bio-science

Gene therapy for haemophilia

Samuel L. Murphy and Katherine A. High

Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

Summary

The ultimate goal of gene therapy is the replacement of

a defective gene sequence with a corrected version to eliminate

disease for the lifetime of the patient. This challenging task is

not yet accomplished, however significant progress is evident.

An initial spate of clinical trials attempting the treatment of

haemophilia with gene transfer primarily resulted in the

demonstration of good safety profiles, but without efficacy.

Subsequent reengineering of vector plasmids and delivery

systems resulted in markedly improved outcomes in animal

models of the disease. The most recent clinical trial for the

treatment of haemophilia B with gene transfer showed

transient achievement of efficacy in the highest dose cohort

tested, but also exposed a previously hidden barrier to the

future success of these treatments. The progress and problems

of gene therapies for haemorrhagic disorders will be discussed.

This review will concentrate on approaches in or near clinical

application.

Keywords: haemophilia, gene therapy, vectors, adeno-associ-

ated virus.

Overview of gene therapy

The ambitious objective of gene therapy is to edit a defective

gene sequence in situ to achieve complete reversion of a disease

phenotype for the lifetime of the patient. In spite of recent

successes in site-specific correction of defective gene sequences,

the focus of most gene therapy strategies to date is on gene

addition rather than gene replacement (Urnov et al, 2005).

This simplified approach relies on a delivery mechanism to

provide a corrected copy of the defective gene without removal

of the error-containing genomic sequence.

While literally hundreds of animal models of disease can

now be effectively treated by gene transfer, a select few diseases

remain the primary focus of much gene therapy research.

A combination of factors including prevalence of disease,

width of therapeutic window, ability to accommodate the

corrected gene sequence in a gene transfer vector, reliability

and availability of animal models of the disease, and funding

and support from disease-specific foundations, all contribute

to the overrepresentation of these few diseases.

Haemophilia A and B are among the most extensively

researched diseases in the field of gene therapy. Small and

large animal models of both diseases are available for

preclinical testing. Importantly, treatment of the disease can

be quantitatively measured through well-defined coagulation

assays, eliminating a problem that plagues gene therapy efforts

for many other disease entities. Another important aspect of

the treatment of haemophilia by gene transfer is that there is

a relatively low threshold for success. If long-term expression

of the defective coagulation factor at 2–3% of wild-type levels

could be achieved, then a substantial reduction in the clinical

manifestations of the disease would be expected (Herzog et al,

1999; Sarkar et al, 2000). Expression of greater than 30% of

the wild-type level of the defective coagulation factor would

result in a phenotypically normal patient under most

circumstances (Pollak and High, 2001), although higher levels

may be required in the face of haemostatic challenge (Plug

et al, 2006).

Another advantage of haemophilia B in the development of

gene therapy strategies is the relatively small size of F9 cDNA

(�1Æ4 kB of coding sequence). This is amenable to insertion into many different gene transfer vectors and allows the

addition of numerous transcriptional regulatory elements to

both improve and restrict transgene expression in select cell

types. F8 cDNA is much larger than that of F9 (>8 kB), and is

not as readily accommodated in gene transfer vectors. Several

strategies have been employed to overcome this difficulty.

A first step towards more efficient packaging of F8 cDNA was

deletion of the non-essential B-domain (Toole et al, 1986;

Eaton et al, 1987). A dual-vector approach, in which the Factor

VIII heavy chain and light chain are separately encoded by two

different vector genomes, is another mechanism by which the

OnlineOpen: This article is available free online at www.blackwell-synergy.com

Correspondence: Katherine A. High, William H. Bennett Professor of

Pediatrics, Investigator, Howard Hughes Medical Institute,

Department of Pediatrics, Children’s Hospital of Philadelphia, 3615

Civic Center Boulevard, Abramson Building Room 302D, Philadelphia,

PA 19104, USA. E-mail: [email protected]

Re-use of this article is permitted in accordance with the Creative

Commons Deed, Attribution 2.5, which does not permit commercial

exploitation.

review

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487 doi:10.1111/j.1365-2141.2007.06942.x

large transgene can be accommodated (Mah et al, 2003).

A third technique, one that also employs two vectors,

fragments two halves of a cDNA and tethers them through

the use of a 3¢ splice donor site in vector genome A and a 5¢ splice acceptor site in vector genome B (Yan et al, 2000;

Lai et al, 2005). In the future, improvements in vector

manufacturing may make it possible to package even the

larger F8 cDNA into the smaller gene transfer vectors without

significantly compromising yield or homogeneity of vector

preparations (Grieger & Samulski, 2005).

Early clinical trials for gene transfer treatment of haemophilia

Between 1998 and 2001 five different Phase I clinical trials were

initiated for the treatment of haemophilia by gene transfer

(Kay et al, 2000; Roth et al, 2001; Manno et al, 2003; Powell

et al, 2003). Several different gene delivery systems were used

in these trials, including a retroviral vector, an adenoviral

vector, two different adeno-associated viral vectors, and a non-

viral gene delivery method. The results from each of these trials

are discussed here with background on each of the vector

delivery systems.

Retroviruses are RNA viruses which use reverse transcrip-

tion to generate a double-stranded DNA intermediate during

replication. Replication-defective retroviral vectors also con-

tain an RNA genome that is reverse transcribed and integrated

into the host genomic DNA. Integration provides the potential

for long-term, persistent gene expression but also increases the

risk of the treatment through the potential for insertional

mutagenesis and/or insertional activation of proximal genes, as

was observed following retroviral transduction of haemato-

poietic cells in a gene therapy trial for X-linked severe

combined immunodeficiency (Hacein-Bey-Abina et al,

2003a,b; Fischer et al, 2004). Viral coding sequences are

provided in trans during the manufacture of the vector and

are not present at detectable levels in the vector itself. Many

retroviruses, including murine leukaemia virus, are incapable

of penetrating the nuclear membrane. These retroviruses and

associated derivative viral vectors can only transduce dividing

cells, requiring the natural breakdown of the nuclear mem-

brane that occurs during cell division in order to enter the

nucleus.

Given this limitation, it is not surprising that the most

successful treatment strategies for liver-directed treatment of

haemophilia using retroviral vectors devised strategies to

induce hepatocyte cell division. One example of this was the

use of a partial hepatectomy immediately preceding vector

infusion (Kay et al, 1993). In this way, hepatocytes were

induced to undergo cycling during the time of vector infusion.

This approach only yielded low levels of Factor VIII expression

(<1%), but partial correction for at least 5 months, in a canine

model of haemophilia B. An alternative approach to target

dividing hepatocytes is to infuse retroviral vectors into

neonates, whose hepatocytes are naturally undergoing rapid

cell division. This approach was successfully employed by

VandenDriessche et al (1999) to fully correct Factor VIII

deficiency in a murine model of haemophilia A. Using an

identical treatment in 13 neonatal mice, 8/13 mice demon-

strated greater than 50% of wild-type Factor VIII activity by

COAtest (chromogenic Factor VIII activity test) assay.

Long-term follow up revealed no significant loss of expression

as long as 15 months after vector treatment. This approach was

successfully extended to the canine model of haemophilia B by

Xu et al (2003), who achieved up to 3Æ5% of normal Factor IX activity levels following retroviral vector transduction of

neonatal haemophilia B dogs.

Previously conducted preclinical studies led to the initiation

of a phase I clinical trial testing a Moloney murine leukaemia

virus-based retroviral vector encoding B-domain deleted

Factor VIII for the treatment of haemophilia A (Greengard

& Jolly, 1999; Roehl et al, 2000; McCormack et al, 2001; Powell

et al, 2003). Doses ranging from 2Æ7 · 107 transducing units (TU)/kg to 4Æ4 · 108 TU/kg were tested. The treatment was well tolerated by all subjects and replication competent virus

was undetectable in all of the collected samples. Factor VIII

levels above 1% were sporadically detected but did not

correlate with the dose administered. Vector DNA was

detectable in peripheral blood mononuclear cells (PBMCs) in

4/8 subjects that received 2Æ8 · 107 TU/kg and 8/8 subjects that received 9Æ2 · 107 TU/kg up to 53 weeks after the infusion. Overall, the signs of clinical improvement following

vector infusion were modest at best. These findings were

consistent with animal studies suggesting that efficient retro-

viral transduction of hepatocytes would require higher doses

and some degree of mitotic induction.

Adenoviral vectors have been frequently used in preclinical

gene transfer studies of haemophilia. Advantages of these

double-stranded DNA vectors are high transgene expression

levels and the ability to transduce hepatocytes efficiently

in vivo. Frequently, however, expression from early generation

vectors was transient, owing to the immunogenic properties of

the adenoviral vectors themselves. In contrast to other vectors

that are devoid of viral gene sequences, early generation

adenoviral vectors encode many viral proteins in addition to

the transgene; these may contribute to the immunogenicity of

these vectors (Schagen et al, 2004). In mice, the immune

response was not sufficient to ablate the effects of the

treatment and curative levels of Factor VIII or Factor IX

could be achieved and sustained for 3–5 months in murine

models of Haemophilia A and B (Smith et al, 1993; Connelly

et al, 1996; Walter et al, 1996). In contrast, large animal

studies demonstrated that the duration of transgene expression

was significantly shorter than that seen in mice and that

hepatotoxicity was correspondingly greater (Kay et al, 1994;

Lozier et al, 1999). In a canine study, a vector dose of

2Æ4 · 1012 plaque forming units (pfu) infused into three haemophilia B dogs resulted in transient expression of Factor

IX at supraphysiological levels. These levels rapidly declined,

reaching 1% of normal by 3 weeks after vector infusion and

Review

ª 2008 The Authors 480 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487

0Æ1% 2 months postvector infusion. A comparable dose of the same vector in mice resulted in sustained levels of Factor IX

expression at 20% of normal for over 4 months (Kay et al,

1994). Further substantiating these findings was a non-human

primate study in which an adenovirus vector was used to

deliver a human Factor IX transgene. In this study, a peak of

80% wild-type Factor IX levels was achieved at the highest dose

(1 · 1011 pfu/kg) followed by a rapid decline to baseline, with human Factor IX levels undetectable by 3 weeks after vector

infusion (Lozier et al, 1999). Moreover, significant hepatotox-

icity was observed. Serum aspartate transaminase (AST) levels

rose above 500 U/ml at both the intermediate dose and the

high dose, and bilirubin levels rose above 85Æ5 lmol/l in the high dose cohort only (Lozier et al, 1999). Thrombocytopenia

was also observed in the intermediate dose and high dose

cohorts. Thrombocytopenia was subsequently linked to

decreased fibrinogen levels and increased platelet clearance

time believed to result from the hepatotoxicity of the vector

(Lozier et al, 1999; Wolins et al, 2003). Recent work by

Othman et al (2007) showed that activation of platelets occurs

following exposure to adenovirus. Activation is, in turn,

followed by upregulation of P-selectin, which is known to

promote platelet clearance (Othman et al, 2007). They also

identified a critical role for von Willebrand factor (VWF) in

the induction of thrombocytopenia following adenovirus

infusion. Using a vector dose that results in thrombocytopenia

in wild-type mice (1 · 1011 pfu), significant thrombocytope- nia was not observed in VWF knockout mice.

Further development of adenoviral vector production

systems allowed complete elimination of viral coding

sequences from the vector. The intent of this manoeuvre

was to minimize the immunogenicity of these vectors. Using

these ‘gutted’ adenoviral vectors, sustained correction of

disease phenotype was observed in haemophilia A mice

infused with 3 · 1012 vector particles (vp)/kg with no apparent hepatotoxicity. Non-human primate studies showed

some dose-dependent hepatotoxicity with the threshold

residing between 1Æ4 · 1012 vp/kg and 4Æ3 · 1012 vp/kg. A Phase I clinical trial was initiated to test the safety of an

adenoviral vector encoding human Factor VIII for the

treatment of Haemophilia A; because of a priori concerns

regarding immunogenicity, the trial was structured to mon-

itor carefully for changes in liver function tests or platelet

count. The first subject, enrolled at the lowest dose of

4Æ3 · 1010 vp/kg, experienced inflammation, fever and myal- gia upon vector infusion; these symptoms are commonly

observed with infusion of adenoviral vectors. The subject also

experienced thrombocytopenia and an elevation in serum

transaminases that peaked 7 d after infusion and returned to

baseline by 19 d postinfusion (Chuah et al, 2004). Due to

safety concerns and a perceived narrow therapeutic index, no

additional subjects were enrolled in this trial.

A third vector modality used in this first group of clinical

trials was the adeno-associated virus (AAV) vector. AAV is

a small, single-stranded DNA virus that is naturally replica-

tion-defective. It relies on functions of the gene products of

helper viruses, such as adenovirus or herpesvirus, in order to

complete its replication cycle. There are no known symptoms

or diseases associated with AAV infection. The small vector

genome results in a small coding sequence capacity for AAV

vectors, making construction of F9 vectors more straightfor-

ward than construction of those for Factor VIII (although

studies employing AAV vectors for the treatment of animal

models of haemophilia A have been successful). Despite the

fact that the vector DNA remains largely episomal (i.e. without

vector genome integration), AAV vectors can nonetheless

direct long-term expression of a transgene, if introduced into a

long-lived, postmitotic (non-dividing) target cell (Nakai et al,

2001; Song et al, 2004). Early work with AAV vectors for the

treatment of haemophilia B used muscle-targeted vectors

encoding the Factor IX transgene under the control of the

cytomegalovirus (CMV) promoter. With this construct, ther-

apeutic levels of serum Factor IX (200–350 ng/ml) were

achieved in mice (Herzog et al, 1997). Unlike other viral

vectors, transduction of muscle cells with AAV vectors resulted

in sustained expression of the transgene in immunocompetent

mice, even when a foreign transgene such as beta-galactosidase

was used (Xiao et al, 1996; Fisher et al, 1997; Monahan et al,

1998). Muscle-directed AAV vectors were subsequently shown

to achieve low but possibly clinically relevant plasma levels

(70 ng/ml) of canine Factor IX in haemophilia B dogs at a

vector dose of 8Æ5 · 1012 vp/kg. This level of expression was sustained for greater than 17 months (Herzog et al, 1999).

In 1999, a Phase I/II clinical trial was initiated to test the

safety and efficacy of intramuscularly delivered AAV vectors

encoding a CMV-driven F9 transgene for the treatment of

haemophilia B. Doses ranged from 2 · 1011 vector genomes (vg)/kg to 1Æ8 · 1012 vg/kg. The vector infusion was well tolerated in all subjects, with no adverse events related to

vector infusion. No toxicity has been observed in over 7 years

of follow-up after vector administration (Kay et al, 2000;

Manno et al, 2003). Importantly, transgene expression was

evident and sustained. Muscle biopsies taken 2 months,

10 months and >3 years after vector administration revealed

the persistence of vector genomes as determined by Southern

blot as well as local expression of Factor IX protein as shown

by immunofluorescent staining (Manno et al, 2003; Jiang

et al, 2006). This was strong evidence that the animal models

of AAV vector-mediated gene transfer were predictive of the

outcome in humans and that sustained transgene expression

was possible. The barrier to the use of this approach as a

treatment for haemophilia was the number of muscle

injections that would be required in an adult haemophilia

patient. Cell culture and animal studies previously established

a limit to the amount of correctly processed, functionally

active Factor IX protein that could be produced in muscle

cells (Arruda et al, 2001; Herzog et al, 2002). This stipulated a

requirement for a larger number of muscle cells to be

transduced by a larger vector dose, rather than the transduc-

tion of the same number of muscle cells with larger doses of

Review

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487 481

vector. Accordingly, more intramuscular injections of the

vector would be required, and the number of injections

thought to be required to reach a therapeutic effect was

deemed clinically impractical.

In addition to viral vector delivery methods, one non-viral

delivery method was also tested in a clinical trial (Roth et al,

2001). This approach, similar in some respects to previous

clinical studies conducted in China (Lu et al, 1993; Qiu et al,

1996), consisted of transplantation of F8-transduced auto-

logous fibroblasts. After isolation from a skin biopsy, patient

cells were transfected with a plasmid encoding a human F8

cDNA ex vivo and stable selection for transfectants was carried

out. Single clones were expanded and tested for Factor VIII

expression level, as well as tumorigenicity and microbial safety,

prior to reimplantation onto the omentum. Animal model

studies were promising for this approach, but data acquired

from the Phase I clinical trial showed only a modest and

temporary indication of positive effects. The treatment was,

however, well tolerated and leaves open the possibility of

future attempts using more potent expression systems for the

ex vivo transduction and selection process. An important step

in advancing this treatment modality will be the determination

of the cause for the apparent loss of expression over time.

Possible obstacles to durable transgene expression include:

senescence of the implanted cells, promoter inactivation,

fibrosis around the transplanted cells and immune responses

to the gene-modified cells.

Recent developments in gene transfer for haemophilia

Adenovirus vectors are still being pursued as a means of

obtaining long-term expression of both Factor VIII and Factor

IX. Improved liver-specific promoters and further redesign of

production methods resulted in long-term expression of

Factor VIII in canine models of haemophilia A, although

some hepatotoxicity remains evident and could complicate

translation into clinical trials (Andrews et al, 2002; Chuah

et al, 2003; Brown et al, 2004). Specifically, inter-subject

variation and a small therapeutic index make the safe use of

adenoviral vectors difficult for stable gene transfer in humans.

As a result of their inherent immunogenicity, adenoviral

vectors are now more frequently used as vaccine delivery

vehicles (Tatsis & Ertl, 2004).

New developments in the field of retroviral vectors are more

promising for application in the treatment of haematological

disorders. One of the most important innovations has been the

development of lentiviral vectors, which have several advan-

tages over the first-generation retroviral vectors. First, they are

capable of transducing non-dividing cells, making them more

suitable for transduction of, for example, hepatocytes and

haematopoietic stem cells. Second, while retroviral vectors

preferentially integrate their genomes near transcriptional start

sites, lentiviral vectors show a random integration pattern into

the open-reading frames of genes (Mitchell et al, 2004). While

this difference does not eliminate the risk of insertional

mutagenesis, it seems likely to mitigate the risk by reducing the

number of full length gene transcripts that might be activated

through vector genome insertion. Improvements in insulator

elements flanking coding sequences within the vector genome

itself further reduced the potential for undesirable insertional

activation events (Chung et al, 1997). Naldini et al showed

that the use of a liver-specific promoter in place of a CMV

promoter could alone be a determinant of stable lentiviral

transduction of hepatocytes (Follenzi et al, 2004). With

a ubiquitous CMV promoter driving expression of either

green fluorescent protein (GFP) or human Factor IX, expres-

sion was short-lived and the loss of expression was accompa-

nied by both antibody formation against the transgene and

T cell infiltrates in the liver. In contrast, use of a liver-specific

promoter resulted in long-term, stable expression of GFP or

human Factor IX in wild-type mice with expression levels of

the latter reaching 200 ng/ml. In a subsequent study, Naldini

et al also showed that the incorporation of four copies of

a microRNA target sequence in the vector genome could

selectively mark vector transcripts for destruction in cells

expressing the corresponding microRNA (Brown et al, 2006).

Using this lineage-specific suppression strategy, selective

downregulation of transgene expression in haematopoietic

cells was achieved. This vector demonstrated decreased

immunogenicity and more robust, stable transgene expression

in immunocompetent mice.

Silencing elements encoded by a lentiviral vector transcript

were also used to demonstrate therapeutic effect in a recent

study of lentiviral-mediated gene transfer for the treatment of

sickle-cell disease. Unlike many other genetic disorders, sickle

cell disease cannot be completely corrected by the addition of

a wild-type copy of the defective gene. The mutant globin

would act as a dominant negative gene product and suppress

the activity of the wild-type therapeutic globin. Similar to the

approach taken by Brown et al (2006), Samakoglu et al (2006)

developed a small interfering RNA (siRNA) element targeting

sickle-globin RNA that was inactive against the vector-encoded

therapeutic gamma-globin transgene RNA. The siRNA was

incorporated into an intron within the gamma-globin trans-

gene. Following transcription of the transgene, both expression

of the therapeutic gamma-globin and post-transcriptional

downregulation of the beta-globin (sickle) RNA levels were

observed. This experiment was conducted in haematopoietic

cells taken from human patients with sickle cell disease, and

provides proof-of-principle that this approach could be

translated into the clinic. Chang et al (2006) recently demon-

strated the potential for lentiviral transduction of haemato-

poietic stem cells for the production of Factor IX for the

treatment of haemophilia B.

Continued advances were also made in animal models of

AAV vector-mediated gene transfer for treatment of haemo-

philia. Substantial improvements in transgene expression levels

were made through the use of liver-specific promoters and

portal vein administration. Therapeutic levels of human Factor

Review

ª 2008 The Authors 482 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487

IX could be expressed and sustained in mice, dogs and non-

human primates (Snyder et al, 1997; Wang et al, 1999; Mount

et al, 2002; Nathwani et al, 2002). In wild-type mice, supra-

physiological levels of 6Æ25 lg/ml were attained in haemophilia B mice from an AAV dose of 5Æ6 · 1011 vg/mouse (�2 · 1013 vg/kg) (Wang et al, 1999). These levels were sustained without any apparent signs of diminution for over

20 weeks. Importantly, these results were scalable to larger

animal models. Haemophilia B canines treated with a vector

dose of 1–2 · 1012 vg/kg responded with expression of circu- lating canine Factor IX between 220 ng/ml and 590 ng/ml (4–

12%) (Mount et al, 2002). Non-human primates infused

through the portal vein with an AAV vector dose of

4 · 1012 vg/kg achieved human Factor IX levels of greater than 400 ng/ml (8%) which were sustained for more than

1 year (Nathwani et al, 2002). None of these studies reported

liver toxicities or abnormalities in serum chemistries.

Further excitement was generated by the discovery of new

AAV serotypes that were isolated from non-human primate

tissues by polymerase chain reaction, using primers designed

within known conserved sequences from previously identified

AAVs (Gao et al, 2002). These new serotypes possessed several

advantages over the more extensively studied, prototypical

human-derived AAV2 serotype. AAV vectors are highly

sensitive to antibody-mediated neutralization in vivo (Scallan

et al, 2006). The frequency of antibodies to these novel AAV

serotypes in the human population was greatly reduced

compared to the frequency of the population carrying

antibodies to the human derived strains of AAV (Gao et al,

2002; Scallan et al, 2006). This would mean the exclusion of

fewer patients should a treatment ultimately be developed.

Another advantage of the new serotypes is that, unlike AAV2

which must be delivered directly into the portal vein to achieve

maximal liver transduction, the novel AAV8 serotype demon-

strates equally efficient transduction of the liver in animals

whether infused directly into the portal vein or into a periph-

eral vein. This finding was initially documented in mice and

has been confirmed in subsequent non-human primate studies

(Sarkar et al, 2004; Davidoff et al, 2005; Nakai et al, 2005).

This provides a less invasive delivery method for clinical

application. Yet another advantage seen with the novel AAV8

serotypes was far superior transduction efficiency in the liver,

resulting in 10–100· greater transgene expression in mice (Gao et al, 2002; Nakai et al, 2005). In contrast to the scalability of

route of administration, however, this finding did not ascend

the evolutionary ladder. Non-human primate studies directly

comparing multiple serotypes showed an attenuated advantage

of the novel serotypes over other serotypes in stable transduc-

tion of hepatocytes (Davidoff et al, 2005; Gao et al, 2006). To

date, neither AAV2 nor any of the novel serotypes have

exhibited any immunogenicity or hepatotoxicity in animals,

either at the time of administration or at later times. In fact,

AAV vector transduction in the liver can result in the

induction of immune tolerance to the transgene product

(Mingozzi et al, 2003). This tolerance was sustained even after

a strong challenge with an adenovirus vector expressing the

identical transgene product.

The promise of animal studies using liver-directed AAV

vectors prompted a second clinical trial testing the safety and

efficacy of an AAV vector for the treatment of haemophilia B

(Manno et al, 2006). In this trial, a liver-specific promoter

was used to drive expression of the F9 transgene. Vector was

infused into the hepatic artery of haemophilia B subjects at

three different doses: 8 · 1010 vg/kg, 4 · 1011 vg/kg and 2 · 1012 vg/kg. At the low and intermediate doses, no Factor IX expression was detected during the course of study. At the

highest dose tested, 1/2 subjects (subject E) experienced a rise in

Factor IX activity levels from an undetectable level at baseline to

a peak of 11Æ2% 2 weeks after vector infusion. This level of Factor IX activity was accurately predicted by the preclinical

animal studies (Mount et al, 2002). Four weeks after vector

infusion, Factor IX activity levels remained above 10%, but at

this time AST/alanine transaminase (ALT) levels rose above

100 IU/ml from a baseline of less then 50 IU/ml. By 5 weeks

after vector infusion the Factor IX activity level declined to

6Æ1% and the AST/ALT levels increased to >200 IU/ml and >500 IU/ml, respectively. From this peak of transaminase

activity, levels slowly declined to reach baseline 14 weeks

postinfusion. Meanwhile, Factor IX activity level also receded to

baseline undetectable levels by 10 weeks postinfusion.

This unexpected sequence of events prompted a thorough

assessment of possible causes for the loss of transgene expres-

sion. A complete workup of the subject was able to rule out any

adventitious infections (e.g. hepatitis B, CMV infection, etc).

No antibody to Factor IX protein was detectable. One possible

explanation for the loss of transgene expression was a T-cell

mediated response to the transduced cells. This phenomenon

had not been observed in any animal model of AAV transduc-

tion, but unlike the animal models, humans are frequently

exposed to wild-type AAV viruses in the context of pathogens

from very early childhood (Blacklow et al, 1968, 1971). These

exposures, because they occur in the context of helper viruses,

such as adenovirus that evoke a strong immune response, may

also evoke an immune response to AAV vector proteins that

could be reactivated by AAV vector infusion. Without serial

PBMC collection, this hypothesis was difficult to test, and with

the observation confined to a single subject, the chance of a

repeat occurrence of this sequence of events was unclear.

Consequently, another subject (subject G) was enrolled at

the intermediate dose (4 · 1011 vg/kg) with a PBMC collection protocol included to allow assessment of T cell responses to

Factor IX and to AAV. The results of this analysis convincingly

demonstrated that a T-cell response to the AAV vector capsid

protein occurred following vector administration. The sub-

ject’s T-cell responses were monitored during this time by

interferon-gamma enzyme-linked immunosorbent assay

(ELISpot), using pools of 15 amino acid peptides overlapping

by 10 amino acids that spanned the entire vector capsid

sequence and the entire F9 sequence. Although T-cell

responses to the vector capsid were undetectable at baseline,

Review

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487 483

responses significantly above background were readily detected

by the first PBMC collection time point, 2 weeks after vector

infusion, and remained positive at each time point tested up to

12 weeks after vector infusion. Simultaneous ELISpot assays to

detect immune responses to the Factor IX protein were

consistently negative. In the 14 weeks following vector admin-

istration, subject G experienced a mild transaminitis (peak

AST and ALT levels of 67 IU/l and 105 IU/l respectively) with

a temporal course closely matched to that experienced by

subject E (Manno et al, 2006). As with the other two subjects

infused at this dose, no Factor IX activity levels >1% were

detected following infusion of the vector. These findings

strongly implicate a T cell response to AAV capsid in the loss

of Factor IX expression in the subject who transiently

expressed therapeutic levels of the transgene.

Epitope mapping using the results from the ELISpot assays

identified a nine amino acid peptide that appeared to be an

immunodominant epitope in the anti-capsid T-cell response of

subject G. This knowledge was used to generate a pentamer

reagent that enabled direct detection of the capsid-specific

CD8+ T-cell population responding to this epitope. The

kinetics of the T-cell response in subject G were mapped with

this reagent and shown to overlap with the peak of serum

transaminases (Mingozzi et al, 2007). Two years after vector

infusion, PBMCs were isolated from subject E. No T-cell

response to AAV capsid was detectable directly ex vivo. Upon

expansion in the presence of AAV capsid peptides, however,

a single immunodominant epitope from the AAV capsid

invoked a significant expansion of T cells from the PBMCs of

subject E. Following two rounds of PBMC expansion in the

presence of this peptide, 8Æ8% of the CD8+ T cells in this culture secreted interferon gamma upon re-exposure to the

epitope (Mingozzi et al, 2007). Also of note from these studies,

immune responses to AAV capsid were detectable in normal,

uninfused subjects in both PBMCs and splenocytes, suggesting

that the events seen in this trial were not aberrant and would

be repeated if additional donors were infused.

Given the likelihood that an immune response to AAV capsid

ablated the transduced cells, and that capsid is present only

transiently before being degraded and cleared from the cells, one

potential solution is to block the immune response to capsid

pharmacologically until capsid has been metabolized and

cleared from the cells. This raises the question of the duration

of persistence of the capsid in the transduced cell, or more

precisely, the duration of persistence of peptide-major histo-

compatibility complexes on the cell surface. Attempts to

generate an animal model that replicates these findings have

so far been unsuccessful (Li et al, 2007a,b; Wang et al, 2007).

Most approaches have relied on a prime-boost regimen with

adenoviral or plasmid vectors expressing AAV capsid, to

generate a robust CD8+ T cell response to AAV capsid. However,

when these immunized mice are challenged with an AAV vector

encoding a human F9 transgene under the control of a liver-

specific promoter (Li et al, 2007b), the transduced hepatocytes

persist even in the presence of AAV capsid-specific T-cells. In

fact, no diminution in transgene expression was detected as

compared to unimmunized mice or control immunized mice.

The next phase in gene transfer for haemophilia

Safe, long-term expression of clotting factors has been

successfully achieved in large animal models of haemophilia

using multiple gene transfer strategies, but these findings have

not yet been translated into success in patients. Ongoing and

proposed clinical studies should help to determine whether

AAV-mediated gene transfer to the liver can achieve success.

The initial liver trial is set to resume, at a dose approximately

half that used in subject E, and with the addition of transient

immunosuppression to block the T-cell response to the

capsid. If a capsid-specific T-cell response was indeed

causative in the loss of transduced hepatocytes in subject E,

an important question remains: will the immunosuppressive

regimen selected be sufficient to block the response? It is also

not entirely clear how long the regimen will need to be

maintained. Alternative hypotheses to explain the findings in

the liver-directed AAV trial have been proposed. One

hypothesis suggests that fragments of the AAV capsid were

expressed in the transduced cells as a result of low-level

packaging of these sequences during the vector production

process (http://www4.od.nih.gov/oba/RAC/meeting.html).

This hypothesis does not adequately explain the difference

in findings between animal models and humans. A second

hypothesis is that an alternate open reading frame contained

within the F9 cDNA encodes an immunogenic protein

sequence, but this hypothesis also fails to explain the

difference between animals and humans (http://www4.od.-

nih.gov/oba/RAC/meeting.html). Other hypotheses focus on

differences in the sensitivity of immune responses in animals

and humans. There is some evidence that T cells in humans

may be more sensitive than T cells in non-human primates as

a result of differential expression of an immunomodulatory

lectin (Nguyen et al, 2006). Yet another possibility is that

a low level of CpG methylated DNA is packaged into the

vector during production and that humans possess more

sensitive innate immune responses to these stimuli. Finally it

has been proposed that there will be differences in capsid

processing and presentation with alternate AAV serotypes,

because of different kinetics of uncoating, differences in

intracellular trafficking, or differences in antigen processing

(Vandenberghe et al, 2006). Based on strong preclinical data

in non-human primates, a proposed trial of AAV8 encoding a

F9 transgene will test this hypothesis (Nathwani et al, 2006).

This next-generation vector carries a self-complementary

expression cassette to enhance expression at lower doses and

encodes a codon-optimized transgene to improve transla-

tional efficiency. Will the muscle ultimately be a better target

for AAV-mediated Factor IX expression? It is clear that

Factor IX transgene expression persisted in subjects injected

with AAV vectors intramuscularly. New systemic approaches

to introduce AAV vector into skeletal muscle have since been

Review

ª 2008 The Authors 484 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487

developed (Arruda et al, 2005). Will these delivery methods

result in stable expression or transient expression of the

transgene? Continuing studies should provide answers to

these questions, and ultimately a safe and effective long-term

treatment for haemophilia.

Acknowledgement

Supported by the National Institutes of Health and the

Howard Hughes Medical Institute.

References

Andrews, J.L., Shirley, P.S., Iverson, W.O., Sherer, A.D., Markovits,

J.E., King, L., Lyons, R.M., Kaleko, M. & Connelly, S. (2002) Eval-

uation of the duration of human factor VIII expression in nonhu-

man primates after systemic delivery of an adenoviral vector.

Human Gene Therapy, 13, 1331–1336.

Arruda, V.R., Hagstrom, J.N., Deitch, J., Heiman-Patterson, T., Camire,

R.M., Chu, K., Fields, P.A., Herzog, R.W., Couto, L.B., Larson, P.J. &

High, K.A. (2001) Posttranslational modifications of recombinant

myotube-synthesized human factor IX. Blood, 97, 130–138.

Arruda, V.R., Stedman, H.H., Nichols, T.C., Haskins, M.E., Nicholson,

M., Herzog, R.W., Couto, L.B. & High, K.A. (2005) Regional

intravascular delivery of AAV-2-F.IX to skeletal muscle achieves

long-term correction of hemophilia B in a large animal model.

Blood, 105, 3458–3464.

Blacklow, N.R., Hoggan, M.D., Kapikian, A.Z., Austin, J.B. & Rowe, W.P.

(1968) Epidemiology of adenovirus-associated virus infection in

a nursery population. American Journal of Epidemiology, 88, 368–378.

Blacklow, N.R., Hoggan, M.D., Sereno, M.S., Brandt, C.D., Kim, H.W.,

Parrott, R.H. & Chanock, R.M. (1971) A seroepidemiologic study of

adenovirus-associated virus infection in infants and children.

American Journal of Epidemiology, 94, 359–366.

Brown, B.D., Shi, C.X., Powell, S., Hurlbut, D., Graham, F.L. & Lil-

licrap, D. (2004) Helper-dependent adenoviral vectors mediate

therapeutic factor VIII expression for several months with minimal

accompanying toxicity in a canine model of severe hemophilia A.

Blood, 103, 804–810.

Brown, B.D., Venneri, M.A., Zingale, A., Sergi Sergi, L. & Naldini, L.

(2006) Endogenous microRNA regulation suppresses transgene

expression in hematopoietic lineages and enables stable gene

transfer. Nature Medicine, 12, 585–591.

Chang, A.H., Stephan, M.T. & Sadelain, M. (2006) Stem cell-derived

erythroid cells mediate long-term systemic protein delivery. Nature

Biotechnology, 24, 1017–1021.

Chuah, M.K., Schiedner, G., Thorrez, L., Brown, B., Johnston, M.,

Gillijns, V., Hertel, S., Van Rooijen, N., Lillicrap, D., Collen, D.,

VandenDriessche, T. & Kochanek, S. (2003) Therapeutic factor VIII

levels and negligible toxicity in mouse and dog models of hemo-

philia A following gene therapy with high-capacity adenoviral vec-

tors. Blood, 101, 1734–1743.

Chuah, M.K., Collen, D. & VandenDriessche, T. (2004) Clinical gene

transfer studies for hemophilia A. Seminars in Thrombosis and

Hemostasis, 30, 249–256.

Chung, J.H., Bell, A.C. & Felsenfeld, G. (1997) Characterization of the

chicken beta-globin insulator. Proceedings of the National Academy

of Sciences of the United States of America, 94, 575–580.

Connelly, S., Gardner, J.M., Lyons, R.M., McClelland, A. & Kaleko, M.

(1996) Sustained expression of therapeutic levels of human factor

VIII in mice. Blood, 87, 4671–4677.

Davidoff, A.M., Gray, J.T., Ng, C.Y., Zhang, Y., Zhou, J., Spence, Y.,

Bakar, Y. & Nathwani, A.C. (2005) Comparison of the ability of

adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8

capsid proteins to mediate efficient transduction of the liver in murine

and nonhuman primate models. Molecular Therapy, 11, 875–888.

Eaton, D.L., Hass, P.E., Riddle, L., Mather, J., Wiebe, M., Gregory, T. &

Vehar, G.A. (1987) Characterization of recombinant human factor

VIII. Journal of Biological Chemistry, 262, 3285–3290.

Fischer, A., Abina, S.H., Thrasher, A., von Kalle, C. & Cavazzana-Calvo,

M. (2004) LMO2 and gene therapy for severe combined immunode-

ficiency. New England Journal of Medicine, 350, 2526–2527.

Fisher, K.J., Jooss, K., Alston, J., Yang, Y., Haecker, S.E., High, K., Pathak,

R., Raper, S.E. & Wilson, J.M. (1997) Recombinant adeno-associated

virus for muscle directed gene therapy. Nature Medicine, 3, 306–312.

Follenzi, A., Battaglia, M., Lombardo, A., Annoni, A., Roncarolo, M.G.

& Naldini, L. (2004) Targeting lentiviral vector expression to

hepatocytes limits transgene-specific immune response and estab-

lishes long-term expression of human antihemophilic factor IX in

mice. Blood, 103, 3700–3709.

Gao, G.P., Alvira, M.R., Wang, L., Calcedo, R., Johnston, J. & Wilson,

J.M. (2002) Novel adeno-associated viruses from rhesus monkeys as

vectors for human gene therapy. Proceedings of the National Acad-

emy of Sciences of the United States of America, 99, 11854–11859.

Gao, G., Lu, Y., Calcedo, R., Grant, R.L., Bell, P., Wang, L., Figueredo,

J., Lock, M. & Wilson, J.M. (2006) Biology of AAV serotype vectors

in liver-directed gene transfer to nonhuman primates. Molecular

Therapy, 13, 77–87.

Greengard, J.S. & Jolly, D.J. (1999) Animal testing of retroviral-med-

iated gene therapy for factor VIII deficiency. Thrombosis and Hae-

mostasis, 82, 555–561.

Grieger, J.C. & Samulski, R.J. (2005) Packaging capacity of adeno-

associated virus serotypes: impact of larger genomes on infectivity

and postentry steps. Journal of Virology, 79, 9933–9944.

Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulf-

fraat, N., McIntyre, E., Radford, I., Villeval, J.L., Fraser, C.C., Cav-

azzana-Calvo, M. & Fischer, A. (2003a) A serious adverse event after

successful gene therapy for X-linked severe combined immunode-

ficiency. New England Journal of Medicine, 348, 255–256.

Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P.,

Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R.,

Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J.I., de

Saint Basile, G., Alexander, I., Wintergerst, U., Frebourg, T., Aurias,

A., Stoppa-Lyonnet, D., Romana, S., Radford-Weiss, I., Gross, F.,

Valensi, F., Delabesse, E., Macintyre, E., Sigaux, F., Soulier, J., Leiva,

L.E., Wissler, M., Prinz, C., Rabbitts, T.H., Le Deist, F., Fischer, A. &

Cavazzana-Calvo, M. (2003b) LMO2-associated clonal T cell pro-

liferation in two patients after gene therapy for SCID-X1. Science,

302, 415–419.

Herzog, R.W., Hagstrom, J.N., Kung, S.H., Tai, S.J., Wilson, J.M.,

Fisher, K.J. & High, K.A. (1997) Stable gene transfer and expression

of human blood coagulation factor IX after intramuscular injection

of recombinant adeno-associated virus. Proceedings of the National

Academy of Sciences of the United States of America, 94, 5804–5809.

Herzog, R.W., Yang, E.Y., Couto, L.B., Hagstrom, J.N., Elwell, D.,

Fields, P.A., Burton, M., Bellinger, D.A., Read, M.S., Brinkhous,

K.M., Podsakoff, G.M., Nichols, T.C., Kurtzman, G.J. & High, K.A.

Review

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487 485

(1999) Long-term correction of canine hemophilia B by gene

transfer of blood coagulation factor IX mediated by adeno-associ-

ated viral vector. Nature Medicine, 5, 56–63.

Herzog, R.W., Fields, P.A., Arruda, V.R., Brubaker, J.O., Armstrong,

E., McClintock, D., Bellinger, D.A., Couto, L.B., Nichols, T.C. &

High, K.A. (2002) Influence of vector dose on factor IX-specific T

and B cell responses in muscle-directed gene therapy. Human Gene

Therapy, 13, 1281–1291.

Jiang, H., Pierce, G.F., Ozelo, M.C., de Paula, E.V., Vargas, J.A., Smith,

P., Sommer, J., Luk, A., Manno, C.S., High, K.A. & Arruda, V.R.

(2006) Evidence of multiyear factor IX expression by AAV-mediated

gene transfer to skeletal muscle in an individual with severe

hemophilia B. Molecular Therapy, 14, 452–455.

Kay, M.A., Rothenberg, S., Landen, C.N., Bellinger, D.A., Leland, F.,

Toman, C., Finegold, M., Thompson, A.R., Read, M.S., Brinkhous,

K.M. & Woo, S.L.C. (1993) In vivo gene therapy of hemophilia B:

sustained partial correction in factor IX-deficient dogs. Science, 262,

117–119.

Kay, M.A., Landen, C.N., Rothenberg, S.R., Taylor, L.A., Leland, F.,

Wiehle, S., Fang, B., Bellinger, D., Finegold, M., Thompson, A.R.,

Read, M., Brinkhous, K.M. & Woo, S.L.C. (1994) In vivo hepatic

gene therapy: complete albeit transient correction of factor IX

deficiency in hemophilia B dogs. Proceedings of the National Acad-

emy of Sciences of the United States of America, 91, 2353–2357.

Kay, M.A., Manno, C.S., Ragni, M.V., Larson, P.J., Couto, L.B.,

McClelland, A., Glader, B., Chew, A.J., Tai, S.J., Herzog, R.W.,

Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A.W. &

High, K.A. (2000) Evidence for gene transfer and expression of

factor IX in haemophilia B patients treated with an AAV vector.

Nature Genetics, 24, 257–261.

Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J.F., Chamberlain, J.S.

& Duan, D. (2005) Efficient in vivo gene expression by trans-splicing

adeno-associated viral vectors. Nature Biotechnology, 23, 1435–1439.

Li, C., Hirsch, M., Asokan, A., Zeithaml, B., Ma, H., Kafri, T. &

Samulski, R.J. (2007a) Adeno-associated virus type 2 (AAV2) cap-

sid-specific cytotoxic T lymphocytes eliminate only vector-trans-

duced cells coexpressing the AAV2 capsid in vivo. Journal of Virology,

81, 7540–7547.

Li, H., Murphy, S.L., Giles-Davis, W., Edmonson, S., Xiang, Z., Li, Y.,

Lasaro, M.O., High, K.A. & Ertl, H.C. (2007b) Pre-existing AAV

capsid-specific CD8 T cells are unable to eliminate AAV-transduced

hepatocytes. Molecular Therapy, 15, 792–800.

Lozier, J.N., Metzger, M.E., Donahue, R.E. & Morgan, R.A. (1999)

Adenovirus-mediated expression of human coagulation factor IX in

the rhesus macaque is associated with dose-limiting toxicity. Blood,

94, 3968–3975.

Lu, D.R., Zhou, J.M., Zheng, B., Qiu, X.F., Xue, J.L., Wang, J.M.,

Meng, P.L., Han, F.L., Ming, B.H., Wang, X.P., Wang, J.B., Liang,

J.J. & Jiang, Z.S. (1993) Stage I clinical trial of gene therapy for

hemophilia B. Science in China. Series B, Chemistry, Life Sciences and

Earth Sciences, 36, 1342–1351.

Mah, C., Sarkar, R., Zolotukhin, I., Schleissing, M., Xiao, X., Kazazian,

H.H. & Byrne, B.J. (2003) Dual vectors expressing murine factor

VIII result in sustained correction of hemophilia A mice. Human

Gene Therapy, 14, 143–152.

Manno, C.S., Chew, A.J., Hutchison, S., Larson, P.J., Herzog, R.W.,

Arruda, V.R., Tai, S.J., Ragni, M.V., Thompson, A., Ozelo, M.,

Couto, L.B., Leonard, D.G., Johnson, F.A., McClelland, A., Scallan,

C., Skarsgard, E., Flake, A.W., Kay, M.A., High, K.A. & Glader, B.

(2003) AAV-mediated factor IX gene transfer to skeletal muscle in

patients with severe hemophilia B. Blood, 101, 2963–2972.

Manno, C.S., Pierce, G.F., Arruda, V.R., Glader, B., Ragni, M., Rasko,

J.J., Ozelo, M.C., Hoots, K., Blatt, P., Konkle, B., Dake, M., Kaye, R.,

Razavi, M., Zajko, A., Zehnder, J., Rustagi, P.K., Nakai, H., Chew,

A., Leonard, D., Wright, J.F., Lessard, R.R., Sommer, J.M., Tigges,

M., Sabatino, D., Luk, A., Jiang, H., Mingozzi, F., Couto, L., Ertl,

H.C., High, K.A. & Kay, M.A. (2006) Successful transduction of liver

in hemophilia by AAV-Factor IX and limitations imposed by the

host immune response. Nature Medicine, 12, 342–347.

McCormack, J.E., Edwards, W., Sensintaffer, J., Lillegren, L., Kozloski,

M., Brumm, D., Karavodin, L., Jolly, D.J. & Greengard, J. (2001)

Factors affecting long-term expression of a secreted transgene

product after intravenous administration of a retroviral vector.

Molecular Therapy, 3, 516–525.

Mingozzi, F., Liu, Y.L., Dobrzynski, E., Kaufhold, A., Liu, J.H., Wang,

Y., Arruda, V.R., High, K.A. & Herzog, R.W. (2003) Induction of

immune tolerance to coagulation factor IX antigen by in vivo hepatic

gene transfer. Journal of Clinical Investigation, 111, 1347–1356.

Mingozzi, F., Maus, M.V., Hui, D.J., Sabatino, D.E., Murphy, S.L., Rasko,

J.E., Ragni, M.V., Manno, C.S., Sommer, J., Jiang, H., Pierce, G.F., Ertl,

H.C. & High, K.A. (2007) CD8(+) T-cell responses to adeno-associ-

ated virus capsid in humans. Nature Medicine, 13, 419–422.

Mitchell, R.S., Beitzel, B.F., Schroder, A.R., Shinn, P., Chen, H., Berry,

C.C., Ecker, J.R. & Bushman, F.D. (2004) Retroviral DNA integra-

tion: ASLV, HIV, and MLV show distinct target site preferences.

PLoS Biology, 2, E234.

Monahan, P.E., Samulski, R.J., Tazelaar, J., Xiao, X., Nichols, T.C.,

Bellinger, D.A., Read, M.S. & Walsh, C.E. (1998) Direct intramus-

cular injection with recombinant AAV vectors results in sustained

expression in a dog model of hemophilia. Gene Therapy, 5, 40–49.

Mount, J.D., Herzog, R.W., Tillson, D.M., Goodman, S.A., Robinson,

N., McCleland, M.L., Bellinger, D., Nichols, T.C., Arruda, V.R.,

Lothrop, C.D., Jr. & High, K.A. (2002) Sustained phenotypic cor-

rection of hemophilia B dogs with a factor IX null mutation by liver-

directed gene therapy. Blood, 99, 2670–2676.

Nakai, H., Yant, S.R., Storm, T.A., Fuess, S., Meuse, L. & Kay, M.A.

(2001) Extrachromosomal recombinant adeno-associated virus

vector genomes are primarily responsible for stable liver transduc-

tion in vivo. Journal of Virology, 75, 6969–6976.

Nakai, H., Fuess, S., Storm, T.A., Muramatsu, S., Nara, Y. & Kay, M.A.

(2005) Unrestricted hepatocyte transduction with adeno-associated

virus serotype 8 vectors in mice. Journal of Virology, 79, 214–224.

Nathwani, A.C., Davidoff, A.M., Hanawa, H., Hu, Y., Hoffer, F.A.,

Nikanorov, A., Slaughter, C., Ng, C.Y., Zhou, J., Lozier, J.N.,

Mandrell, T.D., Vanin, E.F. & Nienhuis, A.W. (2002) Sustained

high-level expression of human factor IX (hFIX) after liver-targeted

delivery of recombinant adeno-associated virus encoding the hFIX

gene in rhesus macaques. Blood, 100, 1662–1669.

Nathwani, A.C., Gray, J.T., Ng, C.Y., Zhou, J., Spence, Y., Waddington,

S.N., Tuddenham, E.G., Kemball-Cook, G., McIntosh, J., Boon-

Spijker, M., Mertens, K. & Davidoff, A.M. (2006) Self-complementary

adeno-associated virus vectors containing a novel liver-specific human

factor IX expression cassette enable highly efficient transduction of

murine and nonhuman primate liver. Blood, 107, 2653–2661.

Nguyen, D.H., Hurtado-Ziola, N., Gagneux, P. & Varki, A. (2006) Loss

of Siglec expression on T lymphocytes during human evolution.

Proceedings of the National Academy of Sciences of the United States of

America, 103, 7765–7770.

Review

ª 2008 The Authors 486 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487

Othman, M., Labelle, A., Mazzetti, I., Elbatarny, H.S. & Lillicrap, D.

(2007) Adenovirus-induced thrombocytopenia: the role of von

Willebrand factor and P-selectin in mediating accelerated platelet

clearance. Blood, 109, 2832–2839.

Plug, I., Mauser-Bunschoten, E.P., Brocker-Vriends, A.H., van Amstel,

H.K., van der Bom, J.G., van Diemen-Homan, J.E., Willemse, J. &

Rosendaal, F.R. (2006) Bleeding in carriers of hemophilia. Blood,

108, 52–56.

Pollak, E.S. & High, K.A. (2001) Hemophilia B: factor IX deficiency.

In: The Metabolic & Molecular Bases of Inherited Disease (eds by C.R.

Scriver, A.L. Beaudet, W.S. Sly & D. Valle), Vol. III, pp. 4393–4413.

McGraw-Hill Medical Publishing Division, New York.

Powell, J.S., Ragni, M.V., White, G.C., 2nd. Lusher, J.M., Hillman-

Wiseman, C., Moon, T.E., Cole, V., Ramanathan-Girish, S., Roehl, H.,

Sajjadi, N., Jolly, D.J. & Hurst, D. (2003) Phase 1 trial of FVIII gene

transfer for severe hemophilia A using a retroviral construct admin-

istered by peripheral intravenous infusion. Blood, 102, 2038–2045.

Qiu, X., Lu, D., Zhou, J., Wang, J., Yang, J., Meng, P. & Hsueh, J.L.

(1996) Implantation of autologous skin fibroblast genetically mod-

ified to secrete clotting factor IX partially corrects the hemorrhagic

tendencies in two hemophilia B patients. Chinese Medical Journal

(England), 109, 832–839.

Roehl, H.H., Leibbrandt, M.E., Greengard, J.S., Kamantigue, E., Glass,

W.G., Giedlin, M., Boekelheide, K., Johnson, D.E., Jolly, D.J. &

Sajjadi, N.C. (2000) Analysis of testes and semen from rabbits

treated by intravenous injection with a retroviral vector encoding

the human factor VIII gene: no evidence of germ line transduction.

Human Gene Therapy, 11, 2529–2540.

Roth, D.A., Tawa, N.E., Jr, O’Brien, J.M., Treco, D.A. & Selden, R.F.

(2001) Nonviral transfer of the gene encoding coagulation factor

VIII in patients with severe hemophilia A. New England Journal of

Medicine, 344, 1735–1742.

Samakoglu, S., Lisowski, L., Budak-Alpdogan, T., Usachenko, Y.,

Acuto, S., Di Marzo, R., Maggio, A., Zhu, P., Tisdale, J.F., Riviere, I.

& Sadelain, M. (2006) A genetic strategy to treat sickle cell anemia

by coregulating globin transgene expression and RNA interference.

Nature Biotechnology, 24, 89–94.

Sarkar, R., Gao, G.P., Chirmule, N., Tazelaar, J. & Kazazian, H.H., Jr.

(2000) Partial correction of murine hemophilia A with neo-anti-

genic murine factor VIII. Human Gene Therapy, 11, 881–894.

Sarkar, R., Tetreault, R., Gao, G., Wang, L., Bell, P., Chandler, R.,

Wilson, J.M. & Kazazian, H.H., Jr. (2004) Total correction of

hemophilia A mice with canine FVIII using an AAV 8 serotype.

Blood, 103, 1253–1260.

Scallan, C.D., Jiang, H., Liu, T., Patarroyo-White, S., Sommer, J.M.,

Zhou, S., Couto, L.B. & Pierce, G.F. (2006) Human immunoglob-

ulin inhibits liver transduction by AAV vectors at low AAV2

neutralizing titers in SCID mice. Blood, 107, 1810–1817.

Schagen, F.H., Ossevoort, M., Toes, R.E. & Hoeben, R.C. (2004)

Immune responses against adenoviral vectors and their transgene

products: a review of strategies for evasion. Critical Reviews in

Oncology/hematology, 50, 51–70.

Smith, T.A., Mehaffey, M.G., Kayda, D.B., Saunders, J.M., Yei, S.,

Trapnell, B.C., McClelland, A. & Kaleko, M. (1993) Adenovirus

mediated expression of therapeutic plasma levels of human factor IX

in mice. Nature Genetics, 5, 397–402.

Snyder, R.O., Miao, C.H., Patijn, G.A., Spratt, S.K., Danos, O., Nagy,

D., Gown, A.M., Winther, B., Meuse, L., Cohen, L.K., Thompson,

A.R. & Kay, M.A. (1997) Persistent and therapeutic concentrations

of human factor IX in mice after hepatic gene transfer of

recombinant AAV vectors. Nature Genetics, 16, 270–276.

Song, S., Lu, Y., Choi, Y.K., Han, Y., Tang, Q., Zhao, G., Berns, K.I. &

Flotte, T.R. (2004) DNA-dependent PK inhibits adeno-associated

virus DNA integration. Proceedings of the National Academy of

Sciences of the United States of America, 101, 2112–2116.

Tatsis, N. & Ertl, H.C. (2004) Adenoviruses as vaccine vectors.

Molecular Therapy, 10, 616–629.

Toole, J.J., Pittman, D.D., Orr, E.C., Murtha, P., Wasley, L.C. & Ka-

ufman, R.J. (1986) A large region (approximately equal to 95 kDa)

of human factor VIII is dispensable for in vitro procoagulant activity.

Proceedings of the National Academy of Sciences of the United States of

America, 83, 5939–5942.

Urnov, F.D., Miller, J.C., Lee, Y.L., Beausejour, C.M., Rock, J.M.,

Augustus, S., Jamieson, A.C., Porteus, M.H., Gregory, P.D. & Holmes,

M.C. (2005) Highly efficient endogenous human gene correction

using designed zinc-finger nucleases. Nature, 435, 646–651.

Vandenberghe, L.H., Wang, L., Somanathan, S., Zhi, Y., Figueredo, J.,

Calcedo, R., Sanmiguel, J., Desai, R.A., Chen, C.S., Johnston, J.,

Grant, R.L., Gao, G. & Wilson, J.M. (2006) Heparin binding directs

activation of T cells against adeno-associated virus serotype 2 capsid.

Nature Medicine, 12, 967–971.

VandenDriessche, T., Vanslembrouck, V., Goovaerts, I., Zwinnen, H.,

Vanderhaeghen, M.L., Collen, D. & Chuah, M.K. (1999) Long-term

expression of human coagulation factor VIII and correction of

hemophilia A after in vivo retroviral gene transfer in factor VIII-

deficient mice. Proceedings of the National Academy of Sciences of the

United States of America, 96, 10379–10384.

Walter, J., You, Q., Hagstrom, J.N., Sands, M. & High, K.A. (1996)

Successful expression of human factor IX following repeat

administration of adenoviral vector in mice. Proceedings of the

National Academy of Sciences of the United States of America, 93,

3056–3061.

Wang, L., Takabe, K., Bidlingmaier, S.M., Ill, C.R. & Verma, I.M.

(1999) Sustained correction of bleeding disorder in hemophilia B

mice by gene therapy. Proceedings of the National Academy of Sci-

ences of the United States of America, 96, 3906–3910.

Wang, L., Figueredo, J., Calcedo, R., Lin, J. & Wilson, J.M. (2007)

Cross-presentation of adeno-associated virus serotype 2 capsids

activates cytotoxic T cells but does not render hepatocytes effective

cytolytic targets. Human Gene Therapy, 18, 185–194.

Wolins, N., Lozier, J., Eggerman, T.L., Jones, E., Aguilar-Cordova, E. &

Vostal, J.G. (2003) Intravenous administration of replication-

incompetent adenovirus to rhesus monkeys induces thrombocyto-

penia by increasing in vivo platelet clearance. British Journal

Haematology, 123, 903–905.

Xiao, X., Li, J. & Samulski, R.J. (1996) Efficient long-term gene transfer

into muscle tissue of immunocompetent mice by adeno-associated

virus vector. Journal of Virology, 70, 8098–8108.

Xu, L., Gao, C., Sands, M.S., Cai, S.R., Nichols, T.C., Bellinger, D.A.,

Raymer, R.A., McCorquodale, S. & Ponder, K.P. (2003) Neonatal or

hepatocyte growth factor-potentiated adult gene therapy with a

retroviral vector results in therapeutic levels of canine factor IX for

hemophilia B. Blood, 101, 3924–3932.

Yan, Z., Zhang, Y., Duan, D. & Engelhardt, J.F. (2000) Trans-splicing

vectors expand the utility of adeno-associated virus for gene therapy.

Proceedings of the National Academy of Sciences of the United States of

America, 97, 6716–6721.

Review

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 479–487 487