Manual Hemofilie3

7/29/2019 Manual Hemofilie3

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

Creation of a mouse expressing defective human factor IX

Da-Yun Jin, Tai-Ping Zhang, Tong Gui, Darrel W. Stafford, and Paul E. Monahan

The majority of cases of human hemo-

philia B are the result of missense muta-tions in the coagulation factor IX gene

and defective circulating factor IX is de-

tectable in most patients. The available

mouse factor IX knockout models of he-

mophilia B (FIXKO mouse) reproduce the

bleeding phenotype of human hemophilia

B, but because the models produce no

factor IX they fail to reproduce the domi-

nant human phenotype. We have created

a human factor IX mouse model of hemo-

philia B (R333Q-hFIX mouse) by homolo-

gous recombination in embryonic stemcells. The mouse expresses no mouse

factor IX, but instead expresses a mis-

sense mutant human factor IX from the

mouse FIX promoter. Mutant human fac-

tor IX mRNA transcript and circulating

human factor IX are detectable through-

out development, but factor IX activity is

less than 1% and the mouse exhibits the

hemophilic phenotype. When R333Q-

hFIXmice were challenged by intramuscu-

lar injection of adeno-associated virus

expressing human factor IX, factor IXexpression without the development of

antibodies was observed. In contrast,

given the same treatment, FIXKO mice

consistently develop antibodies. Our

R333Q-hFIX mice strain will complement

the FIXKO mice for studying factor IX

circulating kinetics and gene therapy.

(Blood. 2004;104:1733-1739)

2004 by TheAmerican Society of Hematology

Introduction

Hemophilia B is an X-linked recessive hemorrhagic diathesisresulting from lack of coagulation factor IX activity. Lack of factor

IX activity may arise from a variety of molecular defects in the

factor IX gene, including base substitutions, deletions, insertions,

and gene inversions. The clinical definition of hemophilia B is

based on the individuals factor IX activity level as mild ( 5%),

moderate (1%-5%), or severe ( 1%). Of mutations resulting in

clinical disease, 96% occur in the 2.2-kb sequence of the factor IX

gene that consists of the promoter region, coding region, and splice

junctions. We, and others, created mice deficient in factor IX by

gene targeting1-3 that proved to be excellent models of hemophilia

B. However, the mice deficient in factor IX were created by

deletion of the promoter as well as a portion of the gene itself and

produced no factor IX antigen.1 Therefore, because most patientswith hemophilia B have circulating levels of defective factor IX

(which is antigenically cross-reacting material, [CRM]) the hemo-

philia B mouse fails to mimic some important characteristics of the

human disease. In particular, when the hemophilia B mice are

treated with exogenous factor IX or by intramuscular gene therapy

to correct their deficiency they usually develop neutralizing

antibodies.4-7 This contrasts with patients with hemophilia B who

rarely develop neutralizing antibodies.

Here we report the creation of a mouse hemophilia B model that

more closely approximates the situation usually observed in

patients with hemophilia B. This mouse expresses human factor IX

R333Q. We chose R333Q because data on several patients with

severe hemophilia B who exhibit this mutation have been re-ported.8 Moreover, these CRM patients have nearly normal levels

of factor IX antigen and their clotting activity is usually less than

1%. To make the mutant mouse more useful we have expressed the

human factor IX R333Q gene as the alanine form of the Ala148Thrdimorphism.9 Because the A-1 antibody binds well to factor IX

with threonine at residue 148 but weakly to the alanine isoform, the

effectiveness of gene therapy can be evaluated by expressing the

threonine isoform and detecting its presence with the A-1

antibody. We anticipate that this mouse model will be an

important tool for gene therapy and for studying the function of

mutant factor IX in vivo.

Materials and methods

Targeting vector for generating the R333Q-human

factor IX mouse

The targeting construct consisted of 1.5 kb of mouse factor IX (5

homology), 2.6 kb of human factor IX cDNA, and a 5.3-kb PacI-BamHI

fragment of mouse gDNA (3 homology region; Figure 1). This construct

was introduced into the osdupdeI plasmid vector (a gift from Dr Oliver

Smithiess laboratory, the Pathology Department, University of North

Carolina at Chapel Hill [UNCCH]) using standard recombinant DNA

techniques.10 In addition to the 5 and 3 homology regions of mouse factor

IX, the targeting construct included a positively selectable neomycin

resistance gene (neo) controlled by the pMC promoter, and a herpes

simplex virus thymidine kinase gene (HSV-tk), driven by the pGK

promoter, as a negative selection marker. The 1.5-kb 5 homology region

contained part of the first intron of mouse factor IX and sequences coding

for the propeptide and first 6 amino acids of the Gla domain of mouse factorIX. The human cDNA was joined to the mouse gene at codons 6 and 7

(human and mouse amino acid sequences are identical through amino acid 7

by creating an XhoI site without changing the amino acid sequence).

Fromthe GeneTherapyCenter and the Departments of Biology and Pediatrics,

University of North Carolina at Chapel Hill.

Submitted January 13, 2004; accepted May 4, 2004. Prepublished online as

BloodFirst Edition Paper, June 3, 2004; DOI 10.1182/blood-2004-01-0138.

Supported by National Institutes of Health grant PO1 HL 66973.

An Inside Bloodanalysis of this article appears in the front of this issue.

Reprints: Darrel W. Stafford, Department of Biology, UNC Chapel Hill, Chapel

Hill, NC 27599; e-mail:[email protected].

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked advertisement in accordance with 18 U.S.C. section 1734.

2004 by The American Society of Hematology

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Site-directed mutagenesis

Two site-specific point mutations, T148A and R333Q, were created in the

human factor IX cDNA by megaprimerpolymerase chain reaction(PCR).11,12

The entire coding region of the human factor IX sequence was determined

by the University of North Carolina-Chapel Hill (UNC-CH) Sequencing

Facility to confirm that the sequence was as expected and no extra

mutations were introduced.

Cell culture and electroporation

TC-1, an embryonic stem (ES) cell line derived from mouse strain

129/SvEvTac FBR,13 was cultured on feeder layers prepared from primary

embryonic fibroblasts, in ES cell medium (Dulbecco modified Eagle

medium-H medium from Gibco BRL [Gaithersburg, MD], with 15% fetal

calf serum, 0.1 mM 2-mercaptoethanol, 0.1 mM L-glutamine). The target-

ing vector was linearized at its unique PmeI site (Figure 1) and electropo-

rated into TC-1 cells. Electroporated cells were cultured in ES cell medium

with G418 (200 g/mL) and ganciclovir (2 M). Nine to 11 days after

electroporation, colonies resistant to G418 and ganciclovir were selected,

expanded, and screened for homologous recombination.

Detecting homologous recombinants

Successful homologous recombination was determined by PCR and

confirmed by Southern blotting. The Clontech Advantage PCR kit (BDBiosciences Clontech, Palo Alto, CA) was used for analysis; the forward

primer, 5-GCACACCCTCACTGTGCTATAACACTC-3, was complemen-

tary to intron 1 of mouse factor IX, whereas the reverse primer, 5-

GGATTGGACTCACACTGGTCACCATC-3, was complementary to hu-

man factor IX cDNA. After an initial incubation at 95C for 1 minute, 35

cycles of 95C for 30 seconds, 68C for 3 minutes, and 68C for 3 minutes

were used. The primer pair gave the expected 1.8-kb PCR product from

targeted DNA. For Southern blot analysis, gDNA from the ES cell clones

was digested with EcoRV. After EcoRVdigestion, DNA was fractionated by

electrophoresis on an 0.8% agarose gel and transferred to a Nytran Super

Charge membrane (Schleicher & Schuell BioScience, Keene, NH). The

DNA probes were designed to hybridize with a 730-bp DNA fragment of

intron 1 or a 680-bp DNA fragment of intron 4 of mouse factor IX (Figure

1). Wild-type cells showed a 17.2-kb fragment with either the 730-bp or

680-bp probe, whereas the targeted cells showed a 13.4-kb fragmentwith the 680-bp probe and a 4.7-kb fragment with the 730-bp probe.

Genotype analysis

PCR and Southern blotting were used for genotyping of tail DNA from

R333Q-human factor IX (R333Q-hFIX) candidate mice. The primer pair

(see Detecting homologous recombinants) was used to screen for

correctly targeted mice. All mice carrying the R333Q human factor IX on

one or both X chromosomes exhibited a 1.8-kb PCR band. Throughout this

report homozygous refers to females carrying 2 R333Q mutant alleles

(H/H, hemophilic female), hemizygous to males with one mutant allele(H/Y, hemophilic male), and heterozygous to females with one copy of the

normal mouse factor IX gene and one copy of R333Q human factor IX

(H/, nonhemophilic carrier female). Mice with murine wild-type factor

IX sequence, including nonhemophilic wild-type females (/), nonhemo-

philic males (/Y), and heterozygous females (as defined), exhibit a 640-bp

PCR fragment from the primer pair, 5-CACACTGGAAACAGCCCAGC-

CAGAGG-3 (forward, at intron 1), 5-GGAGTCACCTCTCTAGTTCCA-

CACTCC-3 (reverse, at intron 2). The 730-bp probe described for

Southern blotting hybridizes to a 17-kb fragment when wild-type murine

factor IX gDNAis present. The same probe reveals a 4.7-kb fragment when

human factor IX cDNA has undergone homologous recombination.

Generation of R333Q-hFIX mice

Targeted ES cells were microinjected into the blastocoele of C57BL/6blastocysts and implanted into a pseudopregnant mouse. Resulting chimeric

males were bred to C57BL/6 females to produce heterozygous (H/)

offspring. These mice were crossed with C57BL/6 males to generate

hemizygous (H/Y) mice expressing human factor IX.

RT-PCR

Total RNA was extracted from liver tissue of hemizygous mice using

TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA was synthe-

sized from 5 g total liver RNA using oligo (dT) primers and 200 units

SuperScript II reverse transcriptase (Invitrogen). The reverse transcription-

PCR (RT-PCR) products from hemizygotes were sequenced to ensure that

human factor IX had no inadvertent mutations introduced.

Quantitation of R333Q-hFIX mRNA by PCR

R333Q-hFIX mRNA levels were determined by quantitative real-time PCR

(qPCR). First-strand cDNA of 6-week-old hemizygous, homozygous,

heterozygous, and wild-type mice was synthesized by RT-PCR. The

primers used, 5-GGAAGCAGTATGTTGATGG-3 (forward) and 5-

TGGTTCACAGGACTTCTGGT-3 (reverse), were complementary to both

human and mouse factor IX. Primers 5-CACAGTGTTGTCTGGTG-

GTA-3 (forward) and 5-GACTCATCGTACTCCTGCTT-3 (reverse) for

the mouse -actin gene were used as a control. Mouse factor IX cDNA was

diluted in series to construct a standard curve. The DyNAmo SYBR Green

qPCR Kit (MJ Research, Boston, MA) was used for qPCR, which was

performed using the DNA Engine Opticon 2 System (MJ Research) and

analyzed according to the manufacturers instructions. Conditions for qPCR

were 95C for 5 minutes, followed by 45 cycles (95C for 10 seconds, 60C

for 20 seconds, 72C for 20 seconds). For each cycle, fluorescence signalswere measured at 72C, 78C, and 84C. For Tm (dl/dT max) measure-

ments, the initial temperature was 55C and the final temperature was

90C. A temperature increment of 0.2C and a hold time of 10 seconds

were set during the melting curve step. Interestingly, there was sufficient

difference in the GC composition of the mouse and human factor IX

cDNAs that differences in Tm were readily observed.

Additional animal care and experiments

C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor,

ME); factor IX knockout mice (FIXKO) were described by our group1 and

were bred in house. FIXKO mice are maintained in colonies of homozygous

knockout (hemophilic) females and hemizygous hemophilic male mice.

The founder mice for the colony were originally backcrossed 4 to 5

generations from the 129 strain background into the C57BL/6 background,so are in a mixed 129/C57BL/6 background. R333Q-hFIX missense mutant

Figure 1. Strategy for targeting the mouse factor IX gene by homologous

recombination. (Top) Endogenous gene locus. Exons 1 to 5 of the endogenous

mouse factor IX gene are shown. (Middle) Targeting construct. The construct

linearized with PmeI and used for homologous recombination is shown. Recombina-

tion between the5 (1.5 kb)and3 (5.3kb) regionsof homologyis depicted. ThePmeI

site is within the plasmid vector, 29 nucleotides from the BamHI site. The human

factor IX cDNA is depicted in black. (Bottom) Targeted gene locus. The mouse factor

IX locus after homologous recombination is shown. The regions from which the

730-bp and 680-bp probes were generated are shown. After EcoRV cleavage of

mouse gDNA, the 730-bp probe gives a 4.7-kb fragment and the 680-bp probe a13.4-kb fragment when homologous recombination has occurred. Mouse gDNA is

shown as a thick line and the inserted neo sequences as a thinner line. B indicates

BamHI; P, PacI.

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mice were generated as described (see Generation of R333Q-hFIX mice).

R333Q-hFIX mice that received gene therapy were F3-F4 generation. All

mice were maintained according to the guidelines of the UNC-CH Animal

Care and Use Committee. Mice were anesthetized using inhaled isoflurane

for all procedures. For gene therapy, 100 L phosphate-buffered saline

(PBS) containing 1 1011rAAV2/hFIX dot blot genomes were injected

into the gastrocnemius muscle (50 L for each hind limb) in 6- to

8-week-old FIXKO mice or 8- to 10-week-old R333Q-hFIX mice. Mice

weighed 20 to 25 g at the time of treatment. Scheduled blood samples were

collected from the retro-orbital plexus and the plasma was stored at 80C

for future enzyme-linked immunosorbent (ELISA) and activated partial

thromboplastin time (APTT) assays.

Determination of hFIX and R333Q-hFIX concentration by ELISA

Plasma samples were collected from(1) 6-week-old untreated homozygous,

hemizygous, heterozygous, and wild-type mice, or from (2) mice following

gene therapy with a factor IX expression vector at the indicated time points.

Initial characterization of the R333Q-hFIX mouse used a sandwich ELISA

using a pair of polyclonal antibodies, as described. 14 The capture antibody,

rabbit antihuman factor IX polyclonal antibody (Dako, Carpinteria, CA)

was diluted at 1:1200 with carbonate buffer. The detecting antibody was a

sheep antihuman factor IX polyclonal antibody SAFIX-APHRP (AffinityBiologicals, Ancaster ON, Canada), which detects factor IX carrying either

of the common polymorphisms at amino acid 148 (Thr148 or Ala148).

To preferentially detect the Thr148 variant of human factor IX, a mouse

antihuman factor IX monoclonal antibody, A-1,15,16 conjugated with

horseradish peroxidase (HRP) was used as the detecting antibody in the

ELISA. The A-1 antibody was originally prepared and characterized by

K. J. Smith with the author (D.W.S.)15,16 and was kindly supplied for the

current studies by A. R. Thomson (University of Washington, Seattle, WA).

The A-1 antibody was conjugated with HRP using an EZ-link Plus activated

peroxidase kit (Pierce, Rockford, IL). The HRP-conjugatedA-1 (A1-HRP, 2

mg/mL) was diluted 1:10 000. Factor IX antigen levels were calculated for

each of the 2 ELISA systems using a human factor IX standard curve

generated from purified recombinant human factor IX. The recombinant

Thr148 factor IX17 was produced in human embryonic kidney 293 cells and

purified using batch adsorption to Q Sepharose as previously described 18,19

Factor IXspecific APTT

Clotting activities of C57BL/6 wild-type, FIXKO, and R333Q-hFIX mice

were measured using citrated plasma collected from the retro-orbital

venous plexus. Untreated mice were sampled at 6 to 8 weeks of age. The

START 4 Coagulation Analyzer (Diagnostica Stago, Asnieres, France) was

used to measure clotting times. Pooled normal human reference plasma

(FACT, George King Biomedical, Overland Park, KS) was serially diluted

with human factor IXdeficient plasma to create APTT standard curves. A

uniform amount of mouse factor IXdeficient plasma was added to each of

the standards to approximate the mouse plasma protein present in the

samples. Each standard and each mouse plasma sample was further diluted

10-fold with diluting buffer (Owen-Koller, Diagnostica Stago). The mixture

of diluted mouse plasma (50 L), human factor IXdeficient plasma (50

L; George King Biomedical), and cephalin/silica activator (50 L;

STAPTT Automate 5, Diagnostica Stago) was incubated at 37C for 3

minutes. Then 50 L 25 mM CaCl2 was added, the clotting time recorded,

and the percentage human factor IX activity calculated from the APTT

standard curve.

In vivo pharmacokinetic studies

Four micrograms recombinant wild-type human factor IX (Thr148 isotype)

was injected into the left jugular vein of wild-type, FIXKO, and R333Q-

hFIX mice (body weight, 25 g; age, 8-10 weeks). Then, 15 L blood from a

tail clip was collected into heparinized capillary tubes at 0.5, 1, 2, 4, 6, 8, 10,

and 15 minutes after injection. The exogenous human factor IX level in the

plasma was measured by the monoclonal antibody A-1 ELISA with arecombinant human factor IX standard.

AAV2 vector construction and rAAV2 production

The plasmid recombinant adeno-associated virus 2 (rAAV2)CBA-hFIX

was generated using standard recombinant DNA techniques10 and contains

a PCR-amplified 1417-bp human FIX cDNA without introns or 5and 3

end untranslated region sequence. Inserted immediately upstream of the

human FIX cDNA is the cytomegalovirus/chicken-actin (CBA) promoter,

digested as an 1869-bp BglII/EcoRI fragment from the plasmid CMV--

actin-hAAT-AAV (a gift from Terence R. Flotte, Biochemistry Department,UNC-CH).20,21 A human -globin/IgG chimeric intron from pCL-neo

vector (Promega, Madison, WI) and a 180-bp murine -globin polyadenyl-

ation signal from PHIH-polyA (a gift from W. Marzluff)22 were added

downstream of the hFIX cDNA. Finally, a 630-bp fragment of noncoding

phage DNA (New England BioLabs, Beverly, MA) was inserted between

the polyadenylation signal and the downstream AAV terminal repeat to

yield an AAV2 expression cassette of the approximate size of the wild-type

AAV genome. The expression cassette is flanked by AAV serotype-2

inverted terminal repeats (ITRs). Recombinant AAV2-CBA-hFIX was

produced, purified, and titered by dot blot assay for vector genome copy

number at the UNC Virus Vector Core Facility using previously described

methods.23

FIX Bethesda inhibitor assay

Inhibitor antibodies against human factor IX were measured by Bethesda

assay. All mouse plasma samples were heated at 55C for 30 minutes to

inactivate endogenous mouse clotting factor or high levels of human factor

IX. The inactivated plasma samples (undiluted or diluted to 1:32 in

heat-treated FIXKO mouse plasma) were incubated with an equal volume

of pooled normal human plasma (George King Biomedical) at 37C for 2

hours and clotting activity assayed by APTT (see Factor IXspecific

APTT). Each Bethesda unit corresponds to the neutralization of 50% of the

factor IX clotting activity in standard normal plasma.

Results

Generation of R333Q-hFIX mice

The targeting construct illustrated in Figure 1 was used to replace

the mouse factor IX gene with human factor IX R333Q cDNA by

homologous recombination. The construct used the signal se-

quence, first intron, and propeptide of mouse factor IX. Approxi-

mately 600 colonies, resistant to both G418 and ganciclovir, were

screened by PCR. One clone, positive by PCR, was confirmed by

Southern blot. The positive clone was microinjected into blasto-

cysts and 8 chimeras (6 male, 2 female) were generated. Two of the

6 male chimeric mice transmitted the ES cell genome to their F1

offspring (indicated by their agouti coat color). The agouti F1 mice

Figure 2. Southern blot analysis of tail DNA from wild-type mice and mice

homozygous (H/H), hemizygous (H/Y), heterozygous (H/) for the R333Q-hFIX

mutation. Tail DNA was digested with EcoRV and hybridized to the 730-bp probe.

Lanes 1 and 7 show a 17.2-kb band from a wild-type female (WT nonhemophilic) and

male (WTnonhemophilic)mouse, respectively. Lanes 3 to 5 show a 4.7-kbbandfrom

female R333Q-hFIX mice (H/H, homozygous hemophilic females) and lanes 6 and 8

also show the 4.7-kb band from male mice that are hemizygous for the mutation (H/Y,

hemizygous hemophilic males). Lane 2 shows 4.7-kb and 17.2-kb bands from theheterozygous mouse (H/ nonhemophilic carrier female).

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are heterozygous for the targeted R333Q-hFIX gene (and the

normal mouse gene) because the human factor IX gene is located

on the X chromosome and the TC-1 ES cells used for electropora-

tion were derived from male mice. Hemizygous R333Q-hFIX mice

were created by backcrossing heterozygous mice to C57BL/6 male

mice. Mice homozygous for human R333Q factor IX were

generated by crossing the F2 heterozygous mice with hemizygous

mice. The genotypes of all mice were confirmed by both PCR (data

not shown) and Southern blot (Figure 2) analysis.

Phenotype of R333Q-hFIX mice

The R333Q-hFIX mice are not morphologically different from

wild-type mice. They survive well if no injury occurs. Litters have

4 to 8 pups with a sex ratio of approximately 1:1. When tails of theR333Q-hFIX mice are cut, they bleed and die unless the wound is

cauterized.

Clearance of human factor IX from wild-type, FIXKO,

and R333Q-hFIX mice

The initial clearance of hFIX from wild-type, FIXKO, and

R333Q-hFIX mice is illustrated in Figure 3. Wild-type mice and

mice expressing R333Q-hFIX showed essentially identical clear-

ance in the first 15 minutes after injection but infused FIX

disappears more rapidly from the knockout mouse.

mRNA and antigen levels of R333Q-hFIX in heterozygous,

hemizygous, and homozygous mice

The amount of mRNA was determined by quantitative PCR as

described in Materials and methods. The level of human

factor IX mRNA in hemizygous and homozygous mice is 2- and2.5-fold lower than that of mouse factor IX in wild-type mice,

whereas the combined level of human and mouse factor IX

mRNA in heterozygous mice is similar to that of factor IX in

wild-type mice (Figure 4).

The antigen level of circulating R333Q-hFIX in mice that were

homozygous, hemizygous, and heterozygous for the R333Q-hFIX

mutation as well as in wild-type (nonhemophilic) mice was

measured by ELISA using paired anti-hFIX polyclonal antibodies.

These antibodies do not cross-react with the endogenous mouse

factor IX because the ELISA detects no antigen in wild-type mice

(Figure 5). Figure 5 also shows that the levels of R333Q-hFIX vary

depending on the genotype. The levels are homozygous, 1.1 0.1

g/mL; hemizygous, 1.3 0.1g/mL; and heterozygous 0.3 0.1g/mL. Factor IX levels are about 0.8 g/mL in the juvenile

R333Q-hFIX mouse and rise to a stable level of approximately 1.2

g/mL in adulthood (Figure 6).

A well-characterized Ala/Thr polymorphism occurs at residue

148 within the activation peptide of human factor IX.9 The

polyclonal antibodies used above recognize both the alanine and

threonine forms. However, the monoclonal antibody A-1 recog-

nizes the threonine isoform preferentially.24 In the original descrip-

tion of A-1 antibody immunoassay of normal human clinical

samples the factor IX levels (background) of the Ala148 isoform

Figure4. Comparisonof mRNAlevelsof R333Q-hFIX and wild-typemice. mRNA

level was measured by qPCR. The results are given as percentage of wild-type

mouse mRNA. Homozygotes (H/H) represent 39%, hemizygotes (H/Y) 48%, and

heterozygotes (H/) 87% of wild-type (WT) mRNA levels. Homozygous refers to the

R333Qmouseandheterozygous refersto a mouse with copiesof humanR333Qandwild-type mouse factor IX. Data represent the mean SD.

Figure 3. Clearance of infused human factor IX. Mice (n 3) producing wild-type

factor IX (F, gray broken line, error bars up only), R333Q-hFIX (, solid line, error

bars down only), or no factor IX (knockout; f, error bars down) were injected at zero

time with 4 g recombinant human factor IX via the jugular vein. Samples were

collected from the tail vein at serial time points for 15 minutes after infusion, and the

survival of the infused Thr148 isoform factor IX was determined by A-1 antibody

ELISA. The concentration at the first time point (30 seconds) is defined as 100%.

Figure 5. Expression of R333Q-hFIX. The concentrations of R333Q-hFIX in

homozygous (H/H), hemizygous (H/Y), heterozygous (H/), andwild-type(WT) mice

are shown in g/mL as assayed by ELISA using paired anti-hFIX polyclonal

antibodies. The levels are homozygous, 1.1 0.1 g/mL (n 4); hemizygous,

1.3 0.1 g/mL (n 3); heterozygous, 0.3 0.1 g/mL (n 6). Wild-type mouse

factor IX is not detectable with this antibody. Data represent the mean SD.

Figure 6. The alanine polymorphism is weakly detected by monoclonal

antibody A-1. All mice in the figure are naive (untreated) mice. The left side of the

figure shows that neither the polyclonal antibody (poly) nor monoclonal antibody A-1

demonstrates background factor IX reactivity in the knockout mouse. In the right side

of the figure the first bar shows the level of factor IX detected by the polyclonal

antibodyin juvenile R333Q-hFIX mice(age 4 weeks).The second barshows thelevel

detected by the polyclonal antibody in adult mice (age 6 weeks). The third bar

demonstrates the low cross-reactivity of A-1 for the alanine polymorphism of the

factorIX expressedin theR333Q-hFIX mice (age 6 weeks). Data representthe mean SD.



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were 1% to 10% of the levels assayed with a factor IX polyclonal

antibody.15 Our values are consistent with these earlier measure-

ments. Figure 6 shows that the low degree of cross-reactivity

between the A-1 antibody and the alanine isoform of factor IX

carried by untreated R333Q-hFIX mice is consistent with the

experience from human samples.

Functional clotting activity of R333Q-hFIX mouse

In clinical practice, severe hemophilia B is defined by a factor IX

activity level of less than 1%, which is considered the lower limit of

reproducible detection of the APTT assay. We modified the factor

IXspecific APTT to detect human factor IX activity in mouse

plasma samples using human factor IXdeficient plasma. Human

plasma standards were diluted in mouse factor IXdeficient plasma

to provide mouse plasma proteins in both the standards and the

samples. The clotting activity of wild-type C57BL/6 mice was less

than 100% in this assay system, suggesting that mouse factor IX is

less active in the human coagulation system than is human factor

IX. The measured activity levels of 0.2% in FIXKO mice were

below the level of reliable quantification ( 1%). It should be

noted that we originally described an APTT factor IX activity levelof 8% for the FIXKO mouse.1 Since that time it has become

obvious that the relative amount of mouse plasmaeven plasma

that is completely deficient in factor IX proteinstrongly affects

the clotting of the human factor IXdeficient plasma in the APTT

assay. The normal APTT of the mouse is markedly shorter than that

in humans.25,26 Including mouse factor IXdeficient plasma in the

standards reduces this artifact and confirms that the plasma of

FIXKO mice does not shorten the clotting time of human factor

IXdeficient plasma. R333Q-hFIX mice had slightly shortened

APTT clotting times when compared to FIXKO mice (122 9

seconds for FIXKO versus 104 8 seconds for R333Q-hFIX

mice); each value corresponds to a factor IX activity of less than

1% (Table 1).

R333Q-hFIX mouse model for hemophilia B gene therapy: lack

of anti-hFIX inhibitor antibody response following hFIX

expression

The value of the R333Q-hFIX mouse as a model for gene therapy

of human CRM factor IX deficiency was assessed by intramuscu-

lar injection of an adeno-associated virus serotype 2 vector

expressing the human factor IX cDNA. A relatively low dose of

1 1011 vector genomes was chosen for 2 reasons. First, doses in

this range have been associated with the development of antibodies

against the human factor IX xenoprotein in several muscle-directed

AAV2 vector studies in wild-type C57BL/6,7,27 wild-type BALB/c,7

wild-type Fv129,7 and FIXKO mice (C57BL/6/129 background).5,28

Second, it was desirable to determine whether even relatively small

degrees of expression from a gene therapy vector could be detected

in the background of this novel CRM

model. Following intramus-cular injection of the rAAV2-CBA-hFIX vector, no human factor

IX could be detected in FIXKO mice before or after infection

(Figure 7 lower line). Nevertheless, human factor IX was clearly

made in response to gene therapy in the FIXKO mice, because

inhibitor antibodies against human factor IX protein were detect-

able by 4 weeks after treatment (Table 2). These inhibitor antibody

titers continued to increase for 3 months following intramuscular

gene therapy, and in 4 of 5 FIXKO mice, the titer rose to more than

5 Bethesda inhibitor units. A Bethesda inhibitor titer of more than 5

defines a clinically high titer inhibitor antibody.29

Gene therapy of R333Q-hFIX mice treated in exactly the same

manner as the FIXKO mice, however, led to an increase in both the

levels of FIX antigen (Figure 7 upper line) and activity (Table 1).

The initial time point of the upper line in Figure 7 shows arelatively high background only because of the low level of

cross-reactivity of the factor IX threonine-specific A-1 antibody

with the factor IX Ala148 isoform of human factor IX that is

expressed in R333Q-hFIX mice. The A-1 antibody assay detects a

concentration of human factor IX of 73 12 ng/mL in the

R333Q-hFIX mice, whereas the actual factor IX level, assayed by a

polyclonal antibody, was 1.2 g/mL. That is, the background

antigen in these experiments was 6%, whereas the background

activity was less than 1%. Furthermore, unlike the knockout

mouse, and consistent with our hypothesis that mice with human

factor IX carrying a missense mutation would exhibit tolerance to

factor IX gene therapy, R333Q-hFIX mice did not develop

antifactor IX inhibitors following intramuscular injection of rAAV2-CBA-hFIX (Table 2).

Table 1. Human factor IX-specific APTT assay of FIXKOand R333Q-hFIX mouse with and without gene therapy

Average factor IX

activity SD, % Range of activities, %

W il d- ty pe , un tr ea te d, C 57 BL/ 6 3 0 8 14-45

FIXKO, untreated 0.2 0.2 0.0-0.7

R333Q-hFIX, untreated 0.9 0.9 0.1-3.2

R333Q-hFIX, 4 mo after treatment 6.6 7.8 0.8-19.8

FIXKO, 4 mo after treatment 0.8 1.0 0.1-2.6

The ability of mouse samples to initiate clotting in human factor IXdeficient

plasma is measured by shortening of the APTT clotting time. Clotting times from

dilutions of human reference plasma are used to derive a standard curve for

calculation of percent human factor IX activity. Mice treated with gene therapy were

tested 4 months after intramuscular administration of rAAV2-CBA-hFIX. Populations:

wild-type, untreated, C57BL/6, n 13; FIXKO, untreated, n 15; R333Q-hFIX,

untreated, n 14; R333Q-hFIX, 4 months after treatment, n 6; and FIXKO, 4months after treatment, n 5.

Figure 7. hFIX expression following gene therapy. Human factor IX in FIXKO

(n 5) and R333Q-hFIX (n 6) mice before and after intramuscular rAAV2-CBA-

hFIX gene therapy, as detected by immunoassay with monoclonal antibody A-1

(specific for the factor IX Thr148 polymorphism). The Thr148 factor IX is incorporated

in the gene therapy vector but is not present in the genome of either strain of mice.

Data represent the mean SD.

Table 2. Antibody formation indicated by the Bethesdainhibitor assay

Mouse Pretreatment 2 wk 4 wk 6 wk 8 wk 10 wk 12 wk

FIXKO no. 1 0 0 2.8 1.7 1.1 2.3 2.5

FIXKO no. 2 0 0 1.5 16.0 16.0 31.2 34.4

FIXKO no. 3 0 0 1.7 9.0 12.8 8.6 16.5

FIXKO no. 4 0 0 2.6 6.6 6.8 4.9 6.0

FIXKO no. 5 0 0 1.0 9.6 17.2 12.0 12.8

All R333Q, n 6 0 0 0 0 0 0 0

Plasma from gene therapytreated mice was incubated with an equal volume of

pooled normal human plasma assayed byAPTT. Numbersgiven in Bethesdainhibitor

units represent the dilution of sample plasma that results in inactivation of 50% of thefactor IX activity of the normal plasma.



6/7

Discussion

The goal of this study was to produce a mouse model that more

accurately reflects the condition usually observed in human hemo-

philia B than do the currently available factor IX knockout mice. In

human factor IX deficiency, lack of expressed factor IX correlates

with an increased risk of inhibitor antibody development. For

example, 17 patients with gross deletions and 12 with early stop

mutations are known to have developed inhibitor antibodies. Of the

41 patients with inhibitory antibodies whose mutations are reported

in the human hemophilia B database, (http://www.kcl.ac.uk/ip/

petergreen/haemBdatabase.html), only 2 have missense mutations.

Missense mutations, however, represent two thirds of the mutations

recorded in the human hemophilia B database,8 and in accordance

with this, the risk of inhibitor antibody formation to human factor

IX is low, about 2% to 5%. A similar situation appears to hold true

for canine hemophilia B. Dogs that express no antigen develop

inhibitor antibodies in response to infused purified canine factor IX

or to canine factor IX delivered by intramuscular gene therapy. 30 In

contrast, canine factor IX gene therapy is generally tolerated indogs with a missense mutation.4,31,32 With specific exceptions, for

example, liver-directed therapy,7,32 the FIXKO mice develop

antibodies subsequent to therapy.5,7,33-35 Because mice are less

expensive to maintain, have shorter gestational periods, and are

easier to breed than dogs, a mouse model that more nearly mimics

the human disease is desirable. Therefore, because 52 individuals

with this mutation have been documented in the Hemophilia B

Mutation Database, we made a mouse model expressing human

factor IX with the missense mutation R333Q. Twenty-three of 43

patients with this mutation have reported factor IX activity levels

less than or equal to 1%, and all but one have reported activity of

less than 5% (the higher values probably represent variability in

assay reproducibility among clinical laboratories). Moreover, all

have factor IX antigen levels more than 30%. Again, most patients

have normal levels and it is likely that the lower reported levels are

the results of laboratory variability.

We confirmed by Southern blot analysis and mRNA and human

factor IX protein determination that human R333Q hFIX replaced

mouse factor IX. Our primers for qPCR were designed to recognize

both mouse and human mRNA. Using these primers, wild-type and

heterozygous females (chimeric for the murine and human se-

quences) had similar amounts of factor IX mRNA. However, the

level of factor IX mRNA was reduced by half in homozygous

R333Q-hFIX females and hemizygous males when compared to

wild-type C57BL/6 mice. The R333Q-hFIX gene includes mouse

intron 1 followed by the cDNA coding for the R333Q-hFIX, and

mRNA processing appears to be less efficient than wild-typeprocessing. The presence of human factor IX in the plasma of the

R333Q-hFIX mouse was confirmed with a variety of monoclonal

and polyclonal anti-FIX antibodies, all specific for the human

protein. R333Q-hFIX antigen in the hemostatically normal heterozy-

gous female is lower than in the hemizygous or homozygous

mouse. This decrease in protein correlates with the reduction of

human mRNA in the heterozygous mice. Wild-type mouse factor

IX is not detectable by the antihuman factor IX antibodies used in

this study.

The antigen level, expressed as g/mL, of human R333Q factor

IX is lower in the mouse than in humans with this mutation. As

mentioned, one explanation may be that the human mRNA with

only one intron is not as efficiently processed as the gene with itsnormal complement of introns. Alternatively, our finding of lower

factor IX in the R333Q-hFIX mouse is consistent with the report of

Mingozzi et al that the normal level of factor IX in mouse plasma is

2.5 g/mL, which is half the normal level of human factor IX.36 It

is possible that less mRNA is made from the mouse FIX promoter

than from the human promoter. Therefore, expression of the

R333Q-hFIX under the control of normal mouse transcriptional

regulatory factors may result in a circulating factor IX level that is

most physiologically relevant for a mouse model of hemophilia. In

any case, the hFIX activity level measured in the R333Q mouse is

less than 1% and, therefore, consistent with that expected from the

human phenotype.

Although factor IX has a relatively long half-life in the

circulation, exogenous factor IX undergoes significant clearance

immediately after infusion in patients with hemophilia B.37 Rapid

distribution from the circulation involves binding to vascular

endothelial cell collagen IV, among other sites, and we have shown

that mutant factor IX molecules lacking collagen IV binding have

decreased initial clearance from circulation of hemophilia B

knockout mice.14,38 It has been reported that the recovery of infused

factor IX in patients with CRM hemophilia B is higher than in

those who are CRM

.39

Consistent with this observation, the initialclearance of infused human factor IX in the CRM R333Q-hFIX

mice is identical to that of wild-type mice with normal circulating

mouse factor IX levels. On the other hand, clearance from the

FIXKO mice that have no circulating factor IX antigen is faster.

However, although there is a measurable difference in clearance, it

is small. Thus, either the number of collagen IVbinding sites is

very large or there are other significant binding sites for factor IX.

The development of inhibitor antibodies is an infrequent but

important complication of current human factor IX protein replace-

ment therapy and frequently complicates animal studies of hemo-

philia gene therapy. Our hypothesis is that gene therapy with

human factor IX in the mouse expressing defective human factor

IX will not elicit inhibitor antibodies. To test this hypothesis, we

challenged the R333Q-hFIX mouse with an AAV vector expressing

functional human factor IX. We chose a vector dose and route of

injection that numerous studies had shown were most likely to

stimulate the production of inhibitory antibodies in FIXKO

mice.5,7,34 Subsequent to gene therapy of R333Q-hFIX mice with

the threonine isoform of normal human factor IX an increase in

both antigen and activity levels of factor IX are observed. The

increase measured by APTT is 6%, whereas the increase measured

by immunoassay is equivalent to 1% of the normal human factor IX

(and 2% of the normal mouse FIX level). Our experience has been

that results from the APTT can appear artifactually high when

assaying factor IX activity of one species mixed with plasma

proteins of another species (P.E.M., T.G., and J. R. Elia, manuscript

in preparation). Nevertheless, for the purposes of characterizinghuman factor IX tolerance in the R333Q-hFIX mouse, the rAAV

dose chosen led to low levels of factor IX produced by gene

therapy. The expression of human factor IX resulted in inhibitory

antibody formation in only the factor IX knockout mice and not in

mice expressing R333Q-hFIX. This observation is consistent with

the outcomes of 2 human clinical trials of AAV human factor IX

gene therapy, in which none of the 14 patients with hemophilia B

(all expressing missense mutations) developed inhibitory

antibodies.40,41

The R333Q-hFIX will be a valuable research resource, espe-

cially for studies in which large numbers of animals are required

(making the use of missense hemophilic dogs impractical). Never-

theless, the use of any animal model to predict antibody responsesin human therapeutic approaches requires caution. Other factors in



7/7

addition to gene defect clearly contribute to the development of

inhibitor antibodies in the setting of coagulation protein replace-

ment. Among these are the individuals immune background (eg,

HLA type) and local or systemic inflammation/illness concomitant

with factor VIII or IX challenge.

In summary, this study describes the generation of a CRM

mouse model of human hemophilia B arising from the expression

of a missense mutant human factor IX gene (R333Q-hFIX). Asexpected, these mice express antigenically detectable but function-

ally deficient human factor IX protein. Furthermore, when infected

with AAV expressing human factor IX, mice expressing defective

human factor IX, as contrasted with FIXKO mice, did not produce

neutralizing antibodies. This suggests that the presence of a

functionally inactive human factor IX present throughout the

development of the mouse results in relative immunologic nonre-

sponsiveness to a human factor IX challenge in the adult R333Q

mouse. Therefore, the R333Q-hFIX model will complement the

available FIXKO model for preclinical studies of factor IX.

Acknowledgments

The authors acknowledge Tomoko Hatada, Hui-Ju Lee, and the

Smithies-Maeda Laboratory for their generous help and discus-

sions and David Straight for reading and discussing the

manuscript.

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