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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3273-3281
Sustained Phenotypic Correction of Murine Hemophilia A by In Vivo
Gene Therapy
By
Sheila Connelly,
Julie L. Andrews,
Angela M. Gallo,
Dawn B. Kayda,
Jiahua Qian,
Leon Hoyer,
Michael J. Kadan,
Mario I. Gorziglia,
Bruce C. Trapnell,
Alan McClelland, and
Michael Kaleko
From the Holland Laboratory, American Red Cross, Rockville; and
Genetic Therapy, Inc, Gaithersburg, MD.
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ABSTRACT |
Hemophilia A is caused by a deficiency of blood coagulation factor
VIII (FVIII) and has been widely discussed as a candidate for gene
therapy. While the natural canine model of hemophilia A has been
valuable for the development of FVIII pharmaceutical products, the use
of hemophiliac dogs for gene therapy studies has several limitations
such as expense and the long canine generation time. The recent
creation of two strains of FVIII-deficient mice provides the first
small animal model of hemophilia A. Treatment of
hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human
FVIII at levels, which declined, but remained above the human
therapeutic range for over 9 months. The duration of expression and
FVIII plasma levels achieved were similar in both hemophiliac mouse
strains. Treated mice readily survived tail clipping with minimal blood
loss, thus showing phenotypic correction of murine hemophilia A by in
vivo gene therapy.
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INTRODUCTION |
HEMOPHILIA A, A COMMON bleeding disorder
affecting one in 5 to 10,000 males in all populations,1 is
caused by a deficiency of blood coagulation factor VIII (FVIII).
Hemophilia A is categorized into severe, moderate, or mild forms, with
over half of the patients manifesting the severe disease.1
Severe hemophiliacs, defined as having less than 1% of normal FVIII
levels, suffer from episodes of spontaneous and prolonged bleeding into
muscles, internal organs, and joints and frequently develop a disabling
arthropathy.1 Current treatment is directed toward
replacing the missing clotting factor in response to bleeding crises
with infusions of plasma-derived or recombinant FVIII.2
While prophylactic treatment of hemophilia A has been shown to reduce
the frequency and severity of bleeding, such therapy is limited by the
availability and high cost of purified FVIII, and the short half-life
of FVIII in vivo.2
Somatic cell gene therapy, which would provide constant blood levels of
FVIII, would be a significant treatment improvement.3 However, expression of FVIII is problematic. The accumulation of FVIII
mRNA is inhibited by sequences present in the coding region,4-8 and secretion of FVIII is
inefficient.9-11 In addition, the FVIII protein must be
secreted directly into the vasculature4,12,13 where the
protein is stabilized by formation of a complex with von Willebrand
factor (vWF).4
Previous studies in gene therapy for hemophilia A had used ex vivo
retroviral transduction approaches. In general, expression of FVIII
protein in genetically modified cells in vitro was
low5,8,14-17 and undetectable in vivo.15,16
Short-term expression of human FVIII in mice was achieved via ex vivo
gene transfer strategies,12,13 although the necessity for
ex vivo cell manipulation and reimplantation is a limitation to the
application of gene therapy in large animal models and humans.
Considerable progress has been made recently in the development of
adenoviral-mediated in vivo gene therapy of hemophilia A.18-21 Adenoviral vectors are an efficient system for in
vivo FVIII gene delivery because a peripheral vein injection in
mice18,22,23 and dogs21 results in efficient
transduction of hepatocytes, cells capable of secreting FVIII directly
into the blood.4 A vector encoding an albumin-promoted
FVIII cDNA resulted in high-level, liver-specific expression of human
FVIII in normal adult mice,19 which was sustained for over
5 months at levels fourfold above the human therapeutic
range.20 Furthermore, administration of a potent
FVIII-encoding adenoviral vector to FVIII-deficient hemophiliac dogs, a
well characterized, large hemophilia A animal model,24,25 resulted in high-level human FVIII expression and complete correction of the coagulation deficiency.21 However, phenotypic
correction in the treated dogs was transient, as the animals developed
a strong antibody response directed to the human protein.21
Therefore, the canine hemophilia A model is not amenable to long-term
expression of human FVIII.
The recent generation of FVIII-deficient mice, by gene disruption
techniques, provides the first small animal model of hemophilia A.26 Two distinct hemophiliac genotypes were developed by
insertion of a neomycin expression cassette into exon 16 or exon 17 of
the murine FVIII gene.26 Similar mutations in humans are
known to cause severe hemophilia A.27 Affected mice of both
genotypes have FVIII activity levels less than 1% of normal and
display lethal bleeding after trauma.26 Therefore, the
phenotype of these mice is similar to that of human
hemophiliacs.1 The murine hemophilia A model provides a
novel tool for the development of hemophilia gene therapy.
In this work, we evaluated adenoviral vectors for the treatment of
murine hemophilia A. Studies were conducted in both hemophiliac mouse
strains and used vectors prepared in two distinct adenoviral vector
backbones. The level and duration of FVIII expression, phenotypic
correction, and the antigenicity of human FVIII in the mouse model was
assessed. We showed expression of functional human FVIII with levels,
which declined, but remained above therapeutic for at least 9 months
and achieved complete phenotypic correction.
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MATERIALS AND METHODS |
Construction of recombinant adenoviruses.
The recombinant adenovirus encoding human FVIII, Av1H8101 (previously
named Av1ALAPH81)19 has been described.19 The
vector was checked for the presence of replication-competent adenovirus contamination by polymerase chain reaction (PCR) directed at E1a sequences,28 and all vector preparations contained less
than 10 plaque-forming units (pfu) of E1a-containing vector per
108 pfu. The  -galactosidase-encoding vector, Av3nBg, has
been described.29 Av3H8101 was generated by cotransfection
of the plasmid, pAvALAPH81,19 and viral DNA from the
vector, Av3nBg,29 into AE1-2a cells29 as
described.29 The Av1H8101 vector concentration was
determined by spectrophotometric analysis30 and by plaque
assay31 on 293 cells. The particle to pfu ratio was 80. Av3H8101 vector concentrations were determined by spectrophotometric
analysis.30 Titers are given as particles per milliliter.
Murine hemophilia A pathophysiology, animal breeding, genotyping,
and manipulations.
Breeding colonies of both genotypes of FVIII knockout mice have been
established.26 Initially, carrier females were bred with
normal C57BL/6 male mice. The genotypes of 4-week-old pups were
determined by PCR analysis of genomic DNA isolated from tail clips.
Briefly, mice were lightly anesthetized with isofluorane, and 1-cm
sections of the tail were collected and frozen on dry ice. The tails
were immediately cauterized to stop bleeding. Notably, approximately
30% of affected mice did not survive tail clipping with cautery. DNA
was isolated from the tail clips using the QIAmp Tissue Kit (Qiagen,
Chatsworth, CA) following the suggested protocol. PCR analysis was
performed as described26,32 using 400 ng of each genomic
DNA and primers specific for the inserted neo gene, exon 16 or
exon 17.32 Subsequently, hemizygous affected males and
homozygous affected females were mated.
The hemophiliac mice are occasionally anemic and bleed severely from
scratches and routine procedures such as ear tagging. Furthermore, the
knockout mice suffer from joint bleeds, subcutaneous bleeding, and
spontaneous death indicating a similarity to the pathophysiology of
human hemophilia A.1 Surprisingly, however, hemophiliac
females of both genotypes survive pregnancy, birth, and the nursing of
pups32 (S.C. unpublished). Breeding of affected males and
females has eliminated the need to genotype the pups by PCR analysis of
genomic DNA.26,32 All mice were confirmed FVIII-deficient
by analysis of plasma levels of functional FVIII using the Coatest
bioassay (see below).
Mice were ear tagged for identification and housed in cages of five to
six each. Occasionally, mice would bleed profusely from ear tagging,
scratches, and injuries incurred from fighting. Topical thrombin
(Thrombostat, Parke-Davis, Morris Plains, NJ) was applied to halt the
bleeding. Approximately 10% of the affected mice died spontaneously.
The tail clip survival study involved lightly anesthetizing the mice
and clipping 2-cm sections of the tail, without subsequent cauterization. After the procedure, mice were checked every 4 hours.
The surviving mice at 24 hours after the procedure were recorded.
Alternative assays to assess bleeding time and/or blood loss
over time were performed before the tail clip survival study. However,
such assays did not distinguish definitively between normal and
hemophiliac mice. Similarly, assessment of the activated partial
thromboplastin time (APTT) of mouse plasma samples did not distinguish
normal from hemophiliac mice (unpublished data). All experiments
involving mice adhered to protocols approved by the Institutional
Animal Care and Use Committee in accordance with the Animal Welfare
Act.
Adenoviral vector administration via tail vein injections and
retroobital phlebotomy were performed as described.18 Mice injected with Av1H8101 received a dose of 4 × 1010
particles per mouse, and mice injected with the Av3 vectors, Av3H8101
and Av3nBg received a dose of 6 × 1010 particles per
mouse. These vector doses yielded equal liver transduction as
determined by Southern analysis (data not shown). Topical thrombin (Thrombostat, Parke-Davis) was applied to the injection site to halt
the bleeding after vector administration. Notably, the hemophiliac mice
did not show any adverse effects from retroorbital bleeding.
FVIII assays, Southern blot, and RNAse protection analyses.
Biologically active human FVIII was measured using the Coatest
chromogenic bioassay (Chromogenix, Mölndal, Sweden) as directed. Coatest measures the FVIII-dependent generation of factor Xa from factor X, with one unit defined as the amount of FVIII activity in 1 mL
of pooled human plasma, 100 to 200 ng/mL.33 Pooled human plasma (George King Bio-Medical, Inc, Overland Park, KS) was used as
the FVIII activity standard to generate a standard curve. Pooled mouse
plasma collected from normal C57BL/6 mice served as the normal mouse
FVIII activity positive control. When compared with human plasma,
normal C57BL/6 plasma FVIII levels showed a median value of 2,500 mU/mL. Pooled plasma isolated from exon 17-disrupted affected mice
served as the negative control (<1% of normal mouse levels) in each
assay performed. FVIII activity values are reported as the mean value
and the standard error of the mean.
The enzyme-linked immunosorbent assay (ELISA) assay, designed to
measure human FVIII-specific antibodies, was performed as follows.
Recombinant human FVIII (Hyland Division of Baxter Healthcare, Glendale, CA), 100 µL/well at 3 U/mL, in 0.05 mol/L
carbonate-bicarbonate buffer pH 9.0, was incubated on 96-well Immunlon
1 pates (Dynatech, Chantilly, VA) at 4°C, overnight. The plate was
washed one time with TBS (20 mmol/L Tris-Cl in 0.9% saline) and 200 µL of blocking buffer (0.17 mol/L H3BO3, 0.12 mol/L NaCl, and 0.5% bovine serum albumin [BSA]) was added for 5 hours at room temperature (RT). Plates were washed three
times with TBS, and 100 µL mouse plasma samples, appropriately
diluted in TBS plus 0.5% BSA, were added to each well and incubated
overnight at 4°C. Plates were washed three times with TBS, and the
detection antibody (100 µL), alkaline-phosphatase conjugated goat
antimouse IgG (Southern Biotechnology Associates, Inc, Birmingham AL)
diluted 1:3,000 in TBS with 0.5% BSA, was added to each well and
incubated at RT for 2 hours. p-Nitrophenyl phosphate (P-NPP;
Calbiochem, La Jolla, CA) 2 mg/mL, in buffer containing 0.1 mol/L
glycine, 1 mmol/L MgCl2, 2 mmol/L ZnCl, pH 10.4, was added
to each well (100 µL) and the absorbance was read at 405 nm using the
Dynatech MR5000 (Dynatech) automated microplate ELISA reader. The
concentration of anti-FVIII antibody was calculated from a standard
curve using a monoclonal mouse antihuman FVIII antibody (MoAb)
413.34 The limit of sensitivity of the ELISA was 50 ng/mL
for mouse plasma samples diluted 1:10.
DNA was isolated from mouse livers using the QIAmp Tissue Kit (Qiagen).
A total of 10 µg of each DNA sample was digested with BamHI
and subjected to Southern analysis.18 The probe, prepared by random oligonucleotide priming, contained FVIII cDNA sequences from
+73 to +1,345.35,36 The copy number control standards were
prepared by adding 600 pg and 60 pg of viral DNA, equivalent to 10 and
1 vector copies per cell, respectively, to 10 µg of control mouse
liver genomic DNA and digesting with BamHI. No vector was
detected in uninjected control mouse liver DNA (data not shown). The
band intensities were quantitated with a Molecular Dynamics' PhosphorImager SF (Sunnydale, CA).
RNA was isolated from mouse livers using the RNAzole B (Tel-Test,
Friendswood, TX) extraction method. RNAse protection analyses were
performed using the RNAse Protection Kit II (Ambion, Austin, TX). For
each sample, 20 to 50 µg of total cellular RNA were hybridized with
an excess of a gel-purified RNA probe (see below), digested with the
RNAse A/T1 solution provided with the kit diluted 1:100, processed as
directed, and analyzed on an 8% polyacrylamide-8 mol/L urea gel
(SequaGel, National Diagnostics, Atlanta, GA). 32P-labeled
fragments from HpaII-digested pBR322 were used as the DNA size
markers. The FVIII probe template, pGemSRpr,19 contains FVIII coding region sequences from (+9) to (+214) and was linearized with HindIII. The mouse glyceraldehyde-3-phosphodehydrogenase (GAPDH)-specific probe template was generated from the
pTRI-GAPDH mouse plasmid (Ambion, Austin, TX) digested with Sty
I. All antisense RNA probes were synthesized with SP6 polymerase and
 -32P-CTP (3,000 Ci/mmole, Amersham,
Arlington Heights, IL).
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RESULTS |
Expression of functional human FVIII in hemophiliac mice.
The first generation, recombinant human FVIII adenoviral vector,
Av1H8101 (previously referred to as Av1ALAPH81)19 contains a mouse albumin promoter, an intron from the human apoliprotein A1
gene, and a human B-domain deleted (BDD) FVIII cDNA. Absence of the
B-domain has no effect on FVIII function, activity, or immunogenicity.11,37,38 The vector backbone was derived
from adenovirus serotype 5 (Ad5) and is devoid of the E1 and E3
regions. A second human FVIII-encoding adenoviral vector, Av3H8101, was generated with the identical FVIII expression cassette and an Ad5 viral
backbone with an additional deletion of the E2a region.29 Attenuation of E2a gene expression has been reported to prolong transgene expression in some model systems.39-41
A breeder colony of FVIII knockout mice has been established, and we
confirmed that the affected mice exhibit FVIII activity levels less
than 1% of normal and bleed acutely in response to trauma.26 To assess human FVIII expression in the
hemophiliac mice, Av1H8101 and Av3H8101 were administered via tail vein
injection to groups of five exon 17-disrupted mice (see Materials and
Methods). Plasma levels of biologically active human FVIII were
measured using the Coatest chromogenic bioassay. Before vector
treatment, all mice showed FVIII plasma levels at less than 1% the
amount detected in a normal, C57BL/6 mouse plasma sample, 2500 mU/mL. Two weeks after vector treatment, FVIII plasma levels were increased to
mean values of 960 ± 68 and 946 ± 170 mU/mL in the Av1 and Av3
vector-treated groups, respectively. Human physiologic levels of FVIII
are defined as 1,000 mU/mL, and therapeutic levels, the amount of FVIII
necessary to convert a severe hemophiliac to a mild or moderate
hemophiliac condition, are 50 mU/mL.33 Therefore, the mice
treated with either the Av1 and Av3 vectors expressed human physiologic
levels of biologically active FVIII, showing directly that adenoviral
vector-mediated expression of FVIII resulted in secretion of functional
FVIII protein. Furthermore, removal of the E2a region had no effect on
vector transduction efficiency or transgene transcriptional activity
(data not shown), indicating that the Av1 and Av3 vectors were equally
efficacious.
Time course of human FVIII expression in the hemophiliac mice.
To assess the time course of human FVIII expression with Av1 and Av3
vectors in exon 17-disrupted hemophiliac mice, four animals were
treated with Av1H8101 and eight were treated with Av3H8101. Plasma was
obtained at the indicated times and assayed for FVIII activity
(Fig 1A). The Av1 and Av3 vectors mediated
expression of supraphysiologic levels of FVIII with peaks of 2,100 and
2,550 mU/mL, respectively, at 1 to 2 weeks postinjection. FVIII
expression was maintained at levels above the human therapeutic range
for at least 40 weeks. With both vectors, the decline in FVIII
expression was greatest between 10 to 20 weeks with more stable
expression observed in some animals thereafter (Fig 1A).
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| Fig 1.
Time course of FVIII expression in hemophiliac mice. The
adenoviral vectors Av1H8101 (4 × 1010 particles/mouse) or
Av3H8101 (6 × 1010 particles/mouse) were administered via
tail vein injection to groups of 4 or 8 exon 17-disrupted hemophiliac
mice, respectively. These vector doses yielded equal liver transduction
as determined by Southern analysis (data not shown). At the indicated
time points, plasma samples were collected and FVIII biological
activity was quantitated. (A) Mean plasma levels of biologically active
FVIII. ( ) Mice that received Av1H8101. ( ) Mice that received
Av3H8101. Data are plotted as a mean value and the standard error of
the mean at each time point. The dotted line represents the human therapeutic level of FVIII, 50 mU/mL.33 (B) FVIII plasma
levels of individual Av3 vector-treated mice. One mouse () died
between 34 and 40 weeks.
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Examination of the time course data from individual mice showed an
initial decline between 10 to 20 weeks. Subsequently, FVIII levels
plateaued in some animals for at least 40 weeks. Interestingly, some
mice showed a sudden, sporadic decline in FVIII expression. To
illustrate this observation, the FVIII expression levels of the
individual mice that received the Av3 vector are plotted from weeks 12 to 40 (Fig 1B). One mouse of the four that received Av1 (data not
shown) and three of the eight that received Av3 expressed constant
levels of FVIII from weeks 12 to 40 (Fig 1B).
Comparison of FVIII expression in exon 16 and exon 17-disrupted
hemophiliac mice.
Two separate genotypes of the FVIII knockout mice were generated by
disruption of either exon 16 or exon 17.26,32 To show that
long-term expression of human FVIII in the mice was not dependent on a
specific murine FVIII mutation, groups of 12 exon 16- and 11 exon
17-disrupted mice were treated with Av3H8101. High level expression of
biologically active FVIII was detected in both mouse genotypes with
expression sustained for at least 24 weeks at levels well above the
human therapeutic range (Fig 2). There was
no difference in the level or duration of FVIII expression between the
two genotypes.
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| Fig 2.
Comparison of human FVIII expression in exon 16 and exon
17-disrupted hemophiliac mice. The adenoviral vector, Av3H8101
(6 × 1010 particles/mouse) was administered via
tail vein injection to groups of 12 exon 16- or 11 exon 17-disrupted
hemophiliac mice. At the indicated time points, plasma samples were
collected and FVIII biological activity was quantitated. Mice that were
expressing below less than 1% of normal FVIII levels (<25 mU/mL)
were killed at 24 weeks. The FVIII levels in the remaining mice were
assayed for an additional 16 weeks. (A) Mean plasma levels of
biologically active FVIII. () Exon 16-disrupted mice. ( ) Exon
17-disrupted mice. Data are plotted as a mean value and the standard
error of the mean at each time point. The dotted line represents the human therapeutic level of FVIII, 50 mU/mL.33
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Examination of the time courses of FVIII expression for the individual
mice showed the same pattern of stable expression previously shown in
Fig 1B. At 40 weeks, four of the exon 16 mice, and four of the exon 17 mice displayed stable, sustained FVIII expression (data not shown).
Notably, by 52 weeks, three exon 16 mice and one exon 17 mouse
continued to display constant, stable FVIII plasma levels (data not
shown).
Phenotypic correction of murine hemophilia A.
Mice expressing therapeutic levels of FVIII appeared phenotypically
normal, as they were no longer anemic, did not die spontaneously, and
did not bleed profusely from scratches or ear tagging. To show directly
that expression of biologically active human FVIII in the treated mice
resulted in correction of the bleeding defect, a tail clip survival
study was performed (Table 1). Groups of exon 16- and exon 17-disrupted hemophiliac mice were treated with Av3H8101, a -galactosidase-encoding adenoviral vector,
Av3nBg,29 or were untreated. All of the Av3H8101-treated
animals expressed therapeutic levels of FVIII 5 days after vector
administration. On day 6, tail clips were performed on the treated and
untreated hemophiliac mice, as well as normal, age-matched C57BL/6
mice. All of the normal C57BL/6 mice readily survived tail clipping with no evidence of distress. Only 5% to 30% of the untreated and
Av3nBg-treated hemophiliac mice survived tail clipping and the
survivors were moribund at 24 hours. In contrast, all mice that
received Av3H8101 readily survived tail clipping with no evidence of
distress. These data show phenotypic correction of murine hemophilia A.
Assessment of vector persistence and antihuman FVIII antibody
response.
To evaluate the mechanism by which FVIII expression was attenuated in
some mice (see Fig 1), Av1H8101-treated exon 17-disrupted mice were
killed 2 and 16 weeks after vector administration and liver DNA and RNA
were analyzed (Fig 3). Southern analysis
showed an average of six vector copies per cell at 2 weeks and 0.8 vector copies per cell at 16 weeks (Fig 3A). Despite the substantial drop in vector copy number, FVIII RNA levels decreased only twofold to
threefold (Fig 3B). Furthermore, FVIII RNA levels did not parallel or
predict FVIII protein expression in the plasma (Fig 3B). Therefore, loss of FVIII expression at 16 weeks in three of the four mice analyzed
was not due to a complete loss of vector DNA or to complete inactivation of the albumin promoter.
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| Fig 3.
Time course of vector persistence in exon 17-disrupted
hemophiliac mice. Av1H8101 (4 × 1010 particles/mouse) was
administered via tail vein injection to a group of 8 exon 17-disrupted
hemophiliac mice. FVIII biological activity was measured before and at
2 and 16 weeks after vector administration. Groups of four mice each
were killed at 2 or 16 weeks, and DNA and RNA were isolated from each
mouse liver. (A) Southern analysis. Each DNA sample (10 µg) was
digested with BamHI. The arrow designates a 3.4-kb fragment
containing the vector-derived FVIII sequence. The standards (lanes 1 and 2) were generated by digesting purified Av1H8101 viral DNA in
amounts equivalent to 10 and 1 vector copies per cell. Lanes 3 through
6 and 7 through 10 represent liver DNA from mice treated with Av1H8101
or Av3H8101, respectively. No vector was detected in uninjected control
mouse liver DNA (data not shown). (B) FVIII protein expression and
RNAse protection analysis. Plasma levels of biologically active FVIII protein measured in each mouse are displayed above the lanes. FVIII
levels at 2 weeks and 16 weeks are displayed above lanes 4 through 7, and 8 through 11, respectively. For the RNAse protection analysis, 50 µgs of total cellular RNA isolated from the mouse livers were used in
each reaction. The arrow labeled FVIII designates the 212-nt human
FVIII-specific protected probe fragment. Lane 2 contains undigested
full-length probe. Lane 3 contains liver RNA isolated from an
uninjected control exon 17-disrupted hemophiliac mouse. Lanes 4 through 7 and 8 through 11 represent RNA from mice at 2 or 16 weeks
after vector treatment, respectively. Lanes 1 and 12 contain
32P-labeled DNA molecular-weight markers. The lower panel
displays a separate RNAse protection assay using 20 µg of total
cellular mouse liver RNA and an antisense RNA probe encoding a portion of the mouse glyceraldehyde 3-phosphodehydrogenase (GAPDH) cDNA. The
arrow labeled GAPDH designates the 134-nt mouse GAPDH-specific protected probe fragment.
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A second hypothesis to explain the sudden, sporadic decline in FVIII
plasma levels observed in some mice (see Fig 1) was the development of
a humoral immune response directed against the human FVIII protein. An
ELISA designed to detect antihuman FVIII antibodies was used to assay
plasma from the exon 16 and exon 17 mice represented in Fig 2 before
vector administration and at various time points thereafter
(Table 2). In all cases, antibody levels
were either undetectable or low, ranging from below the limit of
sensitivity, 50 ng/mL, up to 97 ng/mL. There was no case in which the
drop in FVIII plasma levels could be attributed to the generation of a
strong antibody response. Interestingly, Qian et al42
showed that hemophiliac mice of both genotypes treated with a single
intravenous administration of purified full-length FVIII protein
rapidly developed a strong immune response (approximately 40,000 ng/mL)
to the human protein.
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DISCUSSION |
In this work, we showed long-term phenotypic correction of hemophilia A
by in vivo gene therapy in a clinically relevant small hemophilia A
animal model. Whereas sustained human FVIII expression was demonstrated
previously in normal mice20 and short-term phenotypic
correction was achieved in hemophiliac dogs,21 the FVIII-deficient hemophiliac mouse26 has several advantages
over these animal models for the study of hemophilia A gene therapy and
FVIII in vivo function. In contrast to normal mice, the biologic activity and function of the human FVIII protein expressed in hemophiliac mice can be measured directly, and phenotypic correction of
the bleeding defect assessed. In addition, both FVIII-deficient mouse
genotypes were developed as a mixture of two mouse strains, C57BL/6 and
129SV,26 providing a more diverse genetic background than
inbred strains for gene therapy studies. Compared with the canine
hemophilia A model, the knockout mice provide the only available small
animal model of hemophilia A. Small animal models facilitate the
performance of multiple studies with large cohorts to better
characterize vector function and FVIII protein expression.
An important attribute that distinguishes the hemophiliac mouse model
from that of normal mice is the ability to show phenotypic correction.
The murine hemophiliac phenotype is characterized by subcutaneous,
intraperitoneal, intrathoracic, and joint bleeds. The mice bleed
extensively after minor lacerations, are occasionally anemic, and
exhibit a high spontaneous mortality rate. We initially observed that,
after vector treatment, the mice appeared normal and the bleeding
phenotype resolved. To obtain an objective assessment of phenotypic
correction, a tail clip survival study, which clearly distinguished
hemophiliac from normal mice, was used. This assay showed that human
FVIII expressed by an intravenously administered adenoviral vector is
biologically active, functional in mice, and therapeutic.
The time course of FVIII expression in individual mice showed an
intriguing pattern. With both the Av1 and Av3 vectors, treated mice
displayed an initial drop in FVIII plasma levels, probably due to a low
level of vector-mediated toxicity as previously described with normal
mice.20 FVIII expression in several of the mice subsequently stabilized, showing that a single adenoviral vector administration has the potential to achieve long-term, constant-level expression. Furthermore, long-term FVIII expression was not dependent on a specific murine FVIII mutation, as a similar expression pattern was observed in both hemophiliac mouse genotypes. However, sustained expression was not uniformly achieved, as FVIII levels rapidly and
sporadically declined in some mice.
The mechanism by which FVIII expression levels declined was
investigated by DNA, RNA, and human FVIII-specific antibody analyses. Over time, vector DNA levels declined substantially, while FVIII RNA
levels showed only a minimal decrease. Furthermore, FVIII RNA levels
were similar in mice with widely divergent FVIII plasma levels. Thus,
the decline in plasma levels could not be completely attributed to
either vector loss or transcriptional inactivation. The development of
an immune response to human FVIII seemed a likely explanation for the
loss of FVIII expression. Little or no human FVIII-specific antibody
response was detected in either strain of hemophiliac mice before or
after attenuation of expression, suggesting that a humoral immune
response was not responsible for the loss of FVIII. However, we cannot
rule out the possibility that loss of expression resulted from a shift
in the subclass or quality of a low titer antibody. A cell-mediated
immune response directed toward hepatocytes expressing human FVIII
could also explain the decline in FVIII expression, but the elimination
of hepatocytes would have resulted in a parallel loss of FVIII RNA, which was not observed. Presently, the explanation for the rapid loss
of expression in some mice remains unclear. Further investigation of
this phenomenon may lead to a better understanding of vector and FVIII
physiology and to the development of vectors which more uniformly
remain efficacious.
A major issue broadly relevant to the field of gene therapy is the
potential immunogenicity of endogenously expressed foreign proteins.
Ten percent to 30% of human hemophiliacs treated with intermittent,
intravenous FVIII protein administration develop FVIII inhibitory
antibodies.1,43 Hemophiliac mice injected intravenously
with human full-length FVIII develop a potent anti-FVIII antibody
response,42 and a T-cell response directed to the
full-length protein.44 However, hemophiliac mice treated
with an adenoviral vector encoding human BDD FVIII generated little or
no antibody response to human BDD FVIII. The induction of a cytotoxic
T-lymphocyte (CTL) response was not investigated.
Furthermore, the low antibody levels detected in either strain of
hemophiliac mice indicate that the lack of FVIII immunogenicity was not
dependent on the specific FVIII mutation. These observations represent
preliminary evidence to suggest that constant level, endogenous
expression of human FVIII may be less immunogenic than intermittent,
intravenous protein administration. Verification of this hypothesis
will require a controlled study in which the immunogenicity of BDD
FVIII protein is compared directly with vector-mediated expression of
BDD FVIII. Notably, vector-mediated expression of BDD FVIII can result
in the generation of antibodies against the human protein, as was observed in vector-treated hemophiliac dogs.21 Because the
murine immune system is well studied and easily manipulated,
hemophiliac mice represent an excellent model for characterizing the
immune response to FVIII. Furthermore, this model can be used for
evaluating gene therapy strategies to treat FVIII inhibitory
antibodies, a significant clinical problem in hemophilia.
Two human BDD FVIII-encoding vectors were evaluated in this study. The
first generation Av1 vector is composed of an adenoviral backbone from
which the E1 and E3 regions were removed. The third generation Av3
vector contains, in addition, the deletion of the E2a region. Av3
vectors are completely replication defective in vitro and do not
express detectable levels of hexon capsid protein.29 In
some animal models, attenuation of E2a gene expression results in
prolonged transgene expression and a reduction in the host immune
response to the vector.39-41 However, in other mouse
strains and hemophilia B dogs, the inclusion of a temperature sensitive mutation or removal of the E2a gene had no effect on vector
persistence.45,46 In this work, treatment of hemophiliac
mice with either the Av1 or the Av3 FVIII vectors resulted in similar
liver transduction efficiency, transgene transcriptional activity, and
FVIII expression levels. Both Av1 and Av3 vectors achieved sustained
expression with attenuation in the first 20 weeks followed by stable
FVIII plasma levels in some animals for over 9 months. The finding that Av3 vectors did not provide for greater efficacy in the hemophiliac mice is consistent with the hypothesis that the immune system did not
play a major role in limiting the duration of expression in this model.
Furthermore, the attenuation of FVIII expression in the first 20 weeks
was probably due to direct vector-mediated liver
toxicity,20,46 which is similar with both Av1 and Av3 vectors (T.A.G. Smith, personal communication, August
1997) and may be related to the E4 gene,47-50
which remains in both backbones. The removal of additional backbone
genes may decrease toxicity, minimize the attenuation of expression in
the first 20 weeks, and enable more sustained expression.
The data presented in this work support the use of recombinant
adenoviruses for the treatment of hemophilia A. Prolonged curative therapy was achieved with a single vector administration in a hemophiliac animal model. However, clinical benefit in humans will
require therapy that extends for years, necessitating repeated treatments or more sustained expression. Current studies to allow vector readministration51-53 together with the development
of improved, more attenuated adenoviral vectors54-59 may
soon provide long-term correction of hemophilia A.
| |
FOOTNOTES |
Submitted August 14, 1997;
accepted December 10, 1997.
Address reprint requests to Michael Kaleko, MD, PhD,
Genetic Therapy, Inc, 938 Clopper Rd, Gaithersburg, MD 20878.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs Martin Woodle and Theodore A.G. Smith for critical review
of the manuscript, Dr Theodore A.G. Smith for communication of data
before publication, Dr Russette Lyons and Christoph Wey for assistance
with the animal procedures, and Adam Shoemaker for vector production
and quality control.
| |
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Abstract
Introduction
Abstract