreads to the gonads by hematogenous
dissemination is lost over time, probably by either degradation or by
washout.

A major safety issue to be addressed is whether harm may arise from
PCR-detectable vector sequences in the semen. The major risks are
presumably the risk of horizontal transmission, and the risk of vertical
transmission. Studies by others indicate that the risk of horizontal
transmission is low. Favre et al. (40) showed using an infectious center
assay in non-human primates that had been injected IM with rAAV, that
body fluids collected from the animals never contained infectious
virions at time points beyond 72 hrs after injection. The risk of
vertical transmission will depend on whether vector integrates in motile
sperm. From the available data, this would appear to be unlikely since
the motile sperm fraction has been negative for vector sequences.

The cumulative biodistribution data indicate that AAV vector
disseminates to multiple tissues following hepatic artery
administration, but is predominantly found in the target tissue, liver,
at a level of 0.6 copies per diploid genome following administration of
1 x 10^13^ vg/kg. Vector DNA is at least 1000 times less abundant in
gonadal tissue. The level of vector sequences in tissue is dose
dependent and clears with time. Rabbits have thus far proven the most
useful species in studying the significance of vector in semen. Low but
detectable levels of vector have been detected in rabbit total semen at
early time points following infusion of high doses of vector, but has
not been reproducibly observed in motile sperm. Immunofluorescence
studies of murine and non-human primate testicular tissue indicates that
spermatogenic cells do not express HSPG, the receptor for AAV, and FISH
analysis indicates that while AAV sequences are associated with gonadal
tissue, they remain bound to the testicular basement membrane.
Furthermore, direct exposure of mouse sperm cells to high concentrations
of AAV failed to result in transduction. These data suggest that
germline transmission of AAV is unlikely, but additional experiments are
underway to assess the kinetics of vector shedding in semen and to
determine the cellular distribution of AAV in semen.

## Immune Responses Against the Expressed Coagulation Factor

The risk of inhibitor formation from protein- based therapies of the
hemophilias is lower with hemophilia B than with hemophilia A. High and
colleagues (The Children's Hospital of Philadelphia) have carried out
detailed studies of the immune response against Factor IX following gene
and protein-based treatment in both hemophilic and normal mice (41,42).
Intramuscular administration of AAV-hFIX vector in hemophilia B mice on
a C57Bl/6 background shows that human FIX elicits a T cell response in
these mice, with neutralizing antibody formation by day 14, while murine
FIX results in a less marked T cell response with reduced titers of
neutralizing antibody that are not detectable until 4 weeks after
injection. Anti-murine FIX titers are much higher in hemophilia B mice
on a CD-1 background, indicating that strain variations influence immune
response. Since all these animals have a complete null mutation, data
obtained with a human FIX transgene are likely just as valid as data
obtained with murine FIX.

Intramuscular administration of AAV-human FIX into hemophilia C57Bl/6
mice results in a neutralizing inhibitory antibody response, primarily
of IgG~1~ subclass, as expected for a secreted protein. Intravenous
infusion of recombinant human FIX (two infusions, two weeks apart)
causes a similar IgG~1~ response. Therefore, IM injection of vector and
IV infusion of protein give similar results in these animals.

Portal vein infusion of AAV-human FIX vector in these mice gives a weak,
non-neutralizing antibody response (IgG~2b~) that can be detected by
Western blot or ELISA but not by Bethesda assay. Thus, liver-directed
gene therapy may be [less]{.underline} immunogenic in C57Bl/6 mice than
is protein infusion. While these results are certainly influenced by
choice of mouse strain, and cannot be extrapolated to humans directly,
they nonetheless suggest that liver-directed gene therapy is not
[more]{.underline} immunogenic than protein infusion. It is worthwhile
to note that hemophilia B dogs with a CRM-negative missense mutation
injected with up to 4.6 x 10^12^ AAV particles did not develop
inhibitors against the canine transgene product (31,32)**.**

Additional studies were done in a colony of hemophilia B dogs maintained
at Auburn University. These animals have a 5bp deletion followed by a
single base change that results in a frame shift and early stop codon
leading to an unstable transcript (43). These animals have been found to
be more prone to inhibitor formation. Some of these animals have an
additional genetic defect related to their gene for red cell pyruvate
kinase (PK) leading to a hemolytic anemia, and extensive iron deposition
in the liver. Dogs were infused with vector via the mesenteric vein with
doses of AAV-canine F.IX ranging from 1.2-3.4 x 10^12^ vg/kg. A single
dog with a combined PK/Hemophilia B lesion developed an inhibitor
following an infusion of 3.4 x 10^12 ^vg/kg into the portal vein. Two
additional dogs without the PK mutation, and one Hemophilia dog with a
missense mutation (Chapel Hill colony) did not develop inhibitors
following vector delivery at doses up to 1.2 x 10^12^ vg/kg via the
portal vein, although high levels of factor IX expression (up to 700
ng/ml) were observed (30). Whether the sole case of inhibitor
development following portal vein infusion was a function of dose of
vector or underlying PK deficiency (and resulting hepatic iron overload)
is not clear at this time.

In the hemophilia B mice, neither portal vein nor IM administration of
the AAV vector causes inflammation or activation of cytotoxic T
lymphocytes.

## Summary of Planned Studies

The studies described below have been initiated to evaluate the
potential for both horizontal and vertical transmission of AAV vectors.

Development of an Infectious AAV Assay

Since detecting AAV sequences in the semen of the first two subjects
enrolled in the hepatic artery trial, an unresolved question that
remains is whether these sequences represent infectious AAV. To address
this we have developed a cell based assay to detect infectious AAV in
semen samples. The assay requires 200 l of semen and has a sensitivity
of approximately 100-1000 units. Once optimized, the assay will be used
in the experiment using rabbits (described below) and in human semen
samples.

Rabbit Study

Studies described above have demonstrated that rabbits are a good model
to study gonadal distribution of AAV following intravenous delivery.
Because the initial rabbit studies did not answer all the relevant
questions regarding gonadal distribution, another study has been
initiated. This study is designed to determine a) the kinetics of vector
shedding in semen, and the persistence of vector DNA in serum, and white
blood cells, b) which fraction of the semen contains vector sequences,
and c) whether the vector found in the semen is infectious. Twelve
rabbits have been injected with 1x10^11^, 1x10^12^, or 1x10^13^ vg/kg (4
animals per group). Semen will be collected at weekly time points, and
DNA will be extracted from a portion of the total semen for PCR
analysis. A portion will be fractionated into motile sperm, seminal
fluid and other cellular components, and fractions will be analyzed by
PCR. A third portion will be analyzed for infectious vector using the
assay described above. In addition, both serum and WBC will be analyzed
for vector sequences.

Direct exposure of mature sperm to AAV

In collaboration with Dr. Jon Gordon, Mt Sinai School of Medicine, we
have initiated a study designed to evaluate the potential for vertical
transmission of AAV. In this experiment, mouse sperm will be exposed to
high doses of AAV-hFIX and then used for in vitro fertilization. The two
stage embryos will be implanted into pseudopregnant females and fetuses
will be harvested shortly before birth. DNA will be extracted from the
fetuses and analyzed for vector DNA by Southern blot analysis. Detection
of single copy AAV in the mouse genomic DNA will be used as evidence of
germ line transmission.

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