Human Molecular Genetics, 2016, Vol. 25, No. R1
R36–R41
doi: 10.1093/hmg/ddv475
Advance Access Publication Date: 27 November 2015
Invited Review
INVITED REVIEW
Adeno-associated viral vectors for the treatment
of hemophilia
Katherine A. High* and Xavier M. Anguela
Spark Therapeutics, Inc., 3737 Market St, Suite 1300, Philadelphia, PA, USA
*To whom correspondence should be addressed. Email: [email protected]
Abstract
Gene transfer studies for the treatment of hemophilia began more than two decades ago. A large body of pre-clinical work
evaluated a variety of vectors and target tissues, but by the start of the new millennium it became evident that adeno-associated
viral (AAV)-mediated gene transfer to the liver held great promise as a therapeutic tool. The transition to the clinical arena
uncovered a number of unforeseen challenges, mainly in the form of a human-specific immune response against the vector that
poses a significant limitation in the application of this technology. While the full nature of this response has not been
elucidated, long-term expression of therapeutic levels of factor IX is already a reality for a small number of patients. Extending
this success to a greater number of hemophilia B patients remains a major goal of the field, as well as translating this strategy to
clinical therapy for hemophilia A. This review summarizes the progress of AAV-mediated gene therapy for the hemophilias,
along with its upcoming prospects and challenges.
Introduction
Hemophilia is the X-linked bleeding diathesis caused by mutations in the genes encoding factor VIII (FVIII) or factor IX (FIX), respectively the cofactor and the enzyme responsible for catalyzing
the conversion of factor X to activated factor X in the intrinsic
pathway of the coagulation cascade. The disease is characterized
by recurrent bleeds, primarily into the joints and soft tissues, but
bleeding into other closed spaces such as the intracranial space
may also occur and may be associated with considerable morbidity or mortality (1). Hemophilia A and hemophilia B are indistinguishable clinically and were first distinguished in the clinical
coagulation laboratory in the 1950s (2,3). The incidence of hemophilia is ∼1 in 5000 male births (4), hemophilia A being about four
times as common as hemophilia B. Clinically, patients are classified as severe, moderate or mild; severely affected patients
constitute the largest group, and have <1% normal circulating
levels of FVIII or FIX. Mildly affected patients have ≥5% of normal
levels, and are free of the spontaneous bleeding episodes that
characterize severe disease; moderately severe patients have factor levels between 1 and 5%, and their clinical presentation is also
intermediate between severe and mild. Currently hemophilia is
managed by intravenous infusion of clotting factor concentrates,
which can be given prophylactically, or ‘on demand’, i.e. in response to a bleeding episode. Most moderate or severe patients
administer factor somewhere between 20 and 100+ times/year.
Gene Therapy for Hemophilia: Rationale and
Early Trials
Since the isolation of the genes encoding FVIII (5) and FIX (6),
hemophilia has been an attractive target for investigation of
gene therapy approaches, and the level of activity in terms of
clinical trials of gene therapy for hemophilia reflects this (www.
clinicaltrials.gov). Characteristics that support the attractiveness
of this target include: (i) latitude in the choice of the target tissue.
Biologically active clotting factors can be synthesized in a range
of cell types, and will be effective so long as the gene product
reaches the circulation. (ii) Wide therapeutic window. Most individuals with hemophilia are severely affected, with <1% of normal levels of clotting factor activity, but raising levels even
modestly into the moderately severe range (>1, <5%) will markedly
improve the clinical phenotype; raising levels into the mild range
(≥5%) will prevent spontaneous bleeding episodes and greatly
Received: October 23, 2015. Revised and Accepted: November 16, 2015
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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reduce the patient’s dependence on exogenously infused clotting
factor. On the upper end, raising the level to 100% still leaves the
patient within the normal range. Thus, a wide range of transgene
expression falls into the therapeutic window. (iii) The existence
of small (genetically engineered mice) and large (naturally occurring dog) animal models of hemophilia (reviewed in 7). This has
meant that most strategies can be evaluated in animal models
prior to clinical trials in humans. (iv) The transgene product is
easy to measure (in any hospital coagulation laboratory) from a
blood sample and is an accepted endpoint for product registration since it correlates well with the severity of the disease and
clinical outcome in terms of the annualized bleeding rate.
The size difference between the cDNA for FIX (2.8 kb if
the long 3′UTR is included) and FVIII (∼4.4 kb even for the
B-domain-deleted construct) explains the differences in vector
choice in the early trials. The first wave of gene therapy trials
for hemophilia A, starting in 1998, utilized retroviral (8), adenoviral (sponsored by GenStar Therapeutics, unpublished) and
plasmid vectors (9). Retroviral and adenoviral vectors were delivered intravenously whereas plasmid vectors were ex vivo electroporated into autologous fibroblasts, which were then implanted
on the patient’s omentum in a laparoscopic procedure. The
initial trials for hemophilia B (vide infra), both used adenoassociated viral (AAV) vectors, delivered to either skeletal muscle
or to the liver via infusion into the hepatic artery in the interventional radiology suite. All of these trials were first in class, and all
appeared generally safe, but none achieved long-term expression
at therapeutic levels. However, infusion of an AAV vector into the
liver in a subject with severe hemophilia B (10) clearly resulted in
therapeutic levels of expression (>10% normal) for a period of
several weeks, and laid the groundwork for the current generation of trials, which all involve hepatic transduction by AAV
vectors infused intravenously.
AAV Vectors for Hemophilia B
AAV vectors are engineered from a parvovirus (11). The recombinant vector has tropism for a range of target tissues including
the liver, cell types in the retina and the central nervous system,
skeletal muscle, and cardiac muscle, among others (reviewed
in 12). The DNA sequences carried by recombinant AAV vectors
are stabilized predominantly in an episomal form so that longterm expression can occur only with delivery into long-lived,
post-mitotic cell types; the vector DNA integrates at a very low
frequency and is typically lost from replicating cells (13). One of
the main limitations of AAV vectors is that they cannot package
inserts of more than ∼5 kb (Fig. 1) (14); this explains the initial
focus on hemophilia B in the AAV work. Studies in the large
animal model of hemophilia B (15) established clear proof of concept, showed a favorable safety profile and accurately predicted
dosing requirements in human subjects. Based on these data,
eight subjects were enrolled in the first muscle-directed, AAVbased clinical trial for hemophilia B (16, 17). Importantly, no
vector-related toxicity was observed, and there was evidence of
FIX protein expression in muscle cells up to 10 years after
AAV2-FIX administration (18). However, circulating FIX failed to
rise to >1% and disease phenotype was not improved, suggesting
that the secretion of the synthesized transgene product into the
circulation was not efficient.
In the first liver-directed AAV trial for hemophilia B, a singlestranded AAV2 vector expressing human FIX was infused via the
hepatic artery into seven subjects (10). Efficacy was observed in
the first of the two subjects that received the highest vector
dose of 2 × 1012 vg/kg, with peak FIX levels reaching ∼10% of
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normal. Unexpectedly, an asymptomatic, self-limited rise in hepatic transaminases was observed around week 4 after vector infusion that coincided with the onset of a gradual loss of FIX activity.
Both of these events were attributed to the destruction of transduced hepatocytes by AAV capsid-specific memory CD8+ T cells
(19). This observed immunogenicity against the capsid had not
been predicted by any animal model, and several hypotheses
were formulated to explain it. Among others, uptake by dendritic
cells of the AAV2 virion in a process mediated by binding to heparan sulfate proteoglycans followed by the activation of capsid-specific T cells (20) or the presence of alternative open
reading frames in the FIX coding sequence (21) were proposed
as the culprits. Notably, after a decade of intense work, the immune response against the capsid remains a poorly understood
phenomenon that is not well-modeled in mice (22). The other
subject in the high-dose cohort yielded the second valuable lesson learned from that trial, i.e. pre-existing anti-AAV neutralizing
antibodies (NAbs), even at modest titers, are able to prevent successful liver transduction after systemic vector administration.
The second liver-targeted AAV trial for the treatment of
hemophilia B, conducted by investigators at St Jude Children’s
Research Hospital and University College London, differed from
the first study in two main aspects: (a) it utilized a self-complementary vector genome that was (b) packaged into an AAV8
capsid, administered by peripheral vein infusion. Based on the
pre-clinical data available at the time, both modifications were
expected to result in significantly higher FIX levels although the
extent of any contribution of these two factors is now unclear
(23,24). Three vector doses were used (2 × 1011, 6 × 1011 and 2 ×
1012 vg/kg), with the high-dose mediating peak expression levels
at 8–12% of normal (25). More recently, data from 10 patients were
reported, with a follow-up period of up to 4 years (26). Several
observations of paramount importance were made in this
study. First, all patients achieved long term, stable FIX expression
with average FIX levels of ∼5% of normal in all six patients in the
high-dose cohort. Secondly, in four of these six patients, a transient increase in LFTs was observed between weeks 7 and 10 after
AAV administration, likely as a result of a T-cell response against
the AAV8 capsid. Notably, the prednisolone treatment was able to
control this response and serum alanine aminotransferase (ALT)
levels returned to normal within days. Elevated ALT episodes
were not recurrent and no late toxicity was reported, establishing
a favorable safety profile for this gene transfer protocol. Undoubtedly, these successful results represent a milestone in the gene
therapy field, and a goal of much ongoing work is to replicate
and extend them.
While the clinical improvement in patients who achieved
stable FIX levels of ∼5% of normal is indisputable, risk for excessive hemorrhage after trauma or surgery would be significantly
reduced if stable levels were close to 50%. A more recent Phase
1/2 trial sponsored by Baxalta (clinical trials identifier no.:
NCT01687608) also utilized an AAV8 capsid packaging a selfcomplementary cassette, but expressing FIX Padua. This naturally occurring FIX variant has an activity-to-antigen ratio of
around 8–9 (27). A total of seven patients have been treated: two
at 2 × 1011 vg/kg, three at 1 × 1012 vg/kg and two at 3 × 1012 vg/kg
[World Congress of the International Society of Thrombosis and
Haemostasis (Toronto, Canada, June 2015)]. Peak FIX activity
values in the third cohort subjects reached 30–60% of normal,
highlighting the potential of the Padua variant. However, expression declined sharply around week 6, coinciding with an elevation in ALT levels. One subject in the medium-dose cohort has
had sustained FIX activity levels of 20–25% for a year, whereas
FIX antigen levels in the two other subjects declined over time.
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| Human Molecular Genetics, 2016, Vol. 25, No. R1
Figure 1. Potential limitations in AAV therapy. (A) All viral sequences except the ITRs are replaced by an expression cassette, with a maximum capacity of around 4.7 kb. (B)
Thus far, the AAV-based hemophilia trials have targeted either the muscle or the liver. Pre-existing anti-AAV neutralizing antibodies, even at modest titers, are able to
prevent successful transduction after vector administration through the circulation. As a consequence, as many as 40% of adult hemophilia patients may be ineligible to
participate in liver-directed AAV trials. (C) Once within the cell nucleus, the majority of AAV genomes are stabilized predominantly in an episomal form, which makes
them susceptible to dilution if the cell divides. Episomes will integrate at a very low frequency and thus the potential risk of insertional mutagenesis exists. The capsid
proteins presented on the cell surface may also flag the transduced cells for destruction. (D) Finally, a humoral immune response against the transgene product, the AAV
capsid or both may be mounted.
Finally, the first treated subject in the low-dose cohort showed no
detectable FIX activity whereas FIX activity remains stable at 3%
in Subject 2 (thus, antigen levels must be <1% of normal). The reasons behind the differences in the outcome of these two somewhat similar trials (both utilized the same AAV8 capsid and a
self-complementary genome configuration) remain to be determined. A potential explanation could lie in differences (however
minor) in vector design or manufacturing, which may significantly alter the kind and/or the magnitude of the immune response.
This underscores the need to develop relevant pre-clinical
models to build a more complete understanding of vector immunogenicity associated thus far with AAV gene delivery in
humans.
The T-cell response against the capsid is not the only limitation that the immune system imposes on AAV-based treatments
for hemophilia. As mentioned above, even low levels of preexisting circulating NAbs against the vector can completely
inhibit liver transduction after systemic administration (28).
This means that as many as 40% of adult hemophilia B patients
may be ineligible to participate in liver-directed AAV trials.
Human Molecular Genetics, 2016, Vol. 25, No. R1
Recent data with phylogenetically distant capsids suggest that
switching serotypes may not offer a significant improvement
(29). Several strategies have been devised to overcome the presence of NAbs. We have shown that empty capsids may be used
as decoys (30) and that B-cell depletion using rituximab can decrease anti-AAV antibodies titers in rheumatoid arthritis patients
(31). Others have suggested plasmapheresis (32), chemical or
genetic modification of the AAV capsid (33,34), saline flushing
prior to portal administration (35) and even using naturally enveloped AAV vectors with extracellular vesicles (36).
In addition to the short-term risks posed by the immune response, which primarily relate to efficacy rather than safety,
there is the potential risk of late adverse events related to insertional mutagenesis. Although AAV vectors are stabilized mainly
in an episomal form, it is clear that some low-level of integration
into the host cell genome occurs (37). There have been reports of
an increased incidence of hepatocellular carcinoma (HCC) in
mice injected with high doses of AAV vectors in the neonatal period (38,39), and integration of sequences derived from wild-type
AAV2 has been found in human HCC tissue samples (40). In mice,
the incidence of HCC is highly dependent on the AAV vector dose,
the ability of the regulatory elements to promote increased transcription of proximal genes and the timing of vector delivery
(neonatal versus adult administration) (41). Thus, using the lowest effective dose of a vector devoid of enhancers would seem the
safest approach to minimize risk. Long-term surveillance of large
animals injected with vector, as well as pharmacovigilance in
human subjects, will be required to address the likelihood of
this risk in subjects injected as adults. Importantly, tumors
have not been observed in human subjects injected as long ago
as 2001 (42).
AAV Vectors for Hemophilia A
The development of AAV-based therapies for the treatment of
hemophilia A is still at an early stage, with one trial sponsored
by BioMarin (clinical trials identifier no.: NCT02576795) recently
begun. One of the main challenges is fitting the FVIII expression
cassette within the restricted packaging limits of AAV. At ∼7 kb,
the cDNA for FVIII exceeds the packaging capacity of adenoassociated vectors, which is ∼5 kb (14). The use of a B-domain
deleted form of FVIII (4.37 kb in size) circumvents this limitation,
but imposes a stringent constraint on the size of the regulatory
elements that control FVIII expression. In addition, FVIII
expression is significantly lower than that of similarly sized
clotting factors such as factor V (43). Codon-optimization of the
coding region has been reported to increase FVIII expression
over 40-fold compared with the wild-type sequence (44). Moreover, circulating FVIII levels may be further increased by using
a variant that contains a 17 amino acid synthetic sequence
flanked by 14-aa SQ residues from the N- and C-terminal ends
of the B domain (45). While the presence of the synthetic spacer
allows for an increase in circulating hFVIII levels, the use of a
non-wild-type FVIII sequence in hemophilia A patients raises
concerns about increasing the risk of inhibitor development
due to its potential neo-antigenicity. Generating an AAV-FVIII
vector capable of expressing therapeutic levels of FVIII at a
clinically relevant dose without adding any neo-antigens to the
protein remains an unmet goal.
The most difficult problems in hemophilia care currently
revolve around patients who develop inhibitors, to infused factor.
These occur in as many as 20–30% of all hemophilia A patients
(46); they are much less frequent among those with hemophilia
B. Currently, the standard of care for these individuals is a so-
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called immune tolerance induction regimen (ITI) in which
patients are infused with large doses of clotting factor daily, a
strategy that results in eradication of the inhibitor and restoration of normal FVIII pharmacokinetics in ∼74% of cases (47).
Although the mechanism by with ITI eradicates the inhibitor is
not worked out, this would seem to be an excellent setting for a
gene therapy strategy, which exposes the patient to continuous
levels of FVIII without the need for daily infusions. Indeed studies
in hemophilia A dogs that had developed inhibitors demonstrated eradication of inhibitors following AAV-mediated, liverdirected gene therapy (48). These strategies have not yet been
tested clinically.
Conclusion
These recent clinical trials using AAV-mediated gene transfer have
underscored the tremendous potential of gene therapy for the treatment of hemophilia. However, an unanswered question is whether
episome-derived liver expression will be sustained in a setting of
substantial liver proliferation, as in pediatric patients [the liver
quadruples in size during the first 4–5 years of development (49)]
or those with liver disease (e.g. hepatitis and/or cirrhosis). For
these groups of patients, the integration of the transgene to avoid
AAV dilution and loss of expression could be especially beneficial.
Long-term expression of FIX in hemophilic dogs has been recently
described (50). It has also been shown that in vivo site-specific genome editing can be applied successfully with therapeutic benefit,
achieving long-term expression of human coagulation factors in
mice (51,52). These studies used AAV to deliver zinc-finger nucleases to the target hepatocytes; the extension of the strategy to
large animals has been reported in abstract form (53). Plans are
underway to investigate this strategy clinically (September 9, 2015
—RAC meeting: http://videocast.nih.gov/launch.asp?19141).
A proof-of-concept for the long-standing goal of achieving
sustained and therapeutic levels of a clotting factor after a single
vector administration has been fulfilled in the case of hemophilia
B (26), and a number of trials have been initiated over the past
year (clinicaltrials.gov) with the objective of confirming and consolidating this landmark result. Replication of this success in larger patient cohorts is an immediate goal, followed closely by
efforts to extend the approach to hemophilia A (45), an objective
that has not yet been realized. Extension to those with liver disease, anti-AAV NAbs, FVIII and FIX inhibitors, as well as the pediatric population, are longer term goals for investigation.
Conflict of Interest statement. K.A.H. and X.M.A. are employees of
Spark Therapeutics, Inc. The company’s hemophilia B program
is partnered with Pfizer Inc.
Funding
Previous work was supported by the Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia,
the Howard Hughes Medical Institute and the US National Institutes of Health (grants HL64190, HL078810 and HV78203). Current
work is supported by Spark Therapeutics, Inc.
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