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Hemophilia Gene Therapy: Ready for Prime Time?



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 (AAV) and lentiviral vectors. More recently, gene editing approaches based on designer 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 comprehensively assessed. In the past two decades several phase I 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 sub-therapeutic 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 synthetic FVIII or FIX transgenes have been employed. Alternatively, we and others have shown that the incorporation of hyper-active 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.
Hemophilia Gene Therapy: Ready for Prime Time?
Thierry VandenDriessche
*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:;
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
immunoglobulin domains.
Consequently, these
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
expression cassettes
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.
These pro-
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
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
mouse models.
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
(see below).
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
protein levels.
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-
quent studies.
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
reviewed elsewhere.
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-
gene product,
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
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
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
transfected cells.
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.
To achieve
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.
Inadvertent expres-
sion of FVIII or FIX in APCs increases the risk of
developing inhibitory antibodies that preclude
their long-term expression and renders gene ther-
apy ineffective.
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
tolerance instead.
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.
We have
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-
tiviral vectors.
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
pro-inflammatory cytokines.
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
et al.
and Vandendriessche et al.
). Trans-
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
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
genomic integration.
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.
This implies
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
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
dog models.
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
) before
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-
ticularly AAVs.
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.
However, in
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-
sient immunosuppression.
The risk of inhibitor
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).
Although antigen
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
vg/kg), a
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
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.
Consequently, this
justified the use of a hyperfunctional FIX-R338L
Padua variant to treat hemophilia B patients by
gene therapy.
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
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
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-
aminase kinetics.
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
these diseases.
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,
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
financial support.
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,
Bayer, Pfizer).
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Received for publication June 28, 2017;
accepted after revision July 28, 2017.
Published online: August 3, 2017.
... 6,7 Gene therapy on the other hand offers the prospect of a functional cure with a single therapeutic dose and might have, in addition to providing substantial and constantly present FVIII levels, a tolerizing effect, which might reduce the risk of inhibitor development. [8][9][10][11][12] The results from gene therapy clinical trials using AAV to treat hemophilia B are encouraging 13,14 and the recent release of preliminary data for an AAV5-FVIII human clinical trial demonstrates sustained FVIII activity with no significant adverse events reported. 6,15,16 More recently however, three years follow up data also identified a decline in FVIII expression levels over time, suggesting that this therapy may not last lifelong 17 and causing the FDA to ask for two years of data from the phase 3 trial to show substantial evidence of a durable effect. ...
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One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.
... Acute joint hemorrhage is a common symptom. Patients often suffer from repeated bleeding and chronic joint injury [4]. In addition, in 1-4% of patients, intracranial hemorrhage could be the first symptom [5]. ...
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Background: Congenital hemophilia A is a recessive inherited hemorrhagic disorder. According to the activity of functional coagulation factors, the severity of hemophilia A is divided into three levels: mild, moderate and severe. The first bleeding episode in severe and moderate congenital hemophilia A occurs mostly in early childhood and mainly involves soft tissue and joint bleeds. At present, there are limited reports on severe congenital hemophilia A with low factor XII (FXII) activity during the neonatal period. Case presentation: A 13-day-old neonate was admitted to the hospital with hematoma near the joints of both upper arms. Coagulation tests showed he had low activity of factor VIII (FVIII) and FXII. He was diagnosed with congenital hemophilia A and treated with human coagulation factor VIII (recombinant FVIII). Although the hematoma became smaller, FVIII activity was only increased to a certain extent and FXII activity decreased gradually. Unfortunately, the child responded poorly to recombinant human coagulation factor VIII and his guardian rejected prophylactic inhibitors and genetic testing and refused further treatment. Three months later, the child developed intracranial hemorrhage (ICH) due to low FVIII activity. Conclusions: In hemophilia A, the presence of FVIII inhibitors, drug concentration and testing are three important aspects that must be considered when FVIII activity does not reach the desired level. Early positive disease treatment and prophylaxis can decrease the frequency of bleeding and improve quality of life. We recommend that pregnant women with a family history of hemophilia A undergo early prenatal and neonatal genetic testing.
... While the gains made over the past century have greatly improved longevity and quality of life for hemophiliacs, more recent advancements have focused on treating the diseases at their roots by gene therapy [94,95]. The methods and modalities by which gene therapy have been conducted have varied, ranging from viral vector delivery [96], stem cell transplant [97,98], CRISPR/Cas editing [99,100], and synthetic transgene development (all primarily in animal models to date) [101]. ...
Hematologic diseases include a broad range of acquired and congenital disorders, many of which affect plasma proteins that control hemostasis and immune responses. Therapeutic interventions for these disorders include transfusion of plasma, cryoprecipitate, immunoglobulins, or convalescent plasma-containing therapeutic antibodies from patients recovering from infectious diseases, as well as concentrated pro- or anticoagulant factors. This review will focus on recent advances in the uses of plasma and its derivatives for patients with acquired and congenital hematologic disorders.
Haemophilia is an inherited bleeding disorder in which the haemostatic defect results from deficiency of coagulation factor VIII (FVIII) in haemophilia A or factor IX (FIX) in haemophilia B. Traditional treatments for haemophilia have largely worked by directly replacing the missing coagulation factor, but face challenges due to the short half‐life of FVIII and FIX, the need for frequent intravenous access and development of neutralising antibodies to coagulation factors (inhibitors). Recent advances in haemophilia therapy have worked to eliminate these challenges. Half‐life extension of factor concentrates has lengthened the time needed between infusions, enhancing quality of life. Subcutaneous administration of therapeutics utilising alternative mechanisms to overcome inhibitors have expanded the options to prevent bleeding. Finally, initial successes with gene therapy offer a cautious hope for durable cure. In the present review, we will discuss currently available treatments, as well as highlight therapeutics in various stages of clinical development for the treatment of haemophilia A and B. In this review, we present therapies that are currently clinically available and highlight therapeutics that are in various stages of clinical development for the treatment of haemophilia A and B.
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While platelet-specific gene therapy is effective in inducing immune tolerance to a targeted protein, how the reactivity of pre-existing immunity affects the efficacy, and whether CD8 T cells were involved in tolerization is unclear. In this study, OVA was used as a surrogate protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1⁺ cells from either wild-type (WT)/CD45.2 or OT-II/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy irradiation. Sustained platelet-OVA expression was achieved in >85% of OVA-primed recipients but abolished in animals with high-reactive pre-existing immunity. As confirmed by OVA rechallenge and skin graft transplantation, immune tolerance was achieved in 2bOVA-transduced recipients. We found that there is a negative correlation between platelet-OVA expression and the percentage of OVA-specific CD4 T cells and a positive correlation with the OVA-specific Treg cells. Using the OT-I/WT model, we showed that antigen-specific CD8 T cells were partially deleted in recipients after platelet-targeted gene transfer. Together, our studies demonstrate that robust antigen-specific immune tolerance can be achieved through platelet-specific gene therapy via peripheral clonal deletion of antigen-specific CD4 and CD8 T effector cells and induction of antigen-specific Tregs. There is an antagonistic dynamic process between immune responses and immune tolerance after platelet-targeted gene therapy.
Introduction: Gene therapy clinical trials with adeno-associated virus (AAV) vectors report impressive clinical efficacy data. Nevertheless, challenges have become apparent, such as the need for high vector doses and the induction of anti-AAV immune responses that cause the loss of vector transduced hepatocytes. This fostered research focusing on development of next-generation AAV vectors capable of dealing with these hurdles. Areas Covered: While both the viral vector genome and the capsid are subjects to engineering, this review focuses on the latter. Specifically, we summarize the principles of capsid engineering strategies, and describe developments and applications of engineered capsid variants for liver-directed gene therapy. Expert Opinion: Capsid engineering is a promising strategy to significantly improve efficacy of the AAV vector system in clinical application. Reduction in vector dose will further improve vector safety, lower the risk of host immune responses and the cost of manufacturing. Capsid engineering is also expected to result in AAV vectors applicable to patients with pre-existing immunity towards natural AAV serotypes.
La hemofilia B o enfermedad de Christmas se diferenció por primera vez de la hemofilia A en 1947. Su forma clásica consiste en un trastorno hereditario de la coagulación causado por mutaciones en el gen F9, que codifica para el factor IX de la coagulación. Su herencia está ligada al cromosoma X; las mujeres son portadoras, pero se manifiesta clínicamente en hombres, aunque se han descrito casos de mujeres portadoras sintomáticas. El factor IX activado es una proteína dependiente de vitamina K, sintetizada en el hígado, que forma parte del complejo tenasa, cuya función es formar la mayor cantidad de trombina en el nuevo modelo de la coagulación basado en células. De acuerdo a la actividad del factor IX, su deficiencia se puede clasificar en leve (5% a 40%), moderada (1% a 5%), o severa (<1%). Su diagnóstico se realiza con la presencia de un TPT alargado que corrige con plasma normal y con la determinación del nivel funcional del factor IX, y se confirma con el estudio molecular que demuestra la mutación en el gen F9. Su diagnóstico diferencial incluye otras patologías como la hemofilia A. El tratamiento con factorIX recombinante es el más utilizado en la actualidad, pero se vienen desarrollando nuevas terapias con virus adeno-asociados recombinantes que prometen mejorar la calidad de vida para algunos pacientes afectados. La profilaxis juega un papel fundamental, en particular en los casos de enfermedad moderada y severa.
Hemophilia A (HA) is a hereditary hemorrhagic disease caused by a deficiency of coagulation factor VIII (FVIII) in blood plasma. Patients with HA usually suffer from spontaneous and recurrent bleeding in joints and muscles, or even intracerebral hemorrhage, which might lead to disability or death. Although the disease is currently manageable via delivery of plasma-derived or recombinant FVIII, this approach is costly, and neutralizing antibodies may be generated in a large portion of patients, which render the regimens ineffective and inaccessible. Given the monogenic nature of HA and that a slight increase in FVIII can remarkably alleviate the phenotypes, HA has been considered to be a suitable target disease for gene therapy. Consequently, introduction of a functional F8 gene copy into the appropriate target cells via viral or nonviral delivery vectors, including gene correction through genome editing approaches, could ultimately provide an effective therapeutic method for HA patients. In this review, we discuss the recent progress of gene therapy for HA with viral and nonviral delivery vectors, including piggyBac, lentiviral and adeno-associated viral vectors, as well as new raising issues involving liver toxicity, pre-existing neutralizing antibodies of viral approach, and the selection of the target cell type for nonviral delivery.
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Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
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Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.
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Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Summary: Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.
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Factor VIII (FVIII) is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity.
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In a recent Nature Genetics letter, entitled “Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas,” Nault and colleaguesdocument that of 193 patients with hepatocellular carcinoma (HCC), 11 contained an integrated genome sequence of the wild-type adeno-associated virus 2 (AAV2), and suggest that AAV2 is associated with oncogenic insertional mutagenesis in human HCC. Because AAV2 has long been known to be a nonpathogenic human parvovirus and, in fact, has been shown to possess antitumor activity, it is critical that the scientific and clinical implications of these studies be rigorously assessed to justify their conclusions. We have carefully analyzed the data presented by Nault and colleaguesand reached a conclusion that is at variance with that of the authors.
2201 von Willebrand factor (VWF) is a carrier protein for FVIII and protects FVIII from protease degradation. Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with VWF in platelet α-granules and that platelet-FVIII (2bF8) corrects murine hemophilia A phenotype even in the presence of high titer anti-FVIII inhibitory antibodies. While ectopic expression of FIX in platelets is also trafficked and stored in α-granules and corrects the bleeding diathesis in hemophilia B mice, the efficacy of platelet-FIX is limited in the face of anti-FIX inhibitors, possibly due to the lack of protective carrier protein like VWF for FVIII. In the current study, we wanted to explore the role of VWF in platelet-derived FVIII gene therapy of murine hemophilia A with inhibitory antibodies. We immunized transgenic mice in which 2bF8 transgene was on a FVIII and VWF double knockout background (2bF8tg+/−FVIIInullVWFnull), with recombinant human B-domain deleted FVIII (rhFVIII) to induce inhibitory antibody development. Inhibitor titer was determined by Bethesda assay and phenotypic correction was assessed using tail clip survival tests. The results demonstrated that only 18% (n = 11) of VWF-deficient animals with inhibitor titers between 3 and 8000 BU/ml survived the tail clip challenge. In contrast, 82% (n = 22) of immunized 2bF8tg+/−FVIIInull transgenic mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10 – 50,000 BU/ml (P < 0.001). All 2bF8tg+/−VWFnullFVIIInull mice (n = 12) without inhibitors survived tail clipping and no (n = 6) VWFnullFVIIInull mice survived this challenge. Since VWF is synthesized by endothelial cells and megakaryocytes and distributes in plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF (plasma-VWF vs. platelet-VWF) in platelet-FVIII gene therapy of hemophilia A with inhibitors. To address the effect of plasma-VWF, FVIIInull mice were immunized with rhFVIII to induce inhibitor development and then they received bone marrow transplantation (BMT) from 2bF8tg+/−FVIIInullVWFnull mice. To study the effect of platelet-VWF, VWFnullFVIIInull mice were immunized followed by BMT from 2bF8tg+/−FVIIInullVWF+/+ mice. After at least 6-weeks of BM constitution, mice were analyzed. Viable BMT engraftment in recipients was confirmed by PCR and platelet lysate FVIII activity assay. The levels of VWF in plasma and platelets were quantitated by ELISA. The phenotypic correction was assessed by the tail clip survival test. In the group with plasma-VWF, 5 of 12 (42%) mice survived tail clipping with inhibitor titers between 22 – 1200 BU/ml. In the group with platelet-VWF, 6 of 12 (50%) mice survived tail clipping with inhibitor titers of 5 – 810 BU/ml. As controls, all recipients (6 mice in each group) without inhibitors survived the tail clip challenge. To further investigate the dose effect of inhibitors on platelet-FVIII gene therapy of animals that only have plasma-VWF, we infused varied levels of inhibitory plasma from immunized VWFnullFVIIInull mice into FVIIInull mice that received BMT from 2bF8tg+/−FVIIInullVWFnull, followed by tail clip survival tests. Four of 6 mice with 2.5 BU/ml of inhibitors, 2 of 6 mice with an inhibitor titer of 25 BU/ml, and 1 of 6 mice with an inhibitor titer of 250 BU/ml survived tail clipping. Taken together, in this acute inhibitor model 7/18 (39%) mice with inhibitors between 2.5 – 250 BU/ml survived tail clipping. This survival rate is significantly lower than the group with normal VWF (P < 0.05). These results demonstrate that VWF, including both platelet-VWF and plasma-VWF, is required for optimal platelet-derived FVIII gene therapy of hemophilia A in the presence of inhibitory antibodies. Disclosures No relevant conflicts of interest to declare.
Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. Summary: Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc-/-(NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc-/-(NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein secretion from autologous B cells in vivo in the treatment of hemophilia and other acquired or inherited diseases.
A surge in therapeutic clinical trials over recent years is paving the way for transformative treatment options for patients with hemophilia. The introduction of recombinant factor concentrates in the early 1990's facilitated the use of prophylactic replacement as standard care for hemophilia rather than on-demand treatment. This has revolutionized health outcomes for hemophilia patients, enabling participation in physical activities and reducing debilitating, chronic joint damage. Challenges of prophylactic factor infusion include the frequency of infusions needed to maintain factor levels greater than 1%, patient adherence, reliable intravenous access, and development of neutralizing alloantibodies (“inhibitors”). Novel therapeutics seek to improve upon current factor concentrates by several different mechanisms: 1) extending the half-life of circulating exogenous factor protein, 2) replacing the gene necessary for production of endogenous factor protein, 3) employing bispecific antibody technology to mimic the coagulation function of factor VIII, 4) disrupting anti-coagulant proteins such as tissue factor pathway inhibitor (TFPI) or antithrombin (AT3) with antibodies, aptamers, or RNA interference technology. Emerging treatment options may reduce the frequency of (extended half-life products) or eliminate (gene therapy) the need for scheduled factor concentrate infusions, or provide a subcutaneous administration option (bispecific antibody, AT3 and TFPI targeting therapies). Additionally, the non- factor replacement strategies provide a promising treatment option for patients with inhibitors, presently the greatest unmet medical need in hemophilia. This review highlights current and recently completed clinical trials that are driving a paradigm shift in our current approach to hemophilia care for patients with and without inhibitors. This article is protected by copyright. All rights reserved.
The development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by using a computational strategy that increased factor IX (FIX) levels 11- to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyperfunctional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. This resulted in sustained supraphysiologic FIX activity (400%), correction of the bleeding diathesis at clinically relevant, low vector doses (5 × 10(10) vector genomes [vg]/kg) that are considered safe in patients undergoing gene therapy. Moreover, immune tolerance could be induced that precluded induction of inhibitory antibodies to FIX upon immunization with recombinant FIX protein.