bio-science

C

Gene therapy for hemophilia

Katherine P. Ponder

Purpose of review

This review will highlight the progress achieved in the past

2 years on using gene therapy to treat hemophilia in animals

and humans.

Recent findings

There has been substantial progress in using gene therapy

to treat animals with hemophilia. Novel approaches for

hemophilia A in mice include expression of Factor VIII in

blood cells or platelets derived from ex-vivo transduced

hematopoietic stem cells, or in-vivo transfer of transposons

expressing Factor VIII into endothelial cells or hepatocytes.

Advances in large-animal models include the demonstration

that neonatal administration of a retroviral vector expressing

canine Factor VIII completely corrected hemophilia A in

dogs, and that double-stranded adeno-associated virus

vectors resulted in expression of Factor IX that is 28-fold

that obtained using single-stranded adeno-associated virus

vectors. In humans, one hemophilia B patient achieved 10%

of normal activity after liver-directed gene therapy with a

single-stranded adeno-associated virus vector expressing

human Factor IX. Expression fell at 1 month, however, which

was likely due to an immune response to the modified cells.

Summary

Gene therapy has been successful in a patient with

hemophilia B, but expression was unstable due to an

immune response. Abrogating immune responses is the

next major hurdle for achieving long-lasting gene therapy.

Keywords

adeno-associated virus vector (AAV vector), adenoviral

vector, gene therapy, hemophilia, retroviral vector

Curr Opin Hematol 13:301 – 307. � 2006 Lippincott Williams & Wilkins.

Departments of Internal Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA

Correspondence to Katherine P. Ponder, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA Tel: +1 314 362 5188; fax: +1 314 362 8813; e-mail: [email protected]

Current Opinion in Hematology 2006, 13:301 – 307

Abbreviations

AAV a

opy

deno-associated virus

FIX F

actor IX

FVIII F

actor VIII

HSC h

ematopoietic stem cells

� 2006 Lippincott Williams & Wilkins 1065-6251

right © Lippincott Williams & Wilkins. Unauth

Introduction This review will summarize the major advances made

over the past 2 years in the use of gene therapy to treat

hemophilia. A brief introduction of hemophilia, gene

therapy vectors, and target organs will be given. New

approaches that have been evaluated in mouse models for

the treatment of hemophilia A or B will follow. Since

success in mouse models does not necessarily translate

into large animals, a summary of progress in the use of

gene transfer in large animal models will be provided.

The status of the liver-directed adeno-associated virus

(AAV) vector-mediated gene therapy trial in humans with

hemophilia B will be presented. Finally, concerns about

the risks of gene therapy will be discussed.

Overview of hemophilia and gene therapy Hemophilia is due to a deficiency in a coagulation factor

that results in the inability of the blood to clot efficiently.

Hemophilia A is due to Factor VIII (FVIII) deficiency

and occurs in 1 in 5000 males, while hemophilia B is due

to Factor IX (FIX) deficiency and occurs in 1 in 30 000

males [1]. Hemophilia can also be due to very rare

autosomal recessive deficiencies of other coagulation

factors. Patients with hemophilia experience spon-

taneous bleeding into joints, soft tissues, and other sites.

Hemophilia is treated with intravenous infusion of the

appropriate coagulation factor. Purified preparations of

FVIII and FIX are readily available in developed

countries, although access is limited in developing

countries. Purified factors are not available for the treat-

ment of the rare hemophilias, and so prothrombin com-

plex concentrates or plasma, which have a higher risk of

viral transmission or other adverse effects, have to be

used to treat bleeding episodes. Inhibitors are antibodies

that block the function of a coagulation factor and make

bleeding episodes difficult to treat [2,3].

For hemophilia, gene therapy usually involves transfer of

a wild-type or minimally modified gene into cells in the

body, which results in secretion of a functional protein

into the blood [4 – 8]. Viral vectors contain viral proteins

that bind to receptors on the outside of cells and facilitate

efficient delivery, and are modified to contain the thera-

peutic gene. Commonly used viral vectors include retro-

viral (both gamma retroviral and lentiviral), AAV, and

adenoviral vectors. Retroviral vectors integrate into the

chromosome, which ensures stable maintenance of the

DNA, but can result in insertional mutagenesis. Although

AAV and adenoviral vectors do not usually integrate, they

orized reproduction of this article is prohibited.

301

C

302 Haemostasis and thrombosis

are generally maintained in non-dividing cells. All of

these vectors can be engineered to lack viral coding

sequences and to be replication-incompetent. Plasmid

DNA vectors are often perceived to be safer than viral

vectors, although those that integrate non-specifically can

cause insertional mutagenesis. Plasmid vectors do not

enter cells efficiently.

The most effective target cells for gene therapy for hemo-

philia have been liver and muscle. The liver is transduced

efficiently after parenteral injection, which can result in

long-lasting expression even with non-integrating vectors,

as adult hepatocytes have a low rate of turnover. The

muscle has been another popular target for gene therapy

for hemophilia, due to its accessibility via an intramuscular

injection. Limitations are difficulties in distributing the

vector throughout the muscle and the diffusion barriers

imposed for secretion into the blood. Although successful

muscle-directed gene therapy has been achieved for the

55 kDa FIX protein, it has not been reported for the

�200 kDa FVIII protein.

Progress in gene therapy for hemophilia in mouse models Mouse models are generally evaluated first, due to their

small size and low costs. A variety of vectors and

approaches have resulted in therapeutic expression of

FIX in mice. Since expression of therapeutic levels of

FVIII without inhibitor formation has been more diffi-

cult, this review will focus on progress in gene therapy for

hemophilia A.

Hematopoietic stem cells (HSC) can be re-infused into

patients after ex-vivo transduction. In addition, tolerance

can be achieved by expressing a variety of genes in blood

cells, which may involve central tolerance due to expres-

sion in the thymus. Previous attempts to perform gene

therapy in HSC did not achieve therapeutic expression

[9,10]. More recently, murine HSC were modified ex vivo

opyright © Lippincott Williams & Wilkins. Unautho

Table 1 Summary of advances in gene therapy for hemophilia A in

Age at transfer Vector Gene

Blood cells derived from HSC [11�,12�]

Adult Retroviral Human FV

Porcine F

Platelets derived from HSC [15��]

Adult Transgenic mice Human FV

Endothelial cells [16��] Newborn Plasmid with transposase

Human FV

Hepatocytes [17�] Adult Plasmid with transposase

Human FV

Hepatocytes [18��] Newborn Retroviral Canine FV

B cells [19�] Adult Retroviral A2 and C of huma

FVIII, Factor VIII; HSC, hematopoietic stem cells.

with retroviral vectors expressing human [11 � ] or porcine

[12 � ] FVIII, as summarized in Table 1. Transduced cells

were infused into mice with hemophilia A whose bone

marrow was partially ablated with irradiation or busulfan.

Recipients of retroviral vectors expressing human or

porcine FVIII achieved 25% and 100% of normal FVIII

activity respectively, without inhibitor formation, and the

former group was tolerant to human FVIII protein chal-

lenge. The porcine FVIII has a higher specific activity

than human FVIII, and might be used in humans that

have already developed inhibitors to human FVIII.

A very novel approach to achieving hemostasis despite

the presence of inhibitors is to express FVIII in a-granules

of platelets. Since platelet granules are released at the site

of injury, expression of FVIII in platelets of transgenic

mice can prevent bleeding in mice with hemophilia A

[13,14]. A recent study [15] extended this result by show-

ing that transfer of platelets from transgenic to hemophilia

A mice resulted in achievement of hemostasis in animals

with very high inhibitor titers. In addition, ex-vivo HSC

transduction with a lentiviral vector expressing human

FVIII from a platelet-specific promoter could prevent

bleeding in mice with hemophilia A (R.R. Montgomery,

Medical College of Wisconsin and Blood Research Insti-

tute, Milwaukee, WI, USA, personal communication).

This could serve as an effective treatment in patients

who have already developed inhibitors.

Another approach to treating hemophilia A is to transpose a

plasmid into the host chromosome. Endothelial cells

express von Willebrand factor, and can secrete functional

FVIII into the medium in culture. A recent study [16 ��

]

described the injection of two plasmids with the cationic

polymer polyethylenimine into newborn mice. The first

plasmid expressed the Sleeping Beauty transposase, which

can integrate a gene flanked by transposition sites into a

chromosome. The second plasmid contained the hu-

man FVIII cDNA downstream of an endothelial-specific

rized reproduction of this article is prohibited.

mouse models

Result Comments

III 25% of normal activity without inhibitors

Partial bone marrow ablation; integration

VIII 100% of normal activity without inhibitors

Partial bone marrow ablation; integration

III Achieve hemostasis even with high inhibitors

Bone marrow ablation; integration

III 10% of normal activity Toxicity of poly- ethylenimine; integration

III 10–100% of normal, but need to tolerize with neonatal FVIII protein

Hydrodynamic injection; integration

III 139% of normal activity without inhibitors

Integration

2 domains n FVIII

Reduces inhibitor formation Duration of response unclear; integration

C

Gene therapy for hemophilia Ponder 303

promoter, and was flanked by the transposition sequence.

This resulted in endothelial-specific expression and FVIII

activity that was 10% of normal, without inhibitor for-

mation. Another study used the Sleeping Beauty transpo-

sition system delivered by hydrodynamic injection to

adults to express human FVIII from a ubiquitous promoter

in the liver of mice with hemophilia A [17 � ]. This resulted

in 10–100% of normal FVIII activity and prevention of

bleeding. Since human FVIII protein is antigenic in adult

mice, animals were tolerized with a neonatal injection of

human FVIII protein.

Another study took advantage of the immaturity of the

newborn immune system to achieve tolerance to canine

FVIII in mice [18 ��

]. Newborn mice with hemophilia A

were injected intravenously with a retroviral vector

expressing canine FVIII. They achieved 139 � 22% of normal activity without inhibitor formation and did not

bleed.

A third approach for inducing tolerance to FVIII involved

transfer of a retroviral vector expressing a fusion protein

between domains of human FVIII and IgG into B-cell

blasts [19 � ], an approach that has been shown to induce

tolerance to other proteins. B-cell blasts were transduced

ex vivo with retroviral vectors expressing the C2 or the A2 domain of human FVIII in-frame with an IgG heavy-

chain backbone, and injected into hemophilia A mice.

Animals were then challenged with human FVIII protein

starting 1 week later. Prior injection of transduced B cells

reduced inhibitor titers to �1% of the value in mice that were immunized in a similar fashion, but did not receive

transduced B cells. The tolerized mice still produced

substantial levels of anti-C2 domain antibodies, however,

as determined by immunoassay, demonstrating that tol-

erance was not complete.

There have also been advances in gene therapy for

hemophilia B. AAV vectors expressing human FIX

proteins that were modified to have low affinity for the

extracellular matrix, or to have a higher specific activity,

resulted in FIX activity that was 2 – 5-fold higher after

intramuscular injection than was observed with a similar

dose of a vector expressing wild-type FIX [20 � ]. Mucosal

administration of the immunodominant peptide from

human FIX to mice with hemophilia B reduced inhibitor

formation on subsequent challenge with an AAV2 vector

expressing human FIX [21 � ], although some animals still

produced inhibitors; this approach may be difficult to

apply in humans with marked heterogeneity in their

major histocompatibility complexes.

Progress in gene transfer in large-animal models Gene therapy approaches that are effective in inbred

mice often fail in humans. This may relate to difficulties

opyright © Lippincott Williams & Wilkins. Unauth

in scaling up to larger animals, or to the biology of animals

with a longer life span. In addition, immune responses are

more potent in outbred large animals than in inbred mice,

making it likely that responses in large animals will be

more predictive for humans. For these reasons, many

investigators have evaluated gene transfer in large ani-

mals, primarily dogs and non-human primates, as sum-

marized in Table 2.

Although AAV vectors with alternative capsid proteins

are more effective than AAV2 vectors in mice, these have

been disappointing in large animals. The capsid proteins

on the outside of AAV particles bind to cell surfaces,

which facilitates entry into the cell. The first vectors that

were developed used AAV2 capsid proteins. Pseudo-

typed vectors with capsid proteins from other AAV ser-

otypes are more efficient at transducing liver in mice than

are AAV2 vectors [22,23 � ,24,25]. In contrast, neither

AAV6 nor AAV8 vectors were more effective than

AAV2 at expressing canine FVIII in dogs with hemophilia

A in one study [26 � ]. In another study [23

� ], expression of

canine FIX from an AAV8 vector was �2-fold that from an AAV2 vector in dogs with hemophilia B, while expres-

sion from AAV5 was lower than that from AAV2. Sim-

ilarly, AAV5 or AAV8 vectors expressing human FIX were

not more effective than AAV2 vectors at transducing

hepatocytes in Rhesus macaque monkeys [27 � ,28

�� ].

Although alternative capsid proteins do not improve

transduction of hepatocytes in large animals, they can

allow transduction to be achieved when anti-serotype

neutralizing antibodies are present [23 � ,26

� ,27

� ], which

is a common problem for AAV2 [29].

Utilization of double-stranded, or so-called self-comple-

mentary, AAV vectors has markedly increased the expres-

sion of FIX over that observed with single-stranded AAV

vectors in mice and non-human primates [28 ��

,30]. AAV

has a single-stranded DNA genome that is packaged as

either a negative or a positive strand, with inverted

repeats at the ends that form a hairpin. Upon entering

a cell, expression requires that the DNA be copied into

double-stranded DNA, or that negative and positive

strands from different particles anneal. A recent advance

was the development of AAV vectors with DNA that is

double-stranded at the time of gene transfer, which

results in more rapid and higher levels of expression than

vectors with single-stranded DNA. Use of a double-

stranded AAV8 vector markedly increased expression

in Rhesus macaques as compared with a single-stranded

AAV8 vector, resulting in 21% of normal human FIX

levels with a relatively low dose [28 ��

].

Therapeutic expression of FVIII in large-animal models

has been very difficult to achieve. In a recent study [18 ��

]

a retroviral vector expressing canine FVIII was injected

into newborn dogs with hemophilia A. This resulted in

orized reproduction of this article is prohibited.

C

304 Haemostasis and thrombosis

T a

b le

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w e re

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w o

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5 –

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) �

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p e r

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A A

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n o

t b

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th a n

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; s o

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it o

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C M

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opyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

C

Gene therapy for hemophilia Ponder 305

FVIII activity that was 116 � 5% of that in normal dogs, and no bleeding episodes. In contrast, the size-con-

strained AAV vectors have only achieved �5% of normal FVIII activity using a very small promoter [26

� ] or with

two vectors expressing the heavy and light chains sepa-

rately [31 � ].

Although highly deleted adenoviral vectors have gener-

ally resulted in only transient expression in large animals

[32 – 34], a recent study [35 � ] demonstrated that stable

and therapeutic expression of canine FIX could be

achieved with this vector. In this study, a very high

dose (3 � 1012 vector particles/kg) was injected into adult dogs with hemophilia B, resulting in FIX activity of 2 – 5%

of normal. Problems with this approach include the

induction of an inflammatory response, and the likeli-

hood that expression will decline further over time.

Improved methods for dissemination of an AAV vector

throughout muscle have been described. In the human

muscle-directed AAV2 gene therapy trial, up to 100

separate injections were required for a high dose of

AAV2 [36]. Regional delivery of AAV to muscle was

achieved by cannulating the femoral artery and vein to

create a closed system, and injecting an AAV2 vector and

agents that increase permeability [37 � ]. More recently,

AAV vectors were injected intravenously into a leg where

the blood flow was occluded with an external tourniquet.

This resulted in efficient transduction of muscle without

administration of a permeability agent [38], and achieved

15% of normal canine FIX levels at a dose of 3 � 1012 vec- vector genomes of an AAV2 vector/kg (V. Arruda, Chil-

dren’s Hospital of Philadelphia, Philadelphia, PA, USA,

personal communication).

Gene therapy in humans There have been five gene therapy trials for treatment of

hemophilia in humans. Ex-vivo transduction of fibro-

blasts [39] or intravenous injection of a retroviral vector

[40] for hemophilia A had at most a marginal effect on

FVIII levels, and these approaches are no longer being

pursued. Intravenous administration of a helper-depen-

dent adenoviral vector expressing FIX induced the acute-

phase response and was abandoned due to safety con-

cerns [41]. The muscle-directed AAV vector-mediated

gene therapy trial for hemophilia B did not provide

convincing evidence of expression [36], and enrolment

has been stopped.

The liver-directed AAV-vector-mediated gene therapy

trial was considered very promising, as this approach

resulted in �10% of normal FIX levels in dogs with hemophilia B [42]. Indeed, one of the patients who

received the highest dose (2 � 1012 vector genomes/kg) achieved �10% of normal activity during the first month, and had reduced factor needs [43

�� ]. The FIX activity fell

opyright © Lippincott Williams & Wilkins. Unauth

at 1 month after transduction, however, in conjunction

with an increase in liver enzymes, which was believed to

be due to a cytotoxic T-lymphocyte response directed

against AAV capsid proteins. This trial will be modified to

include immunosuppression at the time of gene transfer

(K.A. High, Children’s Hospital of Philadelphia, Phila-

delphia, PA, USA, personal communication).

Risks of gene therapy Inhibitor formation is a very important concern for gene

therapy. Although Chapel Hill dogs with a missense

mutation in their FIX gene that received liver-directed

gene therapy have generally not developed inhibitors,

some dogs that were treated with muscle-directed gene

therapy have done so [37 � ,44], suggesting that a muscle

approach may be more immunogenic than a liver

approach. On the other hand, some Rhesus macaques

have developed inhibitors to human FIX despite the use

of a liver-restricted promoter [28 ��

], and there are only 11

amino acid differences between the human and the

Rhesus macaque FIX sequences [45]. Neonatal admin-

istration of protein [17 � ] or gene [16

�� ,18

�� ] therapy

induced tolerance to human or canine FVIII in mice;

however, one out of five dogs that received neonatal gene

therapy with a human FVIII cDNA developed high-titer

inhibitors (K.P. Ponder, unpublished observation). Thus,

results in mice may not predict results in large animals

and humans. Patients have not developed inhibitors after

gene therapy, although these adults had been treated

extensively with factor without inhibitor formation, and

were likely to be at low risk. Clearly, the problem of

inhibitor development needs to be evaluated further.

A major concern for integrating vectors is the risk of

cancer from insertional mutagenesis. Although the com-

mon gamma chain used for gene therapy in patients with

X-linked severe combined immunodeficiency may have

contributed to the leukemias that developed in �20% of these patients, integration of the retroviral vector near an

oncogene also played a role [46,47]. In addition, integra-

tion within the Evi1 locus or other sites may have promoted clonal expansion of hematopoietic cells in

non-human primates [48] or in humans with chronic

granulomatous disease [49] after ex-vivo transduction

of HSC. Thus insertional mutagenesis continues to be

a very serious concern for HSC transduction.

Most studies have not reported cancers in small or large

animals that received gene therapy with a viral or

plasmid vector to the liver or muscle. One exception

is the report that neonatal intravenous injection of an

AAV2 vector resulted in liver tumors in mice with

mucopolysaccharidosis VII [50]. Another exception is

the demonstration that fetal or neonatal transfer of some

lentiviral vectors resulted in liver tumors in adults,

although administration of other integrating vectors at

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306 Haemostasis and thrombosis

the same ages was not carcinogenic. This suggests that

there was a specific oncogenic element present in the

vectors that caused cancer [51]. It will be important to

obtain long-term data in animals with therapeutic levels

of expression to assess this risk further.

There are other potential adverse effects of gene therapy.

Some studies have used HSC transduction, which

requires at least partial bone marrow ablation to achieve

engraftment, and has substantial morbidity and mortality.

The use of polyethylenimine or hydrodynamic injection

to enhance delivery of plasmids, or injection of highly-

deleted adenoviral vectors, can be toxic.

Conclusion Gene therapy continues to hold promise for the perma-

nent correction of hemophilia. A variety of approaches

have been effective in animal models, and an AAV2

vector resulted in transient expression in one patient.

Future studies need to address immunological and safety

issues.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: � of special interest �� of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 386).

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