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Circulation Research Compendium on Thrombosis
Advances in Thrombosis and Hemostasis: An Introduction to the Compendium
Global Burden of Thrombosis: Epidemiologic Aspects
Systems Analysis of Thrombus Formation
Animal Models of Thrombosis From Zebrafish to Nonhuman Primates
Platelet-Mediated Thrombosis: From Bench to Bedside
Cross Talk Pathways Between Coagulation and Inflammation
Evolving Treatments for Arterial and Venous Thrombosis: Role of the Direct Oral Anticoagulants
Evolving Treatments for Acute Ischemic Stroke
Gene Therapy for Coagulation Disorders
Jeffrey I. Weitz and John W. Eikelboom, Editors
Gene Therapy for Coagulation Disorders
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Laura L. Swystun, David Lillicrap
Abstract: Molecular genetic details of the human coagulation system were among the first successes of the genetic
revolution in the 1980s. This information led to new molecular diagnostic strategies for inherited disorders of
hemostasis and the development of recombinant clotting factors for the treatment of the common inherited bleeding
disorders. A longer term goal of this knowledge has been the establishment of gene transfer to provide continuing
access to missing or defective hemostatic proteins. Because of the relative infrequency of inherited coagulation
factor disorders and the availability of safe and effective alternative means of management, the application of
gene therapy for these conditions has been slow to realize clinical application. Nevertheless, the tools for effective
and safe gene transfer are now much improved, and we have started to see examples of clinical gene therapy
successes. Leading the way has been the use of adeno-associated virus–based strategies for factor IX gene transfer
in hemophilia B. Several small phase 1/2 clinical studies using this approach have shown prolonged expression of
therapeutically beneficial levels of factor IX. Nevertheless, before the application of gene therapy for coagulation
disorders becomes widespread, several obstacles need to be overcome. Immunologic responses to the vector and
transgenic protein need to be mitigated, and production strategies for clinical grade vectors require enhancements.
There is little doubt that with the development of more efficient and facile strategies for genome editing and
the application of other nucleic acid–based approaches to influence the coagulation system, the future of genetic
therapies for hemostasis is bright. (Circ Res. 2016;118:1443-1452. DOI: 10.1161/CIRCRESAHA.115.307015.)
Key Words: bleeding
■
coagulation
■
factor IX
■
factor VIII
T
he human coagulation system has evolved over the past
450 million years to function through a complex coordinated interaction involving the vessel wall, circulating cells,
and a protein cascade of procoagulants that are regulated by
a series of anticoagulant mediators.1 In normal hemostatic
responses, these various components combine to generate a
protective fibrin clot, limiting blood loss from the circulation. In pathological states, deficiencies, dysfunction, or aberrant regulation of these hemostatic elements result in either
bleeding or thrombotic phenotypes. These pathologies are the
■
gene therapy
■
gene transfer
■
hemophilia
consequence of either rare genetic traits or, more often, a complex interaction of genetic and acquired influences.
Current Therapeutic Approaches
to Coagulation Disorders
Current treatments for bleeding and thrombotic diseases comprise either targeted single factor approaches in which specific
elements of the hemostatic process are replaced or inhibited
or combination strategies that either restore or interfere with
multiple components of the hemostatic response. Although an
Original received October 5, 2015; revision received December 9, 2015; accepted December 16, 2015.
From the Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario, Canada.
Correspondence to David Lillicrap, MD, Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen’s University, Kingston,
Ontario, Canada K7L 3N6. E-mail [email protected]
© 2016 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.307015
1443
1444 Circulation Research April 29, 2016
Nonstandard Abbreviations and Acronyms
AAV
adeno-associated virus
ADAMTS13 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13
FVIII
factor VIII
FIX
factor IX
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initial vasoconstrictive response to vascular damage is crucial
for the early phase of hemostasis (eg, reduced blood flow,
platelet margination, and unfolding of von Willebrand factor),
specific therapies aimed at this early element of hemostasis are
not routinely used. In contrast, cellular therapies to enhance
the contributions of platelets (adhesion and aggregation) and
red cells (contributing to platelet margination and fibrin clot
strength)2 to the generation of an optimal hemostatic response
are frequently applied to the treatment of bleeding. Currently,
most therapies used for coagulation disorder management are
aimed at replacing, enhancing, or inhibiting components of
the procoagulant protein cascade and the corresponding anticoagulant and fibrinolytic response.
Procoagulant Treatment Approaches
Interventions aimed at preventing or treating bleeding can involve either single factor strategies or combination replacement therapy. The more common inherited bleeding disorders,
hemophilia A (factor VIII [FVIII] deficiency) and B (factor IX
[FIX] deficiency), von Willebrand disease, and factor XI deficiency, can be treated with single factor replacement therapies
through the infusion of protein concentrates derived from either plasma or recombinant DNA technology.3
In contrast, most acquired bleeding disorders, such as
vitamin K deficiency, liver disease, and consumptive coagulopathies, are managed with the infusion of either defined multifactor concentrates (eg, prothrombin complex concentrates,
factors II, VII, IX, and X,4 and cryoprecipitate, von Willebrand
factor, factors VIII, XIII, and fibrinogen5) or plasma.
The efficacy of these various treatment regimens can be
monitored through clinical assessment and by the performance of either global tests of hemostasis, such as the prothrombin, partial thromboplastin, and thrombin times, or by
quantification of single factor levels as in the replacement of
FVIII and FIX in the hemophilias.
Gene Therapy Concepts and Historical Context
The concept of using genetic approaches to treat and potentially cure disease originated in the early 1980s with the
cloning of the first human genes. The first, approved, and successful gene therapy study in a human was conducted in 2 patients with inherited adenosine deaminase deficiency in 1990.6
Since then, the field of gene therapy has had a chequered history, but in 2012, the European Medicines Agency approved
for the first time a gene therapy drug Glybera, a gene therapy
treatment for inherited lipoprotein lipase deficiency.7 Now in
2015, there is realistic optimism that this therapeutic modality
can offer significant opportunities for long-term benefits in a
range of disorders.
The coagulation genes were among the first to be characterized in the 1980s, with major contributions derived
from Earl Davie’s laboratory at the University of Washington
(Figure 1A).8–11 These discoveries confirmed that the coagulation proteins were derived from genes which encoded modular
domains that were shared between related families of coagulation factors (eg, the vitamin K–dependent factors and factors V and VIII). Soon after the cloning of the FVIII and FIX
genes, translational application of this knowledge was initiated through the introduction of molecular diagnostics,12 the
development of recombinant clotting factors for replacement
therapy,13 and the initial preclinical trials of gene therapy.
The hemophilias are an ideal model genetic disease for the
application of gene therapy. They result from recessive mutations in 2 well-characterized genes with minimal influence
from other genetic modifiers. Furthermore, it was well recognized from the natural history of moderately severe and mild
hemophilia (clotting factor levels between 1% and 40%) that
small increments in plasma clotting factor levels would result
in significant benefits in reducing the risk of bleeding. Thus
began the 30-year quest to convert what seems to be a simple
therapeutic goal, to deliver a normal gene copy to compensate
for the existing mutant gene.
Spectrum of Gene Therapy Initiatives
for Coagulation Disorders
It is clear, for the reasons detailed earlier, that the hemophilias have been leading candidates for the application of gene
therapy since the dawn of molecular medicine. However, advances in the development of gene therapy strategies for other
pro- or anticoagulant deficiencies has been modest, a fact that
relates to a combination of factors, including the infrequent
incidence of monogenic inherited bleeding and thrombotic
disorders, the existence of safe and effective alternative therapies, and in some instances, inadequate biological knowledge
to effectively design gene therapy strategies.
Aside from FVIII and FIX gene therapy, the only other
procoagulant targets that have been explored to any extent
in preclinical studies have been von Willebrand factor14 and
factor VIIa.15 The former target might find a place for the
treatment of the rare type 3 form of von Willebrand disease
(incidence 1 per million), but even in this population, with
no detectable circulating von Willebrand factor, spontaneous
bleeding rates can often be surprisingly low.16 The limited
activated factor VII gene therapy studies have been focused
predominantly on the potential for developing a sustainable,
long-term strategy for bypassing the presence of FVIII antibodies (FVIII inhibitors) in hemophilia A.
Gene therapy for anticoagulant purposes has been similarly underdeveloped. Again, this relates to the availability of
safe and effective alternative treatments and to the fact that
monogenic traits directly responsible for initiating thrombosis
are rare. Finally, in contrast to the hemophilias, the levels of
transgenic protein expression required to produce a phenotypic benefit are likely to be beyond the capability of current gene
transfer technologies.
One thrombotic disorder where gene therapy has produced promising preclinical results is the inherited form of
Swystun and Lillicrap Gene Therapy for Coagulation Disorders 1445
A
B
Non Viral Gene
Transfer
Hydrodynamic delivery
Targeted nanoparticles
Gene
Therapy
Recombinant Protein
Therapy
Molecular Diagnostics
Viral Gene Transfer
Retrovirus
Adenovirus
Adeno-associated Virus
Basic Science
1984
1986
1990
2013
Coagulation
Factor Gene
Therapy
Mutation Repair
Zinc finger nucleases
TALENs
CRISPR-Cas9
Cell-based Gene Therapy
Embryonic stem cells
Adult stem/progenitor cells
Patient-derived iPS cells
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Figure 1. Gene therapy as a treatment for coagulation factor disorders. A, Gene therapy is the ultimate goal for treatment of
inherited coagulation disorders. Characterization of the clotting factor genes/proteins in the 1980s allowed for the molecular diagnosis
of inherited bleeding and thrombophilic disorders. Subsequently, recombinant protein technologies provided the first clotting factor–
specific replacement therapies. Current strategies for improving treatment of inherited coagulation disorders include improving the
pharmacokinetics of recombinant protein therapies and increasing the safety and efficacy of gene transfer protocols. B, Potential
strategies for gene therapy for coagulation factor deficiencies include viral and nonviral in vivo, methods of delivery of a normal gene
copy, ex vivo modification of host cells to induce normal gene expression, and in situ mutation correction. CRISPR-Cas9, clustered
regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9; iPS, induced
pluripotent stem cells; and TALENS, transcription activator-like effector nucleases.
a disintegrin and metalloproteinase with a thrombospondin
type 1 motif, member 13 (ADAMTS13) deficiency (Upshaw–
Schulman Disease). Studies in the mouse model of this condition, using an adeno-associated viral (AAV) vector–mediated
approach have documented sustained therapeutic levels of
transgenic ADAMTS13 expression and a positive effect on the
clinical features of the disorder.17 However, with safe and effective treatment already available for this rare genetic disease
through infrequent (every 3–4 weeks) infusion of plasma18
and the development of a recombinant ADAMTS13 protein,19
the likelihood of a gene therapy approach reaching the clinic
in the foreseeable future is small.
Gene Therapy Strategies for Coagulation
Disorders
The therapeutic goal for coagulation factor gene therapy is the
delivery of a normal gene copy to produce therapeutically effective levels of the protein that is either deficient or dysfunctional as a result of a germ line mutation (Figure 1B).
Several strategies for genome editing, using technologies
such as zinc finger nucleases and transcription activator-like
endonucleases, have the potential for in situ mutation correction. The application of these approaches has proven effective
in vitro and in animal studies,20,21 but none of the methodologies are close to clinical introduction. Zinc finger nuclease editing has also been applied successfully to target therapeutic
transgene insertion to a safe and transcriptionally favorable
locus.22 The latest technology to be applied to genome editing
uses guide RNA molecules to target the site for revision, and
the Cas9 endonuclease to mediate the double strand cleavage
event required for sequence correction with a normal donor
molecule (clustered regularly interspaced short palindromic
repeats/clustered regularly interspaced short palindromic
repeat–associated 9 [CRISPR-Cas9]).23 There is considerable
enthusiasm in the molecular biology community concerning
the utility of this technology, and the momentum of CRISPRCas9 advances has been dramatic.24 However, despite the
fact that mutant coagulation gene editing has already been
reported in vitro,25 several critical limitations to this technology remain. The achievable efficiency of mutation correction
is still low, and perhaps more problematic is the potential for
damaging off-target editing events elsewhere in the genome.26
Similar weaknesses exist for earlier editing approaches involving zinc finger nuclease repair and TALEN-based strategies.27
For the present, substantial improvements in both efficiency
and safety will need to be developed before any genome editing approach could be considered for clinical application as
a treatment for coagulation factor deficiency or dysfunction.
Current gene therapy initiatives for coagulation pathologies are essentially gene supplementation strategies in which
the mutant gene is untouched and a copy of the normal gene
is added to somatic cells of the patient. By its nature, this type
of approach is best suited to the treatment of recessive traits
in which interference from a dominant mutant product is not
a limitation.
Transgene Delivery Approaches
The first critical hurdle to overcome in any gene transfer
protocol is the efficient delivery of the therapeutic transgene
to somatic cells of the patient (Figure 2A). This goal can be
achieved by either in vivo or ex vivo approaches, and there
are advantages and weaknesses to both strategies. Ex vivo
transgene delivery requires the isolation of a recipient cell
population that can be readily transduced and has the potential
for prolonged survival after delivery back into the recipient.
The advantages of this approach are that efficient rates and
1446 Circulation Research April 29, 2016
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cell-type specificity of transgene delivery can be achieved
(Figure 2B) and that the immunologic complications of in
vivo vector administration are avoided. However, ex vivo
strategies are inherently complex and resource-intensive, and
re-establishment of the genetically modified cells may require
some form of conditioning to ensure engraftment.28 In contrast, in vivo gene transfer is straightforward to perform, but
ensuring efficient transgene delivery to specific cell types,
without inciting immune reactions to the delivery vehicle, is
challenging.
A single human clinical trial for ex vivo delivery of a FVIII
transgene has been performed for hemophilia A. This study,
involving 12 patients, used autologous fibroblasts that were
electroporated ex vivo with a FVIII transgene, selected for optimal FVIII expression, expanded, and subsequently injected
into the omental membrane.29 There were no adverse events
associated with these studies, but also no evidence of sustained FVIII transgene expression, although several patients
showed evidence of transient (2–3 days) minimal increments
of plasma FVIII levels (2%–6%). Other preclinical studies of
ex vivo transgene delivery for hemophilia have involved hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitors. Results from the hematopoietic stem cell
studies have been especially promising30,31 and may advance
to a phase I/II clinical trial in the near future, but the endothelial progenitor investigations have been complicated by the
generation of immune responses to the transgene product.32
One particular ex vivo strategy for ectopic hemophilia gene
therapy deserves further commentary. The generation of FVIII
transgenes that are delivered to and expressed exclusively in
megakaryocytes and platelets has shown promise in promoting an effective hemostatic response in standard animal models of hemophilia A and in the presence of FVIII inhibitory
antibodies.28,33–35 Whether this strategy can be sufficiently optimized in terms of transgene delivery and protein production
A
In Vivo
Gene Transfer
1.
Vector
1.
Isolation autologous
stem cells
(HSC, MSC, EPC)
B
Delivery Virus
(lentivirus)
Organ
(eg. liver)
Delivery
Virus
eg. AAV,
Lentivirus
Modes of Transgene Delivery
The goal of transgene delivery is to introduce the therapeutic
gene construct as efficiently and safely to the recipient, usually to selected types of somatic cells. Importantly, avoidance of delivery to germ cells is a requirement for all clinical
studies because the safety of germ line modification remains
unresolved.
There are essentially 2 modes of transgene delivery: the
application of viral vectors or nonviral vector–mediated strategies. Some of the latter approaches have been used for in vitro molecular biology studies since the 1970s, but in general,
physicochemical protocols are relatively inefficient delivery
approaches for achieving long-term persistence of transgenes.
Electroporation has also been applied in some ex vivo protocols29 but again lacks sufficient efficiency for routine clinical
translation. Finally, localized application of hydrodynamic
gene transfer protocols has been proposed as a further approach to deliver expression plasmids to specific organs or
regions of organs.37,38 However, the inevitable, albeit transient,
vascular and local tissue damage caused by this mode of delivery initiates a strong innate immune response, thus increasing
the likelihood of a secondary adaptive response to the neoantigenic transgene protein.
In marked contrast to the limitations of the various
nonviral modes of transgene delivery, viral vectors use
Ex Vivo
Gene Transfer
Transgene
cDNA
2.
without the requirement of prohibitive conditioning regimens
remains to be established. One potential approach to circumvent the requirement for bone marrow conditioning is the use
of intraosseus vector infusion to mediate in vivo hematopoietic cell transduction. Using a lentiviral-based strategy with a
platelet-specific regulatory sequence, long-term persistence of
intra-platelet FVIII has been demonstrated at sufficient levels
to mediate hemostatic protection in the presence of anti-FVIII
antibodies.36
3.
3. Injection
Muscle
Selection/
Expansion/
Reintroduction
2.
Transduction
Figure 2. Methods for transfer of coagulation factor genes. A, Currently used strategies (clinical or preclinical) for transfer of
coagulation factor genes include in vivo and ex vivo approaches. In vivo methodologies involve inserting the coagulation factor cDNA
into a viral vector genome, with subsequent introduction of the vector into the patient by intravenous or intramuscular injection. Ex vivo
gene transfer involves isolation of autologous stem cells. Isolated cells are transduced with the normal coagulation factor gene, selected
for protein expression, expanded, and then reintroduced into the patient. B, Cellular targets for in vivo transfer of coagulation factor
genes include hepatocytes and skeletal muscle. Cellular targets for ex vivo gene transfer approaches involve isolation and transduction
of autologous stem cells including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and endothelial progenitor cells
(EPCs). AAV indicates adeno-associated virus.
Swystun and Lillicrap Gene Therapy for Coagulation Disorders 1447
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properties that have evolved over millions of years to facilitate the efficient entry of viruses into cells.39 The generation of viral vectors involves the modification of the viral
genome to eliminate the possibility of viral replication and
to provide sufficient space for insertion of the therapeutic
transgene in a viral particle that can still be efficiently packaged. Subsequent to the generation of the modified vector
genome, the vector particles are produced in cell culture,
providing packaging and structural proteins from cotransfected helper plasmids.
Gene therapy clinical trials for hemophilia have used 3
types of viral vector (Figure 3A). One patient received an adenoviral-mediated treatment for hemophilia A; there has been
one trial of retroviral gene transfer of FVIII, and the remaining studies have used AAV vectors to deliver FIX transgenes
(Table).
In contrast to AAV vectors that are discussed later, adenoviral vectors are not appropriate for the delivery of coagulation transgenes, in large part because of the significant
proinflammatory innate immune response that their capsid
proteins incite.48 This response can be limited to the development of fever, thrombocytopenia, and the elevation of liver
transaminases, but can, under certain circumstances, advance
to a fatal systemic inflammatory state such as that which killed
Jesse Gelsinger during a trial of ornithine transcarbamylase
gene therapy in 1999.49–51 Unless this obstacle can be eliminated (which may simultaneously reduce the transduction efficacy of the vector), adenoviral vectors will not find a place in
clinical coagulation factor gene therapy.
The single clinical trial using an FVIII retrovirus in hemophilia A involved the systemic delivery of a classical γretroviral vector that would require cellular replication for
successful transduction.41 None of the 12 patients entered in
this phase 1 study developed complications, but only low level
(≈4%) and transient levels of FVIII were documented, indicating either inadequate transduction and transgene expression.
A
B
Viral Vector
Characteristics
Advantages
Although γ-retroviral vectors are no longer being pursued
for hemophilia therapy, the development of lentiviral vectors,
capable of transducing both dividing and nondividing cells,
has continued. Lentiviral vectors can be used as the means
for transgene delivery either in ex vivo strategies for modifying stem cell populations52,53 or via systemic in vivo delivery.54
Both strategies have been used successfully in animal models
of hemophilia,54,55 and some form of lentiviral-based treatment
may well enter the clinic in the future. Nevertheless, in 2015,
the clearly favored vector system for clinical coagulation gene
therapy was AAV.
AAV-Mediated Gene Therapy for Hemophilia
The development of AAV-based protocols for gene transfer
of FIX has been one of the highlights of clinical gene therapy advances in the past 5 years. AAVs are small infectious
agents (≈20 nm) that belong to the family of parvoviruses.
In humans, infection with AAV is asymptomatic. AAV has a
small single-stranded DNA genome of ≈4.7 kb, with 2 open
reading frames that encode capsid and other structural proteins and proteins facilitating integration of the wild-type
virus into a specific region on chromosome 19. In the construction of AAV vectors, both these coding regions are deleted, allowing the insertion of transgenes of ≤≈5 kb without
adversely influencing the packaging efficiency of vector particles. This packaging limitation has not affected the generation of FIX transgenes, in which the coding sequence spans
≈1.3 kb. In contrast, the FVIII cDNA occupies ≈8.5 kb, and
even after deletion of the nonessential B domain–encoding
sequence, the cDNA is still ≈5 kb. Thus, promoter/enhancer
elements for AAV FVIII constructs have to be compact, and
even then, a reduction in vector packaging efficiency is a
significant concern (Figure 3B).
To date, human clinical trials of skeletal muscle43 and liver-directed44,45 AAV FIX gene therapy have been conducted.
In each instance, strong tissue-specific promoter/enhancer
Limitation
Adenovirus
36 Kb dsDNA
Non-enveloped
Non-integrating
Large genome
Easy to produce high titer
Infects many cell types
High immunogenicity
Retrovirus
(lentivirus)
8 Kb ssRNA
Enveloped
Integrating
Larger genome
High infection efficiency
Stable gene transfer
Insertional mutagenesis
Adenoassociated
virus (AAV)
4.7 Kb ssDNA
Non-enveloped
Non-integrating
Low immunogenicity
Infects many cell types
Long-term gene transfer
Small genome
WT AAV Genome
Rep
ITR
AAV Vector
ITR Enhancer
Codon Optimization/ cDNA contraction ~5 kb ss DNA
Promoter
5’ Regulatory Elements
•
•
4.7 kb ss DNA
ITR
Cap
Constitutive
Regulated
Cell-specific
Developmental-specific
Drug-inducible
Transgene cDNA
ITR
3’ Regulatory Elements
•
•
Enhancer
Poly A tail
Figure 3. Viral vectors for in vivo gene
transfer. A, Gene therapy clinical trials
for hemophilia have used adenovirus (1
patient), γ-retrovirus (12 patients), and
adeno-associated virus (AAV; 31 patients).
B, Adenovirus-associated inflammation and
inadequate transduction and transgene
expression by γ-retrovirus have discontinued
their use in coagulation factor gene therapy.
One challenge of AAV-mediated gene
therapy is the limited packaging size of the
recombinant vector genome. Strategies
for overcoming this limitation include
cDNA contraction and the use of compact
promoter/enhancer elements. ITR indicates
inverted terminal repeat.
1448 Circulation Research April 29, 2016
Table. Summary of Completed and Ongoing Phase I/II Clinical Trials for Hemophilia Gene Therapy
Transfer
Method
Gene
Patients
Treated
Gamma-retrovirus transduction of
autologous fibroblasts
Ex vivo
F9
2
Transient expression (2%)
No adverse effects
reported
Xinfang et al40
Electroporation of autologous
fibroblasts (BDD-FVIII)
Ex vivo
F8
12
Transient expression (4%)
No adverse effects
reported
Roth et al29
Gamma-retrovirus (BDD-FVIII)–IV
In vitro
F8
12
Transient expression (4%)
No adverse effects
reported
Powell et al41
“Gutless” adenovirus
In vitro
F8
1
Transient expression (1%–3%)
Transaminitis,
thrombocytopenia
No published reports42
ssAAV2/2–Intramuscular
In vitro
F9
8
Transient expression (<2%)
No adverse effects
reported
Manno et al43
ssAAV2/2–hepatic artery infusion
In vitro
F9
7
Transient expression
(10%) over 8 wk
Transient transaminitis
Manno et al44
scAAV2/8: (FIXco)–IV
In vitro
F9
10
Persistent expression
(1%–6%) ≤3 y
Transient transaminitis
Nathwani et al45,46
scAAV2/8: (FIX Padua)–IV
In vitro
F9
6
Variable expression
(0.5%–25%) ≤6 mo
No adverse effects
reported
Monahan et al47
Vehicle
Best Outcome
Complications
Reference
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BDD indicates B-domain deleted; FIXco, codon optimized FIX; sc, self-complementary; and ss, single strand.
elements have been used, and the vectors have been delivered
by either intramuscular injection or portal or peripheral vein
injection for the skeletal muscle and liver studies, respectively.
Tissue tropism of the AAV vectors is influenced by the differential efficacy for transduction mediated by serotype-specific
capsids.56 Thus, AAV8 possesses strong hepatotropism and
can be delivered efficiently to the liver via peripheral vein
infusion without the risk of significant transduction of other
tissue types.
Fate of AAV Vectors
Transduction by AAV vectors involves an interaction with
a range of serotype-specific cell surface receptors.56–58 The
vector particles are taken into endocytic vesicles, where, in
the acidic milieu of the endosome, the capsid is removed and
the nucleic acid released for transport to the nucleus.59 The
capsid proteins are then degraded by the proteasome, and
the resultant peptides presented on the transduced cell’s surface, in association with major histocompatibility complex
class I molecules (Figure 4B). The vector genome enters the
nucleus and persists long term, largely as circular extrachromosomal concatemers. However, AAV-based transgenes will
integrate into host cell sequences at sites of natural chromosome fragility, a process that is accompanied by small adjacent insertion/deletion events.60 Although integration into
the host genome is a rare phenomenon for AAV, the large
vector particle doses that are administered in clinical gene
therapy (≈1012 particles/kg) results in millions of transduced
cells with AAV insertions. There has been a single report
of increased tumor incidence (hepatocellular carcinoma) in
a mouse AAV gene transfer model,61 but these observations
were made in old normal and mucopolysaccharidosis VII
mice after injection of the vectors in neonates. This outcome
has not been replicated in any other long-term small or large
animal model of AAV therapy.
Results of AAV-Mediated Gene
Therapy for Hemophilia
To date, ≈31 hemophilia B patients have been treated in 5
AAV gene therapy trials (Table). A clinical study using AAV
to deliver FVIII has just enrolled its first patient. In the hemophilia B trials, initial studies involved intramuscular injection of the vector, showing that long-term expression of the
FIX transgene was achievable.43,62,63 Latterly, hemophilia gene
therapy trials have targeted the liver for transgene delivery
using either portal vein infusion or, more recently, peripheral
vein injection of hepatotropic AAV8 vectors.
The FIX transgene constructs that have been generated
have used small and potent tissue-specific enhancer/promoter elements, and one study has used a codon optimized
FIX cDNA.45 In the most recent FIX clinical trial, the FIX
transgene also contains a gain-of-function missense variation
(Arg338Leu–FIX Padua64,65) that increases the specific coagulant activity of the transgenic protein by ≈7-fold.
For native single-stranded AAV transgenes to mediate
mRNA expression, a second complementary DNA strand has
first to be synthesized. In efforts to accelerate this process,
some investigators have constructed self-complementary AAV
transgenes that mediate earlier expression after delivery.57
Vector doses in the clinical trials have ranged between 2×1011
and 5×1012 vector genomes/kg.
After transgene delivery, FIX plasma levels start to increase after 2 to 3 weeks and have, until the most recent
study using the FIX Padua transgene, resulted in FIX levels
of 2% to 10% in the short term and persistent levels of 1%
to 6%.43,45,46 Preliminary results from the FIX Padua study,
in patients receiving one of the higher vector doses, have
shown transient levels of FIX >50% that have subsequently
fallen to levels similar to the other studies that have been
reported.66 In the context of hemophilia, these relatively
low levels of FIX have had significant benefits in terms of
Swystun and Lillicrap Gene Therapy for Coagulation Disorders 1449
A
B
Complications of Gene Therapy
o
Innate Immunity
Fever, thrombocytopenia, cytokine storm
o
Adaptive Immunity
CD8+
T-cell
Capsid
peptide
presentation
1. Adverse host immune responses
Granzyme B
and Perforin
AAV Vector
Particle
MHC I
Humoral response
eg. Anti-AAV capsid Abs
Cell-mediated response
eg. Anti-AAV capsid cytotoxic T cells
AAV
Receptor
Cytolysis
Capsid
uncoating/
proteolysis
2. Insertional Mutagenesis
FIX
3. Failure of Transgene Expression
o
Too little
o
Not persistent
Nucleus
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Figure 4. Complications of gene therapy. A, Complications of gene therapy include innate and adaptive immune responses, insertional
mutagenesis, and failure of transgene expression. B, Approximately 25% of patients treated with adeno-associated virus (AAV)–mediated
gene therapy develop an anti-AAV capsid cell–mediated immune response. Presentation of AAV capsid protein to cognate CD8+ T-cells
results in cytolysis of transgene-expressing cells. MHC indicates major histocompatibility complex.
reducing bleeding frequency, with patients being able to
stop their routine FIX protein prophylactic infusions. In the
study with the longest follow up of 10 hemophilia B patients,
therapeutically effective levels of FIX between 1% and 5%
have been maintained for a median of 3.2 years after single
peripheral vein vector infusions.46 Follow-up of patients in
all of these trials is ongoing.
Challenges to Gene Therapy for Hemophilia
The first obvious fact is that progress with FIX gene therapy
has been significantly advanced compared with FVIII. This is
because of a combination of limitations presented by FVIII.
First, the FVIII cDNA, even with the B domain–encoding
region deleted, is ≈4.5 kb, and thus, generation of FVIII
transgene constructs that can be efficiently packaged in AAV
particles is difficult. Second, achieving high-level expression
of FVIII has been notoriously problematic, for reasons that
have been only partially explained. Finally, the immunogenic
potential of FVIII is significantly greater than that of FIX, and
preclinical studies of FVIII gene transfer have frequently been
complicated by the development of an anti-FVIII immune response.32,67,68 In contrast, liver-directed AAV gene therapy in
hemophilic dogs with anti-FVIII antibodies has also shown
potential for inducing immunologic tolerance to FVIII.69
Despite these various challenges, a new AAV-mediated FVIII
gene therapy study has just been initiated, and the outcome of
this trial will be eagerly awaited.
In contrast to the challenges presented by FVIII gene
transfer, the studies of FIX delivery have not been complicated by immune responses to the transgenic FIX protein, and in
the latest clinical trials, plasma levels of transgenic FIX have
been adequate to produce a significant long-term clinical benefit. Nevertheless, the predictability and consistency of these
FIX levels are still not well understood.
The most significant problem that has arisen in the recent
AAV clinical trials has been the development, in ≈25% of
patients, of an anti-AAV capsid immune response that has
resulted in transduced hepatocyte cytotoxicity.56,70,71 This
complication is the result of CD8+ cytotoxic T cell responses
to AAV capsid peptides being presented on the surface of
transduced hepatocytes (Figure 4A). The timing of the subsequent transaminitis that develops has varied from as early
as 3 weeks to as late as 10 weeks post vector delivery, probably reflecting either a secondary recall response or a later
primary immune response to the AAV capsid. With the aim
of minimizing this immunologic problem, patients with evidence of preexisting anti-AAV immunity, as demonstrated
by the presence of anti-AAV antibodies, are currently excluded from participation in these clinical trials. This affects
different numbers of patients for the different AAV serotypes
but ranges from ≈30% of the population with antibodies to
AAV8 to ≈60% of the population with preexisting antiAAV2 immunity.72 Unfortunately, even with the generation
of novel AAV capsids, the issue of cross reactivity of the
immune response may still be an obstacle to avoiding the
subsequent cellular immune attack.73
With the development of a cytotoxic T cell response to
AAV capsid peptides, transduced liver cells are killed and the
transgene is lost, thus prevention or rapid intervention of this
immune attack is essential to achieve long-term transgene expression. To date, mitigation and eventual extinction of the
immune response has been possible with transient corticosteroid administration started after early recognition of liver
transaminase increases.45 Although the use of prophylactic immunosuppressive therapy for this complication has been discussed repeatedly, this strategy has not yet been used. Finally,
in addition to the cell-mediated immune response to the AAV
capsid, a potent humoral response is consistently documented
after AAV vector administration that prevents effective repeat
AAV delivery. This obstacle to vector readministration might
be circumvented by strategies such as infusing capsid decoys
to divert the antibody blockade.74
1450 Circulation Research April 29, 2016
Future Considerations for the Application of
Gene Therapy for Coagulation Disorders
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After a 30-year period of in vitro experimentation and preclinical assessment, the field of gene therapy is beginning to
show robust evidence of clinical benefit in a range of genetic
disorders. The recent success of gene transfer for hemophilia
B highlights the potential of this therapeutic modality for
management of coagulation pathologies. As further evidence
of the promise of gene therapy initiatives, the partnership
in hemophilia gene therapy trials by the biopharmaceutical industry has increased dramatically in the past 2 years.
Nevertheless, before gene therapy can be extended to widespread clinical utility, several critical hurdles need to be
overcome. First, is the development of gene therapy vectors
that can be produced in large scale with high and reproducible quality.75 Evidence that the AAV vectors used in a recent
FIX clinical trial contained only ≈10% of transgene containing particles illustrates the need for improved and more efficient vector production protocols. With current clinical trials
being limited to study populations of 5 to 10 patients, there
is a long way to go before the widespread application of gene
transfer can be envisaged.76
Next is the issue of immune obstacles to successful gene
transfer. With levels of preexisting immunity to current AAVbased vectors ranging from 30% to 60%, many otherwise
eligible patients are excluded from this form of treatment.
Whether AAV capsid modifications or the use of novel AAV
serotypes will circumvent this obstacle remains to be seen.
Similarly, the use of other vector types, such as lentiviral constructs, would substantially reduce this problem. Aside from
the problems posed by immune responses to the vector, immune reactions to the novel transgenic protein may also complicate some applications of gene transfer, particularly when
the transgenic protein presents peptide sequences that are
novel to the recipient.
Although immediate adverse effects of gene transfer using AAV and lentiviral vectors have been negligible, the longterm outcome of gene therapy will require formal monitoring,
particularly for genotoxicity outcomes. Studies performed in
small and, more critically, large animal models with greater
longevity have not shown any evidence of an enhanced incidence of chronic pathologies (most importantly, no evidence
of increased cancer development).77 Nevertheless, these observations will need to be strengthened by formal long-term,
multi-year surveillance in human gene therapy recipients.
Finally, the efficacy of gene therapy in contrast to currently
available or next-generation infused factor replacement therapies will need to be evaluated in randomized clinical trials.78
The promise of genetic therapies for improved management of coagulation disorders is now beginning to be realized.
Although gene replacement strategies are the most prominent of these approaches, the application of inhibitory oligonucleotides79 and small inhibitory RNA molecules80 to alter
the hemostatic balance has demonstrated how other nucleic
acid–based strategies have shown considerable potential in
recent clinical trials. With enhanced access to genome editing
technologies, this momentum toward translational benefits is
likely to continue.
Sources of Funding
D. Lillicrap is the recipient of a Canada Research Chair in Molecular
Hemostasis. L.L. Swystun is the recipient of a CIHR fellowship.
Disclosures
None.
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Gene Therapy for Coagulation Disorders
Laura L. Swystun and David Lillicrap
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Circ Res. 2016;118:1443-1452
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