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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1875-1881
The Rhesus Macaque as an Animal Model for Hemophilia B Gene Therapy
By
Jay N. Lozier,
Mark E. Metzger,
Robert E. Donahue, and
Richard A. Morgan
From the National Human Genome Research Institute and the National
Heart, Lung, and Blood Institute, Bethesda, MD.
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ABSTRACT |
We have determined the 2905 nucleotide sequence of the rhesus
macaque factor IX complementary DNA (cDNA) and found it to be greater
than 95% identical to that of the human factor IX cDNA. The cDNA has a
large 3' untranslated region like the human cDNA, but unlike the
human cDNA has two polyadenylation sites 224 nucleotides apart that are
used for transcription of the messenger RNA. The deduced amino acid
sequence is greater than 97% identical to that of human factor IX,
differing in only 11 of 461 amino acids in the complete precursor
protein. We found a single silent polymorphism in the nucleotide
sequence at the third position of the codon for asparagine at position
167 in the secreted protein (AAC/AAT). All residues subject to
posttranslational modifications in the human protein are also found in
the rhesus factor IX sequence. The high degree of homology between the
rhesus and human factor IX proteins suggested the possibility that the
human factor IX protein might be nonimmunogenic in the rhesus. We
tested the immunogenicity of human factor IX in three rhesus macaques
by repeated intravenous injections of monoclonal antibody-purified,
plasma-derived human factor IX over the course of more than a year and
assessed the recovery and half-life of the infused protein, as well as
in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in
the three animals studied, and aPTT mixing studies showed no evidence
for neutralizing antihuman factor IX antibodies. An outbred, nonhuman
primate model that permits assessment of the level and duration of
factor IX expression as well as vector safety would complement the use
of other (mouse and canine) hemophilia B animal models in current use
for the development of gene therapy for hemophilia B.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
HEMOPHILIA B IS A SEX-LINKED, inherited
bleeding disorder caused by deficiency of coagulation factor IX. Factor
IX is a serine protease that activates factor X after its own
activation by factor XI and/or factor VII as part of the blood
coagulation process. Severe disease (<1% of normal factor IX levels)
is associated with spontaneous bleeding (particularly hemarthrosis) and
excessive bleeding with trauma or surgery.1 Because factor
replacement therapy with recombinant or plasma-derived concentrates is
expensive and inconvenient, it has been used clinically in a reactive
mode rather than the more desirable prophylactic mode. Gene therapy of
hemophilia B offers the theoretical advantage of providing a curative
approach in which factor IX could be maintained continuously at
therapeutic levels (at least 1% of normal) and, based on extensive experience with prophylactic factor administration, would prevent spontaneous hemarthroses and preserve normal joints in these
patients.2,3
Gene therapy of hemophilia B using retroviral,4-9
adenoviral,10-12 or AAV-based vectors,8,13-15
is currently under development, and has relied on animal models for
hemophilia B that include knockout mice 16,17 and
hemophilia B canines.18,19 These permit in vivo evaluation
of vector-mediated gene expression in mammals. The underlying
hemophilia makes these animals difficult to maintain, and may pose
difficulties for experiments involving surgical procedures that require
normal hemostasis. Further, the immune response to human factor IX (in
immune competent animals) usually limits the ability to evaluate the
level or duration of factor IX expression from the vectors that would
be used for treatment of humans.11,20,21
It would be advantageous to be able to test long-term expression of
gene transfer vectors that synthesize human factor IX in
immune-competent animals that closely resemble humans before their use
in clinical trials. We therefore began preliminary investigations of
the suitability of the rhesus macaque (Macaca mulatta) for testing of human gene therapy vectors for hemophilia B. We cloned the
rhesus factor IX complementary DNA (cDNA) and determined its sequence
similarity to be greater than 95% identical with the human factor IX
cDNA, and found that intravenously administered human factor IX did not
elicit a significant antibody response in three rhesus macaques.
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MATERIALS AND METHODS |
Animals.
Adult rhesus macaques were housed in a dedicated primate facility under
conditions approved by the National Institutes of Health Institutional
Animal Care and Use Committee, and all protocols for experiments were
approved by the Committee. Rhesus macaques were bred at the National
Institutes of Health (Bethesda, MD) for these and other experiments.
Cloning of the rhesus factor IX cDNA.
Fresh liver obtained from a female rhesus after euthanasia for an
unrelated study was flash frozen in dry ice and stored under liquid
nitrogen before isolation of total RNA for cloning of the factor IX
cDNA. Frozen liver was disrupted with an automated tissue homogenizer,
and total RNA was prepared by extraction with Trizol (Life Sciences,
Bethesda, MD) according to manufacturer's directions. Total RNA was
stored under isopropyl alcohol at  20°C until polyadenylated RNA was purified with the Promega (Madison, WI) Polyattract kit. Polyadenylated RNA was used for first-strand DNA synthesis using reverse transcriptase and second-strand synthesis with Klenow DNA
polymerase, after which the double-stranded DNA fragment was ligated to
adapter-primers (Clontech, Palo Alto, CA) and subjected to polymerase
chain reaction (PCR) amplification using gene-specific oligonucleotide
primers and primers specific for the adapter ligated to the
double-stranded DNA.
5' and 3' rapid amplification of cDNA ends (RACE).
5' and 3' RACE PCR was performed using factor IX-specific
primers complementary to the human factor IX cDNA sequence. All but one
of these proved to be completely conserved after overlapping sequence
for the rhesus factor IX cDNA was determined
(Fig 1).
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| Fig 1.
Strategy for determination of the nucleic acid sequence
of the rhesus macaque factor IX cDNA. (Above) 1% agarose gel
electrophoresis of 5' and 3' RACE PCR fragments obtained by
amplification of adapter-ligated first-strand DNA template with adapter
primer AP1 (Clonetech) and various factor IX-specific
oligonucleotides. Lane 5'8: amplimer obtained with AP1 and H8
primers; lane 5'9: amplimer obtained with AP1 and H9 primers;
lane 5'10: amplimer obtained with AP1 and H10 primers; lane
3'2: amplimer obtained with H2 and AP1 primers; lane 3'4:
amplimer obtained with H4 and AP1 primers. Note the double bands
observed for 3' RACE PCR fragments 3'2 and 3'4, due
to the two different polyadenylation signal sequences, separated by 224 base pairs in the cDNA. (Below) Schematic diagram of rhesus macaque
factor IX cDNA depicting the position of oligonucleotide primers used
to amplify overlapping RACE PCR fragments and determine the nucleic
acid sequence. H series denotes oligonucleotides completely homologous
to human factor IX coding sequence. R series denotes oligonucleotides
with sequence unique to the rhesus macaque factor IX cDNA sequence. Not
shown is the adapter primer that is ligated to each end of the cDNA, to
which oligonucleotide AP1 is complementary. M, Methionine translation
initiation start sites; pA, AATAAA polyadenylation signal sequence.
Drawing not to scale for clarity in depicting oligonucleotide primer
positions.
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TA cloning.
5' and 3' RACE PCR products were subjected to ligation with
the PCR 3.1 plasmid (Invitrogen, La Jolla, CA) and the ligation products were used to transfect competent DH5alpha cells (Life Sciences, Gaithersburg, MD) that were selected on LB-ampicillin plates.
Colonies were screened for inserts by restriction digests with
EcoRI.
Sequence determination.
Automated sequence determination was performed with the ABI Prism model
377 automated fluorescence sequencer (Seqwright, Houston, TX) using the
reverse orientation sequencing primer for pCR 3.1 as well as
gene-specific primers for factor IX. Because the analysis showed novel
sequence data additional primers were designed to "walk" through
the 3' end until both AATAAA polyadenylation sites were located.
Sequence data management, alignment, and analysis was performed with
DNAstar software (Madison, WI). Sequence data was determined at least
twice for each position in the cDNA and in both the sense and antisense
orientation for the protein-coding portion.
Factor IX pharmacokinetics.
Monoclonal antibody (MoAb)-purified human factor IX (Mononine,
Centeon, Kankakee, IL) was injected intravenously into three rhesus
macaques on seven occasions for each animal over the course of a year
at a dose of 25 units/kg. Whole blood obtained at sequential time
points was anticoagulated with 0.105 mol/L sodium citrate and
platelet-poor plasma prepared by centrifugation at 2000g for 20 minutes and frozen until subsequent analysis. The half-life of human
factor IX in the rhesus was determined by plotting the natural
logarithm of the human factor IX level at T = 1, 2, 4, and 8 hours
after injection as a function of time and solving for the time constant
k, in which ln [hFIX] = e kt. The time constant k
was determined by curve-fitting with Deltagraph software (Deltapoint,
Monterey, CA). The half-life was calculated according to the
relationship T1/2 = 0.693/k.
Factor IX immunoassays (enzyme-linked immunosorbent assay
[ELISA]).
An ELISA capable of detecting human factor IX in a background of rhesus
plasma was developed. 96-well, flat-bottomed Immulon 4 plates
(Dynatech, Chantilly,VA) were coated overnight with 0.05 µg of rabbit
polyclonal antihuman factor IX (DAKO A300; DAKO, Glastrup, Denmark) in
0.1 mol/L sodium carbonate buffer (pH 8,8) at 4°C. Plates were then
washed with phosphate-buffered saline (PBS) with 0.05% Tween 20 (Sigma, St Louis, MO) (PBS/Tween) and blocked with PBS/Tween with 6%
bovine serum albumin (BSA) (RIA grade, Sigma; PBS/Tween/BSA) for 1 hour
at 37°C, then used immediately or frozen at 20°C before
use. Rhesus plasma samples were diluted 1:25 with PBS/Tween/BSA and
before loading 50 µL per well. The plate was incubated with samples
and standards (diluted in 6% BSA/PBS/0.05% Tween 20) for 1 hour at
37°C. After washing the plate with PBS/Tween, 100 µL of rabbit
antihuman factor IX conjugated to horseradish peroxidase (DAKO P380;
DAKO) was added and the plate incubated for 1 hour at 37°C. After
washing with PBS/Tween the plate was developed with 100 µL 1 mg/mL
o-phenylene diamine in 0.1 mol/L sodium citrate buffer (pH 4.5)
supplemented with 2 µL 30% hydrogen peroxide per 10 mL solution and
stopped by addition of 100 µL of 1 mol/L hydrochloric acid.
Absorbance at 492 nm was read with a Thermomax Microplate Reader
(Molecular Devices, Menlo Park, CA) to determine the concentration of
human factor IX antigen in each sample.
The cross-reactivity of rhesus and human plasma was examined in detail
and was found to vary with the dilution of the sample. At 1:25
dilutions (used for the assays reported herein) the cross-reactivity of
the sample was about 10%, ie, the amount of factor IX in rhesus plasma
diluted 1:25 was only 10% of that to be expected if there were
complete cross-reactivity between the human and rhesus proteins and
equal concentrations of each in plasma. Background cross-reactivity from endogenous rhesus factor IX in plasma samples was factored out by
adding rhesus plasma to the standards and plate-blank at a
concentration of 4% (vol:vol), ie, a 1:25 dilution. The ELISA was
linear from 30 to 1000 ng/mL of human factor IX when rhesus plasma was
spiked with Mononine, then diluted 1:25 as for the assays reported herein.
Sandwich ELISA for rhesus antihuman factor IX antibodies.
Immulon 4 96-well plates (Dynatech) were coated with 0.1 µg of
MoAb-purified human factor IX (Mononine) per well in 0.1 mol/L sodium
carbonate buffer (pH 8.8) overnight at 4°C, then washed and blocked
with BSA/PBS/Tween for 1 hour at 37°C, and frozen before to use.
Plates were thawed, washed, and 50 µL samples applied to each well
and incubated at 4°C for 1 hour. After washing with PBS/Tween, 100 µL of rabbit antirhesus IgG conjugated to horseradish peroxidase
(Sigma) diluted 1:10,000 were applied to each well and incubated for 1 hour at 4°C. After washing with PBS/Tween, the plate was developed
with 100 µL of 1 mg/mL o-phenylene diamine in 0.1 mol/L
sodium citrate buffer (pH 4.5) supplemented with 2 µL 30% hydrogen
peroxide per 10 mL solution and stopped by addition of 100 µL of 1 mol/L hydrochloric acid. Absorbance at 492 nm was read with a Thermomax
Microplate Reader (Molecular Devices). Dilutions of plasma from a
hemophilia B patient known to have a high-titer antihuman factor IX
alloantibody (a gift from Dr Amy Shapiro, Indiana University Medical
Center, Indianapolis, IN) were included as positive controls;
BSA/PBS/Tween without plasma or serum was included as a negative
control. The titer of a sample was defined as the maximum dilution at
which the absorbance at 492 nm exceeded that of the preimmune serum by
0.05 absorption units.
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RESULTS |
Sequence of rhesus FIX cDNA.
A total of 2.9 kb of cDNA sequence was determined from analysis of the
5' and 3' RACE fragments depicted in Fig 1. The sequence began 19 base pairs 5' to the start of the protein-coding region (Met-46) and extended 1.5 kb past the termination codon at +416. The
5' sequence overlaps 61 nucleotides of rhesus factor IX promoter sequence reported by Pang et al,22 and is in complete
agreement with that data. A 461 amino acid open reading frame encoded a protein with extremely high homology (97% identity) with the human factor IX protein (Fig 2).23 As
expected, the classical factor IX protein domain structure
(Gla-EGF-EGF-Activation peptide-Catalytic domain) was identical to that
of all other factor IX sequences thus far sequenced.
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| Fig 2.
Deduced amino acid sequence of rhesus macaque factor IX
cDNA and alignment with the human factor IX protein sequence.
Differences are denoted by bold typeface. Numbers denote the amino acid
sequence in the translation product and the factor IX domains are
indicated in italics and separated from one another by spaces in the
sequence.
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The pre-pro leader sequence, which directs posttranslational gamma
carboxylation of glutamic acids in the Gla domain, was identical to the
human sequence. All 12 glutamic acids in the Gla domain (potential
substrates for gamma-glutamyl carboxylation) were present in the rhesus
protein. Other key features of the protein that were identical to the
human form were the aspartic acid at position 64 (subject to
 -hydroxylation), the asparagines at 157 and 167 (subject to
N-glycosylation), and serines/threonines at positions 61, 159, 169, and
172, (which in the human protein are subject to O-linked glycosylation)
were also present. Serine 158 and threonine 155, which in the human
protein are phosphorylated and sulfated, respectively,24
were also present in the rhesus protein.
Most of the differences between the human and rhesus factor IX were
substitutions of highly homologous amino acids (eg, Lys/Arg at 37, Glu/Asp at 154, Val/Ile at 216, etc.). There was, however, a basic
amino acid (lysine) in place of glutamic acid at position 96 in the
rhesus that should result in a reversal of charge at that position,
though both are hydrophilic residues with similar characteristics with
respect to secondary structure formation.25
The rhesus factor IX protein sequence has greater homology to human
factor IX than does that of any other known species
(Table 1). The rhesus factor IX protein
also displayed a similar homology to factor IX from various other
species, varying according to phylogenetic relationships.
In humans, the amino acid at position 148 is polymorphic
(Ala/Thr),26,27 whereas in the rhesus there was evidence
only for threonine in the animal studied. There is evidence for a
nucleic acid polymorphism (in the female we analyzed) at Asn 167 in
which different cloned PCR fragments showed either AAC or AAT codons. Direct sequence analysis of multiple uncloned PCR fragments (both in
the forward and reverse orientation) confirmed the presence of C and T
at the third position of the codon.
Like the human factor IX cDNA, the rhesus factor IX cDNA included a
long, 3' untranslated region of approximately equal length to the
protein coding region. The 3' untranslated region bore a high
degree of resemblance (~95% identity) to the human factor IX
3' untranslated region. In contrast to the human factor IX cDNA,
the rhesus factor IX had two AATAAA polyadenylation signals separated
by 224 base pairs at the 3' end of the cDNA. As can be observed
in Fig 1, the 3' RACE fragments amplified as doublets consistent
with two lengths of polyadenylated messenger RNA in approximately
equal proportion, differing by 224 base pairs. The presence of
multiple polyadenylation signals has previously been described as a feature of the human gamma glutamyl carboxylase cDNA as
well.28
Pharmacokinetics and antigenicity of human factor IX in the rhesus
macaque.
To assess the pharmacokinetics and immunogenicity of human factor IX in
the rhesus we repeatedly injected three animals with MoAb-purified,
human factor IX derived from plasma (Mononine) over the course of a
year. We analyzed human factor IX recovery and half-life of elimination
at four different times as shown in Fig 3.
The peak recovery at 30 minutes ranged from 83% to 181% of that
expected after a dose of 25 units/kg body weight and a volume of
distribution of 100 mL per kg body weight, which in the human should
result in a peak level of 1250 ng/mL. The half-life of elimination,
based on first-order elimination from a single compartment, averaged
6.9 hours (SD = 2.1 hours) for the first falloff and 8.6 hours (SD = 2.7 hours) for the fourth falloff (Table
2).
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| Fig 3.
Factor IX falloff studies in three rhesus macaques 84269, RQ 1234, and RQ 1305. Each animal was studied on four occasions by
injecting 25 units Mononine/kg body weight followed by immunoassay for
human factor IX at the indicated time points. Falloff studies were
performed by single injection of factor IX at T = 0, 80 days, and 162 days; three daily injections of factor IX were performed 10 days before
a fourth falloff study at T = 388 days to maximize the
chance that any immune response might be boosted.
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We sought to maximize our ability to elicit an antibody response to
human factor IX protein (without the use of an adjuvant), so 11 days
before the fourth falloff study at T = 388 days we gave three daily
injections of factor IX (25 units/kg). This series of injections of
human factor IX should have boosted any low-titer antibody, and the
resulting antibody (if any) should have been readily observed because
anamnestic antibody responses to factor VIII or factor IX in humans
typically begin to appear 4 to 5 days after re-exposure to antigen and
rises rapidly thereafter. Indeed, the fourth falloff curve remained
superimposable on the prior three for each animal (Fig 3).
In addition, we performed studies of baseline plasma or plasma from the
fourth falloff study mixed with normal human plasma to look for in
vitro evidence of inhibitor antibody formation. These studies
(Table 3) showed no inhibition of factor
IX-dependent coagulation that would have manifested itself as
prolongation of the activated partial thromboplastin time. A plate
ELISA for rhesus antihuman factor IX antibodies was negative for all
postfalloff sera in the three animals studied with the exception of
1:50 titer antibodies observed after the second factor IX injection in
animal 84269 and after the third injection in animal RQ1305; these
animals were negative for antihuman factor IX antibodies in all
subsequent postinjection sera. The positive sera, when heat inactivated
for 30 minutes at 56°C, did not prolong the aPTT of normal human
plasma significantly when compared with the aPTT of normal human plasma mixed with heat-inactivated negative serum (29.5 seconds, SD = 3.3 seconds for seronegative samples, v 26.5 seconds, SD = 4.5 seconds for 84269 post-second falloff and 30.0 seconds, SD = 2.1 for
RQ1305 post-third falloff; P > .05 for both comparisons by t test).
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DISCUSSION |
We have determined the complete cDNA sequence of the rhesus macaque
coagulation factor IX, which is the first complete coagulation factor
cDNA sequence reported from a nonhuman primate. The cDNA and the
derived amino acid sequence is 97% identical to the human factor IX at
the amino acid sequence level. The factor IX protein domain structure
and potential sites for post-translation modification are identical to
that of the human.
The nucleic acid sequence data and the derived amino acid sequence show
the rhesus factor IX protein to be more homologous to the human factor
IX protein than either that of the canine or mouse, both of which have
been used as models for hemophilia B gene therapy, and both of which
are known to make antibodies to human factor IX. It is therefore likely
that human factor IX would be less immunogenic in the rhesus than in
the canine or mouse models used to test gene therapy vectors.
The immunogenicity of factor IX transgenes in the context of viral (or
nonviral) gene transfer is crucial to assess before human trials to
detect the development of antihuman factor IX inhibitor antibodies,
which have severe adverse consequences in patients with severe
hemophilia B. Although inhibitor antibodies are much less frequent in
patients with severe hemophilia B (~3%) than in patients with severe
hemophilia A (~20%), the former are much more serious, having been
associated with anaphylaxis and/or nephrotic syndrome when
factor IX is repeatedly administered in an effort to induce tolerance
for factor IX.29,30
As can be observed in Fig 3, the sequential falloff studies are
essentially superimposable for each individual animal. The half-life of
human factor IX in the rhesus (5 to 11 hours) is shorter than that
observed in humans (18 to 22 hours),1 but is comparable to
that observed for human factor IX in other species, particularly
rodents.11 The shorter half-life of human factor IX in
nonhuman species is perhaps a consequence of differences in
posttranslational modifications (eg, glycosylation, sulfation, phosphorylation) or differing affinity for receptors that bind glycosylated proteins in general (eg, the ASOR receptor on the hepatocyte31) or factor IX in particular (factor IX
receptors on endothelial cells).32,33 Our studies on the
pharmacokinetics of human factor IX in the rhesus show no evidence for
inhibitor antibody formation after repeated injections. It must be
acknowledged that additional injections of human factor IX might have
elicited an antibody response in the animals studied and it is also
possible that if a greater number of animals were treated similarly a
high-titer antibody response might have been elicited in some.
Adjuvants that enhance immunogenicity and antibody formation were not
used in this study because our intent was to administer factor IX in the same manner as hemophilia B patients receive it in the clinical setting. It is also possible that repeated administration of a weakly
immunogenic protein to an adult animal in a "nondanger" setting
may serve to induce tolerance by deleting antihuman factor IX
lymphocytes in the absence of "signal 2".34
There is precedent for a lack of immune response to human factor IX in
certain normal (immune competent) animals, despite differences between
the factor IX amino acid sequences. The C57Bl/6 strain of (normal)
mouse typically does not make antihuman factor IX antibodies even in
the context of adenoviral-mediated gene expression, although other
normal mouse strains (eg, CD1) readily make antihuman factor IX
antibodies.35 In contrast, hemophilia B canines routinely
make antibodies to human factor IX after human factor IX
infusions36,37 or gene transfer with human factor IX
vectors.38
The significance of the transient low-titer antihuman factor IX
antibodies in two of our animals is unclear. These are non-neutralizing (by aPTT mixing studies) and do not affect the pharmacokinetics of
infused human factor IX protein. Transient, noninhibitory, canine
anticanine factor IX antibodies have been detected by Western blotting
after AAV-canine factor IX gene transfer into skeletal muscle of
hemophilia B canines by Herzog et al,39 indicating that
even self-tolerance for factor IX can be broken. The antibodies in the
rhesus monkeys suggest at least that there are
complementarity-determining region sequences in the rhesus genome
necessary for antihuman factor IX antibody production, however an
adjuvant (eg, a viral gene transfer vector) would be required to elicit
a high-titer antibody response to human factor IX.
If a human factor IX vector causes antihuman factor IX antibodies in
the rhesus macaque, the rhesus cDNA could be used to test whether the
viral vector or the transgene causes loss of self tolerance (ie, by
making and administering a rhesus factor IX vector and looking for
autoantibodies to rhesus factor IX). Loss of self-tolerance to
erythropoietin has been shown in mice after administration of
adenoviral vectors encoding the (human) erythropoietin transgene,
despite strong homology between the mouse and human
proteins.40 Such studies will permit a better understanding
of the potential immunogenicity of endogenous proteins expressed by
viral vectors in the future.
Our results show that it should be possible not only to collect vector
safety and toxicity data, but also to analyze the level and duration of
human factor IX expression in a normal, nonhuman primate animal model,
which is available to investigators in the field of gene therapy.
Indeed, we have recently been able to show dose-dependent,
adenovirus-mediated expression of human factor IX in the three rhesus
macaques used in the studies reported herein (data not shown). Studies
of human factor IX vectors will be critical to the development of
hemophilia B gene therapy protocols, and the rhesus macaque should be
considered a potential model for preclinical testing of human factor IX
gene transfer vectors.
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FOOTNOTES |
Submitted July 9, 1998; accepted November 5, 1998.
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.
Address correspondence to Richard A. Morgan, Building 10, Room 10C103,
10 Center Drive, Bethesda, MD 20892-1851.
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