Accepted: 20 March 2018 DOI: 10.1111/hae.13494
ORIGINAL ARTICLE
Haemophilia gene therapy: From trailblazer to gamechanger H. Evens1 | M. K. Chuah1,2 | T. VandenDriessche1,2 1 Department of Gene Therapy & Regenerative Medicine, Faculty of Medicine & Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium 2
Department of Cardiovascular Sciences, Center for Molecular & Vascular Biology, University of Leuven, Leuven, Belgium Correspondence Thierry VandenDriessche and Marinee K. Chuah, Department of Gene Therapy & Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels Belgium. Emails:
[email protected];
[email protected] Funding information FWO; Shire; Bayer; Pfizer; VUB-IOF-GEAR (GENEFIX); Strategic Research Project Grower; AFM; Willy Gepts Fund
Haemophilia is an attractive disease target for gene therapy that fostered the development of the field at large. The delivery of the clotting factor genes into the patients’ cells could be accomplished using different types of gene delivery vehicles or vectors. Adeno-associated viral vectors (AAV) and lentiviral vectors represent some of the most promising gene delivery technologies that allow for a relatively efficient delivery of the therapeutic FVIII and FIX transgenes into the relevant target cells. To reduce the risks associated with insertional mutagenesis due to random vector integration, gene- editing approaches have also been considered based primarily on zinc finger nuclease (ZFN) and CRISPR/Cas. However, comprehensive analysis of off-target effects is still required. It is particularly encouraging that relatively stable therapeutic FVIII or FIX expression levels were reached in severe haemophilia patients in recent clinical trials after liver-directed AAV gene therapy. This success could be ascribed in part to improvements in vector design. In particular, clotting factor levels could be increased by codon optimization of coagulation factor transgenes. Alternatively, incorporation of a hyperactive gain-of-function R338L mutation (FIX Padua) in the FIX gene improved the overall efficacy. However, some patients still show transient liver toxicity, especially at high vector doses, possibly due to inflammatory immune responses, requiring the need for transient immunosuppression. The exact immune mechanisms are not fully understood, but may at least in some patients involve an AAV-capsid specific T cell response. Moreover, there is a need to identify the key factors that contribute to the interpatient variability in therapeutic efficacy and safety after gene therapy. KEYWORDS
adeno-associated viral vectors, coagulation, CRISPR, factor IX, factor IX Padua, factor VIII, haemophilia, lentiviral, zinc finger nuclease
1 | I NTRO D U C TI O N
FIX proteins can develop in some patients, requiring immune toler-
Current treatment for haemophilia is protein substitution therapy
ulation factors, several protein-based next-generation therapeutics
(PST) by administration of recombinant or plasma-derived clotting
have been developed recently. These include PEGylation or fusion
factors. Although PST significantly improved the quality of life and
of the clotting factors with immunoglobulin domains or albumin.1
life expectancy of patients suffering from haemophilia, it has several
These products will reduce the administration frequency of recom-
disadvantages. First, it does not cure haemophilia, because patients
binant clotting factors and provide a new standard for treatment of
are still at risk of bleeding episodes despite the treatment. Second,
haemophilia patients. Despite these advances, the need to develop a
conventional clotting factors have a short half-life, necessitating fre-
long-term cure for haemophilia by gene therapy remains. 2
ance induction or bypass agents. To prolong the half-life of the coag-
quent administration of factor VIII (FVIII) or factor IX (FIX). Finally,
Earlier reviews on the subject3-10 provide a comprehensive
neutralizing antibodies (“inhibitors”) against the recombinant FVIII or
description of all of the possible strategies that were tested in
50 | wileyonlinelibrary.com/journal/hae © 2018 John Wiley & Sons Ltd
Haemophilia. 2018;24(Suppl. 6):50–59.
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EVENS et al.
preclinical animal studies and describe some of the earlier clinical
be used to boost mRNA levels of FVIII and FIX.18-20 Expression can
trials. In this review, we will highlight the most recent advances in
be further increased by combining these promoters with other reg-
gene therapy for haemophilia and address some of the outstanding
ulatory elements (e.g. hepatic control regions (HCR), enhancers).19
questions. We will highlight some of the key concepts and their clin-
Although regulatory elements are typically identified through trial-
ical translation.
and- error, we have developed and experimentally validated an alternative approach to identify robust human tissue-specific cis- regulatory modules (CRMs) by means of genomewide data mining.
2 | G E N E TH E R A PY
FVIII or FIX expression could be robustly increased in different gene therapy vectors when these CRMs were combined with existing
The ultimate aim of gene therapy for haemophilia is to achieve sus-
promoters. 21-23 While augmenting transgene expression, the risk
tained high-level coagulation factor expression and a stable cor-
of insertional oncogenesis of integrating vectors was not increased
rection of the bleeding phenotype. Finding a cure for haemophilia
when using these CRMs, not even in tumour-prone mouse models. 23
requires the delivery of the constructs to the appropriate target cells
Synthetic transcriptional enhancers can also be created de novo by
and the optimization of the coagulation FVIII or FIX expression cas-
randomly ligating synthetic oligonucleotides coding for hepatic tran-
sette. The therapeutic gene can be stably integrated into the target
scription factor binding sites and screening these in vitro in hepatic
cell chromosomes or stay in the nucleus as episomal DNA, depend-
cell lines. 24 Preclinical mouse models indicate that the cellular speci-
ing on the gene delivery vector used. One approach to achieve this is
ficity of the promoter and regulatory elements is important, because
to deliver the FVIII or FIX gene directly in vivo to nondividing post-
they can have consequences on the immune response directed
mitotic cells, like hepatocytes or skeletal muscle cells. An alternative
against the clotting factors (as discussed below). 24,25
approach is to deliver the clotting factor genes into stem/progenitor
To our knowledge, we were the first to explore the use codon-
cells, like haematopoietic stem cells (HSCs). To achieve persisting
optimized coagulation factor genes more than 20 years ago 17 in an
expression in the HSC progeny, integrating vectors should be used.
attempt to augment their expression. The development of codon-
Additionally, the induction of inhibitors by gene therapy should be
optimized FVIII and FIX transgenes led to an increase in protein ex-
avoided, or the therapy should induce FVIII or FIX-specific immune
pression without changing any amino acid sequence of the FVIII or
tolerance, which already has been accomplished in preclinical stud-
FIX protein.18,21,26,27 It is therefore encouraging that all of the recent
ies.11-15 The induction of immune tolerance to the coagulation fac-
successful gene therapy trials for haemophilia A or B relied on codon
tors after gene therapy is dependent on several variables. These
optimization to boost expression levels. Preclinical studies revealed
include the vector design, target cells, transgene product or the mu-
that the outcome of the different codon optimization strategies may
tation causing the haemophilia disease phenotype.
vary, depending on the transgene and or the algorithm used. For example, typically codon optimization resulted in a twofold to three-
2.1 | Designing optimal FVIII and FIX expression cassettes
fold increase in FIX protein expression levels, whereas an increase in FVIII protein expression levels up to 44-fold has been reported. 26 One of the potential advantages of codon optimization is that the
All gene therapy approaches focus on expressing the therapeutic
amino acids remain unchanged (i.e. synonymous mutations) which
protein at high levels while using a low dose of vector to reduce un-
may obviate immune concerns, as opposed to nonsynonymous mu-
wanted immune responses or toxicity related to the vector. Mainly
tations. However, this assumption has recently been challenged as
for FVIII this has been challenging, because of bottlenecks in ex-
synonymous mutations may inadvertently result in conformational
pression at the transcriptional, translational and post-translational
changes in the FIX protein. 28
16
level.
Rationally designed transgenes and expression cassettes are
FVIII expression levels can be enhanced by deleting the B-
needed to overcome these challenges. If improvements can be made
domain (FVIIIΔB), because it increased FVIII mRNA levels compared
at each of these levels, this could eventually lead to substantial dose
to when the full-length FVIII cDNA was used. 29
benefits for haemophilia patients in clinical trials. Instead of using
However, when the B-domain is deleted, post-translation in-
the full-length transgenes of FVIII and FIX, cDNAs of these genes
tracellular FVIII trafficking is compromised because of the loss of
are used in gene therapy. To improve expression levels, sometimes
critical glycosylated residues.30 This defect can be counteracted by
17,18
introns are integrated in the constructs,
which are likely to fa-
reintroducing a 17- amino acid synthetic B- domain–derived poly-
cilitate extra-nuclear transport of the transcripts and subsequent
peptide into the FVIIIΔB cDNA, presumably reconstituting these
expression.
asparagine-linked oligosaccharides, increasing secreted FVIII pro-
For haemophilia gene therapy, the use of the endogenous FVIII
tein levels around 3-fold. 27 Another possibility is to incorporate a
and FIX promoters would seem attractive to mimic natural expres-
furin cleavage site within the B-domain at position R1645H that is
sion as much as possible. However, these may not be very robust
similar to the canine sequence (HHQR vs. RHQR), which increases
and hence not very suitable to maximize expression levels in the
FVIII expression in haemophilia A mice around twofold.31 Porcine-
face of limiting gene transfer efficiencies. Instead, tissue-specific
human hybrid FVIIIΔB transgenes could also increase FVIII pro-
heterologous promoters, like transthyretin or α1-antitrypsin, can
tein levels.32 However, overexpression of FVIII above 200% could
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EVENS et al.
52
potentially lead to cellular stress, which possibly increases the risk of inhibitor development.33 However, we showed that FVIII levels
2.3 | Preclinical and clinical studies
that are up to 75-fold increased compared to physiological levels fol-
To achieve sustained, lifelong therapeutic coagulation factor expres-
lowing liver-directed gene therapy did not result in liver toxicity or
sion without any toxicity or immune responses, a safe and efficient
transaminitis.34
gene therapy system is needed. Several well-suited vectors have
The efficacy of gene therapy vectors could also be improved using hyperfunctional clotting factor genes. We have shown that
been developed for haemophilia gene therapy, each with their own advantages and limitations.
incorporation of a single R338L gain-of-function point mutation in the FIX gene leads to an increase in FIX activity up to eightfold using liver-directed gene therapy. 21,35 This specific mutation (also known
2.3.1 | Nonviral vectors
as FIX-Padua) was first discovered in patients suffering from throm-
Nonviral gene transfer generally relies on DNA-b ased chemical
bophilia.36,37 It is a very suitable candidate to significantly boost
or physical transfection methods. Unlike viral vectors, nonviral
efficiency of haemophilia B gene therapy, with minimal change of
vectors in general do not lead to adaptive immune responses.
the FIX sequence. Independent studies have confirmed the func-
However, DNA can give rise to innate immune responses, typically
14,38,39
after gene ther-
related to Toll-like receptor (TLR) signalling pathways. Compared
apy, and other mutations to enhance FIX activity have also been
to viral vector- m ediated gene therapy, the efficiency of gene
explored.40,41
transfer is in general lower in nonviral gene therapy. Additionally,
tional augmentation of this point mutation
the FVIII or FIX expression is typically short-term after nonviral transfection, except when a selection is used on ex vivo trans-
2.2 | Target cells for haemophilia gene therapy
fected cells
There are several different cell types that are attractive targets to 3,42
47
or when transposons or integrases are used. 23,53-55
In a phase I clinical trial for haemophilia A, autologous fibroblasts
As FVIII and FIX proteins are
were electroporated with FVIII-expressing plasmids, after which
naturally generated by liver sinusoidal endothelial cells (LSECs) and
these were selectively expanded and implanted into the omentum
hepatocytes, respectively, the liver is an obvious target organ for
of patients. Despite the fact that there were no adverse effects,
gene therapy for haemophilia. Both viral and nonviral gene therapy
the therapeutic efficiency was relatively limited and FVIII expres-
treat haemophilia by gene therapy.
systems targeting LSECs and hepatocytes have been explored.
43
sion was not sustained. 56
One of the most important features of gene therapy targeting the hepatic microenvironment is that immune tolerance towards the transgene product could be induced, which may depend partly on
2.3.2 | Adenoviral vectors
regulatory T-cell induction.44-46 In addition to LSECs, other ECs have
The use of adenoviral vectors to treat haemophilia has been ex-
also been explored for haemophilia gene therapy, including blood
plored. However, in the late 1990s, the adenoviral vector trial for
outgrowth endothelial cells (BOECs),47,48 and strategies to enhance
ornithine transcarbamylase deficiency failed, and this research has
their engraftment and persistence after transplantation are being
been halted.57 After systemic administration, there is a risk of un-
explored further.49
controllable inflammatory reactions that can possibly be lethal,
Another target for haemophilia gene therapy is the skeletal muscle cell. These cells are able to perform all the required post-
which is a main drawback for adenoviral gene therapy. We and others have shown that supraphysiological coagula-
translational modifications for a functional FIX protein and also have
tion factor levels can be reached after high-c apacity adenoviral
a relatively robust secretion capacity. Whereas FIX can be easily se-
gene therapy with vectors without any viral genes and encoding
creted by muscle cells into the blood circulation, this is not the case
for the FVIII or FIX gene, without evident toxicity and inflamma-
for FVIII, possibly because of the relatively large size of the FVIII
tion in mouse and dog models of haemophilia. 34,58,59 In severe
protein particularly when a complex is formed with von Willebrand
haemophilia A patients, a phase I clinical trial was performed, in
factor (vWF). It would appear that the risk of provoking a transgene-
which a high-c apacity adenoviral vector encoding for the FVIII
directed immune response is higher following muscle-directed gene
gene was administered systemically. The first treated patient
transfer, unlike in liver-directed approaches.50 Nevertheless, early
seemed to express low FVIII levels above the 1% threshold.
clinical trials revealed that sustained FIX expression was detected
However, the trial was discontinued after that patient showed
in muscle biopsies in patients up to 10 years after muscle-directed
signs of a transient inflammatory response with haematologic
gene therapy, despite the lack of circulating FIX levels.51
and liver abnormalities due to the vector infusion. It is therefore
Finally, haematopoietic stem cells (HSCs) are also investigated
necessary to minimize the interaction between adenoviral vec-
as haemophilia gene therapy targets to express the clotting factor
tors and the innate immune system, e.g. by localizing the delivery
genes in various haematopoietic lineages. FVIII and FIX can be se-
of the vectors. 60 Based on these concerns and given the success
creted into the blood circulation directly following HSC-directed
of AAV-b ased gene therapies, it seems unlikely that adenoviral
gene transfer, especially using HSC- derived erythrocytes, mega-
vectors would become a realistic option for haemophilia any time
karyocytes and their platelet progeny as a delivery platform.52
soon.
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in correction of the haemophilic phenotype in both mouse and dog
2.3.3 | γ-Retroviral and lentiviral vectors
haemophilia models.52,74,75 The primary response to vascular injury
γ-Retroviral vectors have the property to stably integrate into the
is mediated by activated blood platelets; therefore, the storage of
DNA of actively dividing cells. We demonstrated for the first time
FVIII protein within platelets could offer a locally inducible treat-
that gene therapy could cure haemophilia A in 1999 in neonatal hae-
ment for haemophilia A. Interestingly, even in presence of high-titre
mophilia A mice, leading to lifelong FVIII expression.61 As neonatal
inhibitory antibodies after lentiviral platelet-directed FVIII expres-
FVIII-deficient mice have a rapid hepatocyte turn-over, they are
sion, correction of the disease phenotype in haemophilia A mice
permissive for γ-retroviral transduction. Subsequently, this was also
could be accomplished.73,76,77
verified in a neonatal dog model of haemophilia A and haemophilia B.
62,63
Cytotoxic preconditioning regimens, like busulfan, are needed to
Because of the necessity for cell division for this therapy, a
enable engraftment of HSCs and to create a bone marrow “niche.”
phase I clinical trial in adult haemophilia A patients resulted in low
This cannot be achieved without side effects; therefore, it is import-
FVIII levels.64,65 To overcome this problem, lentiviral vectors can be
ant to assess the risk/benefit ratio of HSC-based gene therapy for
used, because they are capable of transducing quiescent nondivid-
haemophilia. One of the methods that has been looked into to avoid
ing hepatocytes, resulting in relatively efficient adult liver transduc-
myeloablative preconditioning is the delivery of lentiviral FVIII vec-
tion.66,67 Lentiviral vectors can not only transduce hepatocytes,
tors directly into the bone marrow. This method led to in situ trans-
but also transduce professional antigen-presenting cells (APCs, e.g.
duction of bone marrow and HSCs and the resulting FVIII production
Kupffer cells) and LSECs.68 If FVIII or FIX is inadvertently expressed
in platelets derived from HSCs.78 An alternative option is to use B
in APCs, the risk of inhibitory antibody development is increased,
cells, which are capable of yielding therapeutic levels of FIX pro-
which prevents long-term expression and makes the gene therapy
tein in vivo in immunodeficient mice receiving xeno-transplantation
unsuccessful.69,70 In recent studies, it has been shown that residual
without necessitating myeloablative preconditioning.79
+
FIX expression in CD11b plasmacytoid dendritic cells (DC) could
The use of γ- retroviral and lentiviral vectors could result in
contribute to this immune reaction, while expression of FIX in con-
the risk of insertional oncogenesis caused by random genomic in-
ventional DCs or LSECs could instead contribute to immune toler-
tegration, which is one of its main safety concerns.67 This risk can
15
The immune tolerance that is induced by hepatic FVIII or FIX
be affected by the vector design or the presence of transcription-
gene transfer by lentiviral vectors typically requires regulatory T-cell
ance.
ally active long terminal repeats (LTRs). This transcriptional activity
induction.
25,46
The use of hepatocyte-specific promoters can avoid
can be reduced by removing around 400 bp in the LTR region (ie
expression in APCs and in this way result in long-term FIX expres-
self-inactivating vector, SIN). Combined with using a promoter in an
sion.69,70 In some circumstances, it was necessary to use micro-RNA
internal position, this can markedly reduce the risk of insertional on-
regulated expression cassettes to tweak the specificity of expres-
cogenesis. Transducing HSCs or hepatocytes using lentiviral vectors
15,24,35,71
in tumour-prone mouse models has confirmed that these SIN vec-
We have shown that introducing a codon-optimized hyperac-
tors are relatively safe.72,80 This suggests that the risk of insertional
tive FIX-R338L (Padua) gene with either integration-defective or
oncogenesis can be significantly reduced, even in tumour- prone
integration-competent lentiviral vectors in haemophilia B mice could
models, by merely changing the vector design. This risk can be fur-
sion to realize long-term expression and immune tolerance.
significantly augment FIX activity.
35
A subsequent study in hae-
ther minimized by integration-defective lentiviral vectors (IDLVs)
mophilia B dogs showed that liver-directed lentiviral gene transfer
that contain an inactivation mutation in the integrase. In comparison
resulted in sustained therapeutic levels of FIX protein, which could
with conventional integrase-competent lentiviral vectors, there was
pave the way for possible clinical translation,72 provided that inac-
a reduction in transgene expression levels.71 However, we showed
tivation of lentiviral vectors by human complement could be min-
that when the FIX-R338L Padua variant was incorporated, relatively
imized. Most individuals have not been pre-exposed to lentiviral
robust levels of FIX activity could be achieved even when IDLVs
vector components, contrary to AAV vectors; therefore, there is no
35
pre-existing vector-specific immunity, which is an advantage when
eventually contribute to a decline in transgene expression remains
using lentiviral vectors. However, lentiviral vectors can still induce
uncertain at this point.
were employed. Whether the slow hepatocyte turn-over would
innate immune responses and the corresponding increase in pro- inflammatory cytokine production, because they are able to efficiently interact with professional APCs.70 To deliver FVIII or FVIII to HSC and other stem/progenitor
2.3.4 | Adeno-associated viral vectors (AAV) Haemophilia B
cell populations, γ-retroviral and lentiviral vectors have been em-
Stable FIX expression could be reached after muscle-directed gene
ployed (reviewed in 66, 67, 73). When HSCs were transduced with
transfer in murine or canine models for haemophilia B that carry a
γ-retroviral or lentiviral vectors containing the FVIII or FIX gene and
missense mutation.81,82 However, when haemophilia B dogs have a
transplanted into myeloablated haemophilic mice, this resulted in
FIX null mutation, the induction of high-titre inhibitors after muscle-
phenotypic correction and immune tolerance induction to the cog-
directed gene therapy limits phenotypic correction.83 The intravas-
nate transgene products. Using this method, the expression of FVIII
cular delivery of AAV-FIX vectors to the muscle, under transient
and FIX could be directed specifically to platelets, which resulted
immune suppression could augment the therapeutic efficacy up
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EVENS et al.
54
to 10-fold.84 Not only the underlying FIX gene mutation, but also
species.103,104 One advantage of the AAV8 serotype is that it shows
vector dose and local FIX antigen doses in the transduced muscle
less cross-reactivity with pre-existing antibodies against AAV2. Also,
determine the risk of inhibitor formation, which could be avoided
preclinical studies have indicated that the uptake of the capsid by
by transient immune suppression or by reducing the vector dose
dendritic cells could be decreased compared to AAV2 vectors, which
per site (reviewed in 85). These preclinical studies were the starting
could reduce T-cell activation.105 Expression levels varied between
point of a phase I clinical trial by Dr. High and Dr. Kay and colleagues,
1 and 6% of normal FIX levels over a period of around 3 years, which
in which severe haemophilia B patients with an underlying missense
had not been shown previously. All 6 patients in the group receiv-
mutation in the FIX gene were administered with AAV-FIX by intra-
ing the highest dose (2 × 1012 vg/kg) reached expression levels up
muscular injections at multiple sites.
86
Even though low or subthera-
to 5% of normal FIX. These patients displayed more than 90% less
peutic circulating FIX levels were reached, the FIX expression could
bleeding episodes and prophylactic treatment with FIX concentrate.
be locally detected in the muscle up to 10 years after injection.51 Because of the requirement of repeated muscle injections and
AAV-specific T-cell responses were also observed in this clinical trial, though not in all subjects.
also the risk of inhibitor development, liver-directed AAV-mediated
The same vector design was used in a more recent clinical trial
gene therapy was explored as another option. Besides AAV2, other
(UniQure; NCT02396342), although here AAV5 was used instead of
naturally occurring serotypes, like AAV5, AAV8 and AAV9, variants
AAV8. Approximately 4% of FIX expression levels was achieved in
of capsids acquired by evolution or selection87,88 or capsids contain-
the patients that received a dose of 5 × 1012 vg/kg of the AMT-060
could be used. Preclinical studies in
vector.106 When a higher dose was given (2 × 1013 vg/kg) to another
haemophilia mouse or dog models and also in nonhuman primates
patient cohort, the FIX expression levels improved to almost 7%.
have shown that sustained therapeutic FIX expression levels can be
These patients showed a reduction in spontaneous bleeding rates
reached.18,20,70,90-95 Even in haemophilia B dogs that harbour a FIX
and FIX prophylactic treatment. Eight of 9 patients receiving pro-
null mutation and are prone to develop inhibitors, phenotypic cor-
phylactic treatment before gene therapy treatment did no longer
rection could be accomplished after liver-directed AAV2-FIX gene
need prophylaxis. Additionally, no AAV- specific T- cell responses
transfer.93 This is consistent with the clinical correlates showing that
were observed during this trial.
ing specific point mutations
89
subjects with CRM-(cross-reacting material negative)/null mutations
Hence, to achieve comparable FIX levels as what had been re-
did not develop inhibitors after liver-directed gene therapy.96,97 This
ported in the UCL/St Jude’s trial, it would appear that a 2.5-to 10-
suggests that liver-directed gene therapy is able to reduce the risk of
fold higher vector dose was used in the UniQure trial. However, it
inhibitor development, possibly as a result of regulatory T-cell (Treg)
remains challenging to draw definite conclusions from these trials
induction.44,98
in the absence of any head-to-head comparison and the low pa-
Because of these successful liver-directed gene therapy studies,
tient numbers. Additionally, there might be other confounding vari-
a phase I clinical trial was carried out using AAV vectors containing
ables that could have an impact on the outcome of the trial, such
the FIX gene in severe haemophilia B patients. In this trial, promising
as variability in viral production methods, the purity of the vectors,
therapeutic FIX levels of almost 12% were reached.99 Unfortunately,
patient- specific features. Remarkably, transaminase levels were
FIX expression was only transient and there was evidence of vector
raised in patients in both the AAV5 and AAV8 trials, which could
dose-dependent hepatotoxicity that coincided with elevated plasma
also be detected in the initial AAV2 trial.99 Consequently, patients
transaminases. This observation could be explained by the “T-cell hy-
were given a transient immunosuppressive treatment with glucocor-
pothesis.” This implied that AAV capsid-derived antigenic peptides
ticoids to attempt to block unwanted immune responses. Although
were possibly presented in association with MHC class I proteins
there is indirect evidence backing up the “T-cell hypothesis” men-
by the transduced hepatocytes to T cells, as a possible explanation
tioned above, not all patients in the AAV8 trial with an increased
for the ELISPOT results obtained from PBMCs from the treated pa-
AAV capsid-specific T-cell response had elevated liver enzyme lev-
tients.99,100 Even though there was only modest antigen presenta-
els. Furthermore, other patients in both the AAV5 and AAV8 trial
tion, the levels could have been high enough to mark the hepatocytes
showed the opposite effect: elevated liver enzyme levels without
for T- cell–mediated destruction. Interestingly, this AAV- specific
an AAV capsid-specific T-cell response. This implies that the pres-
T-cell response was not seen in the various preclinical haemophilia
ence of vector-specific T cells in the circulation might not necessar-
models (murine, canine) nor in nonhuman primates, although several
ily be related to the immune rejection of hepatocytes transduced
efforts have been made to simulate this observation 101,102
with AAV, possibly because the liver microenvironment can have an
Another liver-directed gene therapy clinical trial was performed
effect on the local T-cell response. Another possibility is that the
at University College London (UCL)/St Jude’s Hospital Memphis by
elevated liver enzyme levels are not caused by the AAV-specific T-
Dr. Nathwani and colleagues, in which patients suffering from se-
cell response or the response was not strong enough to clear the
vere haemophilia B were intravenously administered with an AAV8
transduce hepatocytes. These controversies could eventually be
gene therapy vector containing a codon-optimized FIX gene driven
elucidated by mouse models that are able to simulate these immune
by a liver-specific promoter.73,96 AAV8 is able to increase transduc-
responses or by subsequent clinical trials.102
tion in hepatocytes compared to other serotypes, at least in mice,
The previously discussed AAV2, AAV5 and AAV8 trials indicate that
but this does not automatically mean it can be translated to higher
it is still important to further enhance the efficiency of these vectors,
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EVENS et al.
so lower, and thus safer, effective vector doses could be adminis-
In another more recent study, an AAV8 vector with a 5.2 kb ex-
tered, which do not result in transaminitis. The use of a hyperfunc-
pression cassette encoding for a codon-optimized B-domain deleted
tional codon-optimized FIX-R338L Padua variant led to a substantial
FVIII transgene was created. The incorporation of a 17-amino acid
dose advantage with no evidence of an increased risk of thrombosis or
peptide (V3), which contained glycosylation residues, in the FVIIIΔB
immunogenicity in comparison with wild-type FIX after liver-directed
sequence could boost FVIII expression around threefold. In haemo-
Other studies have validated these
philia A mice treated with 2 × 1012 vg/kg, supraphysiological hFVIII
14,37,39
results, also in canine models by Dr. Arruda and colleagues.
expression levels (732 ± 162% of normal) could be obtained. In a non-
Therefore, the hyperfunctional FIX-R338L Padua variant is a very in-
human primate model receiving the same dose, stable hFVIII expres-
teresting treatment option for haemophilia B gene therapy.
sion levels of 15 ± 4% were observed. Three macaques that received
gene therapy in mouse models.
21,35
In two independent recent clinical trials, the FIX- R338L
a higher dose did reach human FVIII expression levels of above 100%,
Padua gene was introduced and showed promising results. The
but this resulted in the development of neutralizing anti-F VIII anti-
first trial (Shire; NCT01687608) had patients treated with a self-
bodies, which could be eliminated by transient immunosuppression. It
complementary AAV8 vector containing a codon-o ptimized FIX-
is not yet known whether this V3 peptide increases the immunogenic
R338L Padua variant (BAX335). In this trial 3 dosing cohorts were
risk in patients. A clinical trial is ongoing to address this.
performed, treating 7 patients in total. Two of the patients showed
Recently, a phase I/II clinical trial was performed (BioMarin;
transient FIX activity levels of more than 50%. One of the patients
NCT02576795), in which 9 patients were treated with an AAV5 vec-
from the medium dose cohort (1 × 1012 vg/kg) showed persistent
tor expressing FVIIIΔB 114 based on encouraging results in primates
FIX levels. None of the patients had developed antibodies against
and haemophilic mice.115 Seven of those patients were treated with
FIX. The second trial (Spark Therapeutics/Pfizer; NCT02484092)
the highest dose of 6 × 1013 vg/kg after a dose escalation study.
97
resulted in a more consistent response in the patients.
The
Six of 7 patients treated with the highest dose of 6 × 1013 vg/
single- s tranded AAV vector (SPK- 9 001) encoded for a codon-
kg showed a gradual increase in FVIII activity levels and reached
optimized FIX-F338L Padua variant and had an alternative mu-
more than 50% of normal FVIII activity levels after 20 weeks, when
tated capsid. Fourteen weeks after receiving the initial dose of
steady-state levels were attained. After 52 weeks, a mean FVIII ac-
5 × 1011 vg/kg, the 10 participants reached a steady-s tate FIX ac-
tivity level of 93 ± 48% was achieved for the patients receiving the
tivity level of around 33.7 ± 18.5% without evidence of inhibitory
highest dose. In four of the patients, levels of more than 150% of
antibodies. The annualized bleeding rate of the patients dropped
normal FVIII activity levels were reached, with peaks ranging from
from 11.1 to 0.4 events after SPK-9 001 administration. Not only
201 to 349% of normal. The six patients of the highest dose that
the annualized bleeding rate, but also the annualized infusion rate
were previously on FVIII prophylaxis showed a reduction in annual-
dropped from 67.5 to 1.2 after vector administration, rendering
ized bleeding rate from 16 to 1 event per year after treatment. The
further FIX prophylaxis unnecessary for these patients. However,
median annualized FVIII infusion rate dropped from 138 infusions
2 of the patients showed a transient elevation of liver enzymes
per year to 2 after gene transfer and was actually 0 since week 2.
and a decrease in FIX activity levels, which could indicate the pres-
However, the patients in the high-dose cohort showed increased
ence of a capsid-specific immune response. Oral corticosteroids
transaminase levels, but these were asymptomatic. These patients
prevented a further reduction of FIX activity levels.
did require glucocorticoid treatment after the gene therapy treatment, which was tapered off successfully for all patients. During
Haemophilia A
the 1-year follow-up, there was no indication of the presence of
One of the limiting steps for haemophilia AAV-based gene therapy is
FVIII inhibitors or T-cell–mediated immune responses to the AAV5
the packaging constraint of these vectors, because of the relatively
capsid. In this trial, the vector doses were substantially higher than
large size of the FVIII transgene. In general, the required vector dose
previously used in AAV-based haemophilia trials, which causes chal-
was relatively high and combining the FVIII transgene with a potent
lenges during vector manufacturing.
hepatocyte-specific promoter in a single vector remained challenging. It is preferred to use a single-chain vector over a dual-chain vector, which separately expresses the heavy and light chain.107-110 To
3 | CO N C LU S I O N S A N D PE R S PEC TI V E S
increase the efficiency of haemophilia A AAV-based gene therapy, small regulatory elements can be used to drive the expression of
The recent results of haemophilia clinical trials yielded the most
a B-domain deleted from of FVIII, codon optimizing the FVIII gene
encouraging results to date, showing sustained FIX and FVIII
and/or integrating specific mutations in the FVIII cDNA. 27,31,110-113
expression levels and a reduced bleeding rate and recombinant
Treatment of haemophilia A dogs with AAV2, AAV6 and AAV8 vec-
coagulation factor use, without requiring further prophylaxis.
tors containing the cFVIII gene resulted in persistent FVIII expres-
In particular, the improvements of vector designs or using im-
sion at a therapeutic level for more than 3 years, without antibody
proved clotting factors (codon-o ptimized or hyperfunctional vari-
development or toxicities. However, in the haemophilia A dogs,
ants) were essential to make advancements in haemophilia gene
there was no difference in liver transduction efficiency between the
therapy. However, patients expressing FIX or FVIII in the lower
different serotypes, in contrast to mice.
range are still at risk of traumatic bleeding. Vector-induced liver
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EVENS et al.
56
inflammation remains a challenge that is not fully understood but could be controlled by transient immune suppression. Although vector capsid-s pecific T-cell immune responses can be detected in at least some patients, it is likely that a more complicated picture will emerge whereby other immune mechanisms are involved that account for the transaminitis and/or the decline in transgene expression. This may possibly be due to innate immune responses that could be related—at least in part—to the presence
AC K N OW L E D G E M E N T S We thank the members of the Department of Gene Therapy & Regenerative Medicine and all our collaborators for their various contributions to some of the work presented in this review. We also wish to thank FWO, Shire, Bayer, Pfizer, VUB-IOF-GEAR (GENEFIX), Strategic Research Project Grower, AFM, Willy Gepts Fund for providing financial support.
of CpG motifs in the vector backbone. In the long run, it would be preferred to adapt the vector to the patient, rather than the other way around, and obviate the need for immune suppression altogether. In addition, controversies about the potential risk of insertional oncogenesis associated with the use of AAV vectors remain.116-123 It is particularly encouraging that preclinical studies in
D I S C LO S U R E S The authors have patent applications and granted patents in the field of gene therapy for haemophilia. They also received industrial grants and/or consultancies from various companies involved in gene therapy for haemophilia (Shire, Bayer, Pfizer).
haemophilia mice, dogs or nonhuman primates did not reveal any increased tumorigenic risk after AAV gene transfer. Nevertheless, in certain animal models, an increase in tumour incidence was apparent that seems to have been associated with the AAV treatment. Several confounding variables appear to be associated with this increased risk, including the vector dose, the vector design and the presence of strong enhancers, the mouse model and age of the recipient mouse. Interestingly, epidemiological studies revealed that Clonal integration of wild-type adeno-associated virus type 2 (AAV2) fragments corresponding to the 3′ untranslated region was found in 11 of 193 hepatocellular carcinomas. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2, TERT, CCNE1, TNFSF10 and KMT2B, leading to their overexpression, consistent with the presence of an enhancer in the 3′ UTR.123 Nevertheless, efficient site-specific integrating vectors based on designer zinc finger nucleases, CRISPR/Cas9 or nuclease-free targeting approaches into “safe harbour” loci could potentially minimize this risk.124-127 These nucleases induce a double-s trand break at the desired locus, increasing the efficiency of directed homologous recombination at least 10 000 to 100 000-fold. These gene-editing approaches had led to sustained clotting factor levels in haemophilic mice by either correcting the mutated clotting factor gene or by integrating a functional gene copy in a safe harbour locus. This opens up new perspectives to possibly treat paediatric subjects by gene editing even in the face of hepatocyte proliferation. Nevertheless, the immune ramifications of expressing designer nucleases and the possible off-t arget effects following gene editing of homologous gene sequences in the genome would need to be addressed. Ultimately, it will be necessary to develop gene therapy strategies that could also be employed in patients with pre-existing immunity to the vectors or even with high-t itre inhibitors or a higher risk of inhibitor development than in the initial patient cohorts in these early phase I/II trials. Eventually pivotal trials are needed for marketing authorization approval by the regulatory authorities. Only then will gene therapy make a real difference for patients suffering from haemophilia and their families.
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How to cite this article: Evens H, Chuah MK, VandenDriessche T. Haemophilia gene therapy: From trailblazer to gamechanger. Haemophilia. 2018;24(Suppl. 6):50‐59. https://doi.org/10.1111/ hae.13494
