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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 3968-3975
Adenovirus-Mediated Expression of Human Coagulation Factor IX in
the Rhesus Macaque Is Associated With Dose-Limiting Toxicity
By
Jay N. Lozier,
Mark E. Metzger,
Robert E. Donahue, and
Richard A. Morgan
From the Clinical Gene Therapy Branch, National Human Genome Research
Institute, Bethesda, MD; and the Hematology Branch, National Heart,
Lung, and Blood Institute, 5 Research Court, Rockville, MD.
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ABSTRACT |
We used a first-generation adenovirus vector (AVC3FIX5) to assess
whether human factor IX could be expressed and detected in the rhesus
macaque, which we have shown does not make high-titer antibodies to
human factor IX protein. Three animals received 1 × 1010
to 1 × 1011 plaque-forming units per kilogram by
intravenous injection. Human factor IX was present within 24 hours of
vector administration and peaked 4 days later at 4,000 ng/mL in the
high-dose recipient, and lower levels were seen in the
intermediate-dose recipient. No human factor IX was detected in the
low-dose recipient's plasma. Serum cytokine analysis and early
hypoferremia suggested a dose-dependent acute-phase response to the
vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared
concomitant with high-titer antihuman factor IX antibody development.
There was substantial, dose-dependent, dose-limiting liver toxicity
that was manifest as elevated serum transaminase levels,
hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting
times. Of particular interest was prolongation of the thrombin clotting
time, an indicator of decreased fibrinogen or fibrinogen dysfunction.
All evidence of liver toxicity resolved except for persistent
hypofibrinogenemia in the high-dose recipient, indicating possible
permanent liver damage. Our data suggest a narrow therapeutic window
for first-generation adenovirus-mediated gene transfer. The development
of antihuman factor IX antibodies and abnormalities of fibrinogen in
the rhesus macaque is of concern for application of adenovirus (or
other viral) vectors to hemophilia gene therapy.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
WE HAVE PREVIOUSLY shown that the rhesus
macaque factor IX cDNA is highly conserved compared with the human
factor IX cDNA and the protein is greater than 97% identical to human factor IX, the highest known homology between human and nonhuman factor
IX proteins.1 Repeated intravenous injections of human factor IX protein in rhesus macaques resulted either in no antibody formation or transient, low-titer, nonneutralizing antibodies that
disappeared despite further administration of protein. These findings
suggested that the rhesus macaque might be a suitable nonhuman primate
animal model for preclinical testing of human factor IX gene transfer.
Hemophilia, especially hemophilia B (factor IX deficiency), has been
considered an ideal candidate for gene therapy, because relatively low
amounts of protein expression would be sufficient for amelioration of
severe disease.2,3 Adenovirus vectors have been studied as
potential vectors for hemophilia A and B gene therapy.4-13
Obvious advantages of adenoviral vectors are efficient in vivo gene
transfer (particularly to hepatocytes) by simple intravenous injection
and the ability to produce high-titer vector stocks for animal studies.
Potential disadvantages of adenovirus vectors include immunogenicity of
the virus, which may cause T-cell-mediated elimination of transduced
cells, thereby limiting transgene expression,14-17 and
concomitant humoral immunity against the vector, which may preclude
repeated administration.9,10,13,17 A related problem is the
potential immune adjuvant effect of the adenovirus, which may serve to
induce antibodies against the transgene of interest (eg, factor IX).
This is especially important, because antibodies against factor IX, for
instance, would preclude its use in hemophilia B and would subject the
patient to the risk of anaphylaxis and nephrotic/nephritic syndromes
with further exposure to factor IX protein
concentrates.18,19 This would be particularly problematic, because alternatives to factor IX therapy (recombinant human factor VIIa, prothrombin complex concentrates, etc) are less predictable in
efficacy and, in the case of recombinant factor VIIa, available (until
recently) in the United States only as an investigational drug.20
We asked whether human factor IX could be expressed in rhesus macaques
using a first generation E1, E3-deleted adenovirus vector to mediate in
vivo gene transfer of the human factor IX gene. Our studies focused on
factor IX expression, the immune response to human factor IX, and
toxicity of the vector in vivo. Dose-dependent expression of human
factor IX was observed in the 3 animals studied, but significant
coagulopathy and liver toxicity was associated with the administration
of the vectors. These findings are especially important for the
evaluation of adenovirus vectors for hemophilia gene therapy.
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MATERIALS AND METHODS |
Animals.
Rhesus macaques weighing 7 to 10 kg (age range, 6 years, 2 months to 7 years, 3 months) were maintained at a primate research facility at the
National Institutes of Health (Rockville, MD). Animal protocols were
reviewed and approved by the Institutional Animal Care and Use
Committee of the Heart, Lung, and Blood Institute. The animals used for
these experiments were the subjects of our previous report on
intravenous injection of human factor IX protein in rhesus
macaques.1
Adenovirus vector.
Adenovirus vector AVC3FIX5 was constructed as described
previously,21 and high-titer preparations were prepared by
infection of HEK293 cells (ATCC, Manassas, VA) at high multiplicity of
infection (MOI) and freeze-thaw of cells with cytopathic
effect 40 hours after infection, followed by ultracentrifugation of
crude viral lysate on cesium chloride. Cesium-chloride banded virus was
dialyzed extensively against 10 mmol/L Tris (pH 7.5), 2 mmol/L
MgCl2, and 10% glycerol before freezing at
 80°C. Titers of 4 × 1011 plaque-forming units (pfu)/mL were documented on HEK293 cells by plaque
titering under soft-agar overlays. Limiting dilution of adenovirus
vector on HEK293 cells was performed and TCID50
(tissue-culture infectious dose, 50%) titers of 2 × 1012 TCID50/mL were demonstrated using Reed-Munch proportional distance factors. The vector preparation was
tested for replication competent adenovirus (RCA) by MA Bioservices (Rockville, MD) and found to be negative at less than 1 RCA per 107 vector TCID50 by testing for cytopathic
effect on human A549 cells at limiting dilutions.
Enzyme-linked immunosorbent assay (ELISA) for human
factor IX antigen.
An ELISA for human factor IX, described as reported
previously,1 was used to measure human factor IX in rhesus
macaques after injections of the AVC3FIX5 vector or human factor IX
protein (Mononine; Centeon, Kankakee, IL).
Anti-AVC3FIX5 antibody titration.
Ninety-six-well polystyrene plates were coated with 3 × 107 pfu of AVC3FIX5 in 100 µL 0.1 mol/L sodium carbonate
buffer (pH 8.8) per well overnight at 4°C and then blocked with 6%
bovine serum albumin (BSA)/phosphate-buffered saline
(PBS)/0.05% Tween 20 for 1 hour at 37°C. Dilutions of
rhesus serum were incubated at 37°C for 1 hour and washed, and
rhesus antibodies were detected by incubating a 1:10,000 dilution of
rabbit antirhesus IgG conjugated to horseradish peroxidase (Sigma, St
Louis, MO), washing with PBS/0.05% Tween 20, and color developed with
1 mg/mL o-phenylenediamine in 0.1 mol/L sodium
citrate (pH 4.5) with 2 µL H2O2 per
10 mL. Color development was stopped with 1 mol/L HCl and then measured with a Thermomax plate reader (Molecular Devices Corp,
Sunnyvale, CA) at 492 nm, and the optical density was compared with
that of equal dilutions of preadenovirus serum.
Antiadenovirus titers were defined as the greatest dilution at which
the absorbance at 492 nm exceeded the identical dilution of baseline
serum by greater than 0.05 absorbance units.
Antihuman factor IX antibody titration.
Ninety-six-well polystyrene plates were coated with 0.1 µg human
factor IX (Mononine) in 100 µL 0.1 mol/L sodium
carbonate buffer (pH 8.8) per well overnight at 4°C and then
blocked with 6% BSA/PBS/0.05% Tween 20 for 1 hour at 37°C.
Various dilutions of rhesus serum were incubated at 37°C for 1 hour
and washed, and antibodies were detected and quantified as described
above for antiadenovirus antibodies.
Coagulation assays.
Prothrombin times were performed with a fibrometer by mixing 0.1 mL
citrated plasma with 0.2 mL thromboplastin reagent (Organon Technica,
Durham, NC) at 37°C. Activated partial thromboplastin times (aPTT)
were performed with 0.1 mL of citrated plasma mixed with 0.1 mL of
factor-sensitive lipid (FSL) partial thromboplastin reagent (Sigma) and
mixed with 0.1 mL of 20 mmol/L CaCl2 to start the
coagulation process. Thrombin clotting time (TCT) assays were performed
by mixing 150 µL of 0.4 U/mL thrombin (Boehringer Mannheim, Indianapolis, IN) diluted in Owren's buffer (Sigma) with an equal volume of citrated plasma. Baseline TCT on normal pooled human or
rhesus plasma were approximately 15 seconds in this assay.
Fibrinogen ELISA.
Ninety-six-well ELISA plates were coated with 100 µL/well of 1:1,000
dilution of goat antihuman fibrinogen (Sigma) in 0.1 mol/L sodium
carbonate buffer (pH 8.8) overnight at 4°C and then blocked with
6% BSA/PBS/0.05% Tween 20 for 1 hour at 37°C. Rhesus plasma (50 µL/well) was then incubated for 1 hour at 37°C and then washed
with PBS/0.05% Tween 20. Fibrinogen was detected by subsequent
incubation with mouse monoclonal antihuman fibrinogen antibodies FG-21
and 85D4 (Sigma) diluted 1:10,000 in 6% BSA/PBS/Tween 20 and then with
goat antimouse IgG conjugated to horseradish peroxidase (Sigma) diluted
1:1,000 in 6% BSA/PBS/0.05% Tween 20. Color was developed with
o-phenylenediamine/hydrogen peroxide and stopped with 1 mol/L
hydrochloric acid, and fibrinogen levels were quantified by comparison
with dilution of baseline citrated plasma from the same animal. Under
these conditions, the ELISA for fibrinogen gave approximately 30%
cross-reactivity between rhesus and human fibrinogen in citrated plasma.
Fibrinogen immunoelectrophoresis.
Immunoelectorphoresis was performed on 4 µL plasma samples using
precast sodium barbital agarose gels (Kallestadt, Chaska, MN) at 200 V
for 45 minutes. After electrophoresis, goat antihuman fibrinogen
antibody (Sigma) diluted 1:1,000 was placed in slots parallel to the
direction of electorphoresis and precipitation was permitted to take
place overnight at 4°C. After 3 days of washing with normal saline,
precipitin arcs were stained with naphthol blue-black dye (Sigma) and
then destained with 5% acetic acid/0.5% glycerol.
D-dimer assay.
Semiquantitative D-dimer latex agglutination testing was performed on
citrated plasma as per the manufacturer's directions (Sigma).
Complete blood counts (CBC) and serum chemistry
determination.
CBC were performed on EDTA-anticoagulated blood on a Cell Dyne 3500 analyzer (Abbott Laboratories, Abbott Park, IL), and
serum chemistry evaluations were performed on serum samples using an Ektachem chemistry analyzer (Kodak, Rochester, NY).
Adenovirus cultures.
Heparinized blood and body fluids were collected at various time points
after adenovirus vector administration, and adenovirus vector was
cultured by plating various dilutions of material on HEK293 cells to
determine TCID50 levels of the vector in the body fluid.
Blood samples were diluted 1:100 in Dulbecco's modified Eagle's
medium (DMEM)-H/10% fetal bovine serum supplemented with 2 mmol/L glutamine and antibiotics and plated directly. Urine and fecal
samples were diluted 1:100 (wt:vol) in PBS and filtered through
0.2-µm filters before plating on HEK293 cells to determine the
TCID50 levels. Sterile cotton swabs passed through the
oropharynx of each animal were then placed in 10 mL PBS and the PBS was
filtered before plating on HEK293 cells.
Human factor IX falloff assays.
Human factor IX (Mononine; Centeon) was reconstituted at 100 U/mL, and
25 U/kg body weight was injected intravenously and citrated plasma was
collected at T = 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours,
12 hours, 24 hours, and 2 weeks after injection for analysis of human
factor IX antigen and/or antihuman factor IX antibodies. Human factor
IX antigen levels were compared with previous falloff studies in the
same animals published previously.1
Bethesda inhibitor assay.
Bethesda inhibitor assays were performed as per the method of Kasper et
al.22 Samples of citrated plasma diluted in Owren's buffer
(Sigma) were incubated with either normal human plasma or normal rhesus
plasma for 2 hours at 37°C, and then residual factor IX activity
was determined with a fibrometer using human factor IX-deficient human
plasma (Sigma). One Bethesda unit was defined as the reciprocal of the
dilution of test plasma at which 50% of factor IX activity is
inhibited. The sensitivity of the assay was 1 Bethesda inhibitor assay
unit (BIAU) per milliliter.
Interleukin-6 (IL-6) cytokine assay.
Serum IL-6 was assayed with a human IL-6 immunoassay (Biosource,
Camarillo, CA) as per the manufacturer's instructions.
Cross-reactivity with rhesus IL-6 derives from its 98% homology to
human IL-6 protein.23
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RESULTS |
Dose-dependent expression of human factor IX after injection of
AVC3FIX5 vector.
Adenovirus vector AVC3FIX5 is a first generation design vector that
uses the cytomegalovirus (CMV) promoter to drive the
expression of a human factor IX cDNA.21 Three rhesus
macaques were injected with 8 × 1010, 4 × 1011, or 8 × 1011 pfu of
AVC3FIX5 in 10 mL of PBS by intravenous injection under sedation. All 3 animals displayed no distress other than mild tachypnea shortly after the injection in the high- and low-dose recipients.
Figure 1 shows human factor IX antigen
levels in 3 rhesus macaques after the administration of different doses
of AVC3FIX5. The recipient of the highest dose (RQ1305) developed peak
levels of factor IX that were 80% of normal human levels (~4,000
ng/mL) at day 4, followed by a gradual decrease to undetectable levels
by 3 weeks after vector administration. The intermediate-dose recipient
(84269) demonstrated peak human factor IX levels of 18% of normal
(~900 ng/mL), which remained approximately at this level for 2 weeks before becoming undetectable at 3 weeks. The low-dose recipient (RQ1234) failed to demonstrate detectable (>30 ng/mL) human factor IX
at any time point after vector administration. Thus, adenovirus vector
administration resulted in dose-dependent expression of human factor IX
in the rhesus macaque, with a threshold of greater than 8 × 1010 pfu (1 × 1010 pfu/kg) for detectable
factor IX expression.
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| Fig 1.
Human factor IX in rhesus plasma after administration of
AVC3FIX5 vector (normal human factor IX plasma level, 5,000 ng/mL).
Each point is the average of ELISA results performed in duplicate.
( ) Low-dose; ( ) intermediate-dose; ( ) high-dose.
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Hepatotoxicity of adenovirus vector.
All 3 animals demonstrated hepatotoxicity that was dose-dependent.
Serum AST was elevated (Fig 2) compared
with baseline levels as soon as 6 hours after vector injection and
increased to peak levels 2 to 19 times baseline from 6 hours after
administration (low-dose and intermediate-dose recipients) to 7 days
after administration (high-dose recipient). The intermediate- and
high-dose recipients demonstrated elevation of serum alkaline
phosphatase of 3 to 8 times normal and elevation of bilirubin as high
as 15 times baseline at day 10 for the high-dose recipient after vector
administration (Fig 2). The high-dose recipient displayed lethargy and
poor appetite at the time of peak transaminasemia and required
intravenous and subcutaneous fluid support and transfusion with rhesus
donor plasma. Serum albumin levels decreased from a baseline of 3.6 g/dL to a nadir of 2.0 g/dL for the high-dose recipient and 2.6 g/dL
for the intermediate-dose recipient (Fig 2). The low-dose recipient did
not exhibit elevation of alkaline phosphatase levels or total bilirubin
or any significant perturbation of albumin levels.
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| Fig 2.
Liver function tests after administration of AVC3FIX5
vector. () Low-dose; () intermediate-dose; () high-dose.
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Blood urea nitrogen (BUN) and creatinine levels remained within normal
limits for all time points tested after vector administration (data not shown).
Coagulation abnormalities after AVC3FIX5 administration.
The high-dose recipient demonstrated a marked prolongation of the
prothrombin time that doubled from its baseline value of 12 seconds to
25 seconds within 48 hours of vector administration. This high level
decreased to a plateau of 15 to 17 seconds at day 10, but did not
normalize until day 31 (Fig 3). The
intermediate-dose recipient displayed a prompt increase in the
prothrombin time from a baseline of 12 seconds to 17 seconds within 48 hours of vector administration, which returned to baseline levels by 10 days after vector administration. The low-dose recipient displayed a
2-second increase in the prothrombin time from baseline and, like the
intermediate-dose recipient, returned to baseline by day 10. The
prolongations of the prothrombin times in the 3 monkeys were
concomitant with the elevated liver transaminases and total bilirubin,
suggesting liver toxicity.
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| Fig 3.
Coagulation studies of recipients of AVC3FIX5 vector.
(Top left panel) Prothrombin time (PT); (bottom left panel) aPTT; (top
right panel) TCT; (bottom right panel) plasma fibrinogen levels
normalized to the percentage of baseline value. () Low-dose; ()
intermediate-dose; () high-dose.
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The aPTT were also elevated in a dose-dependent fashion, as seen in Fig
3. Because this was seen in the low-dose recipient, which had no
demonstrable (<30 ng/mL) human factor IX antigen circulating in
plasma, the possibility of human factor IX interfering with the rhesus
coagulation cascade in vitro to produce a prolonged aPTT is unlikely.
We measured the aPTT of pooled normal rhesus plasma spiked with 50, 100, 500, or 5,000 ng/mL of purified human factor IX (Mononine) and saw
no change in the aPTT from baseline (data not shown). Thus,
interference with rhesus factor IX or the aPTT by human factor IX is
effectively ruled out as an explanation for the prolonged aPTT values.
A particularly striking finding was the biphasic prolongation of the
TCT seen in the intermediate- and high-dose animals after vector
administration, which was suggestive of hypofibrinogenemia and/or
dysfibrinogenemia (Fig 3). The high-dose recipient displayed peak TCT
values of greater than 90 seconds at the highest point (22 days after
vector administration) as compared with a baseline of 12 seconds. In
fact, the high-dose recipient demonstrated a partial recovery of the
TCT before a marked elevation to peak levels subsequently, suggesting a
second, delayed insult or injury resulting in disturbances of
fibrinogen. The first peak coincides with the markers of liver
toxicity; however, the second, more abnormal peak occurs after these
markers of liver injury have largely normalized. A similar, although
less pronounced biphasic pattern is also seen for the intermediate-dose
animal, whereas the low-dose recipient's TCT was only minimally
changed after vector administration. Fibrin D-dimer assays were
negative at days 1, 2, 3, 4, 10, and 14, except for the intermediate-
and high-dose animals, which were weakly positive with undiluted day-4 plasma (but negative at 1:2 or greater dilutions of day-4 plasma).
Effect of adenovirus vector on rhesus fibrinogen levels.
An ELISA for human fibrinogen with 30% cross-reactivity for rhesus
fibrinogen was developed and used to measure fibrinogen antigen levels
in the animals after adenovirus vector administration. Figure 3 shows
plasma fibrinogen antigen levels normalized to pretreatment baseline
levels in the 3 rhesus monkeys. Fibrinogen protein levels vary widely
and reach peak values 3 to 8 times baseline in the period immediately
after vector administration. The high-dose recipient eventually reaches
a steady-state fibrinogen level approximately 20% of normal,
consistent with the persistently elevated TCT shown in Fig 3, whereas
the low- and intermediate-dose recipients demonstrate increased
fibrinogen antigen levels well after normalization of their TCTs.
Antibodies to human factor IX.
We previously demonstrated that these animals produce transient,
low-titer, or no antibodies to intravenously administered human factor
IX protein. As can be seen in Fig 4,
high-titer (>1:10,000) IgG antibodies were produced in all 3 animals
within a few days of vector injection. The relatively rapid IgG
response suggests an anamnestic response to human factor IX to which
these animals had previously been exposed.1 Antihuman
factor IX Bethesda inhibitor assays were also performed to assess
antibodies that inhibit factor IX coagulant activity. As seen in Fig 4,
antibodies that inhibit human factor IX activity were demonstrated in
all 3 animals 8 weeks after vector administration. The Bethesda
inhibitor titers varied according to the dose of the vector
administered. Factor IX falloff studies were performed 80 days after
vector administration to assess the pharmacokinetics of human factor IX
protein in the presence of high-titer antibodies. As can be seen in
Fig 5, no significant difference in
recovery and half-life (8.4 hours) is seen for the low-dose recipient
compared with all previous falloff studies. In contrast, the
intermediate- and high-dose recipients demonstrate a blunted recovery
peak (~50% of expected) followed by a rapid falloff (1.3 and 1.8 hours, respectively) and a second, delayed peak 4 to 8 hours later,
suggesting a biphasic pattern of distribution/redistribution of human
factor IX protein.
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| Fig 4.
Antihuman factor IX antibodies after administration of
AVC3FIX5 vector. Solid symbols represent titer of rhesus antihuman
factor IX IgG determined by microtiter plate immunoassay (ELISA). Open
symbols represent antihuman factor IX neutralizing antibodies
determined by a Bethesda inhibitor assay. () Low-dose; ()
intermediate-dose; () high-dose.
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| Fig 5.
Human factor IX falloff studies, before and after
AVC3FIX5 administration. Twenty-five units per kilogram of Mononine
(Centeon) were injected intravenously and human factor IX plasma
antigen levels were determined by ELISA at the time points shown.
Preadenovirus falloff values are represented with solid symbols and are
the average of 4 pre-AVC3FIX falloff studies (±1 standard deviation)
as described previously by Lozier et al.1 Postadenovirus
falloff values are represented by open symbols and each value is the
average of ELISA results in duplicate for 1 falloff study at day 80. () Low-dose, pre-AVC3FIX5; () intermediate-dose, pre-AVC3FIX5;
() high-dose, pre-AVC3FIX5; ( ) low-dose, post-AVC3FIX5; ( )
intermediate-dose, post-AVC3FIX5; ( ) high-dose, post-AVC3FIX5.
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No evidence for rhesus antirhesus factor IX antibodies was seen in
Bethesda inhibitor assays (sensitive to ~1 BIAU) performed on
citrated plasma. Plasma was heat-inactivated at 56°C for 30 minutes
to destroy any residual rhesus factor IX that might mask antirhesus
factor IX antibodies; no inhibition of rhesus factor IX activity was
seen in heat-inactivated samples.
Indicators of acute-phase response.
Serum IL-6 and serum iron were measured in the first 24 hours after
administration of AVC3FIX5. As seen in Fig
6, IL-6 levels increased sharply in the intermediate- and high-dose
recipients, whereas little or no response was seen in the low-dose
recipient. Conversely, serum iron levels decreased in all 3 animals in
a dose-dependent manner (Fig 6).
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| Fig 6.
Early acute-phase indicators after administration of
AVC3FIX5. Upper panel depicts serum IL-6 cytokine levels. Bottom panel
depicts serum iron levels. () Low-dose; () intermediate-dose;
() high-dose.
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Vector distribution in bloodstream and secretions and antibody
production.
Adenovirus vector levels were assayed in the bloodstream of each animal
at various time points after injection, starting with a time point
immediately after the injection (T = 5 minutes). The vector could be
detected immediately after administration for all 3 animals and at 24 hours in the blood of the intermediate- and high-dose recipients but
not in the low-dose animal. No virus could be detected in any animals'
blood at 48 hours. These data indicate a volume of distribution ranging
from 10 to 14 L (Vd = AVC3FIX5 injected/AVC3FIX5 at T0),
indicating extremely rapid clearance of the vector from the
bloodstream, perhaps in the reticuloendothelial system of the liver,
spleen, and/or lungs. No virus was detected in urine, feces, or oral
swabs at any time point tested up to 2 weeks after injection. Vector
clearance was followed by vigorous antibody production.
Figure 7 shows high-titer
(>1:10,000) antiadenovirus IgG antibodies by 14 days that
persisted for as long as it was measured (>90 days).
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| Fig 7.
Rhesus serum anti-AVC3FIX5 IgG titers after AVC3FIX5
administration. () Low-dose; () intermediate-dose; ()
high-dose.
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DISCUSSION |
Administration of our first generation, E1-deleted,
E3-deleted, human factor IX adenovirus vector to rhesus macaques at
doses of 5 × 1010 pfu/kg to 1 × 1011 pfu/kg led to therapeutic levels of human factor IX
for up to 3 weeks, indicating the utility of the rhesus macaque as a
nonhuman primate animal model for testing safety and (short-term)
efficacy of human factor IX vectors. Expression was dose-dependent and
at the highest dose led to essentially curative (~80% of normal)
human factor IX plasma protein levels in the recipient. Although the
low-dose recipient had active AVC3FIX5 vector in the bloodstream
immediately after injection and would be expected to express
approximately 200 to 400 ng/mL of human factor IX in its plasma by
comparison with the intermediate- and high-dose animals, no plasma
human factor IX could be detected. This is consistent with a threshold
effect for adenovirus transduction, previously observed in hemophilia A
canines and mice subjected to adenovirus-factor VIII vector
transduction and in mice and primates receiving
adenovirus-erythropoietin vectors at similar doses.8,24 The
mechanism for this phenomenon is unclear. It is possible that the
acute-phase response may activate transcription factor NF B to
facilitate CMV promoter-driven expression in the intermediate- and
high-dose recipients, but not in the low-dose recipient.25
The rapid increase in IL-6 (the chief mediator of the early acute-phase
response)26-28 in the intermediate- and high-dose
recipients (but not seen in the low-dose recipient) and the
dose-dependent decrease in serum iron (an inverse indicator of the
acute-phase response)28,29 supports this hypothesis (Fig
6).
The apparent loss of factor IX expression in the intermediate-and
high-dose recipients may be explained largely on the basis of the
high-titer, antihuman factor IX antibodies that were evident as soon as
10 days after treatment, which has been seen in other animal species
after adenovirus-factor IX vector administration in
vivo.9,10,12,13 High-titer antibodies to human factor IX
were not necessarily expected, because these animals previously failed
to develop high-titer antibodies when injected repeatedly with human
factor IX protein.1 In addition to antihuman factor IX
antibodies, silencing of the CMV promoter in adenovirus vectors may be
considered as a reason for diminished transgene expression, especially
in the liver, as demonstrated previously.25,30 Furthermore, loss of nonintegrating, episomal adenovirus vector might be accelerated by hepatocyte turnover/regeneration after liver injury, particularly in
our high-dose recipient.
Administration of the adenovirus vector was associated with
substantial, dose-limiting liver pathology, which is likely due to
direct toxicity to hepatocytes.27 There is only a 10-fold difference in vector dose over which the response ranges from no
detectable human factor IX in plasma to curative levels of factor IX
associated with life-threatening liver toxicity. Such a narrow
therapeutic window may be considered problematic for the design of any
study of adenovirus vectors for use in humans with hemophilia or other
diseases in which liver expression of a particular transgene is
desired. The endosomolytic properties of external adenovirus fiber
proteins can be toxic to cells and might cause significant liver
toxicity in vivo, because approximately 80% of injected adenovirus
vector goes to the liver.5 Expression of adenovirus genes
(eg, E2 and E4) present in our vector also might have contributed to
the liver toxicity that we found in our animals. For example, it has
recently been demonstrated that adenovirus vectors containing the E4
region can induce cell cycle dysregulation, leading to growth arrest
and cell death.31 In this regard, results of gene transfer
in mice using a gutless adenovirus vector expressing an
 -1-antitrypsin gene suggest that liver toxicity can largely be
circumvented by elimination of all adenovirus genes in the
vector.32,33 The chief limitation to such vectors is the
difficulty in preparing large quantities of helper virus-free vector stocks.
It is possible that plasma factor IX levels in the low-dose recipient
were below the sensitivity of the ELISA, which in our hands is
approximately 30 ng/mL of human factor IX protein in a background of
rhesus plasma.1 Levels below this amount (<1% of normal
human levels) are not likely to be therapeutic for patients with
hemophilia B. Antibodies to human factor IX in the low-dose recipient
serve to indicate that there must have been at least some low-level
human factor IX expression (enough to immunize) despite the lack of
measurable protein in plasma.
It is interesting that antibodies to human factor IX are of similar
titer in all 3 animals by microtiter plate immunoassay (Fig 4) but have
different effects on factor IX pharmacokinetics and factor IX activity
assays. High- and intermediate-dose recipients' antibodies alter the
pharmacokinetics of infused human factor IX protein, but the low-dose
recipient's antibodies do not (Fig 5). This may be due to recognition
of different epitopes or perhaps differences in the antibody
subclass/isotype produced in the low-dose recipient. In the low- and
intermediate-dose animals, the aPTT normalized after resolution of
liver toxicity, despite the persistence of antihuman factor IX titers
of 1:10,000 or more (Figs 3 and 4). Although the high-dose animal's
aPTT remained slightly elevated (Fig 3), this is more likely to be due
to liver damage and/or fibrinogen abnormalities in light of the
abnormal PT, TCT, and fibrinogen studies (Fig 3).
Transient canine anticanine factor VIII antibodies have been seen in
hemophilia A canines receiving canine factor VIII adenovirus vectors
(Sheila Connelly, Genetics Therapy Inc [Gaithersburg, MD], personal communication, March 18, 1999) and
transient canine anticanine factor IX antibodies have been described in
hemophilia B canines receiving intramuscular AAV-canine factor IX
vectors,34 which suggests that some viruses may serve as
adjuvants for antibody formation and temporary loss of self-tolerance
when used for in vivo gene transfer. In our limited series, there was
no apparent loss of self-tolerance as reported in these studies or in
studies of erythropoietin gene transfer in mice.35 Our
model differs from other hemophilic animal models in that endogenous
rhesus factor IX is continuously present after vector administration. Endogenous rhesus factor IX might serve to prevent antirhesus factor IX
antibodies (by promoting immune tolerance) or might mask low-titer
antibodies. Antirhesus factor IX activity antibodies were not detected
in any of our animals by an aPTT-based Bethesda inhibitor assay
sensitive to approximately 1 BIAU/mL. It is quite possible that there
were rhesus antirhesus factor IX antibodies that we could not detect on
the basis of a low titer or inability to neutralize factor IX activity
in vitro. However, antibodies that do not neutralize factor IX activity
or significantly alter factor IX metabolism/clearance are probably of
little importance.
The induction of high-titer, antihuman factor IX antibodies after
vector administration suggests that adenovirus might serve as an immune
adjuvant, ie, a danger signal when presented simultaneously with a
potential immunogen.36 In this regard, it is noteworthy that adenovirus vectors expressing  -galactosidase can provoke cell-mediated immunity (against transduced muscle fibers), but adeno-associated viruses expressing -galactosidase do not; this correlates with the ability of adenovirus vectors to transduce antigen-presenting dendritic cells.37 Administration of our vector also caused antiadenovirus antibodies, as expected (Fig 7); such
antibodies are one of the chief limiting features of adenovirus vectors
and have prevented repeated administration of adenovirus vectors in
vivo.9,10,12,13
The coagulopathy that followed adenovirus administration was associated
with a dose-dependent prolongation of the prothrombin time, the aPTT,
and, most dramatically, the TCT. Because the TCT is a direct indicator
of the quantity and function of plasma fibrinogen, it is likely that
the coagulopathy seen after administration of the adenovirus vector is
a consequence of fibrinogen dysfunction and, in the case of the
high-dose recipient, hypofibrinogenemia as a consequence of severe
liver toxicity (Figs 2 and 3). The prolonged prothrombin time and aPTT
may be indirectly due to abnormal fibrin aggregation from an
abnormal/dysfunctional fibrinogen or low levels of fibrinogen, as noted
above. We investigated whether there was an abnormal fibrinogen arc on
immunoelectrophoresis that would indicate dysfibrinogenemia. None of
our subjects had abnormal fibrinogen mobility or additional arcs on
fibrinogen immunoelectrophoresis (data not shown); however, this is a
rather insensitive test due to the minimal electrophoretic mobility of fibrinogen.38 The trace levels of fibrin D-dimers after
vector administration (seen only at one time point) makes disseminated intravascular coagulation an unlikely explanation of the persistently abnormal TCT.
Liver toxicity is not an unexpected finding after gene transfer with
high doses of adenoviruses; however, the coagulopathy seen after
adenovirus-mediated gene transfer has not been previously studied in
detail, although possible hypofibrinogenemia (based on prolongation of
the TCT) was alluded to in an early report of adenovirus-mediated gene
transfer in hemophilia B canines.10 The latter phenomenon
should be carefully evaluated during future development of gene
transfer vectors to be used in patients with hemophilia A or B.
| |
ACKNOWLEDGMENT |
The authors thank James Higginbotham for plaque-purifying the AVC3FIX5
vector before injection into the rhesus macaques and Earl West for
analysis of serum chemistry and CBC on these animals. In addition, we
thank Jim Meade for helpful discussions on factor IX Bethesda inhibitor assays.
| |
FOOTNOTES |
Submitted March 25, 1999; accepted August 30, 1999.
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.
Presented in part at the 40th Annual Meeting of The American Society of
Hematology, Miami Beach, FL.
Address reprint requests to Richard A. Morgan, PhD, Bldg 10, Room
10C103, 10 Center Dr, Bethesda, MD 20892-1851; e-mail:
rmorgan{at}nhgri.nih.gov.
| |
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ABSTRACT
INTRODUCTION