Hemophilia Gene Therapy: Ready for Prime Time?
*and Marinee K. Chuah
Department of Gene Therapy & Regenerative Medicine, Free University of Brussels (VUB), Brussels, Belgium;
Center for Molecular & Vascular Biology, Department
of Cardiovascular Sciences, University of Leuven, Leuven, Belgium.
Hemophilia A and B are congenital, X-linked bleeding disorders caused by mutations in the genes encoding for
the blood clotting factor VIII (FVIII) or factor IX (FIX), respectively. Since the beginning of gene therapy,
hemophilia has been considered an attractive disease target that served as a trailblazer for the ﬁeld at large.
Different technologies have been explored to efﬁciently and safely deliver the therapeutic FVIII and FIX genes
into the patients’ cells. Currently, the most promising vectors for hemophilia gene therapy are adeno-
associated viral vectors (AAVs) and lentiviral vectors. More recently, gene editing approaches based on de-
signer nucleases or CRISPR/Cas, have also been considered to minimize risks associated with random vector
integration and insertional mutagenesis though off-target issues would have to be carefully and comprehen-
sively assessed. In the past two decades, several phase 1 hemophilia gene therapy clinical trials have been
initiated with varying success. In particular, the early gene therapy clinical trials in hemophilia B patients
based on AAV showed either transient or subtherapeutic clotting factor expression levels. This could be
ascribed, at least in part, to suboptimal vector design and/or inadvertent immune consequences triggering
hepatic inﬂammation. Hence, there was an unmet need to further increase vector safety and efﬁcacy in future
trials, preferably by using lower vector doses. It is particularly encouraging that sustained therapeutic FVIII
and FIX expression levels have recently been attained after gene therapy in patients with severe hemophilia
paving the way towards pivotal trials and commercialization. Nevertheless, transient liver toxicity still occurs
and the use of transient immunosuppression was still required to curtail inadvertent immune responses,
especially at high vector doses. To further boost clotting factor expression levels, codon-usage optimized syn-
thetic FVIII or FIX transgenes have been employed. Alternatively, we and others have shown that the in-
corporation of hyperactive gain-of-function R338L mutation in the FIX gene substantially increased the overall
efﬁcacy. It is inevitable that the continued improvements in vector engineering and new insights in the vector–
patient interactions will further beneﬁt the development of a safe and effective cure for hemophilia A and B.
Keywords: factor IX, factor VIII, coagulation, hemophilia, AAV, lentiviral, CRISPR, ZFN, factor IX Padua
HEMOPHILIA A AND B are congenital X-linked bleed-
ing disorders caused by mutations of genes encod-
ing the blood clotting factor VIII (FVIII) or factor IX
(FIX), respectively. Consequently, these disorders
are characterized by a bleeding disorder due to a
deﬁciency in the corresponding clotting factors.
Patients often suffer from recurrent bleeds and
chronic joint damage resulting in a crippling ar-
thropathy. The bleeding could even be fatal in
cases of intracranial hemorrhage. According to the
World Federation of Hemophilia, the diseases af-
fect an estimated 400,000 individuals worldwide,
of which 80–85% are affected by hemophilia A
and the remaining 15% by hemophilia B. Current
treatment consists of protein substitution therapy
(PST) with recombinant or plasma-derived clot-
ting factors. Although PST improved the patient’s
quality of life and signiﬁcantly prolonged life ex-
pectancy, it has several drawbacks. First, this
treatment is noncurative, implying that the patient
remains at risk of developing bleeding episodes
*Correspondence: Dr. Thierry VandenDriessche or Dr. Marinee Chuah, Department of Gene Therapy & Regenerative Medicine, Faculty of Medicine and Pharmacy, Free
University of Brussels, Building D, Room D306, Laarbeeklaan 103 B-1090, Brussels, Belgium. E-mail: email@example.com; firstname.lastname@example.org
ªThierry VandenDriessche and Marinee K. Chuah 2017; Published by Mary Ann Liebert, Inc. This is an Open Access article distributed
under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
HUMAN GENE THERAPY, VOLUME 28, NUMBER 11 DOI: 10.1089/hum.2017.116 j1013
Mary Ann Liebert, Inc.
and/or chronic joint damage. The second constraint
pertains to the clotting factors’ short half-lives,
requiring frequent treatment with prophylaxis or on
demand using relatively large doses of FVIII or FIX.
Finally, some patients develop neutralizing anti-
bodies (clinically deﬁned as inhibitors) against the
injected FVIII or FIX proteins that render further
PST ineffective. Several next-generation protein-
based therapeutics have recently been developed
that prolong the half-life of the clotting factors, for
instance by pegylation or fusion with albumin and
second-generation products require less frequent
administration and represent a new benchmark for
all emerging products to treat hemophilia patients,
including gene therapy. Given the intrinsic limi-
tations of PST, there is still a need to develop a
long-term cure for hemophilia via gene therapy.
The current review celebrates the 25th anni-
versary of the European Society of Gene and Cell
Therapy. Our own personal journey in the ﬁeld of
gene therapy for hemophilia coincidentally also
started exactly 25 years ago. This allowed us to
witness ﬁrsthand the exciting developments in
gene therapy for hemophilia that had an impact on
so many other diseases since then. In this review,
we will highlight the most relevant and most recent
developments in the ﬁeld of gene therapy for he-
mophilia A and B. We will also discuss some of the
outstanding questions and remaining challenges
that would still need to be addressed. It is beyond
the scope of the current review to provide a com-
prehensive description of all of the possible strat-
egies and animal studies that have been published
relating to hemophilia gene therapy in the past 30
years, which have been covered by other earlier
reviews on the subject.
Instead, we will focus on
highlighting a few key concepts with speciﬁc em-
phasis clinical translation.
The main objective of developing gene therapy
for hemophilia is to achieve prolonged high-level
expression of clotting factors and stably correct the
bleeding disease. Essentially, there are two dis-
tinct approaches to achieve this objective. The ﬁrst
approach is FVIII or FIX gene delivery to long-lived
post mitotic cells, such as hepatocytes or skeletal
muscle cells. In this case, the therapeutic genes
should either integrate into the target cell genome
or persist as episomal DNA. Alternatively, FVIII or
FIX genes could be delivered to stem/progenitor
cells, such as hematopoietic stem cells (HSCs),
using integrating (viral or nonviral) vectors in
order to achieve long-lasting expression through
their progeny. Designing the optimal FVIII and
FIX expression cassette and delivering these con-
structs into the appropriate target cells in a safe
and efﬁcient manner will be essential toward
achieving a cure for hemophilia. Ideally, gene
therapy should not result in the induction of in-
hibitors or should enable clearance of pre-existing
inhibitors through induction of FVIII- or FIX-
speciﬁc immune tolerance, which has been accom-
plished in preclinical studies.
The ability to in-
duce immune tolerance to FVIII or FIX after gene
therapy varies depending on several confounding
variables, including the transgene product, the
vector design, the target cells, and the underlying
mutation of the endogenous FVIII or FIX gene.
Designing optimal FVIII and FIX
As with any gene therapy approach, it is criti-
cally important to express high levels of the thera-
peutic protein using the lowest possible vector dose
to minimize untoward vector-associated immune re-
sponses or toxicity. This has been particularly chal-
lenging since there are several bottlenecks at the
transcriptional, translational and post-translational
level, especially for FVIII.
It is therefore necessary to
overcome each of these limiting steps using rationally
designed transgenes and expression constructs. In-
cremental improvements at the transcriptional,
translational, and/or post-translational levels may
ultimately translate into signiﬁcant dose beneﬁts in
clinical trials in hemophilia patients. All of the gene
therapy approaches rely on the use of FVIII and FIX
cDNAs instead of the full-length transgenes per se.
Nevertheless, in many cases, introns were incorpo-
rated in the constructs
to further boost expression
levels that act possibly, at least in part, by facilitating
extra-nuclear transport of the cognate transcripts.
The endogenous FVIII and FIX promoters are
not particularly robust and are therefore not ide-
ally suited for gene therapy for hemophilia. In-
stead, to boost the FVIII and FIX mRNA levels,
we and others have explored the use of robust
tissue-speciﬁc heterologous promoters, such as a
1-antitrypsin or transthyrethin.
moters could be combined with other regulatory
elements (e.g., HCR, enhancers) to further boost
More recently, we have developed a
novel approach to identify robust human hepatocyte-
speciﬁc cis-regulatory modules (CRMs) using
genome-wide computational strategies instead of the
conventional trial-and-error approaches. The use of
these CRMs, in conjunction with existing promoters,
resulted in relatively robust increases in FIX or
1014 VANDENDRIESSCHE AND CHUAH
FVIII gene expression when used in the context of
different gene therapy vectors.
It is reassuring
that these CRMs increased expression of hetero-
logous transgenes, including FVIII and FIX,without
increasing the risk of insertional oncogenesis of inte-
grating vectors, even in highly sensitive tumor-prone
Alternatively, synthetic transcrip-
tional enhancers were generated de novo by random
ligation of synthetic oligonucleotides coding for bind-
ing sites of hepatic transcription factors and in vitro
screening in hepatic cell lines.
The speciﬁcity of the
promoter and regulatory elements can have impor-
tant consequences for the immune response directed
against the transgene products
More than 20 years ago, we pioneered the use of
codon-optimization of clotting factor genes in at-
tempt to increase their expression.
Since then, we
and others have continued to develop codon-
optimized FVIII and FIX transgenes
resulted in higher levels of protein expression for
the same amount of mRNA. One of the main ad-
vantages of codon optimization is that it does not
alter any of the amino acids encoded by the FVIII or
FIX transgene obviating possible immunogenicity
concerns. Typically, codon-usage optimization re-
sulted in a 3-fold increase in FIX protein expression
levels, but in the case of FVIII an unexpectedly
greater impact on FVIII protein levels was appar-
ent (up to 44-fold).
Deletion of the B-domain
(FVIIIDB) was also shown to be essential in
boosting FVIII expression levels, by increasing the
FVIII mRNA levels compared with the full-length
Nevertheless, deletion of the entire
B-domain compromises speciﬁc post-translational
intracellular FVIII trafﬁcking due to the loss of
critical glycosylated residues.
To compensate for
this defect, a synthetic B-domain–derived 17-
amino acid polypeptide was reintroduced in the
FVIIIDBcDNA that presumably reconstitutes
these asparagine-linked oligosaccharides, consis-
tent with a *3-fold increase in the secreted FVIII
Alternatively, incorporation of a
furin cleavage site within the B domain (position
R1645H) that mimics the canine sequence (histidine-
histidine-glutamine-arginine [HHQR] vs. human
arginine-histidine-glutamine-arginine [RHQR]) in-
creased FVIII expression *2-fold in hemophilia
FVIII expression levels could also be
increased by generating porcine-human hybrid
It has been shown that
overexpression of FVIII above 200% may provoke
cellular stress which may in turn increase the risk of
It may therefore be prudent to restrict
FVIII expression levels to minimize the risk of such
untoward cellular stress responses. However, it is
reassuring that FVIII levels up to 75-fold higher
than physiologic levels did not result in detectable
transaminitis or liver toxicity.
As an alternative to codon optimization, we
showed, for the ﬁrst time, with liver-directed gene
delivery that a relatively robust increase in FIX
activity (by up to 8-fold) could be obtained by in-
corporating a single R338L gain-of-function point
mutation in the FIX gene.
This point mutation
(designated as FIX-Padua) was initially discovered
in thrombophilic patients
and this could be har-
nessed to signiﬁcantly augment the efﬁcacy of gene
therapy for hemophilia B with minimal sequence
perturbation. The functional enhancement of this
speciﬁc point mutation was conﬁrmed in subse-
Several other mutants have
been explored that enhance FIX activity.
Target cells for hemophilia gene therapy
Different cell types have been explored for gene
therapy of hemophilia and have been extensively
Some of the most relevant
cell types are discussed in more detail below.
The liver is an attractive target organ for both
hemophilia A and B gene therapy since FVIII and
FIX are naturally produced by liver sinusoidal
endothelial cells (LSECs) and hepatocytes, re-
spectively. Consequently, LSECs and hepatocytes
have been targeted by different viral and nonviral
systems for gene therapy. Perhaps most
importantly, the hepatic niche may favor the in-
duction of immune tolerance towards the trans-
which may depend, at least in part,
on the induction of regulatory T-cells.
Skeletal muscle cells have a relatively robust se-
cretory capacity and are equipped with the necessary
cellular machinery enabling the post-translational
modiﬁcations required to generate functional FIX.
Though FIX could be secreted into the circulation
when expressed in the muscle, off-loading FVIII in
the circulation is impaired compared with the liver.
This may be due to the relatively large size of FVIII
particularly when complexed with vWF. Unlike liver-
directed gene transfer, it would appear that muscle-
directed approaches appear to have a higher risk of
inducing immune response against the transgene
Despite these limitations, early clinical
trials showed that muscle can give rise to sustained
FIX expression in patients up to 10 years after gene
Hematopoietic stem cells (HSCs) have also been
explored as targets for hemophilia gene therapy
to speciﬁcally express FVIII or FIX in different he-
matopoietic lineages. In particular, HSC-derived
erythrocytes, megakaryocytes and their platelet
HEMOPHILIA GENE THERAPY 1015
progeny could serve as a delivery platform to secrete
the clotting factors directly in the circulation fol-
lowing HSC-targeted gene transfer.
Preclinical and clinical studies
Gene therapy for hemophilia A and B requires
an efﬁcient and innocuous gene transfer system
that should preferably give rise to sustained, life-
long therapeutic FVIII or FIX expression without
any immune or toxic side effects. Several vectors
have been developed that are well suited for gene
therapy of hemophilia, each with their own ad-
vantages and limitations.
Nonviral vectors typically rely on DNA-based
physical or chemical transfection methods. Though
these methods typically result in no adaptive im-
mune responses, as opposed to when viral vectors
are employed, it is well established that DNA can
evoke innate immune responses in vivo, typically
involving Toll-like receptor signaling pathways.
The gene transfer efﬁciencies are generally lower
compared with most viral vector-mediated gene
transfer approaches. Moreover, nonviral transfec-
tion of FVIII or FIX-containing constructs typically
results in short-term expression of the transgene
product, unless selection is applied on ex vivo
A phase 1 clinical trial for he-
mophilia A had previously been conducted with
stably transfected autologous ﬁbroblasts that were
electroporated with FVIII-expressing plasmids.
After selective expansion, the transfected ﬁbroblasts
were implanted into the patient’s omentum. Though
no adverse events were noted, the therapeutic efﬁ-
cacy was modest and no sustained FVIII expression
could be attained.
We and others have shown that
the efﬁciency of stable genomic integration and FIX
or FVIII expression levels could be substantially
increased using transposons or integrases, consis-
tent with sustained phenotypic correction of the
bleeding phenotype in hemophilic mice.
Gamma-retroviral and lentiviral vectors
Gamma-retroviral vectors integrate stably into
the chromosomes of actively dividing cells. In 1999,
we had established the ﬁrst proof-of-concept that
hemophilia A could be cured by gene therapy in a
preclinical model of hemophilia A.
this, neonatal FVIII-deﬁcient mice were used since
they are permissive for c-retroviral transduction by
virtue of the rapid hepatocyte turn-over. This was
subsequently conﬁrmed in neonatal hemophilia A
and B dogs.
A phase 1 clinical trial was con-
ducted in adult hemophilia A patients but FVIII
levels were low due to the requirement for cell
In contrast, lentiviral vectors could
transduce quiescent noncycling hepatocytes, lead-
ing to relatively efﬁcient transduction in adult
livers (reviewed in Matrai et al.
). We demon-
strated that lentiviral vectors could also transduce
professional antigen-presenting cells (APCs) (i.e.,
Kupffer cells) and LSECs.
sion of FVIII or FIX in APCs increases the risk of
developing inhibitory antibodies that preclude
their long-term expression and renders gene ther-
Recent studies revealed that
residual FIX expression in CD11b
dendritic cells contributes to this immune re-
sponse, whereas FIX expression in conventional
dendritic cells or LSECs contributes to immune
The mechanism of immune tol-
erance induction following hepatic FVIII or FIX
delivery with lentiviral vectors requires induction of
regulatory T cells.
Long-term FIX expression
could be achieved by preventing expression in
APCs through the use of hepatocyte-speciﬁc pro-
In some cases, ﬁne-tuning the speciﬁcity
of expression using microRNA-regulated expression
cassettes was required to achieve long-term ex-
pression and immune tolerance.
shown that FIX activity could be further increased
by using a codon-optimized hyperactive FIX-R338L
(Padua) after transduction in hemophilic mice with
integration-competent or integration-defective len-
Moreover, sustained therapeutic
FIX expression levels could be attained after liver-
directed lentiviral transduction in hemophilic
paving the way toward eventual clinical
translation. One advantage of lentiviral vectors is
the absence of pre-existing, vector-speciﬁc immu-
nity since most individuals have not been naturally
pre-exposed to the lentiviral vector components, in
contrast to adeno-associated viral (AAV) vectors (see
below). Nevertheless, by virtue of their ability to
efﬁciently interact with professional APCs, lenti-
viral vectors are capable of inducing innate immune
responses, consistent with increased production of
Gamma-retroviral and lentiviral vectors have
been employed to deliver FVIII or FIX genes to
HSCs and other stem/progenitor cells popula-
tions (reviewed in previous publications by Matri
and Vandendriessche et al.
plantation of HSC transduced with FVIII or FIX
c-retroviral or lentiviral in myeloablated recipient
mice resulted in phenotypiccorrection in hemophilic
mice and induction of immunological tolerance to
the transgene products. One of the attractive fea-
tures of lentiviral transduction of HSCs is that
1016 VANDENDRIESSCHE AND CHUAH
FVIII and FIX expression can be speciﬁcally di-
rected to platelets resulting in hemostatic correction
in both mouse and dog models of hemophilia.
As activated blood platelets mediate the pri-
mary response to vascular injury, storage of FVIII
within platelets may provide a locally inducible
treatment to maintain hemostasis for hemophilia
A. Remarkably, phenotypic correction of the bleeding
disease in hemophilia A mice could even be achieved
in the presence of high-titer inhibitory antibodies
after lentiviral platelet-directed FVIII expression,
which requires von Willebrand factor.
strategy could be especially beneﬁcial to treat he-
mophilia patients with pre-existing inhibitors.
To facilitate HSC engraftment and create a
‘‘niche’’ in the bone marrow, it is necessary to use
busulfan or other cytotoxic preconditioning regi-
mens. However, since preconditioning is not with-
out side effects, the risk/beneﬁt ratio would need to
be carefully assessed in the context of HSC-based
hemophilia gene therapy. To obviate the need for
myeloablative preconditioning, direct intraosseous
delivery of lentiviral-FVIII vectors has been ex-
plored. This resulted in bone marrow and HSC cell
transduction in situ and subsequent FVIII pro-
duction in HSC-derived platelets.
B cells could be employed, since they yielded ther-
apeutic FIX levels in vivo in xeno-transplantated
immunodeﬁcient mice without preconditioning.
One of the main safety concerns related to c-
retroviral and lentiviral vectors relates to the risk
of insertional oncogenesis resulting from random
The vector design or the
presence of transcriptionally active long terminal
repeat can inﬂuence this genotoxic risk. Removal of
approximately 400 bp in the long terminal repeat
region to abolish its transcriptional activity (i.e.,
self-inactivating vector design) coupled with the
use of a promoter in an internal position substan-
tially lowers the risk of insertional oncogenesis.
The relative safety of these self-inactivating vec-
tors has been conﬁrmed experimentally in sensi-
tive tumor-prone mouse models using lentivirally
transduced HSCs or hepatocytes.
that the vector design itself can dramatically de-
crease the risk of insertional oncogenesis even in
tumor-prone mouse models that overestimate this
risk. Integration-defective lentiviral vectors that
harbor an inactivating mutation in the integrase
further minimize the risk of insertional oncogene-
sis. We showed that transgene expression levels
were reduced in comparison with conventional
integrase-competent lentiviral vectors.
theless, relatively robust FIX activity levels could
be obtained with integration-defective lentiviral
vectors when the FIX-R338L Padua variant was
Adenoviral vectors have been extensively stud-
ied for hemophilia gene therapy but were largely
abandoned after the failed adenoviral vector trial
to treat ornithine transcarbamylase deﬁciency in
the late 1990s. Their main disadvantage pertains
to the risk of uncontrollable inﬂammatory reac-
tions following systemic administration that can
potentially be fatal. Nevertheless, we and others
have shown that high-capacity adenoviral vectors
devoid of any viral genes and encoding FVIII or FIX
gave rise to robust supraphysiologic clotting factor
expression levels, with limited toxicity and no evi-
dence of inﬂammation in hemophilic mouse and
A phase 1 clinical trial was
conducted in severe hemophilia A patients, based
on the systemic administration of a high-capacity
adenoviral vector encoding FVIII. Although very
low FVIII levels (approximately 1%) may have been
obtained, the trial was stopped when a transient
inﬂammatory response with hematologic and liver
abnormalities became apparent after the ﬁrst
patient was treated. Though the latest-version
high-capacity adenoviral vectors result in reduced
adaptive immune responses and improved stability
of transgene expression, the innate immune re-
sponse in human subjects is difﬁcult to predict and
can still be a serious issue. Consequently, it may
not be straightforward to simulate the adenoviral
vector-induced innate immune response in pre-
clinical animal models. Current efforts are aimed
at minimizing the interaction between high-
capacity adenoviral vectors with the innate im-
mune system (e.g., by localized delivery
contemplating future clinical trials with this type
of vector for hemophilia gene therapy. However,
this will be a tough call given the promising de-
velopments with other safer vector systems, par-
Adeno-associated viral vectors
Hemophilia B. AAV2 vectors can transduce a
broad variety of tissues, including muscle and liver.
Muscle-directed gene transfer in a mouse or canine
hemophilia B model with a missense mutation
resulted in stable FIX expression.
dogs with a FIX null mutation, high-titer inhibi-
tors could be induced curtailing phenotypic cor-
The overall therapeutic efﬁciency was
increased up to 10-fold by delivering the AAV-FIX
vectors to the muscle intravascularly, under tran-
The risk of inhibitor
HEMOPHILIA GENE THERAPY 1017
formation was determined by the underlying mu-
tation in the FIX gene, vector dose, and local FIX
antigen doses in the transduced muscle and could
be prevented by transient immune suppression or
by limiting the vector dose per site (reviewed in
Wang et al.
). Based on these preclinical studies, a
phase 1 clinical trial was initiated whereby severe
hemophilia B patients with a missense FIX muta-
tion were injected at multiple intramuscular sites
with AAV-FIX vectors.
Though circulating FIX
levels were low or subtherapeutic, FIX expression
was detectable locally in the muscle for at least 10
years with no evidence of anti-FIX antibodies.
The need for repeated muscle injections, and the
increased inhibitor risk, justiﬁed exploring AAV-
mediated hepatic gene transfer as an alternative.
Initially AAV2 was used, followed by other natu-
rally occurring serotypes (i.e., AAV8, AAV9, AAV5)
or capsid variants obtained by evolution and se-
or containing speciﬁc point mutations.
Preclinical studies with the AAV vectors in murine
and canine models of hemophilia or in nonhuman
primates have demonstrated persistent therapeu-
tic FIX expression, leading to partial or complete
correction of the bleeding phenotype.
Long-term correction of the bleeding disease could
be achieved after liver-directed AAV2-FIX gene
therapy even in inhibitor-prone hemophilia B dogs
harboring a FIX null mutation.
It would appear,
therefore, that the risk of inhibitor development is
reduced after liver-directed gene therapy compared
with muscle-targeted therapy. This may possibly
be due to the induction of regulatory T cells.
These liver-directed preclinical gene therapy
studies justiﬁed the use of AAV vectors in a phase 1
clinical trial in patients suffering from severe he-
mophilia B. It was particularly encouraging that
therapeutic FIX levels, reaching nearly 12% of
normal levels, could be achieved following liver-
directed delivery of AAV-FIX.
However, FIX ex-
pression was transient and resulted in vector dose–
dependent hepatotoxicity consistent with plasma
elevation of transaminases. One possible hypothe-
sis (designated hereafter as the ‘‘T-cell hypothesis’’)
that could account for this observation is that the
transduced hepatocytes were able to present AAV
capsid–derived antigens in association with major
histocompatibility complex class 1 to T cells, which
would explain the enzymed-linked immunoSpot
(ELISPOT) results from patient peripheral mono-
nuclear blood cells (PMBCs).
presentation was modest, it may have been sufﬁ-
cient to ﬂag the transduced hepatocytes for - cell–
mediated destruction. Though attempts were made
to simulate this AAV-speciﬁc T-cell response in
various model systems in vitro and in vivo,
remains somewhat puzzling why this has not been
observed in more conventional preclinical disease
models (mouse, dogs) or in nonhuman primates.
In a second liver-directed gene therapy trial,
severe hemophilia B patients were injected intra-
venously with an AAV8 vector expressing a codon-
optimized FIX from a liver-speciﬁc promoter.
This trial was based, at least in part, on the ratio-
nale that AAV8 allows for a substantial increase in
hepatic transduction compared with other sero-
though this may not necessarily translate
to higher species, including humans. This AAV8
serotype exhibits reduced cross-reactivity with pre-
existing anti-AAV2 antibodies. Interestingly, its
uptake by dendritic cells may be reduced compared
with conventional AAV2 vectors, potentially result-
ing in reduced T-cell activation based on preclinical
The AAV vector used in this trial was
based on a so-called self-complementary design,
which was believed to overcome one of the limiting
steps in AAV transduction, namely the single-
stranded DNA to double-stranded transcription-
competent double-stranded DNA conversion.
Nevertheless, its effect on expression may vary de-
pending on the expression cassette. The treated
subjects expressed FIX above the therapeutic 1%
threshold for several years after vector administra-
tion, yielding sustained expression levels ranging
between 1% and 6% of normal levels over a median
period of 3.2 years. This demonstrated, for the ﬁrst
time, that sustained therapeutic FIX levels could be
achieved after gene therapy in hemophilia B pa-
tients. In the high-dose group (2 ·10
consistent increase in the FIX level to *5% was
observed in all 6 patients, which resulted in a re-
duction of more than 90% in both bleeding episodes
and the use of prophylactic FIX concentrate. In this
trial, AAV-speciﬁc T-cell responses could be de-
tected, but not in all of the patients.
A recent trial based on the same vector design
(UniQure; NCT02396342) but using another cap-
sid (AAV5) essentially conﬁrmed these results. In
those patients (n=5) who received the initial dose
level (5 ·10
vg/kg) of the AMT-060 vector, an
average of *4% FIX was attained. In the higher-
dose cohort (2 ·10
vg/kg) (n=5), FIX expression
increased to almost 7%. This was consistent with a
reduced spontaneous bleeding rate and FIX usage
to the extent that eight out of nine participants who
were on FIX prophylaxis initially did not require
prophylaxis any longer. In this trial, AAV-speciﬁc
T-cell responses could not be detected.
Based on these trial results no apparent beneﬁt
of using AAV5 over AAV8 was apparent in terms of
1018 VANDENDRIESSCHE AND CHUAH
efﬁcacy. Nevertheless, it is challenging to directly
compare these trail results since there were no
head-to-head comparisons and there may be many
confounding variables that impact on the trial
outcome, besides the capsid itself (i.e., ratio of
functional to total vector particles, differences in
manufacturing and vector purity, patient-
dependent characteristics, etc.). Strikingly, in both
these AAV8 and AAV5 trials, elevated transami-
nase levels were apparent, consistent with the re-
sult obtained in the initial AAV2 trial.
were therefore treated by transient immune sup-
pression with tapering doses of glucocorticoids in
the hope of blocking any inadvertent immune re-
sponses. Though there is circumstantial evidence
supporting the ‘‘T-cell hypothesis,’’ some patients
in the AAV8 trial had no elevated liver enzyme
levels despite an increased in AAV capsid-speciﬁc
T-cell response. Other patients in both the AAV8 or
AAV5 trial had an increase in liver enzyme levels,
without any supporting evidence of an AAV capsid-
speciﬁc T-cell response. This suggests detecting
vector-speciﬁc T-cells in the peripheral blood may
not necessarily be associated with the immune re-
jection of AAV-transduced hepatocytes, perhaps
due to the impact of the liver microenvironment on
the local T-cell response. Alternatively, the liver
enzyme elevation in some of the subjects may have
been unrelated to an AAV- speciﬁc T-cell response
or the T-cell response may not have been sufﬁ-
ciently robust to clear the transduced hepatocytes.
The availability of new mouse models that mimic
these responses may ultimately shed light on these
Based on these AAV2, AAV8, and AAV5 trials,
there is a need to further improve the overall efﬁ-
cacy to the extent that lower and safer effective
vector doses could be used that, ideally, would not
result in transaminitis. We had initially demon-
strated that liver-directed gene transfer of a codon-
usage optimized, hyperfunctional FIX R338L Padua
variant resulted in a signiﬁcant dose advantage
without any apparent increased thrombotic or im-
munogenic risk compared with wild-type FIX.
This was independently conﬁrmed in other studies,
including canine models.
justiﬁed the use of a hyperfunctional FIX-R338L
Padua variant to treat hemophilia B patients by
It is particularly encouraging that the superi-
ority of the FIX-R338L Padua was recently sup-
ported by two independent clinical trials. In the
ﬁrst trial (Shire, NCT01687608), patients were
treated with a scAAV8 vector encoding a codon-
optimized FIX-R338L Padua variant (BAX335).
Seven patients were treated in three dosing co-
horts, and 2 patients had transient FIX activity
>50%. FIX levels persisted in 1 patient from the
medium dose cohort of 1 ·10
vg/kg. None of the
patients developed FIX inhibitors. In a second trial
(Spark Therapeutics/Pﬁzer), a more consistent re-
sponse among the trial subjects was apparent. The
single-stranded AAV vector genome in the SPK-
9001 vector (NCT02484092) contained a codon-
optimized FIX-R338L Padua variant and was
packaged using an alternative mutated capsid
mutant. The mean steady-state FIX activity level
for the 10 participants, 12 weeks treated at the
initial dose level (5 ·10
vg/kg), was sustained at
33% with no evidence of inhibitory antibodies.
Annualized bleeding rate among the 10 patients
was reduced by 96% to a mean of 0.39 annual
bleeds, compared with 9.2 bleeds before SPK-9001
administration. In addition, annualized infusion
rates were reduced 99% to a mean of 0.98 annual
infusions, compared with 68.5 infusions before
SPK-9001 administration. FIX prophylaxis was
discontinued. However, 2 patients who showed a
transient asymptomatic elevation in liver enzymes
and drop in FIX activity, potentially indicating an
immune response, were treated with oral cortico-
Hemophilia A. The packaging constraints of
AAV initially hampered vector production for he-
mophilia A gene therapy due to the relatively large
size of the FVIII transgene. Typically, relatively
high vector doses were required and it remained
challenging to accommodate the FVIII transgene
with a potent hepatocyte-speciﬁc promoter in a
single vector. The use of single-chain was preferred
over dual-chain vector expressing the heavy and
light chain separately.
The efﬁcacy of gene
therapy for hemophilia A using AAV vectors could
be enhanced by using small regulatory elements to
drive expression of a B-domain–deleted form of
FVIII by using codon-optimized FVIII cDNA and/or
incorporating speciﬁc mutations in the FVIII
Hemophilia A dogs that received
AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII per-
sistently expressed therapeutic levels of FVIII,
without antibody formation or other toxicities, for
more than 3 years. However, in contrast to mice, liver
transduction efﬁciencies are similar between AAV2,
AAV6, and AAV8 serotypes in hemophilia A dogs.
In a more recent study, an AAV8 vector was
constructed that contained a 5.2 kb vector ge-
nome encoding a codon-optimized FVIIIDBcDNA.
A 17-amino-acid peptide (V3) containing glycosyl-
ation residues was incorporated in the FVIIIDB
HEMOPHILIA GENE THERAPY 1019
sequence that typically boosted FVIII expression
*3-fold. Supraphysiologic hFVIII expression lev-
els (732 –162% of normal) could be attained in he-
mophilia A mice that were injected with 2 ·10
kg. Stable hFVIII expression at 15 –4% of normal
was observed at this dose in a nonhuman primate
receiving the lowest vector dose (2 ·10
However, human FVIII expression above 100%
was observed in three macaques that received a
higher dose resulting in neutralizing anti-FVIII
antibodies that were abrogated with transient im-
munosuppression. Whether this V3 element will
increase the risk of inhibitors in patients with he-
mophilia A remains to be seen.
A phase 1/2 clinical trial was recently initated
(BioMarin, NCT02576795) based on an AAV5 vec-
tor (designated as BMN270) that expressed a
B-domain deleted FVIII (identical to Refacto
A total of 9 patients have received a single dose of
the the BMN270 vector, and 7 of those have been
treated at the highest dose. Out of 9 patients, 6
patients who received the highest dose of 6 ·10
vg/kg attained FVIII levels above 50%, and one had
FVIII levels above 10%, after a follow-up of 16
weeks. The therapeutic outcomes improved over
time, with 4 patients reaching 146% FVIII level by
week 20. For the 6 patients at the high dose and
previously on a FVIII prophylactic treatment reg-
imen, the mean annualized bleeding rate dropped
91% from 16.3 before receiving BMN 270 to 1.5 two
weeks after receiving the therapy. For those same 6
patients, the mean annualized FVIII infusions fell
98% from 136.7 to 2.9. Nevertheless, those patients
receiving high doses of vectors required a steroid
regimen for up to 2 weeks after the gene therapy,
whereas the maximum transaminase levels were
about twice the upper limit of normal at about 12
weeks after the gene delivery, which then declined
over the next few weeks. The vector doses used in
this trial were substantially higher than what had
been used in the AAV-based hemophilia trials. This
poses signiﬁcant vector manufacturing challenges
and may have accounted for the unusual trans-
CONCLUSIONS AND PERSPECTIVES
Is gene therapy for hemophilia now ready for
prime time? This is the ‘‘million-dollar question’’—
literally and ﬁguratively, given the high cost of
current treatment (PST costs an estimated average
$300,000/year per patient). The prospect for a real
cure for hemophilia patients has never looked more
promising than it does today. This was made pos-
sible through a concerted effort of many investi-
gators that contributed to improved vector
development and design, vector manufacturing,
basic studies in coagulation, immunology and vec-
tor–host interactions at the molecular and cellular
level, and proof-of-concept studies in preclinical
disease models. This required an unprecedented
multidisciplinary effort involving basic scientists,
clinicians, regulators, and stakeholders from in-
dustry that fueled the gene therapy pipeline. The
advances in the clinic progressed through several
logical stages starting with the demonstration that
transient therapeutic clotting factor levels could
be attained, to more sustained and higher FVIII
and FIX expression levels and activities, reducing
bleeding frequency and clotting factor usage, even
obviating the need for prophylaxis. In particular,
the use of bioengineered clotting factors (i.e., based
on codon-optimized FVIII or FIX and/or hyper-
functional FIX variants) and improved vector de-
signs was important to bring gene therapy for
hemophilia to the next level. Nevertheless, there are
still some issues that will need to be addressed.
Traumatic bleeds still occur in patients, and the is-
sue of vector-induced liver inﬂammation will need to
be better understood and overcome. Moreover, the
potential risk of insertional oncogenesis continues
to raise controversy.
Nevertheless, the emer-
gence of efﬁcient site-speciﬁc integrating vectors
based on designer zinc ﬁnger nucleases, CRISPR/
Cas9, or nuclease-free targeting approaches into
‘‘safe harbor’’ loci opens new perspectives to further
minimize this risk.
The careful selection of
potent cis-regulatory elements that boost FIX or
FVIII expression without any apparent increased
risk of insertional oncogenesis is warranted.
An adequate balance between efﬁcacy and safety
will need to be continuously assessed in the face of
new insights in this rapidly developing ﬁeld, par-
ticularly if the number of patients are expected to
substantially increase in the coming years as gene
therapy trials move into phases 3 and 4. Eventual
marketing authorization approval by regulatory
authorities depends on the successful completion of
these larger trials. Only then will gene therapy for
hemophilia make a concrete difference in the lives
of patients and their families that are affected by
We thank the members of the Department
of Gene Therapy and Regenerative Medicine and
all of our collaborators for their various contri-
butions to portions of the work presented in this
review. We also wish to thank Fonds voor We-
tenschappelijk Onderzoek (FWO), Shire, Bayer,
1020 VANDENDRIESSCHE AND CHUAH
Pﬁzer, Agentschap voor Innovatie door We-
tenschap en Technologie (IWT), Vrije Universiteit
Brussel Industrieel Onderzoeksfonds Groups of
Excellence in Applied Research (VUB-IOF-GEAR)
(GENEFIX), Strategic Research Program (SRP)-
Groeier, and the Willy Gepts Fund for providing
The authors have patent applications and
granted patents in the ﬁeld of gene therapy for
hemophilia. They also received industrial grants
and/or consultancies from various companies in-
volved in gene therapy for hemophilia (Shire,
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Received for publication June 28, 2017;
accepted after revision July 28, 2017.
Published online: August 3, 2017.
HEMOPHILIA GENE THERAPY 1023