British Journal of Haematology, 2001, 115, 744±757
Review
GENE THERAPY FOR HAEMOPHILIA
The possibility of a `cure' for haemophilia A and B remains the
ultimate goal for patients with haemophilia. The cloning of the
factor VIII and IX genes in the 1980s and subsequent
production and introduction of recombinant factor VIII and
IX over the last decade have continued to promise the prospect of
a gene therapy treatment for haemophilia. In recent years,
much work has been focused on developing strategies for gene
therapy treatments, and we are now seeing phase I trials for both
haemophilia A and B. How close are we to realizing our goal?
CONCEPTS OF GENE THERAPY
Gene therapy, in the broadest sense, is the introduction of
foreign genetic material into a cell with therapeutic intent.
The ultimate gene therapy for haemophilia A and B would be
therapeutic gene replacement and/or direct correction of the
molecular defect in the mutated factor VIII or IX gene. Direct
genomic modification, using chimeric RNA/DNA oligonucleotides (Kren et al, 1998, 1999), has been demonstrated,
but for haemophilia A and B therapeutic gene replacement
and/or direct correction of the molecular defect remain a
long way in the future for clinical application.
Gene therapy for haemophilia today therefore relies upon
the addition of normal functional coagulation factor DNA
sequences with appropriate promoter and enhancer elements to the cells of a patient with a defective coagulation
factor gene, so that the modified cells can produce
functional protein as directed by the inserted DNA.
HAEMOPHILIA AS A TARGET FOR GENE THERAPY
Although the haemophilias have long been seen as a good
clinical model for the development of gene therapy for
single-gene disorders, it is now a major target for gene
therapy, as it has several fundamental advantages over
other single-gene disorders: there is a simple cause and
effect relationship between coagulation factor deficiency
and disease phenotype/clinical symptoms. Tissue-specific
expression of the transgene and precise regulation are
probably unimportant; suitable well-characterized small and
large animal models are available for preclinical studies. In
clinical trials, efficacy can be established and assessed easily
using both clinical and laboratory end-points.
From a clinical point of view, haemophilia gene therapy
would be a major step forward. Replacement therapy with
Correspondence: Professor John Pasi, Division of Haematology,
University of Leicester, Robert Kilpatrick Clinical Science Building,
Leicester Royal Infirmary, PO Box 67, Leicester LE2 7LX, UK. E-mail:
[email protected]
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factor VIII and IX products is now safer than ever but
continues to be less than ideal. Although there is increasing
use of recombinant products, with virus-inactivated plasma
products, there remain ever-present concerns about bloodborne infection and contamination with infectious prions
causing variant Creutzfeldt±Jakob disease (vCJD) (Will et al,
1996; Evatt, 1998). Even with state-of-the-art prophylactic
regimes, replacement treatment must be administered
intravenously at frequent intervals, which is often problematic in young children.
There have been major advances in the field in recent years
but, with present technology, gene therapy is unlikely to be
able to provide a total `cure' for haemophilia. Technology
presently available is still not sufficiently advanced to effect a
lifelong gene transfer that ensures continuing production of
coagulation factor to normalize levels. Despite this, perhaps
the most important single aspect of haemophilia that makes
it such an excellent target for gene therapy is the fact that
even a small rise (1±2% of physiological levels) in circulating
factor VIII or IX would have a significant beneficial
therapeutic effect, protecting against spontaneous bleeding
and potentially transforming the lives of patients with severe
haemophilia. In essence, endogenous factor production to
afford such small rises in factor levels would achieve the goals
of prophylaxis without regular infusions of concentrate.
Although in concept gene therapy is equally applicable to
haemophilia A and B, there are some important differences in
terms of the practical aspects of developing a gene therapy
treatment for each disorder. Factor IX cDNA is only 3 kb,
whereas factor VIII cDNA is significantly larger at 8´8 kb.
Smaller partially deleted factor VIII genes that do not contain
the large internal B domain of the protein reduce the size of
the factor VIII cDNA by < 30%. Such B-domain-deleted
factor VIII remains fully functional in vivo, as the B domain is
not required for coagulant activity (Berntorp, 1997).
However, the large size of the factor VIII gene has placed
constraints upon the choice of gene delivery system for use
in haemophilia A models and has led to different approaches
being developed for haemophilia A and B. In addition,
although a 1±2% increase in plasma level would have
beneficial effects in both diseases, it must be borne in mind
that the normal plasma level of factor IX (5 mg/ml) is some
50 times higher than for factor VIII (100 ng/ml). Successful
gene therapy approaches for haemophilia B may therefore
not work well for haemophilia A and vice versa.
EX VIVO VERSUS IN VIVO MODIFICATION
Cells, isolated from the patient, can be genetically modified
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(transduced) in culture, expanded and then returned to the
patient. This ex vivo approach enables transfection conditions to be carefully controlled and optimized. In addition,
unmodified cells can be eliminated before transplantation.
Effective return of modified cells to the body can, however,
be difficult. For transduced blood or haemopoietic cells,
there is little problem, as these can be returned directly to
the bloodstream. However, it is necessary for other cells to
have an appropriate support matrix and vascular supply.
Site of implantation may be a major determinant in
providing the right local environment for the effective
function of transplanted cells (Zatloukal et al, 1994).
The alternative to ex vivo systems, modification of cells
within the body (in vivo modification), eliminates problems
associated with transplanting cells. It provides the advantage of apparent simplicity and ease with which it could be
applied to the treatment of patients, in addition to costeffectiveness. In its simplest form, it would involve the
injection of a vector containing the appropriate genetic
material into the bloodstream or tissues that need to be
modified. However, to be functional, such an in vivo system
needs to be extremely efficient, not be inactivated in vivo by
antibodies or an induced immune response and, if injected
systemically, targeted to particular tissues. Any host
immune response that develops on exposure to the vector
itself may also preclude vector readministration. Germline
gene transfer cannot be excluded after systemic administration of a vector. Safety in this regard is an important
prerequisite for any in vivo vector system before clinical use.
However, in vivo modification systems provide the ultimate
gene therapy goal.
GENE DELIVERY SYSTEMS
The most challenging aspect of gene therapy for haemophilia remains the production and development of a suitable
gene delivery system, which will lead to high-level longterm expression of coagulation proteins. Numerous technologies, both viral and non-viral, have evolved as possible
vector systems for effecting genetic modification. Viruses
with potential for use as vectors in haemophilia models
include retroviruses, adenoviruses, parvo/adeno-associated
viruses and lentiviruses. Common to all these viral vector
systems is the ability to transfer genetic material as part of
the infectious process. Most viruses used for gene transfer
have had genes that confer virulence removed with
coagulation factor VIII or IX genes substituted.
Retroviral vectors
Moloney murine leukaemia virus (MoMLV)-based retroviral
vectors have commonly been used for gene therapy
protocols and extensively studied as vector systems in
haemophilia. Modified defective MoMLV-based vectors have
been popular, as they can be produced at high titre, have the
capacity to infect a wide variety of cells, integrate into the
host genome (so allowing possible long-term maintenance
of the transgene in daughter cells) and are relatively nonimmunogenic (McCormack et al, 1997). However, cellular
division is required to allow such vectors to transduce and
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integrate, thereby limiting retroviral gene therapy to
actively dividing target cells. Gene therapy for haemophilia
using MoMLV-based vectors therefore requires that, for
direct in vivo transduction, cells are either naturally dividing
or induced to divide.
Concerns about safety have been expressed as a result of
integration of genetic material into the host genome and the
possibility of insertional mutagenesis as well as concerns
about the presence of replication-competent retrovirus
contaminating preparations. Rare clonal transformation
has only been observed in severely immunocompromised
primates treated with preparations contaminated with high
levels of replication-competent retrovirus (Donahue et al,
1992). Although these risks cannot be excluded completely,
there have been no cases reported in over 1500 patients
treated in clinical trials to date (Anderson, 1998).
An additional potential stumbling block in the use of
retroviral vectors is their susceptibility to inactivation by
primate complement (Takeuchi et al, 1996). This could limit
their utility in clinical practice. Recent results suggest that
human cells, if used to form packaging cell lines, can
produce a complement-resistant vector (Greengard & Jolly,
1999).
Adenoviral vectors
Adenoviruses are double-stranded DNA viruses with a
36 kb genome. Adenoviruses infect non-dividing cells and
allow larger genes to be transferred than can be achieved
using retroviruses. In addition, they may transfer multiple
copies of the gene of interest and can effect in vivo gene
transfer. There are at least 50 different human serotypes.
Most adenoviral vectors currently used are derived from
serotypes 2 and 5, which are endemic and cause upper
respiratory tract infection. Most individuals have become
immunized by natural infection during childhood. This may
potentially lead to impairment in humans of adenoviral
transduction in vivo. They have a long safety record, having
been used in live virus vaccines studies for many years
without problems.
Two generations of adenoviral vectors have been used in
the context of haemophilia. First-generation adenoviral
vectors have two early native genes deleted (E1A and E1B)
to accommodate the genes of interest and render the
modified virus replication incompetent. Production of
infective virus for use as a vector requires the use of a
complement-packaging cell line.
Recombinant adenoviral vectors commonly only express
transferred gene product for a short period. This arises from
two predominant mechanisms. The adenovirual DNA is not
integrated into the host genome after infection and remains
episomal. As a result of this, the transferred genes are only
transiently maintained within the cells and are eliminated
at cell division. Secondly, a host immune response is elicited
that limits the duration of transgene expression and
readministration of vectors. In the modified E1-deleted
adenovirus, much of the adenovirus genetic structure is
maintained. Proteins expressed from such native genes are
immunogenic and, even if expressed at low level, are
believed to contribute to an antiviral immune response
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(cytotoxic T-cell response) that results in the clearance of
transduced cells (Yang et al, 1994a). Similarly, readministration of the vector will be hampered.
Further adenoviral vectors have been developed that have
E2 and/or E4 functions inactivated, in addition to E1
deletion. It is uncertain whether these vectors have any
significant advantages over E1 alone deleted vectors (Yang
et al, 1994b; Krougliak & Graham, 1995).
In an attempt to overcome some of these problems, a
second generation of adenoviral vectors has been developed,
in which almost all viral coding sequences have been deleted
using partially deleted ancillary adenovirus with a complementing cell line. These `gutless' or mini-Ad systems
should overcome some of the more problematic limitations
of the first-generation adenoviral vectors (Chen et al, 1997;
Morsy et al, 1998).
Adeno-associated viral vectors
Adeno-associated virus (AAV) is a single-stranded DNA
human parvovirus with a 4´7 kb genome. Although it is not
known to cause human disease, 85% of the adult
population is seropositive to AAV capsid proteins. AAV is a
naturally defective virus, requiring a helper virus, usually
adenovirus, to complete its life cycle and generate infectious
particles. In AAV vectors, usually based on serotype 2, all
viral coding sequence is removed and replaced by the
chosen transgene. As all native viral genes are removed, the
immune response to AAV is eliminated. Wild-type AAV
transfects non-dividing cells and integrates at preferential
sites in chromosome 19 (Kotin et al, 1990), which is
mediated by rep gene products (Weitzman et al, 1994). As all
native viral genes are eliminated in recombinant AAV
(rAAV), although rAAV similarly transfects non-dividing
cells, it integrates more randomly throughout the genome
(Ponnazhagan et al, 1997).
The presence of residual immunogenic adenovirus in
stocks of AAV vector has potentially been a problem,
negating the relative non-immunogenicity of rAAV vectors.
However, it is now possible to produce rAAV vectors without
the use of an adenoviral helper (Matsushita et al 1998; Xiao
et al, 1998).
Lentiviral vectors
As outlined above, MoMLV-based retroviral vectors have a
number of limitations. In order to overcome these limitations, retroviral vectors derived from lentiviruses have been
developed, with human immunodeficiency virus (HIV-1) as
prototype. Like all retroviral vectors, lentiviral vectors will
integrate into the genome. However, unlike MoMLV-based
vectors, lentiviral vectors will infect non-dividing cells.
MoMLV-based vector nucleoprotein complexes can only
reach the target cell nucleus when the nuclear membrane is
disrupted during mitosis. Lentiviral nucleoproteins, however, contain nuclear localization signals, which mediate
their active transport through nuclear membrane pores into
the nucleus during cell interphase (Bukrinsky et al, 1993;
Lewis & Emerman, 1994).
Lentiviruses, such as HIV, normally have a limited range
of cell targets and are major pathogens. Simple vectors,
derived from HIV-1, carry an unacceptable risk of recombination (to produce infectious virus) from elements used
within the constructs to generate the vector. These
difficulties have been overcome by the production of hybrid
vector systems. Such hybrids use an HIV-1-derived backbone that contain elements required for the ability to infect
non-dividing cells, integrate and express transgene but not
any viral (virulence) genes. Typically, the envelope is derived
from another virus. This allows a broader range of cells to
be targeted, according to the envelope used, and prevents a
wild-type lentivirus being generated as the envelope gene of
the parent virus is absent in all components used to produce
the vector (Naldini, 1999).
Physical systems
Physical gene delivery systems are attractive alternatives to
recombinant viral vectors. However, the development of
such systems has been repeatedly hampered by very poor
transduction efficiency. The genes transferred using physical
methods remain episomal and so potentially short-lived
within the cells. Methods that have been explored in
haemophilia include direct gene transfer by injection,
liposome encapsulation and receptor-mediated transfer.
ANIMAL MODELS
Individual approaches to gene therapy for haemophilia A
and B might appear to be promising in vitro. However, they
can only really be assessed for potential viability as a
therapeutic system by animal testing. Although murine
models of severe haemophilia A (Bi et al, 1995) and B (Lin
et al, 1997; Wang et al, 1997) have been developed, there
appears to be a degree of interstrain variability in terms of
immune response to gene therapy systems. Potential
problems in mice, such as antibody responses to nonhomologous transgenes (e.g. human factor VIII in a murine
model) are predictable and increasingly appreciated. These
can be overcome to some extent in vivo in immunodeficient
strains, but are more effectively dealt with using homologous factor VIII and IX genes. Small animal models are
crucial, allowing optimization before scale up and assessment in a large animal system. Large animal models are
essential for studying the efficacy of present and future
approaches to gene therapy. For both haemophilia A and B,
well-characterized canine models are available that mirror
the human condition. Canine models, using specific canine
factor VIII and factor IX cDNA to avoid issues of
confounding (inhibitory) alloantibody response, will also
provide an indication of clinical efficacy of the proposed
gene therapy as affected animals suffer with frequent
bleeding problems.
GENE THERAPY STUDIES IN HAEMOPHILIA B
Factor IX, unlike factor VIII, is relatively easy to express. It is
widely distributed and can equilibrate between vascular and
extravascular compartments (Liles et al, 1997). Unlike
factor VIII, therefore, for successful gene therapy, factor IX
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production does not necessarily need to be from sites such
that it is directly secreted into the circulation (see later).
Retroviral systems
Multiple different cell lines have been transduced with factor
IX including primary fibroblasts (St Louis & Verma, 1988;
Palmer et al, 1989; Axelrod et al, 1990), myoblasts (Dai et al,
1992; Yao & Kurachi, 1992; Yao et al, 1994), hepatocytes
(Armentano et al, 1990), haemopoietic stem cells (Hao et al,
1995), endothelial cells (Yao et al, 1991), gut epithelial cells
(Lozier et al, 1997) and keratinocytes (Gerrard et al, 1993,
1996; Page & Brownlee, 1997, White et al, 1998) ex vivo
using factor IX-containing retroviruses. Functional factor IX
is produced by many cell types despite the need for complex
post-translational gamma carboxylation required for full
function of factor IX.
Early gene therapy studies used human factor IXtransduced fibroblasts transplanted back into rodents (St
Louis & Verma, 1988; Palmer et al, 1989). Expression of
factor IX declined over a few weeks as a result of
neutralizing antibodies (St Louis & Verma, 1988) and in
vivo inactivation of viral promoters in the transplanted
fibroblasts (Hoeben et al, 1991). Human factor IX-transfected murine myoblasts transplanted into skeletal muscle in
mice was more successful, with factor IX expressed for up to
6 months or longer although at a very low level (Dai et al,
1992; Yao & Kurachi, 1992; Yao et al, 1994). However,
similar studies in dogs were much less successful with only
10 d expression of factor IX at levels that would be
insignificant in clinical terms (unpublished observations).
The first gene therapy trial for haemophilia used a similar
MoMLV-based approach. Performed in China in 1993, skin
fibroblasts from two boys with moderate haemophilia B
were ex vivo transduced with a factor IX retroviral vector (Lu
et al, 1993). Transduced cells were shown to produce factor
IX in vitro and transplanted subcutaneously into the boys.
One boy was reported to have had a response in factor IX
activity and a reduced requirement for replacement therapy.
The other boy had no discernible benefit. Although
encouraging, this study did not unequivocally support this
approach to gene therapy for haemophilia B in humans.
Early in vivo retroviral studies were similarly disappointing. Retrovirus, containing canine factor IX, was infused via
a splenic vein catheter into dogs with haemophilia B that
had undergone a partial hepatectomy to induce division of
cells to allow retroviral infection of hepatocytes (Kay et al,
1993). This procedure led to a very low-level expression of
factor IX (0´1 unit/dl) for 9 months.
Adenoviral systems
Although partial correction was observed with retroviral
constructs, the degree of correction was clearly insufficient
to be of clinical value in humans. More promising results
have been obtained using adenoviral vector systems. These
vectors, which do not require cells to be dividing, show a
strong tropism for liver (although they may also transduce a
number of other tissues) and can be administered intravenously peripherally. Adenoviral systems have now been
shown to express factor IX in a number of both haemophilic
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dogs (Kay et al, 1994) and normal (Walter et al, 1996) and
haemophilic mice (Kung et al, 1998). These experiments
produced supraphysiological levels of factor IX. However,
the correction was short term. This decline resulted from
both cell-mediated and humoral immune response. Readministration of the adenoviral vector into adult animals
failed to produce further factor IX, presumably because of
high levels of neutralizing antibody (Kay et al, 1994; Walter
et al, 1996). Adenoviral vectors used for factor IX transfer
have now been shown efficiently to activate factor IXspecific cytotoxic T-lymphocytes (CTLs) and T helper cells of
both Th1 and Th2 subsets, leading to inflammation and
destruction of transduced tissue and activation of B cells
(Fields et al, 2000).
To circumvent the immune response, adenoviral gene
transfer has been repeated using either immunosuppression,
such as cyclosporin A and cyclophosphamide, or modified
adenoviral vectors. Experiments using immunosuppressive
regimes have shown significantly higher levels of factor IX
activity and at therapeutic levels for longer periods but,
again, the levels progressively declined over the ensuing
weeks to pretreatment levels (Dai et al, 1995; Fang et al,
1995). Similarly, experiments using second-generation
adenoviral vectors have failed to improve duration of
expression significantly (Fang et al, 1996). Animals were
again refractory to repeated administration of the vector,
because of immune-mediated clearance, although combination immunosuppression given at first exposure was shown
to allow a second exposure (Smith et al, 1996). In terms of
human clinical application, the methods and degree of
immunosuppression required to allow persistent vector
expression and/or readminstration are unacceptable.
Adeno-associated virus
The smaller size of the factor IX gene has allowed the use of
adeno-associated virus as a vector system for haemophilia B.
This has been the most promising approach to gene therapy
for haemophilia to date. Recombinant AAV (rAAV) has been
used for both liver- and muscle-based gene therapy
approaches to haemophilia B. rAAV is known to transduce
muscle fibres (Xiao et al, 1996; Fisher et al, 1997) after
direct injection. Using an rAAV vector containing factor IX
driven by a cytomegalovirus (CMV) promoter, stable
expression of factor IX at therapeutic levels of 250±
350 ng/ml (5±7% of normal) has been demonstrated in
Rag-1 immunodeficient mice for over 12 months (Herzog
et al, 1997). Higher levels of factor IX can be expressed by
promoter modifications using skeletal muscle-derived elements (Hagstrom et al, 2000).
Transduction of liver by rAAV was initially thought to be
significantly less efficient than muscle transduction and
required additional modifications such as wild-type virus
and irradiation applied to the liver (Koeberl et al, 1997).
However, intraportal infusion of rAAV vectors appears to
lead to improved transduction efficiency and expression of
factor IX (Snyder et al, 1997; Nakai et al, 1998), with factor
IX levels of 1±3 mg/ml (20±60% of normal). Initial poor
expression and efficiency may have resulted from use of the
CMV promoter, which is rapidly shut down in liver (Nakai
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et al, 1998). Infusion of factor IX rAAV has been able to
correct murine models of haemophilia B phenotypically
(Snyder et al, 1999), with supraphysiological levels (15±
20 mg/ml) of factor IX, dependent upon the promoter used
(Wang et al, 1999).
Both intramuscular and portal vein administration of
rAAV lead to the expression of factor IX activity, but it would
appear that there are some clear distinctions between the
two approaches. For any given dose, portal vein infusion
appears to yield higher levels of factor IX (Nathwani et al,
2001), although it is a more complex route of administration. The role of route of administration in terms of the
immune response remains somewhat uncertain (see below).
Experiments on dog models of haemophilia using rAAV
have represented a major challenge with a scale up of 400±
800 times on a weight-for- weight basis. Factor IX AAV
vectors have been infused into the portal circulation (Snyder
et al, 1999; Wang et al, 2000) and also injected
intramuscularly (Chao et al, 1999; Herzog et al, 1999).
All studies documented systemic expression of factor IX and
did not show any significant toxicity. In the dogs that were
infused into the portal vein up to 250 ng/ml factor IX (5%
physiological activity), expression was seen, although this
varied according to dose of rAAV infused (Snyder et al,
1999; Wang et al, 2000). In the animals that were injected
intramuscularly, expression of factor IX was variable (3±
70 ng/ml), again dependent upon the dose of rAAV
administered (range 101121013 vector particle/kg) (Chao
et al, 1999; Herzog et al, 1999). Expression was stable for
over 2 years with no clear signs of decline.
Interestingly, although rAAV does not activate factor IXspecific CTLs, intramuscular injection of rAAV can lead to a
factor IX-specific B-cell response (Fields et al, 2000) and has
been associated with factor IX antibody (inhibitor) development (Monahan et al, 1998) in dogs. However, only one of
the dogs, in the larger more recent study, injected with
rAAV developed an inhibitor to factor IX, which was
transient (Herzog et al, 1999). The discrepancy probably
results from the use of human factor IX gene (Monahan et al,
1998) compared with canine factor IX more recently (Chao
et al, 1999; Herzog et al, 1999).
Lentiviral systems
The use of lentiviral vectors has been reported recently. In
these preliminary studies, 50±60 ng/ml factor IX has been
expressed in normal mice using an EF1a enhancer/
promoter-driven human factor IX gene after intraportal
administration, although there was evidence of self-limiting
lentiviral-induced hepatic injury (Park et al, 2000). Expression of factor IX could be significantly increased (<300 ng/
ml) by the induction of hepatic proliferation (partial
hepatectomy). It is possible to speculate that the induction
of liver injury, by the vector itself, may be partially involved
in the improved efficiency of lentiviral transduction.
Although lentiviral vectors can produce therapeutic levels
of factor IX, they require either induction of hepatocyte
division or use at doses that result in liver injury. For
haemophilia B, further work will be required to improve
such vectors to achieve therapeutic levels at doses that are
considered safe.
GENE THERAPY STUDIES IN HAEMOPHILIA A
Factor VIII is significantly more difficult to express in gene
therapy systems than factor IX for a variety of reasons. The
factor VIII gene is a complex 26-exon gene of over 186 kb.
The cDNA at 8´8 kb is also too large to be packaged by
many vector systems. As outlined earlier, a B-domaindeleted version has been used in the majority of studies
looking at gene therapy treatments for haemophilia A.
Deletion of the B domain also has the added benefit of
increasing factor VIII expression, compared with the fulllength clone. Not only is factor VIII cDNA a problem by
virtue of its size, but it is also known to contain sequences,
particularly within the A2 domain, that repress its
expression, resulting in low levels of factor VIII mRNA
(Lynch et al, 1993; Hoeben et al, 1995; Koeberl et al, 1995;
Fallaux et al, 1996). Some sequences inhibit transcriptional
elongation (Lynch et al, 1993; Koeberl et al, 1995), whereas
others inhibit transcriptional initiation (Hoeben et al, 1995;
Fallaux et al, 1996). This latter effect is thought to be
mediated by sequences within the factor VIII cDNA that are
a 10/11 match with consensus seen in autonomously
replicating sequences (ARS) in yeast. Such ARSs have been
implicated in modulating the activities of transcriptional
silencers and enhancers (Hoeben et al, 1995; Fallaux et al,
1996).
Factor VIII is a large and relatively unstable protein that is
naturally confined to the circulation. Unless it is bound to
von Willebrand factor (VWF), it is highly susceptible to
proteolytic degradation. For factor VIII gene therapy to be
successful, therefore, factor VIII must be secreted directly
into the circulation to allow rapid binding to circulating
VWF. Choice of target tissues and cells to express factor VIII
transgenes is consequently more restricted than for factor IX
and haemophilia B.
Retroviral systems
As with factor IX, expression of human factor VIII has been
reported in a wide variety of cell types and cell lines. It has
been shown by various groups that functional human factor
VIII could be detected in cell culture media after MoMLV
retroviral transfer of factor VIII into human skin fibroblasts
(Hoeben et al, 1990), murine skin fibroblasts (Israel &
Kaufman, 1990), endothelial cells (Lynch et al, 1990) and
murine bone marrow (Hoeben et al, 1992).
However, low levels of MoMLV-based vector production
(up to 1000-fold lower than expected) and low expression of
factor VIII were frequent problems. These difficulties were
presumed to be the result of the repressive sequences noted
in the factor VIII cDNA, as outlined above. Conservative
mutation of such sequences failed to improve the production
of vector or the expression of factor VIII. However,
incorporation of an intron upstream of the factor VIII
sequence leads to significant improvements in both vector
titre and factor VIII expression (Chuah et al, 1995; Dwarki
et al, 1995).
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As attainable titres of retrovirus were low, initial studies
for haemophilia A gene therapy used an ex vivo approach
with reimplantation of transduced cells. Implanted transduced bone marrow and human primary fibroblasts
(Hoeben et al, 1992, 1993) failed to produce detectable
factor VIII. However, using an improved MFG backbone,
retroviral vector system-transduced human fibroblasts,
transplanted into immunodeficient mice, expressed factor
VIII close to normal values found in man (Dwarki et al,
1995). In these experiments, the transduced fibroblasts
were implanted as neo-organs of collagen-coated PTFE fibre
lattices with modified fibroblasts incorporated into them.
Expression was transient (<1 week) and also highly
dependent on the site of transplantation of transduced
cells. These studies showed that the implantation site and
local environment of the graft site, such as vascular access,
target cell type and vector design, were critically important
in obtaining detectable factor VIII levels in the circulation.
As factor VIII expression was low, alternative targets for
retroviral gene therapy were sought. Bone marrow stroma
has been seen as an attractive target. It is relatively easy to
obtain and could be simply transduced ex vivo by MoMLVbased vectors. Transduction of bone marrow stroma with
factor VIII-containing retroviral vectors has been shown to
produce high levels of factor VIII in vitro (Chuah et al, 1998)
and, after transplantation, non-myeloablated immunodeficient mice produce factor VIII activity of up to 20 ng/ml.
Factor VIII expression declined as a result of LTR promoter
inactivation (Chuah et al, 2000). Transduced bone marrow
stroma may be an ex vivo option for haemophilia A gene
therapy, although issues concerning choice of promoter
need to be addressed.
In vivo gene therapy, aiming to target hepatocytes, is the
major target for retroviral vectors. Recent work has focused
on technological improvement to develop this possibility and
overcome previous experimental problems. For retroviral
vectors to be effective and to transduce hepatocytes in vivo,
the cells need to be naturally cycling/dividing or induced to
divide. However, other important likely factors that have
contributed to previous problems are low viral titre, a
threshold effect of dose and unawareness of a lag phase
between injection and factor VIII production (Greengard &
Jolly, 1999). With improvements in retroviral design,
production, purity and titre, there became a possibility of
an in vivo retroviral gene therapy. Titres of virus are now
possible that are many fold better than first-generation
MoMLV-based vectors. In addition, encapsulating retrovirus
in the G-glycoprotein of the vesicular stomatis virus (VSV),
pseudotyping, has improved tropism for hepatocytes. The
use of growth factors that either induce proliferation or
improve the efficiency of retroviral transduction has been
shown significantly to improve retroviral-mediated hepatocyte transduction and may have an increasing role in
developing retroviral gene therapy (Bosch et al, 1996; Patijn
et al, 1998).
High-titre VSV pseudotyped factor VIII vectors have been
injected into neonatal haemophilia A mice (VandenDriessche et al, 1999). Factor VIII levels of . 20%
physiological were detected in < 50% of the animals, with
749
some animals expressing extremely high/supranormal levels
of factor VIII. The remaining animals developed inhibitory
antibodies. In these experiments, factor VIII expression was
sustained for more than 12 months and demonstrated for
the first time that haemophilia A could be cured in an
animal model by a gene addition approach. The predominant expression of factor VIII in these experiments was in
the liver and may result from higher hepatocyte turnover
rates in young animals compared with adults.
Similar in vivo retroviral transduction has also been
shown in immunocompetent juvenile (6-week-old) rabbits
with long-term expression of factor VIII, < 75% of normal
for 6 months (Greengard & Jolly, 1999). Higher levels were
obtained in the juvenile rabbits compared with the adult
rabbits, again probably because of higher hepatocyte
turnover rates. Dogs with haemophilia have also been
treated with this system using a human factor VIII gene
(Greengard & Jolly, 1999). Development of the anti-human
anti-factor VIII antibodies in this model system has
confounded analysis, although enzyme-linked immunosorbent assay (ELISA) did show production of factor VIII.
Importantly, despite antiretroviral antibodies, animals were
not refractory to further administration of vector.
These data suggest that MoMLV-based systems may be
therapeutically useful in haemophilia (Greengard & Jolly,
1999).
Adenoviral systems
Adenoviral systems have been used for factor VIII gene
therapy models. Peripheral vein administration of human Bdomain-deleted factor VIII adenoviral constructs in mice,
dogs and primates results in efficient transduction of
hepatocytes. Although other tissue may also be transduced,
the use of liver-specific promoter elements has allowed the
restriction of expression to the liver. High-level factor VIII
production is seen in normal adult mice (Connelly et al,
1995, 1996a,b) with . 100% normal factor VIII levels in
some experiments. Phenotypic correction has also been
shown in haemophilic mice (Connelly et al, 1998; 1999)
and dogs (Connelly et al, 1996c). In mice, expression has
been maintained for over 12 months using human (Connelly et al, 1996a) and canine (Gallo-Penn et al, 1999)
factor VIII genes. However, in haemophilic dogs, expression
is short term (Connelly et al, 1996c), predominantly because
of a humoral immune response to human factor VIII.
Murine factor VIII has been used in the murine haemophilia
A model using an E1/E3-deleted adenoviral vector, but this
also led to short-term expression as a result of speciesspecific transgene humoral and cellular immune responses
(Sarkar et al, 2000).
Experiments in primates have shown that a tagged factor
VIII is expressed at therapeutic levels (. 20 ng/ml) after
injection of an E1-, E2- and E3-deleted vector (Brann et al,
1999). The duration of expression in this model is not clear,
as the experiment only lasted 1 week, and it was shown that
factor VIII was only expressed at higher doses of vector
(3 1012 particles/kg). Transient elevations of liver
enzymes, histological evidence of liver inflammation,
anaemia, thrombocytopenia and lymphoid and marrow
q 2001 Blackwell Science Ltd, British Journal of Haematology 115: 744±757
750
Review
hyperplasia were noted, particularly at the high dose level.
Similar dose-dependent toxicity has been observed independently for factor IX-containing adenoviral vectors (Lozier
et al, 1999). Of importance, factor VIII expression using
typical adenoviral vectors appears to have a dose threshold
effect in vivo, independent of toxicity effects or immune
response (Bristol et al, 2000). These findings together would
have important implications for defining the possible
therapeutic window of adenoviral vector for clinical use.
The induction of a host immune response and the
consequent short-term expression of the factor VIII transgene in the adenoviral system continue to limit the potential
use of adenoviral vectors. To overcome these difficulties, the
so-called `gutless' adenoviral vectors that completely eliminate viral coding sequences have been studied. MiniAdFVIII
contains only minimal viral cis-elements that are required
and contains the full-length factor VIII cDNA under the
control of a liver-specific albumin promoter (Balague et al,
2000). This vector, injected via a tail vein at 2 1011
particles per mouse or more (a relatively high dose), led to
sustained expression of human factor VIII in haemophilic
mice at physiological levels (. 200 ng/ml, . 100% normal)
with complete phenotypic correction. Expression gradually
declined in some mice as a result of the production of antihuman factor VIII antibodies, although it persisted in those
that did not. No significant toxicities were noted. A similar
threshold effect to `whole' adenoviral vectors was also
observed.
Studies in non-human primates have shown the expression of low levels of factor VIII (up to 0´8 units/dl) and
correction of the whole-blood clotting time in a haemophilic
dog (Fang et al, 2001). However, transient haematological
and hepatic toxicities were also observed.
Adeno-associated virus
Use of AAV for haemophilia A had initially been seen as
problematic because of the small capacity of rAAV and the
relatively large size of the factor VIII gene. As an alternative
approach, the factor VIII gene was delivered by dual
infection, using two rAAV vectors, of its component heavy
and light chains (Burton et al, 1999). The component
chains reassociated in vivo after transduction of hepatocytes
(normal mice via portal vein infusion) to produce greater
than physiological levels of factor VIII (. 200 ng/ml).
Although demonstrating potential, questions remain as to
whether unbalanced production of light or heavy chains, in
cells not co-transduced, has any effect on immune response
and the development of inhibitors to factor VIII.
A simpler approach would be the design of a factor VIII
expression cassette that could fit within AAV and not
compromise rAAV production or transgene expression. This
has been a major challenge. Novel B-domain-deleted factor
VIII mutants have recently been developed that can be
efficiently packaged in AAV, although they are slightly
larger than wild-type AAV (107% and 109%) (Gnatenko
et al, 1999; Chao et al, 2000). Intraportal injection of such
factor VIII rAAV in non-haemophilic severe combined
immunodeficient (SCID) mice led to the production of factor
VIII with detectable plasma levels < 50 ng/ml (<25%
physiological) and predictable anti-human factor VIII
antibodies in immunocompetent strains (Chao et al,
2000). Expression was long term. These data provide
encouraging results for the potential use of rAAV for
haemophilia A as well as haemophilia B.
Lentiviral systems
The use of lentiviral vectors has also been reported recently
for haemophilia A (Park et al, 2000). In contrast to factor IX
expression, < 30 ng/ml factor VIII (15% physiological) was
expressed in normal mice, but this response was again
predictably transient as a result of the development of antifactor VIII antibodies in immunocompetent mice (Park et al,
2000). Immunodeficient mice demonstrated sustained
factor VIII expression. As for haemophilia B, the use of
lentiviral vectors is in its early stages.
NOVEL GENE DELIVERY SYSTEMS FOR
HAEMOPHILIA A AND B
This review has largely focused upon the use of viral vectors
to transfer factor IX and VIII genes. However, a non-viral
gene transfer technology would be preferable, simplifying
concerns about safety, vector-induced immune responses,
recombination events and potential cost and labour
problems
with
large-scale
industrial
production.
Approaches used include naked DNA by direct injection or
in conjunction with liposomes, polymers or polypeptides.
However, early studies using such non-viral systems have
been problematic because of the low efficiency of transduction and transient expression of the transgene as a result of
the transgene remaining episomal and not integrating.
In haemophilia-related studies, although a B domainless
factor VIII construct could be transfected efficiently into
fibroblasts and myoblasts ex vivo using a receptor-mediated
system (Zatloukal et al, 1994), transduced cells, once
transplanted into the liver or spleen, could significantly
elevate systemic levels of factor VIII for less than 48 h.
More recent work has again allowed the prospect of a
non-viral delivery system to be considered. It has been
shown previously that the intramuscular injection of naked
plasmid DNA enables foreign gene expression in muscle
(Wolff et al, 1992). Further studies showed that the
intravascular delivery of naked plasmid DNA also enabled
high levels of expression not only in muscle but also in
hepatocytes, but that this technique required injection
directly into the hepatic vessels with outflow occlusion. By
rapidly injecting plasmid DNA in large fluid volumes, high
levels of transgene expression from hepatocytes could be
obtained (Zhang et al, 1999). Using a similar technique, a
factor IX plasmid (containing a hepatic apolipoprotein E
locus control region, a factor IX intron and untranslated
region) could be transiently expressed from hepatocytes
(0´5±2 mg/ml for 225 d) (Miao et al, 2000). Although not
directly applicable to clinical gene delivery, these studies
demonstrate that direct injection of suitable plasmid DNA
constructs could overcome some of the earlier limitations of
non-viral gene delivery.
A more ambitious approach has been the use of
q 2001 Blackwell Science Ltd, British Journal of Haematology 115: 744±757
Review
transposon technology to support stable chromosomal
integration. Transposons are naturally occurring genetic
elements capable of moving from one chromosome location
to another. A synthetic transposable element, `Sleeping
Beauty', which encodes a transposase enzyme, has been
shown to insert foreign genes into chromosomes (Ivics et al,
1997; Luo et al, 1998). Using this technology, it has been
possible to facilitate somatic integration of naked DNA into
mouse chromosomes to produce long-term factor IX
expression (<150 ng/ml) in normal and haemophilic mice
(Yant et al, 2000). Five to six per cent of mouse liver cells
had DNA inserted into them after tail vein injection, a
similar level to the use of lentivirus or rAAV systems.
Readministration of the vector also led to increased
expression of factor IX. Clearly, the use of such bacterial
transposase is in its infancy, and the toxicity and complication profile of these systems remains to be defined.
The ultimate target in gene therapy would be to repair the
molecular defect that leads to haemophilia. Direct genomic
modification, using chimeric RNA/DNA oligonucleotides to
alter the factor IX gene, has been demonstrated in mice
(Kren et al, 1998), as has correction of model gene defects in
murine Criggler±Najjar syndrome (Kren et al, 1999). As
there is a large a range of mutations in haemophilia, such
approaches would require tailor-making of therapy for each
patient and would not correct the common intron 22
inversion in severe haemophilia A. However, this strategy
has shown, in principle, that it may be possible to
manipulate mutated genes directly to correct haemophilia.
CLINICAL TRIALS
Selection of patients
As genetic material is the putative therapeutic agent, gene
therapy may be seen as qualitatively different from other
forms of treatment. However, somatic gene therapy reflects a
natural progression in the application of biomedical science
to medicine. Gene therapy has historically been seen to be
applicable to diseases for which current therapeutic
approaches are ineffective or where the prospects for
effective treatment appear to be exceedingly low. Although
disruptive and restrictive, the safe and effective therapies
that exist today make haemophilia different. This may alter
the ethical issues and considerations that arise in evolving
clinical trials.
Clearly, the patients most likely to benefit from a gene
therapy approach to treatment are those with severe
haemophilia (factor level , 1 units/dl). However, what
other characteristics should define a trial population?
Children could be seen to have the most to gain from a
gene therapy treatment. However, is it ethical to test a novel
and potentially unknown treatment on young children?
This is particularly important when considering potential
alterations to the immature germline and issues of consent
to such a potentially radical treatment with unknown and
unpredictable side-effects. Patients considered for participation in clinical trials should, therefore, be adults until gene
therapy is proven to be safe and effective.
Many risks and unknowns exist with gene therapy. Issues
751
for each trial will vary according to the vector system, route
of administration, target tissue and co-morbid states that
exist in the patients. For each individual study, the potential
risk of inhibitory alloantibody development, alteration of the
natural history of any co-morbid condition, germline
transmission, mutagenesis and immune response to vector
(rendering the patient refractory to future treatments) must
be considered and balanced against potential outcome.
Patient selection and entry criteria may therefore vary
between clinical trials.
To address these concerns, patients should be heavily
pretreated with factor concentrates, have no detectable or
history of inhibitory alloantibodies and have a clearly
documented molecular defect (to allow for assessment of
inhibitor risk).
The presence of co-morbid conditions such as HIV and
hepatitis C (HCV) in any study population is thorny. Patients
with HIV infection who are severely immunocompromised
may not develop inhibitory alloantibodies, and a limited life
span would hamper the collection of long-term safety data.
Conversely, patients with HIV infection with well-maintained CD4 counts may be suitable candidates. Can patients
on highly active antiretroviral therapy safely stop medication in order to participate in retroviral or lentiviral gene
therapy trials? With regard to HCV infection, will HCV
infection preclude inclusion in liver-targeted gene therapy?
The potential role of viral vectors inducing alterations in
cytokine/lymphokine profiles and their influence upon the
progression of liver disease is unknown. Finally, should trials
be restricted to men who are sterile (vasectomy or other
documented medical condition) until there is extensive data
on germline safety in relation to the chosen vector? This is
not an exhaustive list, and other topics for concern may
emerge.
Clinical trials
Three phase 1 clinical trials for haemophilia A and B have
been initiated and reported. These studies use a variety of
gene transfer systems and vectors. Although promising data
have been obtained, no trial carried out to date has been so
significantly successful as to allow further studies to proceed
forward in the same format.
The first results to be reported have been obtained using
an intramuscular injection of factor IX-containing rAAV
(under cover of factor IX concentrate) in adult patients with
severe haemophilia B and is based on the extensive
preclinical animal work outlined above. In the reported
eight patients so far enrolled, this dose escalation study
(three dose cohorts starting at 2 1011 vector genomes/kg,
increasing in half-log increments) has shown no toxicity
(except for one case of transient thrombocytopenia), no
germline transmission of vector sequences nor production of
inhibitory anti-factor IX antibodies (Kay et al, 2000; Manno
et al, 2000, 2001). Muscle biopsy performed 2 months after
vector administration showed the presence of vector genome
(polymerase chain reaction and Southern blot) and the
expression of factor IX in muscle fibres and the extracellular
matrix. Preliminary data also suggest modest increases in
plasma factor IX level in two of the six subjects analysed to
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752
Review
date (. 1 units/dl but , 2 units/dl factor IX rise) and a
reduction in the number of factor IX concentrate infusions
in three of the six subjects (Kay et al 2000; Manno et al,
2001). Data from this study have suggested that dose
calculations based on animal data may have overestimated
the dose required in humans to achieve therapeutic levels. A
similar rAAV study is also proposed using an intrahepatic
infusion route.
For haemophilia A, two systems are presently being
investigated using ex vivo transfection of autologous
fibroblasts and in vivo retroviral transfection systems. In
the fibroblast protocol, cells are removed from the patient,
cultured and transfected with a B-domain-deleted factor VIII
gene by electroporation. A clonal population of cells that
expresses factor VIII is then isolated before injection of the
autologous cells into the patient. Preclinical assessments
have demonstrated safety and durable expression of human
factor VIII (. 5% of normal) for . 1 year in mice after a
single treatment (Roth et al, 2000), although full preclinical
data are yet to be published. To date, six adult patients have
been treated with transfected cells administered by laparoscopic omental injection with a published follow-up of
12 months (Roth et al, 2001). There have been no serious
toxicities, long-term complications or inhibitory anti-factor
VIII antibodies, four of the six patients demonstrating
repeated factor VIII activity levels modestly above baseline
(largest recorded increase in VIII:C activity 3´5 units/dl on
one occasion). In the two out of four patients who kept
diaries of bleeds and treatment, this increase in factor VIII
activity coincided with a decreased bleeding frequency and
factor VIII concentrate use (Roth et al, 2001). No
improvement in factor VIII level lasted beyond 10 months
after implantation. Although theoretically safe, being an ex
vivo modification, as a cell-based therapy, this system is
highly labour intensive and would be difficult to apply to
large patient populations.
The second haemophilia A clinical trial under way uses
an MoMLV retroviral vector expressing B-domain-deleted
factor VIII. In this open-label dose escalation study, vector is
infused intravenously daily for three successive days. This
study is based on the preclinical efficacy data derived from
the intravenous use of high-titre retroviral vector systems in
rabbits and haemophilic dogs (Greengard & Jolly, 1999).
Preliminary data in 13 enrolled patients suggest safety at
the doses used in the study, no significant adverse events
and no evidence of replication-competent retrovirus.
Although no subject had sustained repeated levels
. 1 unit/dl, six patients showed factor VIII . 1 units/dl
on at least two occasions. Most elevated levels were in the
range of 1´0±1´8 units/dl, although isolated higher levels
were reported for three subjects (Powell et al, 2001).
A phase I dose escalation study using MiniAdFVIII
(gutless adenovirus) has recruited one patient to date.
Future enrolment is on hold pending analysis of the initial
data and toxicity profile.
To date, therefore, over 25 patients with haemophilia
have been treated with gene therapy protocols. All three
studies have failed to show conclusively that therapeutic
levels of factor VIII and IX can be obtained reliably. However,
importantly to date, these studies have not identified
significant safety concerns. It is most likely that these
present protocols will require revision and, even if successful
when modified, it is most likely that such protocols will lead
at best to only modest increases in circulating factor VIII
and IX levels, and patients will continue to require
treatment with concentrate for surgery and trauma. It is
also of note that all these human studies have been preceded
by animal trials that have generally shown higher rises in
plasma factor VIII and IX than have been seen in the human
trials. This re-emphasizes that animal studies can only be a
rough guide to human response.
CONCLUSIONS
Several preclinical studies have shown that long-term
therapeutic levels of factor VIII or factor IX can be achieved
in both haemophilic mice and dogs. These advances in viral
and non-viral gene transfer technology hold real promise for
the development of gene therapy treatments for haemophilia A and B.
Although gene therapy strategies based on MoMLV-based
retroviral vectors initially produced no, poor or short-term
expression of factor VIII or factor IX, they have now evolved
such that MoMLV-based systems can cure murine models of
haemophilia A, achieve stable therapeutic factor VIII levels
in rabbits and partial correction of canine haemophilia A.
This has been achieved through a combination of improved
vector design, the ability to generate high titres and better
expression cassettes. Today, we see a phase I clinical trial in
severe haemophilia A patients using similar MoMLV-based
vectors.
Although seen as a theoretical improvement upon
MoMLV-based systems, the continued evaluation of efficacy,
safety and toxicity of lentiviral vectors will be essential
before they can be considered for the treatment of
haemophilia.
Adenoviral vectors remains the most efficient vectors in
achieving high-efficiency transduction in vivo. However, the
continuing development of retroviral, lentiviral and AAV
vectors is reducing this difference. Despite the use of
stronger expression cassettes that allow lower doses of
vector and reduce hepatotoxicity, the immune response
against adenoviral gene products per se continues to hamper
their use. So-called `gutless' adenoviral systems, which do
not contain adenoviral genes, may provide a solution to this
problem, although these systems are yet to be fully proven
as effective in the haemophilic dog model (Fang et al, 2001).
The use of AAV for haemophilia gene therapy has been
very promising initially for factor IX and now for factor VIII.
A complete cure of murine haemophilia B can be achieved
by intrahepatic administration of factor IX rAAV and partial
phenotypic correction of canine haemophilia B by intraportal or intramuscular injection of factor IX rAAV. The
phase I clinical trial of intramuscular injection of factor IX
rAAV has shown modest improvement in clinical endpoints, which is encouraging. Future rAAV phase I studies
using an intrahepatic route are planned.
In clinical terms, a continuing unanswered question
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remains the inhibitor response in haemophilic patients
treated with gene therapy systems. The assessment of
inhibitor development has been clouded in animal models
by the use of non-homologous genes that has led to the
production of alloantibody. With the increasing use of
homologous genes in animal models of haemophilia, these
issues may now be partially clarified. However, in the
human setting, whether gene therapy would increase or
decrease the likelihood of inhibitor formation compared
with concentrate therapy is unknown. Concerns remain
that viral vectors per se or impurities in vector preparations
may facilitate inhibitor formation. Equally, it is not clear
whether or not gene therapy could break tolerance in
patients. This might be of particular importance when
factor VIII or IX is produced from ectopic engineered sites
and potentially presented in association with class I and II
major histocompatibility complex (MHC) at these sites.
However, arguably rather than lead to or promote an
immune response that produces inhibitory alloantibodies,
continuous production of factor VIII and factor IX in vivo
after gene therapy may actually induce immune tolerance.
The history of gene therapy for haemophilia has, in the
past, been oversold. Overzealous presentation of initial gene
therapy studies obscured their exploratory nature. This has
led to the widely held, but mistaken, perception that gene
therapy in its broadest context was already highly successful. Research in gene therapy for haemophilia is now
catching up with the expectations of what it can deliver.
However, despite the tremendous progress in the field over
the past few years, many questions, such as the inhibitor
issues, remain largely unexplored and unanswered. With
three clinical trials now reported, expansion of these studies,
albeit with modified protocols, is inevitable. As new
haemophilia gene therapy trials are instituted, patient
safety must remain a priority.
In utero temporary correction of murine haemophilia A
and B models is possible (Lipshutz et al, 1999; Schneider
et al, 1999) and raises the prospect of lifetime cure of
haemophilia before birth. However, advances in gene
technology will soon make it possible to introduce targeted
alterations into the germline in mice. It is possible to
conceive that such technology may promise the possibility of
correcting the affected coagulation factor gene in the
germline of haemophilic patients and so `prevent' haemophilia in future generations. Although perhaps an unrealistic goal at present, the ethics of these potential aspects of
gene therapy need to be fully and widely debated, as they
have wide implications for genetic manipulation in general
(Billings et al, 1999).
Today, 80% of the world's population is without access to
effective therapy for haemophilia. Severe haemophilia
remains a crippling and fatal disorder in these areas. Even
with the increasing availability of factor concentrates,
present-day approaches using standard replacement therapy are impracticable in many areas of the third world, not
least because of population numbers and cost. Gene therapy,
if available as a simply administered treatment, offers a real
practicable alternative therapy. Our target should therefore
be gene therapy protocols that are applicable to the vast
753
majority, if not all, people with haemophilia at a cost that
can be afforded.
Division of Haematology, University of
Leicester, Robert Kilpatrick Clinical Science
Building, Leicester Royal Infirmary,
Leicester, UK
K. John Pasi
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Keywords: gene therapy, haemophilia, factor VIII, factor IX.
q 2001 Blackwell Science Ltd, British Journal of Haematology 115: 744±757