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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 784-790
Human Factor IX Corrects the Bleeding Diathesis of Mice With Hemophilia
B
By
Szu-Hao Kung,
J. Nathan Hagstrom,
Darrell Cass,
Shing Jen Tai,
Hui-Feng Lin,
Darrel W. Stafford, and
Katherine A. High
From the Departments of Pediatrics and Pathology, and the Department
of Surgery, University of Pennsylvania and the Children's Hospital of
Philadelphia, Philadelphia, PA; and the Department of Biology, the
University of North Carolina at Chapel Hill.
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ABSTRACT |
Mice with hemophilia B have been engineered using gene targeting
techniques. These animals exhibit severe factor IX deficiency and a
clinical phenotype that mirrors the human disease. We have bred the
founder animals onto two different strains of mice, C57B1/6 and CD-1,
and have sought to determine whether adenoviral vectors expressing
human factor IX could correct the bleeding diathesis of mice with
hemophilia B. Initial experiments showed that purified plasma-derived
human factor IX added to murine factor IX-deficient plasma resulted in
complete correction of the activated partial thromboplastin time
(aPTT), and that injection of 1011 particles
of an adenoviral vector expressing human factor IX resulted in
normalization of a modified aPTT in mouse plasma. As an additional
method of assessing the function of human factor IX in the murine
coagulation system, bleeding times were performed in normal,
hemophilic, and adenoviral-treated hemophilic mice. By two different
bleeding-time techniques, the treated hemophilic mice gave values
identical to normal littermate controls, whereas the untreated
hemophilic mice exhibited heavy blood loss and prolonged bleeding.
There was a marked difference in antibody formation in the two strains
of mice; 100% of the hemophilic CD-1 mice formed antibodies to human
factor IX, but none of the C57B1/6 mice did. These data suggest that
the C57B1/6 hemophilic mice will be more useful for gene transfer
studies, while the CD-1 hemophilic mice may be of greater utility in
studying the development of inhibitors.
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INTRODUCTION |
HEMOPHILIA B is the bleeding diathesis
resulting from a deficiency of blood coagulation factor IX (F.IX).
Genetically engineered hemophilia B mice have recently been produced by
gene targeting experiments in ES cells.1 These mice exhibit
a severe bleeding diathesis including exsanguination after tail
transection, premature death, severe bleeding after trauma, and
evidence for spontaneous bleeding in the foot pads that mirrors the
disease seen in humans, and should prove useful as a model in efforts
to establish an experimental basis for gene therapy of hemophilia in
humans.
Despite a high degree of conservation at both the DNA and protein
levels, clotting factors from one species are not always fully
functional in plasma from another species. Thus, for example, human
F.VIIa does not interact with murine tissue factor.2,3 Most
currently available gene therapy vectors express human F.IX; new
vectors can be constructed expressing murine F.IX, but this is a
time-consuming process, and analysis of results would be hampered by
the absence of commercially available antibodies to murine F.IX,
precluding assays such as enzyme-linked immunosorbent assay (ELISA) and
immunofluorescence. Thus, we sought to determine whether vectors
expressing human F.IX could correct the bleeding diathesis of mice with
hemophilia B. Our results show that human F.IX is functional in the
murine coagulation system and that it completely corrects the bleeding
diathesis in hemophilia B mice. We also demonstrate a strain-dependent
variation in the development of antibodies to human F.IX, suggesting
that some strains will be more useful than others in most experimental
protocols.
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MATERIALS AND METHODS |
Recombinant adenoviral vector.
An E1 gene deleted adenoviral vector (Ad-F.IX) containing human F.IX
cDNA including the 3 untranslated region driven by a cytomegalovirus
(CMV) immediate early gene promoter/enhancer was constructed as
previously described.4 The particle:plaque-forming units
ratio of the Ad-F.IX is 30:1 as determined in a plaque assay as
previously described.5 The viral preparations were stored at  80°C in phosphate-buffered saline (PBS) containing 3% sucrose.
Animal procedures.
All procedures involving mice were approved by the Institutional Animal
Care and Use Committee at The Children's Hospital of Philadelphia in
accordance with the Animal Welfare Act. Hemophilia B carrier females
were bred with normal male mice of either C57B1/6 or CD-1 background.
Hemophilic offspring were identified by Southern blot on tail DNA and
by aPTT-F.IX assays.1 Eight- to 9-week-old hemophilia B
mice were injected with 1011 particles of Ad-F.IX via the
tail vein. Blood was collected directly into citrated tubes (mixed with
citrate within 3 seconds) following tail transection performed under
anesthesia. Hemostasis was obtained by ligating the tip of the tail
with 4-0 Vicryl suture (ETHICON, Inc, Somerville, NJ). For
immunofluorescence staining, animals were killed and the left lobe of
the liver was removed and frozen in OCT embedding compound (Sakura) in
a methylbutane dry ice bath.
Assays for antigen levels and biological activity of human F.IX.
Human F.IX (hF.IX) antigen in mouse plasma was determined by ELISA as
previously described.4 A polyclonal rabbit anti-human F.IX
antibody (Dako Corp, Carpenteria, CA) was used as primary antibody, and
a polyclonal goat anti-human F.IX antibody coupled to horseradish
peroxidase (Affinity Biologicals, Hamilton, Ontario, Canada) was used
as the secondary antibody. The linear range of the standard curve was
from 3 to 100 ng/mL. This ELISA does not cross-react with mouse F.IX.
All samples were measured in duplicate; the intra- and inter-assay
coefficient of variation was 8.7% and 15%, respectively.
APTT-F.IX assays were performed to determine the clotting activity of
hF.IX expressed in the treated mice in the presence of hF.IX deficient
plasma (Organon Teknika Corp, Durham, NC). In this assay,
50 µL of aPTT reagent (Organon Teknika Corp) and 50 µL
of hF.IX-deficient plasma were incubated with 50 µL of a 1:5 dilution
of sample mouse plasma.
CaCl2was added and time to clot formation was measured with a fibrometer
(Fibrosystem; BBL, Cockeysville, MD).1 This
assay was developed to limit the amount of mouse plasma required for
evaluation.
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| Fig 1.
Time course of aPTT-F.IX assays in hemophilia B mice
following injection of 1 × 1011 particles of an
adenoviral vector expressing human F.IX. Citrated plasma was collected
and aPTT-F.IX assays performed at the indicated time points. Each
symbol represents an individual animal, with each point an average of
duplicate measurements. (A) C57B1/6 hemophilic mice. (B) CD-1
hemophilic mice. Mouse 7 is a normal littermate treated with the same
dose of vector. ( ), 1; ( ), 2; ( ), 3; ( ), 4; ( ), 5;
( ), 6; ( ), 7.
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| Fig 2.
Time course of plasma concentration of hF.IX in
hemophilia B mice following intravenous injection of 1011
particles Ad-F.IX. Plasma samples were collected at the indicated time
points and hF.IX levels determined by ELISA. Each point is an average
of duplicate measurements. (A) C57B1/6 hemophilic mice and 1 normal
littermate (no. 7). (B) CD-1 hemophilic mice and 1 normal littermate
(no. 7). In both cases the normal littermate was treated with the same
dose of vector. (), 1; (), 2; (), 3; (), 4; (), 5;
(), 6; (), 7.
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| Fig 3.
Tail bleeding time by a filter paper method. Data are
shown for (A) a normal mouse, (B) a C57B1/6 hemophilic mouse, (C) a treated C57B1/6 hemophilic mouse 1 week after injection of Ad-F.IX vector.
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| Fig 4.
Total and neutralizing antibodies to human F.IX in
hemophilic CD-1 mice. Plasma samples were evaluated by (A) ELISA, (B)
Western blot, and (C) Bethesda assay at indicated time points. (A)
Plates were coated with purified plasma-derived hF.IX, plasma samples from treated mice were applied, and rabbit anti-mouse IgG conjugated with horseradish peroxidase served as a detection antibody. Each result
represents duplicate measurements from a single animal. (B) Lanes 1 through 6 represent an immunoblot time course on a single animal, weeks
0, 1, 2, 3, 5, and 7, respectively, after Ad-F.IX injection. Lanes 7 through 9 represent three additional CD-1 hemophilic mice at week 2 postinjection. (C) The presence of antibodies that interfere with
coagulation was assessed by the Bethesda assay and reported as Bethesda
units. A time course in four representative animals is shown. ( ), 1;
(), 2; ( ), 3; (), 4; (), 5; ( ), 6.
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| Fig 5.
Immunofluorescence staining for human F.IX on liver
sections from treated and untreated hemophilic mice. (A) Untreated
C57B1/6 hemophilic mouse. (B) Treated C57B1/6 hemophilic mouse 2 weeks after Ad-F.IX injection. (C) Treated CD-1 hemophilic mouse 2 weeks after Ad-F.IX injection. Original magnification ×200.
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To determine if hF.IX can correct mouse F.IX deficient plasma in vitro,
F.IX-deficient plasma was collected from F.IX knockout mice after tail
transection. Fifty microliters of murine F.IX-deficient plasma was
reconstituted with 1 µL of plasma-derived F.IX (Mononine; Armour
Pharmaceutical Co, Kankakee, IL) to reach final
concentrations of 5, 0.5, 0.05, 0.005, and 0.0005 µg/mL, representing
100%, 10%, 1%, 0.1%, and 0.01% of the physiological range of
hF.IX. Fifty microliters of reconstituted plasma was incubated with 50 µL of aPTT reagent for 3 minutes in the absence of hF.IX-deficient
plasma and time to clot formation after addition of CaCl2
was measured as described above.
To determine the functional activity of the hF.IX expressed in vivo, 50 µL of diluted plasma from treated hemophilia B mice, diluted to an
antigen concentration of 200 to 400 ng/mL in imidazole buffer, was
added to 50 µL of hF.IX-deficient plasma, and an aPTT performed as
described above. The specific activity level was determined using a
standard curve of purified plasma-derived F.IX added at serial
dilutions to imidazole buffer containing the same concentration of
untreated hemophilia B mouse plasma as treated plasma in the test
sample.
Assays for circulating antibody against hF.IX.
An ELISA was used as previously described to determine the presence of
antibodies to hF.IX.6 These data were confirmed with a
Western blot as previously described.6
Modified Bethesda assay for hF.IX inhibitors.
A modification of the originally described Bethesda assay7
was performed to screen for the presence of antibodies that interfere
with coagulation (inhibitors). Ten microliters of serially diluted
sample plasma and 10 µL of pooled normal human plasma (Verify 1;
Organon Tenika Corp) was added to 30 µL of dilution buffer and
incubated at 37°C for 2 hours. This mix was then added to 50 µL of
aPTT reagent and 50 µL of human F.IX-deficient plasma and incubated
at 37°C for 3 minutes. CaCl2 was added and time to clot
formation measured. Pooled normal human plasma incubated in dilution
buffer without the test sample was used as a control. The dilution with
residual activity closest to 50% was used to calculate the inhibitor
titer, in which 50% residual F.IX activity equals 1 Bethesda unit
(BU)/mL.
Immunofluorescence staining of liver.
Cryosectioned (6 µm) liver tissues were subjected to an indirect
immunofluorescence staining protocol.6 The primary antibody was an affinity-purified goat anti-hF.IX antibody (Affinity
Biologicals) and the secondary antibody was a rabbit anti-goat IgG
(Dako, Carpinteria, CA) conjugated with fluorescein
isothiocyanate (FITC).
Bleeding time assay.
Mice were anesthetized using metofane gas, and positioned horizontally
on a platform that allowed the tail to drop about 2 cm from the top of
the platform. The tail was pulled through a template with fixed sizes
of openings until snug and then transected by cutting the face of the
template using a no. 21 surgical blade. This technique results in
wounds of identical cross-section of approximately 1.5-mm diameter. To
evaluate bleeding from the incision, Whatman filter paper
(Whatman International Ltd, Maidstone, UK) was applied to the edge of
the forming clot every 30 seconds, taking care not to dislodge the
clot. Blood that continued to flow from the cut during the 30-second
interval was allowed to fall on the filter paper at the same point. In
another set of experiments, blood was collected into a citrated
Eppendorf tube for a duration of 5 minutes after the
transection. The total volume of whole blood was then measured. For
both protocols, hemostasis was achieved following the procedure by
closure with Vicryl suture.
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RESULTS |
Breeding and handling of hemophilia B mice.
Carrier females (ES 129-derived) were bred with males of two different
strains, C57B1/6 and CD-1. Breeding into these strains has now occurred
for four generations of offspring; complete conversion of one strain to
another requires approximately 20 generations. Of 143 mice from the
F1-F4 generations, 29 mice (20.3%) were shown to have hemophilia B by
Southern blot genotyping (Table 1). This is slightly
lower than the expected number (25%), and suggests that there may be
some fetal wastage for the affected mice. The number of carrier females
was as predicted; these mice appear unaffected clinically. Both
hemophilic males and hemophilic females (obtained by breeding carrier
females with hemophilic males) are competent breeders, but the
mortality rate from bleeding is high among the affected mice,
especially in the neonatal period and in association with invasive
procedures required for genotyping.
Human F.IX corrects murine F.IX-deficient plasma in an in vitro
clotting assay.
Plasma was prepared from three mice with hemophilia B and used to
generate pooled murine F.IX-deficient plasma. Purified plasma-derived human F.IX in varying concentrations was added back to F.IX-deficient plasma (either human or murine) and aPTTs determined (Table
2). Partial correction of the aPTT of murine
F.IX-deficient plasma was demonstrated at concentrations as low as
0.001 U/mL (0.1% of normal human plasma concentration), and the aPTT
progressively shortened as a function of the amount of human F.IX
added. Thus, human F.IX corrects murine F.IX-deficient plasma in an in
vitro clotting assay. For comparison, an identical experiment was
performed using human F.IX-deficient plasma as substrate; these results are also shown in Table 2 and are similar to those given for murine
F.IX-deficient plasma. The clotting time of 25 seconds in murine plasma
reconstituted to 1.0 U/mL of human F.IX is consistent with our
observation that the aPTT of pooled normal murine plasma is shorter
than that of pooled normal human plasma (data not shown).
In vivo correction of the bleeding diathesis in the hemophilia B
mouse model by intravenous injection of an adenoviral vector expressing
human F.IX.
Although coagulation tests are routinely used to assess hemostasis, the
process of coagulation in vivo involves a number of interactions that
are not modeled in in vitro testing, including interaction with
specific cellular receptors,8-11 and clearance from the
circulation.12,13 We sought to assess correction of coagulation in vivo after injection of an adenoviral vector expressing human F.IX (Ad-F.IX). Two strains of mice, CD-1 and C57B1/6, were injected at 8 to 9 weeks of age with 1011 particles of
Ad-F.IX, a dose determined in previous studies in normal CD-1 mice to
produce supratherapeutic levels of F.IX in the plasma. Blood was
obtained at weekly intervals for determination of clotting times and
human F.IX antigen levels in treated mice. In addition, bleeding times
were determined in hemophilic, normal, and treated mice (1 week after
treatment in the latter group).
Clotting times were determined in a modified aPTT assay referred to as
an aPTT-F.IX1; this assay is used because it requires only
very small amounts of plasma. The baseline clotting time of the
hemophilic mice in this assay is 59.9 seconds ± 1.0 seconds and of
normal mice is 33.5 seconds ± 3.1 seconds. After a single intravenous
injection of Ad-F.IX, the aPTT-F.IX in hemophilic C57B1/6 mice falls
into the normal range and remains at that level for the duration of the
experiment (Fig 1A).* On the
other hand, in hemophilic CD-1 mice the clotting time shortens
transiently but then returns to baseline (Fig 1B). Similar results are
seen with the hF.IX ELISA, ie, supraphysiologic levels persist
(although they are gradually declining) in the C57B1/6 mice for the
duration of the experiment (Fig 2A), whereas in the
hemophilic CD-1 mice F.IX levels decrease to the undetectable range 2 weeks after injection (Fig 2B).
Tail bleeding times provide an additional measure of coagulation in
vivo, since, unlike template bleeding times in humans, tail bleeding
times in mice are sensitive to levels of coagulation factors.14-17 Indeed, the first indication that the
hemophilia A knockout mice manifested a bleeding phenotype was their
100% mortality rate after tail transection for
genotyping.18 We used two different methods to assess
bleeding times in the mice. In the first, the tail was transected at a
specified diameter (1.5 mm) and filter paper was used to assess blood
oozing from the wound without disturbing the forming clot (Fig
3A-C). Extensive blood loss in the hemophilic mice
necessitated stopping the test after a period of ~7 to 8 minutes, to
prevent exsanguination, but the differences in the findings for normal,
hemophilic, and treated mice are readily apparent from visual
inspection of the filters. As a second, more quantitative measure of
the bleeding time, total blood volume lost over 5 minutes was measured
in 12 mice (Table 3). These data document that blood
loss in the treated hemophilia mice is similar to that of normal mice,
and is 10-fold less than in untreated hemophilia B mice.
The highest levels of F.IX expression, in the range of 50 to 90 µg/mL, are 10- to 20-fold higher than physiological levels. Whether
the hepatocyte can synthesize fully active F.IX at these supraphysiologic levels is unknown. We assessed the specific activity of the transgene product in a clotting assay in which serial dilutions of treated hemophilic mouse plasma (containing human F.IX as the transgene product) were added to human F.IX-deficient plasma. Results
are compared to a standard curve, and show that, at plasma concentrations in the 20 to 50 µg/mL range, only about half of the
material is biologically active (data not shown). These findings are in
agreement with our previous studies in hemostatically normal CD-1
mice.4 We have never observed levels of expression of biologically active material of greater than 25 µg/mL, and suspect that this represents an upper limit for biosynthesis of F.IX in the
murine liver.
Duration of expression of human F.IX in hemophilic mice is
strain-dependent and is limited in CD-1 mice by rapid formation of
antibodies to human F.IX.
The structure of the murine F.IX gene deletion in the hemophilia B mice
suggests that no protein will be produced.1 In humans with
hemophilia B, mutations associated with an absence of any protein
production are more likely to result in antibody production when
patients are exposed to human F.IX.19,20 We sought to
determine whether hemophilic mice from the two strains studied here
develop antibodies to human F.IX, and whether strain-dependent differences in antibody formation could account for the difference in
duration of expression of the transgene. In the hemophilic C57B1/6
mice, antibodies were undetectable for the duration of the experiment.
These data are consistent with previous work in hemostatically normal
C57B1/6 mice,21,22 which have not generally produced
antibodies to human F.IX after intravenous vector injection (vide
infra). The results were quite different in CD-1 hemophilic mice.
Figure 4A shows the time course of appearance of
antibodies to human F.IX as measured in an ELISA. Data are shown for
six mice at weeks 1 and 2, and four mice at later time points; one mouse was killed for immunofluorescence studies and a second mouse died. Antibodies are initially detected 1 week after injection, are
present in high-titer beginning 2 weeks after injection, and remain
present at high levels for the duration of the experiment. Western blot
analysis for the presence of murine antibodies to human F.IX (Fig 4B)
confirms these findings and shows that all CD-1 mice tested have
readily detectable antibody 2 weeks after injection of Ad-F.IX.
Finally, a Bethesda assay on plasma from treated CD-1 mice (Fig 4C)
demonstrates the presence of antibodies which inhibit coagulation
function (ranging from 6 to 13.1 BU) as early as 3 weeks after
injection, with a steady increase documented during the course of the
experiment.
Site of transgene expression.
Previous work has shown that intravenous injection of adenoviral
vectors into adult mice results in expression primarily in liver.4,21,23 Transgene expression in the liver was also documented in the hemophilia B mice (Fig 5, see page
787). In an immunofluorescence assay using antibodies
that do not cross-react with murine F.IX (see Materials and Methods),
we show the presence of human F.IX in liver sections from both C57B1/6
and CD-1 hemophilic mice 2 weeks after treatment with Ad-F.IX (Fig 5B
and C). Note that hF.IX immunofluorescence staining is comparable for
the two strains of mice, despite the fact that human F.IX is not
detectable in the plasma of CD-1 mice at this time point. These data,
coupled with those shown in Fig 4, suggest that the absence of
detectable expression in the plasma of the hemophilic CD-1 mice results
from formation of antibodies to human F.IX in this strain. Human F.IX was not detected in liver sections from untreated mice (Fig 5A) or from
mice injected with AAV-lacZ (data not shown).
| |
DISCUSSION |
The hemophilia B mice are quite fragile in terms of bleeding, and in
this respect seem to differ from published reports describing the
hemophilia A knockout mice.18,24 As noted earlier, the risk
of bleeding in the hemophilia B mice was increased in the neonatal
period and in association with invasive procedures required for
genotyping, but even routine handling such as occurs in changing cages
sometimes led to lethal hemorrhage in these animals. Modifications of
routine procedures that resulted in improved survival included deferring tail cutting for genotyping until 8 weeks of age; placing mature males into separate cages, since fighting can result in death
from bleeding; avoiding excessive use of the heat lamp (before tail
vein injection or blood withdrawal), because hyperactivity can also
result in traumatic bleeds; and suturing the tail after tail-cutting,
to reduce bleeding from the wound. The reasons for the difference in
phenotype between the hemophilia A mice and the hemophilia B mice are
unclear. In humans, the two entities are indistinguishable clinically,
a fact that is at least partly due to the kinetics of the IXa-catalyzed
activation of F.X, the reaction in which F.VIIIa serves as a cofactor.
The reaction can be catalyzed by IXa, phospholipid and calcium alone,
but the catalytic efficiency is accelerated by more than 4 orders of
magnitude when F.VIIIa is present.25 In
physiologic terms, then, the reaction does not proceed without the
presence of the cofactor. If the murine system is less dependent on the
presence of the cofactor, then it may be that small amounts of Xa can
be generated in the hemophilia A mice, which lack the cofactor but not
the enzyme, whereas the hemophilia B mice, completely lacking the
enzyme, will have a virtual absence of function in the intrinsic
pathway. Studies are currently underway to assess this possibility by
determining levels of activated clotting factors in normal mice and in
mice with hemophilia A and hemophilia B.
The major purpose of this study was to determine whether human F.IX can
correct the bleeding diathesis of hemophilia B mice as measured by in
vitro and in vivo clotting assays. The results indicate that human F.IX
is fully functional in the murine clotting system. In in vitro clotting
assays, purified human F.IX corrects the clotting time of murine
F.IX-deficient plasma. In addition, hemophilic mice injected with an
adenoviral vector expressing human F.IX exhibit normalization of the
aPTT-F.IX. However, coagulation in the whole organism involves a number
of interactions that are not tested in in vitro plasma-based clotting
assays. Interactions of F.IX/F.IXa with endothelial cell
receptors,10 with monocytes,26 and with
putative platelet receptors11 all contribute to the in vivo
process of coagulation, as do factors such as volume of distribution
and mechanisms of clearance. One cannot necessarily assume that human
F.IX or IXa will interact with appropriate murine cellular receptors to
effect hemostasis in hemophilic mice. To assess this we carried out
bleeding times in treated and untreated hemophilic mice. The treated
mice received an intravenous injection of an adenoviral vector
expressing human F.IX, which had been previously shown to direct
high-level expression of human F.IX in hemostatically normal
C57B1/6,21 (and unpublished observations, April 1996) and CD-1 mice.4 In the current study, both
strains of mice demonstrated correction of plasma-based clotting times and of bleeding times, and both showed phenotypic correction of bleeding, although correction was only transient in the CD-1 mice, due
to formation of antibodies to human F.IX.
A wealth of data have shown that hemostatically normal C57B1/6 mice do
not generate antibodies to human F.IX21 (and
unpublished data, April 1996) or canine F.IX27 when
injected intravenously with vectors expressing these transgenes, but
duration of expression is limited because of the immune response to
adenoviral proteins. (Interestingly, the route of administration of the
vector plays a role in whether antibody formation occurs, because
C57B1/6 mice do develop antibodies to human F.IX after intramuscular injection of adenoviral or AAV vectors expressing human
F.IX.6) In hemostatically normal CD-1 mice, we have
previously shown a duration of expression of ~13 to 16 weeks (during
which levels gradually decline) following tail vein injection of
Ad-F.IX, and we have documented the presence of antibodies to
adenoviral proteins and, based on fall-off curves, the absence of
neutralizing antibodies to human F.IX.4 The findings are
somewhat different in hemophilic mice of these two strains. Like their
hemostatically normal counterparts, the hemophilic C57B1/6 mice do not
generate antibodies to human F.IX, but the hemophilic CD-1 mice rapidly develop antibodies to human F.IX, in marked contrast to the
hemostatically normal CD-1 mice and to the hemophilic C57B1/6 mice. One
may infer from these findings that the presence of murine F.IX, which
shares 80% sequence conservation with human F.IX,28
produces some level of tolerance, which is strain-dependent to the
human F.IX protein. Interestingly, in the CD-1 hemophilic mice,
antibodies to the protein were detected by ELISA as early as 1 week
after injection, whereas antibodies interfering with function in
coagulation were not detected until 2 weeks later.
In humans with hemophilia B, antibodies are more likely to arise in
individuals with large gene deletions or nonsense mutations that result
in an absence of protein production,19 but the nature of
the mutation is clearly not the only determinant of whether antibodies
occur, because there are many documented cases where an inhibitor
occurs in only a few members of a large cohort all affected with the
same mutation.29 The F.IX knockout mice described here have
a deletion of genomic sequences containing exons 1-3 of the murine
gene; they are thus comparable to humans with large gene deletions, and
exhibit a similar phenomenon, ie, in mice with the identical mutation,
some (CD-1) develop antibodies whereas others (C57B1/6) do not. A
strain-related variability in response to adenoviral vectors has been
documented previously, and shown not to correlate with H-2
type.30 We document in these two strains of hemophilic mice
a strain-related variability in antibody response to the transgene
product. Our findings suggest that C57B1/6 hemophilia B mice will be
more useful for studies of gene transfer, whereas the CD-1 strain of
hemophilic mice may be of greater utility for studying the development
of inhibitors.
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FOOTNOTES |
Submitted September 24, 1997;
accepted November 17, 1997.
*
The aPTT-F.IX in treated hemophilic mice is shorter than values for
normal mice. This is attributed to the higher concentrations of F.IX in
the treated mice (samples measured at a 1:5 dilution), and to the
presence of human F.IX versus murine F.IX in the assay.
Supported by National Institutes of Health Grants No. R01 HL53668 and
P50 HL54500 to K.A.H. and F32 HL09397 to J.N.H.
Address reprint requests to Katherine A. High, MD, Division of
Hematology, 310A Abramson Research Center, The Children's Hospital of
Philadelphia, 34th St and Civic Center Blvd, Philadelphia, PA 19104.
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.
| |
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Abstract
Introduction
Abstract