Download Megakaryocyte- and megakaryocyte precursor

From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Review Series
MEGAKARYOCYTES TO PLATELETS IN HEALTH AND DISEASE
Megakaryocyte- and megakaryocyte precursor–related gene therapies
David A. Wilcox
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI; Children’s Research Institute, Children’s Hospital of Wisconsin, Milwaukee, WI;
and Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI
Hematopoietic stem cells (HSCs) can be
safely collected from the body, genetically
modified, and re-infused into a patient
with the goal to express the transgene
product for an individual’s lifetime. Hematologic defects that can be corrected with
an allogeneic bone marrow transplant can
theoretically also be treated with gene
replacement therapy. Because some genetic disorders affect distinct cell lineages, researchers are utilizing HSC gene
transfer techniques using lineage-specific
endogenous gene promoters to confine
transgene expression to individual cell
types (eg, ITGA2B for inherited platelet
defects). HSCs appear to be an ideal target
for platelet gene therapy because they can
differentiate into megakaryocytes which
are capable of forming several thousand
anucleate platelets that circulate within
blood vessels to establish hemostasis by
repairing vascular injury. Platelets play an
essential role in other biological processes
(immune response, angiogenesis) as well
as diseased states (atherosclerosis, cancer, thrombosis). Thus, recent advances in
genetic manipulation of megakaryocytes
could lead to new and improved therapies
for treating a variety of disorders. In summary, genetic manipulation of megakaryocytes has progressed to the point where
clinically relevant strategies are being
developed for human trials for genetic
disorders affecting platelets. Nevertheless, challenges still need to be overcome
to perfect this field; therefore, strategies
to increase the safety and benefit of megakaryocyte gene therapy will be discussed.
(Blood. 2016;127(10):1260-1268)
Overview: normal megakaryocytopoiesis, platelet production, and function
Hematopoietic stem cells (HSCs) are not only self-replicating but they
are also pluripotent, thus capable of differentiating into 2 major lineages
known either as lymphoid or myeloid blood cells.1 The myeloid lineage
further differentiates into 3 distinct cell types known as myelocytes,
erythrocytes, and megakaryocytes. Figure 1 is a schematic diagram that
depicts the HSCs that become the progenitor cell committed to megakaryocyte differentiation, which remains capable of mitotic cell
division2 although the proliferating diploid megakaryocyte
progenitor cell gradually loses its capacity to divide and undergo
endomitosis. Unique among the other cell types, a maturing
megakaryocyte retains its ability to replicate DNA but neither the
nucleus, cytoplasm, nor cell membrane divides resulting in a unique
multiploidy (8N-128N) nucleated cell with a very complex
internal membrane system, granules, and organelles. 3 A mature
megakaryocyte enters an apoptotic stage where the nucleus
degrades and the cytoplasm and cell membrane fragments into
#5000 proplatelets per cell. 4 This ultimately leads to formation
of circulating blood platelets that are small (3-mm diameter),
anucleate, and discoid-shaped entities that play a fundamental
role in hemostasis.5
Evolution of megakaryocyte manipulation
A significant advance in furthering our understanding of platelets was
realized with the use of molecular biological techniques for transfer of
recombinant DNA for expression of platelet proteins within oncogenic
promegakaryocyte transformed cell lines and nonmegakaryocytic cell
lines transfected with recombinant DNA.6 It has been 30 years since
Submitted July 1, 2015; accepted September 30, 2015. Prepublished online as
Blood First Edition paper, January 19, 2016; DOI 10.1182/blood-2015-07607937.
1260
researchers attempted to alter tissue-cultured megakaryocytes
derived from transfection of HSCs.7 This work occurred
simultaneously with reports of groundbreaking preclinical studies
demonstrating gene transfer techniques useful for correction of
inherited disorders.8-11 Results of the first clinical gene transfer
trials for an immunologic disorder (adenosine deaminase–severe
combined immunodeficiency) began 20 years ago,12 whereas it
has only been 15 years since reports of successful genetic
manipulation of human megakaryocytes were first published and
clinical trials targeting the megakaryocyte lineage are still in
the planning stages.13-15 This discrepancy of time is attributed to
a scarcity of megakaryocytes in vivo which made it difficult
to propagate and examine these cells in vitro. The ability to
manipulate megakaryocytes was improved greatly when researchers developed methods to propagate and differentiate a
sufficient number of megakaryocytes and platelets in vitro and in
vivo as a result of the discovery of the c-mpl ligand (aka, thrombopoietin,
megakaryocyte growth, and development factor).16,17 Initial preclinical research focusing on megakaryocyte manipulation used
early-generation recombinant viral vectors and nonviral gene
transfer techniques that proved moderately effective at genetically modifying tissue-cultured HSCs and megakaryocytes
derivatives. Today, technological advances that proved successful for recent human gene therapy of other hematologic
disorders18,19 are being adopted to help design clinical protocols
for the first human trials aimed at modifying human megakaryocytes to correct inherited bleeding disorders (eg, Glanzmann
thrombasthenia [GT], Bernard-Soulier syndrome [BSS], and
hemophilia, which will be discussed in greater detail in this
review).
© 2016 by The American Society of Hematology
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
PLATELET-TARGETED THERAPY
1261
Figure 1. Promising strategies for megakaryocyte
gene transfer. Displayed is a schematic diagram that
summarizes 3 strategies currently being examined for
megakaryocyte modification including: transplantation
of cytokine-mobilized CD341 PBSCs transduced with a
lentiviral vector; direct injection of lentiviral vector into
the bone marrow space to transduce HSCs; and
lentiviral vector transduction of iPSCs dedifferentiated
from peripheral blood mononuclear cells followed by
transfusion of genetically altered platelets into the
patient. Although each method focuses on modification
of the HSCs with a lentiviral vector under the transcriptional control of a megakaryocyte-specific gene promoter, there is significant contrast in procuring the HSC
target cell as well as different strategies for accomplishing megakaryocyte manipulation. HSC differentiation
along the megakaryocyte lineage is depicted by stage
from HSCs within the bone marrow to formation of
proplatelets, preplatelets, and finally mature platelets
within the vascular space. The cartoon person illustrates the routes of collection and transfusion of
genetically modified cells and a potential point of
injection for LV within the bone marrow space. PBSCs
are all HSCs. BM, bone marrow; LV, lentivector.
Gene therapy targeting the
megakaryocyte lineage
Genetic therapies aimed at modifying megakaryocytes can potentially be used to improve a wide variety of disorders because
platelets play a role in several physiological events (eg, hemostasis,
immune response, and wound healing) as well as pathological
conditions (eg, atherosclerosis, cancer, sepsis, and thrombosis).20
To participate in these biological processes, platelets use a plethora
of biologically active molecules on the surface, in the cytoplasm,
and within granular compartments that mediate platelet function.6
Platelets normally circulate quiescently (nonreactive with other blood
cells or the vasculature) until they become activated upon stimulation
by physiological agonists of platelet activation (eg, adenosine
59-diphosphate, epinephrine, thrombin), adhere to subendothelial
matrix molecules (collagen, von Willebrand factor) at the site of a
vascular injury, change shape, and release the contents of their granules.
Because platelets are anucleate, a strategy aimed at replacing or editing
defective genes involved in normal platelet function or inducing ectopic
expression of genes to manipulate platelet function would have to be
focused on altering the genetics of HSCs, progenitor cells, or megakaryocytes depending upon the optimal length of time the gene product
should be expressed. For example, a strategy aimed at using platelets to
deliver an antioncogenic agent to treat cancerous tumors may try to
provide a chemotherapy treatment of a limited time. In contrast, a
protocol with the goal of long-term correction of an inherited platelet
defect may likely strive for permanent genetic manipulation of the HSC
because human megakaryocytes have a lifespan of 5 days when
undergoing polyploidization, maturation, and platelet formation.21
There are several megakaryocyte-specific gene promoters that
could potentially direct transgene transcription including: members of
the glycoprotein (GP) GPIBA-GPIX-GPV complex,22 ITGA2B (aka,
integrin aIIb, GPIIb),23 GPVI,24 c-mpl,25 and platelet factor 4 (PF4).26
These promoters bind GATA-1, Ets (Fli-1), and FOG-1 factors that
induce transcription in early and mid stages of megakaryocytopoiesis.27,28
For example, PF4 is expressed at high levels during megakaryocytopoiesis and stored within platelet a-granules.29 Its gene promoter26 and distal
regulatory regions30 are well characterized and have been shown to be
useful for controlling megakaryocyte-specific transgene expression. To
date, the most common megakaryocyte-specific gene promoters used
within gene transfer vectors used for megakaryocyte modification include:
GP1BA, ITGA2B, and PF4 because they have been shown to consistently
drive moderate- to high-level protein expression preferentially
within megakaryocytes (see Table 1).31-33 These gene promoters
could potentially be used to drive megakaryocyte-specific transgene
expression within the confines of a variety of gene transfer vectors
that have been characterized for their ability to efficiently and safely
express the transgene product including: recombinant lentiviral, adenoviral, and adeno-associated viral vectors as well as plasmid DNA.34
Each gene transfer technique has unique advantages and disadvantages depending partially upon vector used and the disorder that is being
treated (summarized in Table 1). This report will focus mainly on use of
a recombinant lentiviral gene transfer vector because it has been shown
to deliver remarkable results in human clinical trials that target HSCs.
This vector can accommodate relatively large complementary DNA
(cDNA) cassettes (10 kb), integrate stably into the genome within
nondividing HSCs, and appears relatively safe and efficient for
sustained gene transfer for a variety of inherited genetic disorders.18,19
Correction of inherited platelet defects
Defects of distinct platelet proteins are very rare (1:1 000 000
individuals); however, taken collectively, a molecular genetic defect in
a gene that plays a role in normal platelet function occurs in 1:10 000
individuals usually manifesting itself in the form of uncontrolled
bleeding.35 A recent review by Nurden and Nurden elegantly illustrates
several known genetic defects affecting platelet proteins that may
be ideal candidates for gene therapy including surface molecules
(TMEM16F, Scott syndrome), cytoplasmic (STIM1/ORAI, Stormorken
syndrome) and structural proteins (WASP, WIPF1, Wiskott-Aldrich
syndrome) as well as granule constituents (NBEAL2,GFI1B, gray
platelet syndrome).36 Disorders resulting from platelet defects are
commonly referred to as “benign” because uncontrolled bleeding
frequently responds favorably to treatment with platelet transfusions,
use of antifibrinolytic agents, and recombinant factor VIIa (FVIIa).37
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1262
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
WILCOX
Table 1. Advantages and disadvantages of new strategies to correct hemophilia A
Gene vector
AAV
Target
Gene promoter
FVIII
Reference
Endothelium liver
Liver specific
BDD FVIII
61
modified
Advantages
Disadvantages
1No pre-tx conditioning
2Vector size limitation
1Shown safe in humans
2Use only in patients without AAV inhibitor and
1No reports of mutagenesis
2Cell death ends treatment
1Use with AAV inhibitors
2Submyeloablative conditioning required
without FVIII inhibitor
LV
HSC CD341
PBSCs
LV
HSC CD341
Nonspecific
CMV
Meg-GPIBA
PBSCs
BDD FVIII
63
2Mutagenesis risk
modified
BDD FVIII
67
modified
1Theoretically 1 treatment
2Use only without FVIII inhibitor
1Use with AAV and FVIII
2Submyeloablative conditioning required
inhibitors
1Theoretically 1 treatment
2Mutagenesis risk
2GPIBA gene promoter associated with low plt
production
LV
HSC BM
Meg-GPIBA
BDD FVIII
72
1No pre-tx conditioning
2Target cell not purified for transduction
2Mutagenesis risk
1Use with AAV and FVIII
inhibitors
LV
HSC CD341
Meg-ITGA2B
BDD FVIII
31
PBSCs
2GPIBA gene promoter associated with low plt
production
1Theoretically 1 treatment
2Feasibility in humans?
1Use with AAV and FVIII
2Submyeloablative conditioning required
inhibitors
1Normal plt production
2Mutagenesis risk
BM, bone marrow; CMV, promoter of the cytomegalovirus; LV, lentiviral vector; Meg, megakaryocyte-specific; plt, platelet; tx, transplant.
However, patients can become refractory to platelet transfusions and
infusion of these medications can be costly and short-lived.38 Allogenic
bone marrow transplant has been used successfully to correct dogs and
humans with GT,39,40 although transplant-related complications (eg,
graft-versus-host disease, graft failure) has limited the use of this
treatment.41,42
Gene transfer into cytokine-mobilized CD341 peripheral blood
stem cells (G-PBSCs) from 2 GT patients served as the first model to
test the feasibility of correcting a platelet defect with HSC gene
transfer using a retroviral vector under the transcriptional control of a
megakaryocyte-specific ITGA2B gene promoter driving expression of a
normal human ITGB3 replacement cDNA gene cassette.43 Results
from that study showed that retroviral vector transduction of CD341
G-PBSCs, leading to expression of 34% of normal levels of a functional
integrin aIIbb3 receptor on the surface of 19% progeny megakaryocytes, was sufficient to permit cells to mediate retraction of a fibrin clot
in vitro.43 The ability of HSC gene replacement therapy to correct
a platelet defect in vivo was observed when hemostasis was improved
in mice affected with GT following expression of 10% of normal
integrin receptor levels of a functional hybrid murine aIIb-human b3
complex on the surface of 50% of megakaryocytes derived from a
transplant of Itgb3(2/2) bone marrow transduced with the ITGA2B
gene promoter-controlled retroviral vector encoding human b3 into
mice preconditioned with irradiation causing complete myeloablation.44 Because GT is considered a benign disorder, a mild (clinically
relevant) strategy using submyeloablative pretransplant conditioning
was developed. It was observed that hemostasis could be established for
at least 5 years in a canine (large animal) model of GT following
autologous transplant of CD341 G-PBSCs transduced with an ITGA2B
gene promoter-controlled lentiviral vector driving expression of human
aIIb coupled with a drug-selection gene methyl-guanine methyltransferase (MGMT) under the transcriptional control of a tissuenonspecific gene promoter.45 Thus, HSCs expressing MGMT were
enriched in vivo by treatment of animals with Carmustine (O6-benzyl
guanine), which ultimately resulted in de novo expression of 6% of
normal levels of a functional human aIIb-canine b3 hybrid receptor on
the surface of 10% circulating blood platelets.45 These modest levels of
gene transfer allowed platelets to adhere to the receptor’s major ligand
(fibrinogen), form measurable aggregates, and mediate retraction of
a fibrin clot in vitro. Remarkably, improved hemostatic function
was evident with observations of 135-fold reduced blood loss and
improved buccal bleeding time decreased to 4 minutes compared with
.20 minutes (experimental end point for untreated GT dogs) for at least
5 years after HSC transplant.
A second gene replacement strategy for GT has been recently
reported that used gene transduction of induced pluripotent stem cells
(iPSCs) with a vector expressing human b3 under the transcriptional
control of the megakaryocyte-specific Gp1BA gene promoter. The
iPSCs were dedifferentiated and immortalized from peripheral blood
derived from 2 GT patients.32 Similar to HSC gene transfer and
transplant, this approach resulted in the de novo expression of 50% of
normal levels of a functional aIIbb3 receptor on the surface of
megakaryocytes in vitro.32 The results from this work indicate that
transfusion of gene-modified autologous platelets derived from
iPSCs could provide additional elements of safety for a gene transfer
protocol because the potential for the patient to develop mutagenesis
and oncogenesis due to pretransplant conditioning reagents and
random insertion of a gene transfer vector into the genome is greatly
reduced by transfusion of anucleate platelets. In addition, transplant
of autologous platelets with 1 new receptor may decrease immunemediated recognition and destruction of transfused platelets as
observed with transfusions from unrelated donors. However, treating
uncontrolled bleeding events with this strategy would likely require a
lifetime of multiple transfusions with a sufficient quantity of platelets
derived from gene-modified iPSCs, whereas in contrast, HSC gene
transfer and transplant should theoretically only require 1 transfusion.
The feasibility of HSC gene therapy aimed at modifying
megakaryocytes was confirmed useful for another rare inherited
platelet defect causing BSS. The investigators first used an ITGA2B
gene promoter-controlled lentiviral vector encoding GP1ba in tissuecultured human cells in vitro.46 This was followed by work that showed
improved platelet structure and function following GP1BA lentiviral
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
vector gene transduction of bone marrow that was transplanted into a
murine model of BSS.47
Arterial occlusive disorders are a leading cause of human morbidity.
Thus, platelets may also be useful for delivery of antithrombotic agents
to sites of occlusion. There is 1 report of the ectopic expression of
urokinase-type plasminogen activator in platelets of mice affected with
the inherited platelet defect know as Quebec platelet disorder.48 This
group showed that expression of fibrinolytic proteins in platelets could
be used to favorably alter the hemostatic balance at sites of thrombosis
via ectopic expression of murine urokinase-type plasminogen activator
within transgenic mice where transgene expression was driven by the
megakaryocyte-specific PF4 gene promoter. Results showed that
targeted expression and storage of urokinase in the platelets of transgenic mice altered platelet biology and a bleeding diathesis similar to
that seen in patients with Quebec platelet disorder. This confirmed the
role of ectopic urokinase expression as the etiology of this inherited
disease. Remarkably, the mice were resistant to the development of
occlusive carotid artery thrombosis in the absence of systemic
fibrinolysis and displayed rapid resolution of pulmonary emboli.
Furthermore, transfusion of urokinase-expressing platelets into wildtype mice prevented formation of occlusive arterial thrombi. In
summary, results of that study indicate feasibility for delivering
fibrinolytic agents to sites of thrombus formation following targeted
storage of urokinase in platelets indicating a potential viable strategy to
prevent thrombosis and hemorrhage.
Ectopic delivery of coagulation factors
from platelets
Hemophilia A is a severely debilitating and deadly hemorrhagic
disorder (incidence 1:5000 males) linked to molecular genetic
defects in the plasma protein, coagulation FVIII.49,50 Plasma-derived
and recombinant factor concentrates are currently available from
numerous sources for administration (frequently several times per
week) to treat serious bleeding episodes. Infusion of $1% of normal
FVIII levels has shown dramatic improvement in hemostasis. In
addition, nonfactor products including desmopressin have also been
used, depending on the clinical situation and severity of FVIII
deficiency. New treatment strategies are being investigated that use
permanent transfer of a replacement gene transfer into HSCs to provide
continuous production of FVIII secreted precisely at the site of vascular
injury from activated platelets (Table 1). This result could dramatically
improve the quality of life for patients and may substantially reduce the
cost of clinical care for spontaneous uncontrolled bleeding events.51
Initial human trials for FVIII gene transfer involving the implantation of transformed fibroblasts and infusion of recombinant viral
vectors targeting the liver failed to produce sustained correction of the
hemophilia A bleeding diathesis.52,53 However, recent success of gene
therapy for hemophilia B treatment with adeno-associated viral vector
(AAV)-mediated factor IX (FIX) expression within the liver for
secretion into the blood indicates that it may be feasible to develop a
similar strategy for treatment of hemophilia A.54 Gene transfer for
hemophilia A appears to be a greater challenge than hemophilia B for a
variety of reasons including (1) locating a gene transfer vector that can
accommodate the large B-domain deleted (BDD) FVIII cDNA (4 kb)55
compared with the small FIX cDNA (1.5 kb), (2) achieving adequate
levels of transgene expression, and (3) preventing/averting a frequent
complication of the development of anti-FVIII immunity as well as
immune recognition of the coat protein of the AAV gene transfer vector
(which is also a potential problem for FIX gene transfer). Table 1 is a
PLATELET-TARGETED THERAPY
1263
summary of some of the advantages and disadvantages for a few of the
recent strategies that have been proposed that promote expression of
human FVIII for correction of hemophilia A. There have been reports
that inducing de novo FVIII expression within the liver endothelial cells
by either IV infusion of naked DNA56 or a new generation of AAV
equipped with less immunogenic coat proteins and a vector encoding
small active forms of FVIII cDNA (that can meet the 4.4-kb packaging
capacity of AAV) shows promise for endothelial synthesis and
secretion of FVIII into the plasma to treat hemophilia A.57-61
Nonetheless, as with AAV clinical trials for hemophilia B, targeting the
liver for FVIII expression will likely exclude hemophilia A patients
with preexisting antibodies to the AAV viral capsid (40%),
individuals who have already developed or could develop inhibitory
antibodies to plasma FVIII (30%), and persons with preexisting liver
disease or damage due to acquisition of hepatitis or HIV from
contaminated blood products from factor replacement therapy.51 Thus,
as a potentially viable alternative approach to advert the challenges
imposed by AAV-mediated liver-targeted therapies for hemophilia A,
we and others hypothesize that autologous transplant of HSCs
transduced with a lentiviral vector (10-kb DNA packaging capacity)
encoding replacement FVIII cDNA may be an ideal strategy for
correction of hemophilia A within humans (Figure 1). One group
proposes to transduce HSCs with a lentiviral vector under the transcriptional control of a tissue-nonspecific gene promoter that expresses
FVIII within all hematopoietic cell lineages.62,63 Because activated
blood platelets mediate the primary response to vascular injury by
adhering to a wound site and secreting biologically active proteins,20 it
is speculated that use of a megakaryocyte-specific gene promoter that
confines synthesis and storage of FVIII within platelets may be a more
tailored approach for providing continuous, locally inducible treatment of maintaining hemostasis precisely at the site of vascular injury
for hemophilia A. Support for this approach has been observed in
preclinical studies that showed HSC gene transfer targeting expression
within human megakaryocytes resulted in the trafficking of a biologically active form of human FVIII into the a-granule compartment
of human platelets derived from HSCs xenotransplanted in mice.64
Further investigations of HSC lentiviral-mediated gene transfer
demonstrated that platelet FVIII is capable of establishing hemostasis
in murine and canine models of hemophilia A (even in the presence of
inhibitory antibodies in mice and without eliciting the formation of
antibodies in a line of hemophilia A dogs known to readily form
inhibitors to human FVIII).31,65-67 These results suggest that platelets
engineered to store FVIII have great potential for establishing
hemostasis in humans with hemophilia A. The data indicate that
platelet FVIII may be essential for patients affected with inhibitory
antibodies to FVIII, which would likely cause their exclusion from
clinical trials for liver-targeted genetic therapy or HSC tissue
nonspecific expression of FVIII.68 It is noteworthy that Du et al have
shown that a lentiviral vector using the ITGA2B gene promoter driving
expression of a molecule encoding a fragment of the VWF propeptide
and D2 domain (which helps traffic the molecule for storage within
a-granules) fused to BDD FVIII successfully confined a majority of
FVIII specifically within the platelet a-granule compartment of
platelets to establish hemostasis for canine hemophilia A.31,69,70 These
results indicate that the addition of an a-granule targeting peptide may
add an additional level of safety for treating patients with preexisting
inhibitory antibodies because sequestration of FVIII within platelet
a-granules may help to concentrate FVIII within granules and prevent
leakage of FVIII into the plasma from the cytoplasmic compartment.
This result is consistent with studies performed in mice affected with
von Willebrand disease that stored FVIII less efficiently within
a-granules due the absence of von Willebrand factor (carrier protein for
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1264
WILCOX
FVIII), which helps traffic FVIII into the granule compartment and
protects FVIII from degradation.71
Wang et al have preliminary data in mice showing that it may be
feasible to directly inject bone marrow with lentivector encoding FVIII
under the transcriptional control of the megakaryocyte-specific GPIBA
gene promoter (Figure 1).72 One advantage of this approach is that the
mice did not require pretransplant conditioning with submyeloablative
reagents (Table 1). Albeit, because the marrow cavity consists of
several cell types, it is likely difficult to control the precise cell type that
the viral vector will transduce. It also remains to be seen whether large
animals and hemophilia patients (who encounter serious bleeding from
the joints) could tolerate direct injection of vector into their bones.
Secretion of antioncogenic agents
from platelets
Platelets have been observed to play a significant role in cancer. They
can adhere directly to solid tumors leading to evasion from recognition
and destruction by immune cells. Platelets have also been observed to
release growth factors and proangiogeneic agents that promote tumor
growth and metastasis.73 Thus, there is growing interest in developing
strategies that permit platelets to synthesize, store, and deliver
antioncogenic agents to cancer cells in an effort to confine chemotherapy to the tumor site. One report has demonstrated positive results
following transfusion of platelets that were preloaded with chemotherapeutics or nanoparticles that inhibit oncogenesis.74 Another group
showed that CD341 HSCs transduced with a lentiviral vector under
the transcriptional control of a tissue nonspecific (CMV) gene promoter driving expression of cDNA encoding an antiangiogenic agent,
tumstatin. The HSCs successfully differentiated into megakaryocytes
that synthesized, stored, and released tumstatin from platelet a-granules
that caused an antiangiogenic effect on lung (A543) tumor cells in
vitro.75 A recent report showed that the use of platelet interleukin-24
derived from HSC gene transfer was associated with the inhibition of
melanoma solid tumor growth in mice.76 Each study demonstrates
the “proof-of-concept” that it is feasible to equip platelets with
antioncogenic agents to inhibit tumor growth. Because cancer is an
acquired disease, gene transfer strategies using platelets engineered to express antioncogenic agents could be improved greatly
with the ability to discontinue the treatment. Specifically, transfusion of an individual with genetically engineered platelets (with
a life span of 10 days) may be preferable to infusion of HSCs
modified to express the antioncogenic agent because it appears
logical that the treatment should only be given as long as the cancer
persists. Thus, recent studies describing transfusion of platelets
derived from iPSCs or HSCs modified with gene transfer vectors
indicate the potential feasibility toward accomplishing this
goal.32,77,78 Current analysis of the field indicates that it is
essential to achieve further improvement for in vitro production of
platelets (especially to increase the number and quality of platelets
for transfusion) to make this strategy clinically feasible. 79 The
authors of that article surmised that it may be more advantageous
to infuse megakaryocytes (rather than platelets) derived from
iPSCs. The megakaryocytes were observed to congregate within
murine lungs where the maturation into platelets occurred.
However, transplantation of genetically altered megakaryocytes
could potentially pose a risk for insertional mutagenesis and
oncogenesis resulting from infusion of a nucleated cell that has
been genetically altered by integration of a lentiviral vector
randomly into the genome. Furthermore, mutagenesis appears less
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
of a risk by transfusion of anucleate platelets rather than infusion of
megakaryocytes derived from iPSC cells that have been created by
dedifferentiation of mature peripheral blood mononuclear cells to
iPSCs with oncogenes. Nevertheless, optimism is high that these
technical issues will be resolved.
Current challenges for modifying
megakaryocytes and precursor cells
There are several issues that researchers had or will have to
contend with to translate platelet gene therapy from bench to
bedside. Some of the challenges are common to protocols that use
lentiviral vector-mediated gene transfer (eg, concern of potential
side effects from pretransplant conditioning regimen, insertional
mutagenesis, development of an acquired immune response to
the transgene product). Other challenges reported appear to be
specifically associated with targeted genetic modification of the
megakaryocyte lineage (eg, resulting in altered platelet function
and decreased platelet production related to ectopic transgene
expression).80
Submyeloablative pretransplant
conditioning regimen
Some of the first successful gene therapy trials for X-linked severe
combined immunodeficiency syndrome (X-SCID) involved the
transfer of a gene (gc) that imparted a survival advantage to HSCs for
genetic disorders affecting a patient’s ability to mount an immune
response.81 This permitted the trial participants to forego pretransplant conditioning with chemotherapeutic agents that destroy
untransduced stem cells to create a niche in the bone marrow for
transplanted cells, thus improving the transduction efficiency and
long-term gene marking in vivo. It is noteworthy that most of the
genes examined in preclinical trials for modification of megakaryocytes for inherited bleeding disorders do not appear to impart a
survival advantage to HSCs.6,34 Thus, preclinical trials in large
animal models for GT and hemophilia A have used submyeloablative pretransplant conditioning with reduced-intensity total body
irradiation or chemotherapeutic agents (eg, busulfan) with limited
toxicity to HSCs as has been used for allogeneic “mini” bone marrow
transplant protocols to help improve the percentage of HSCs carrying
the transgene that engraft, while simultaneously limiting the risk of
death from complete marrow ablation.31,45 The large animals (dogs)
that received an autologous transplant of lentiviral vector-transduced
CD341 G-PBSCs appeared healthy at least until the experimental
end point of 3 to 5 years after transplant, suggesting that the use of
the submyeloablative conditioning protocol is likely safe for use in
humans. Concerns have been expressed against the use of chemotherapeutic pretransplant conditioning regimens for HSC gene
therapy for inherited bleeding disorders considered to be “benign.”68
Thus, they are likely the first clinical trials that demonstrate success
will be performed on adult hemophilia A patients who have no
treatment options for severe bleeding episodes due to development
inhibitory antibodies to FVIII. A positive outcome in these patients
showing low incidence of side effects as a result of the protocol will
likely help to diminish concerns for use of a submyeloablative
conditioning regimen.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
Insertional mutagenesis
The risk vs benefit ratio for genetic modification for megakaryocytes for
“benign” inherited bleeding disorders requires evidence of greater
benefit and/or reduced risk of harm from the HSC gene transfer and
transplant protocol compared with treatment offered by previous gene
transfer trials for patients with inherited disorders with no treatment
options.82 Retrovirus-based vectors have been improved and used for
platelet gene therapy protocols because these constructs mediated the
first successful human gene therapy trials in individuals affected with a
hematologic disorder, X-SCID.83 Because all retroviral vectors incorporate randomly into the host genome, use of these constructs places the
patient at some risk for insertional mutagenesis. For example, 3 of 10
patients treated with gene therapy for X-SCID developed leukemia as a
result of mutagenesis of the patient’s genome.84 For 2 of the patients, an
oncoretroviral vector (based upon the Moloney murine leukemia virus)
inserted into and activated a T-cell proto-oncogene (LMO-2).85
X-SCID may be particularly susceptible to leukemogenesis because
correction of the g (c) gene defect confers a survival advantage to the
transduced cells; this, plus alterations in genes such as LMO-2 that
control cell division, may result in uncontrolled growth of cells that
have a survival advantage.85,86 Another limitation of oncoretrovirus
vectors is that they only integrate into cycling cells, and a majority of
normal HSCs cycle very slowly (30 days per cycle on average).
Because the efficiency of HSCs transduced with this system is low, it
is not surprising that the first successful gene therapy clinical trial was
for a disorder in which the transgene product provided a growth
advantage for transduced HSCs.87 Recombinant lentiviral vectors
(eg, HIV-1) are unique retroviruses that have become more useful for
gene therapy of hematologic disorders because they can transduce
nondividing cells.18,19 Second, compared with early retroviral
vectors, lentiviral vectors appear to have improved safety with more
of a propensity to insert into nontranscribed regions of the genome.88
Third, the use of endogenous gene promoters rather than viral gene
promoters and enhancers appears to have helped increase safety as
supported by analysis of the latest HSC human gene therapy trials,
which have yet to report any adverse side effects due to insertional
mutagenesis leading to oncogenesis.18,19
Immune response to transgene product
The introduction of a normal replacement gene or protein always raises
the concern for the development of an acquired immune response to the
transgene product. One mouse (n 5 21) and 1 dog (n 5 3) transplanted
with HSCs transduced with lentiviral vector encoding b3 and aIIb,
respectively, developed an immune response to the integrin receptor
that led to severe destruction of genetically altered platelets.44,45 This
outcome demonstrates that expressing foreign proteins within platelets
does not guarantee tolerance or privilege against immune recognition.
Fortunately, a standard treatment of diminishing immune-mediated
destruction of human platelet transfusions (transient infusion of IV
g globulin “IVIgG” and corticosteroids) effectively diminished clearance
platelets in animals that developed an immune response to de novo
synthesis of aIIbb3 on the surface of platelets derived from lentiviral
vector transduced HSCs.33,34 Although this indicates that IVIgG may
subside an immune response if it occurs during human clinical trials to
correct GT, it is likely that an ideal candidate for the first clinical trial
would be an individual who has not generated an immune response
following multiple transfusions of donor platelets or a GT patient
PLATELET-TARGETED THERAPY
1265
classified as type II or variant (who expresses residual levels of a
dysfunctional form of aIIbb3) because their immune system has been
previously exposed to the aIIbb3 without consequence. It appears
remarkable that 20 of 21 mice and 2 of 3 dogs did not generate an immune
response to de novo aIIbb3 expression, indicating that it may be
sufficient to express the least alloantigenic form of aIIbb3 rather than
custom design integrin subunits to match the genotype of each patient.
Other preclinical data indicate that immune-mediated recognition of
genetically altered platelets is potentially a greater risk for correction of
GT with aIIbb3 (expressed on the surface) compared with correction
of hemophilia A where platelet-derived FVIII (sequestered within the
cytoplasm and a-granules) has not been observed to elicit a detectable
immune response in mice and dogs.31,66 This result is further supported
by work demonstrating that platelet FVIII has been shown to improve
hemostasis in hemophilia A mice that were conditioned to develop high
titer inhibitory antibodies to FVIII.65,89 In summary, previous studies
indicate that physical location of the transgene product could be related to
the relative risk of developing immune response leading to destruction of
genetically altered platelets; however, the precise mechanism for these
phenomena remains to be determined.
Transgene expression causing altered
platelet function
There has been concern raised that platelet-derived FVIII can affect
normal platelet function by causing the formation of unstable blood
clots. One report examined clot response to laser injury in both
cremaster arterioles and venules in mice either infused with FVIII or
transgenic for platelet-derived FVIII.90 In both sets of vessels, platelet
FVIII was equally as effective as infused plasma FVIII. Temporal and
spatial differences were observed in fibrin and platelet accumulation
within clots depending on how FVIII was delivered. These differences were attributed to the temporal and spatial distribution of the
a-granular–released FVIII within a developing clot that resulted in an
increased frequency and size of embolic events seen with platelet FVIII.
That result may have negative implications for the use of platelet FVIII
in gene therapy for hemophilia A. However, although there were no
formal analyses performed to detect emboli, there were no reported
observations of increased size or frequency of embolic events occurring
in mice or dogs affected with hemophilia A following successful
transplant with HSCs transduced with a lentiviral vector expressing FVIII under the transcriptional control of the ITGA2B gene
promoter.31,66
Transgene expression causing decreased
platelet production
There has been at least 1 report of decreased platelet production
following HSC gene transfer targeting ectopic expression of proteins
within the megakaryocyte lineage of mice.80 This observation could
be potentially of great significance because use of a megakaryocytespecific or lineage-nonspecific gene promoter that induces synthesis
of high levels of foreign proteins within megakaryocytes may have a
negative effect on platelet production with effects that are not fully
realized. The researchers of that study concluded that it may be more
beneficial to express low levels of a protein of high specific activity.
Consistent with that hypothesis, each study using a lentiviral vector
driven by the ITGA2B gene promoter for platelet-targeted (ITGA2B,
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1266
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
WILCOX
ITGB3, FVIII, FIX) transgene expression within murine and canine
models for GT and hemophilia demonstrated very modest to moderate
levels of protein synthesis, resulting in the ability to establish hemostasis within these animals for a prolonged period whereas platelet
production levels were not affected.31,43,45,66 This modest level of FVIII
synthesis could be attributed to the use of a human gene promoter
within animal models, although use of the same ITGA2B gene
promoter-controlled lentivectors within primary human HSC cells in
tissue culture also resulted in modest levels of transgene expression.13,43,64
Based upon previous results, it is likely that use of the ITGA2B gene
promoter will continue to drive moderate transgene transcription
within human megakaryocytes in vivo that should be sufficient to
improve hemostasis, whereas megakaryocytopoiesis and platelet
production should not be affected with use of this vector in humans.
Summary
Significant advances in gene therapy (resulting from improved methods
for HSC collection, transduction, and transplantation), as well as the
development of a better understanding of the process of megakaryocytopoiesis, platelet production, and lineage-specific gene transfer,
have all impacted current research investigations that aim to manipulate
megakaryocytes and HSC precursors. Currently, there are at least 3
plausible strategies that target HSC gene transfer aimed at specifically
modifying the megakaryocyte lineage, including lentiviral transduction
of HSCs in the form of CD341 PBSCs, bone marrow, and iPSCs
(Figure 1).31,32,45,72 This work has furthered our efforts to achieve the
ultimate goal of correcting inherited genetic defects of platelets and
permitting platelets to deliver therapeutic agents directly to the site of
injury. There remains an endless challenge to increase the safety and
benefit from HSC gene transfer that will likely concentrate on
developing strategies that obtain high transduction and gene marking
efficiencies with low frequency for insertional mutagenesis. There is
also a goal to decrease the potential for deleterious side effects through
the development of pretransplant conditioning regimens that use mild
but effective reagents that obtain optimal engraftment of genetically
modified HSCs. There are also aspirations to produce a greater number
of high-quality platelets from iPSCs that should increase the feasibility
of this strategy to be used for clinical trials. Genetic therapy aimed at the
manipulation of the megakaryocyte lineage will continue to strive for
adequate protein synthesis and storage within progeny platelets without
affecting the delicate balance of normal platelet production and
function. Notwithstanding the current issues of the field, it is undoubtedly an exciting time to be involved in research aimed at megakaryocyte
and megakaryocyte precursor gene therapy because the first human
clinical trials targeting platelets appear to be on the near horizon.
Acknowledgments
Some of the research studies described in this review were
supported by the following grants: National Institutes of Health–
National Heart, Lung, and Blood Institute (NIH-NHLBI) R01 HL68138 (D.A.W.), NHLBI Gene Therapy Resource Program (Indiana
University Lentiviral Vector Production Laboratory) RSA 1253
(D.A.W.), and Production Assistance for Cellular Therapies (Boston
Children’s Hospital Laboratory) NHLBI 00085/Wilcox; American
Heart Association (Northland Affiliate) Beginning Grant-in-Aid
0160441Z and Grant-in-Aid 0755827Z (D.A.W.); and generous gifts
from the Children’s Hospital Foundation (D.A.W.), Midwest
Athletes Against Childhood Cancer Fund (D.A.W.), John B. &
Judith A. Gardetto (D.A.W.), Glanzmann Research Foundation
(D.A.W.), and Jamie Swain/Voya (D.A.W.).
Authorship
Contribution: D.A.W. wrote the manuscript.
Conflict-of-interest disclosure: D.A.W. has a patent application
pending as a co-inventor entitled, “Platelet Targeted Treatment” (US
provisional patent application no. 61/717 951; international patent
application no. PCT/US2013/066651).
Correspondence: David A. Wilcox, Department of Pediatrics,
Medical College of Wisconsin, MFRC #6014, 8701 Watertown
Plank Rd, Milwaukee, WI 53226; e-mail: [email protected].
References
1. Breton-Gorius J, Reyes F. Ultrastructure of
human bone marrow cell maturation. Int Rev
Cytol. 1976;46:251-321.
2. Ebbe S. Biology of megakaryocytes. Prog Hemost
Thromb. 1976;3:211-229.
3. Italiano JE Jr, Lecine P, Shivdasani RA, Hartwig
JH. Blood platelets are assembled principally at
the ends of proplatelet processes produced by
differentiated megakaryocytes. J Cell Biol. 1999;
147(6):1299-1312.
4. Patel SR, Hartwig JH, Italiano JE Jr. The
biogenesis of platelets from megakaryocyte
proplatelets. J Clin Invest. 2005;115(12):
3348-3354.
5. Italiano JE Jr, Shivdasani RA. Megakaryocytes
and beyond: the birth of platelets. J Thromb
Haemost. 2003;1(6):1174-1182.
6. Nurden AT. Platelet membrane glycoproteins: a
historical review. Semin Thromb Hemost. 2014;
40(5):577-584.
7. Block KL, Ravid K, Phung QH, Poncz M.
Characterization of regulatory elements in the
59-flanking region of the rat GPIIb gene by studies
in a primary rat marrow culture system. Blood.
1994;84(10):3385-3393.
8. Chowdhury JR, Grossman M, Gupta S,
Chowdhury NR, Baker JR Jr, Wilson JM. Longterm improvement of hypercholesterolemia after
ex vivo gene therapy in LDLR-deficient rabbits.
Science. 1991;254(5039):1802-1805.
9. Yao SN, Wilson JM, Nabel EG, Kurachi S,
Hachiya HL, Kurachi K. Expression of human
factor IX in rat capillary endothelial cells: toward
somatic gene therapy for hemophilia B. Proc Natl
Acad Sci USA. 1991;88(18):8101-8105.
10. Anderson WF, Goldberg S, Kantoff P, Berg P,
Eglitis M, Humphries RK. Attempts at gene
therapy in beta-thalassemic mice. Ann N Y
Acad Sci. 1985;445:445-451.
11. Steinmetz M. Immune response restored by gene
therapy in mice. Nature. 1985;316(6023):14-15.
expression of gene products in megakaryocytes
derived from retrovirus-transduced human
hematopoietic cells. Proc Natl Acad Sci USA.
1999;96(17):9654-9659.
14. Shiraga M, Ritchie A, Aidoudi S, et al. Primary
megakaryocytes reveal a role for transcription
factor NF-E2 in integrin a IIb b 3 signaling. J Cell
Biol. 1999;147(7):1419-1430.
15. Faraday N, Rade JJ, Johns DC, et al. Ex vivo
cultured megakaryocytes express functional
glycoprotein IIb-IIIa receptors and are capable of
adenovirus-mediated transgene expression.
Blood. 1999;94(12):4084-4092.
16. Choi ES, Nichol JL, Hokom MM, Hornkohl AC,
Hunt P. Platelets generated in vitro from
proplatelet-displaying human megakaryocytes are
functional. Blood. 1995;85(2):402-413.
12. Blaese RM, Culver KW, Miller AD, et al.
T lymphocyte-directed gene therapy for ADASCID: initial trial results after 4 years. Science.
1995;270(5235):475-480.
17. de Sauvage FJ, Hass PE, Spencer SD, et al.
Stimulation of megakaryocytopoiesis and
thrombopoiesis by the c-Mpl ligand. Nature. 1994;
369(6481):533-538.
13. Wilcox DA, Olsen JC, Ishizawa L, Griffith M,
White GC II. Integrin alphaIIb promoter-targeted
18. Aiuti A, Biasco L, Scaramuzza S, et al. Lentiviral
hematopoietic stem cell gene therapy in patients
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
with Wiskott-Aldrich syndrome. Science. 2013;
341(6148):1233151.
19. Biffi A, Montini E, Lorioli L, et al. Lentiviral
hematopoietic stem cell gene therapy benefits
metachromatic leukodystrophy. Science. 2013;
341(6148):1233158.
20. Leslie M. Cell biology. Beyond clotting: the powers
of platelets. Science. 2010;328(5978):562-564.
21. Machlus KR, Italiano JE Jr. The incredible
journey: from megakaryocyte development to
platelet formation. J Cell Biol. 2013;201(6):
785-796.
22. Roth GJ, Yagi M, Bastian LS. The platelet
glycoprotein Ib-V-IX system: regulation of gene
expression. Stem Cells. 1996;14(suppl 1):
188-193.
23. Uzan G, Prenant M, Prandini MH, Martin F,
Marguerie G. Tissue-specific expression of the
platelet GPIIb gene. J Biol Chem. 1991;266(14):
8932-8939.
24. Holmes ML, Bartle N, Eisbacher M, Chong BH.
Cloning and analysis of the thrombopoietininduced megakaryocyte-specific glycoprotein VI
promoter and its regulation by GATA-1, Fli-1, and
Sp1. J Biol Chem. 2002;277(50):48333-48341.
25. Kaushansky K, Drachman JG. The molecular and
cellular biology of thrombopoietin: the primary
regulator of platelet production. Oncogene. 2002;
21(21):3359-3367.
26. Ravid K, Beeler DL, Rabin MS, Ruley HE,
Rosenberg RD. Selective targeting of gene
products with the megakaryocyte platelet
factor 4 promoter. Proc Natl Acad Sci USA. 1991;
88(4):1521-1525.
27. Romeo PH, Prandini MH, Joulin V, et al.
Megakaryocytic and erythrocytic lineages share
specific transcription factors. Nature. 1990;
344(6265):447-449.
28. Wang X, Crispino JD, Letting DL, Nakazawa M,
Poncz M, Blobel GA. Control of megakaryocytespecific gene expression by GATA-1 and FOG-1:
role of Ets transcription factors. EMBO J. 2002;
21(19):5225-5234.
29. Doi T, Greenberg SM, Rosenberg RD. Structure
of the rat platelet factor 4 gene: a marker for
megakaryocyte differentiation. Mol Cell Biol.
1987;7(2):898-904.
30. Zhang C, Thornton MA, Kowalska MA, et al.
Localization of distal regulatory domains in the
megakaryocyte-specific platelet basic protein/
platelet factor 4 gene locus. Blood. 2001;98(3):
610-617.
31. Du LM, Nurden P, Nurden AT, et al. Platelettargeted gene therapy with human factor VIII
establishes haemostasis in dogs with haemophilia
A. Nat Commun. 2013;4:2773.
32. Sullivan SK, Mills JA, Koukouritaki SB, et al. Highlevel transgene expression in induced pluripotent
stem cell-derived megakaryocytes: correction of
Glanzmann thrombasthenia. Blood. 2014;123(5):
753-757.
33. Nguyen HG, Yu G, Makitalo M, et al. Conditional
overexpression of transgenes in megakaryocytes
and platelets in vivo. Blood. 2005;106(5):
1559-1564.
34. Wilcox DA, White GC II. Gene therapy for
platelet disorders: studies with Glanzmann’s
thrombasthenia. J Thromb Haemost. 2003;1(11):
2300-2311.
35. Nurden AT, Nurden P. Congenital platelet
disorders and understanding of platelet function.
Br J Haematol. 2014;165(2):165-178.
PLATELET-TARGETED THERAPY
and perioperative management in patients with
Glanzmann’s thrombasthenia. Expert Rev
Hematol. 2014;7(6):733-740.
1267
mediated gene transfer in hemophilia B. N Engl J
Med. 2011;365(25):2357-2365.
38. White GC II. Congenital and acquired platelet
disorders: current dilemmas and treatment
strategies. Semin Hematol. 2006;43(1 suppl 1):
S37-S41.
55. Pittman DD, Alderman EM, Tomkinson KN,
Wang JH, Giles AR, Kaufman RJ. Biochemical,
immunological, and in vivo functional
characterization of B-domain-deleted factor VIII.
Blood. 1993;81(11):2925-2935.
39. Flood VH, Johnson FL, Boshkov LK, et al.
Sustained engraftment post bone marrow
transplant despite anti-platelet antibodies in
Glanzmann thrombasthenia. Pediatr Blood
Cancer. 2005;45(7):971-975.
56. Ye P, Thompson AR, Sarkar R, et al. Naked DNA
transfer of Factor VIII induced transgene-specific,
species-independent immune response in
hemophilia A mice. Mol Ther. 2004;10(1):
117-126.
40. Niemeyer GP, Boudreaux MK, Goodman-Martin
SA, Monroe CM, Wilcox DA, Lothrop CD Jr.
Correction of a large animal model of
type I Glanzmann’s thrombasthenia by
nonmyeloablative bone marrow transplantation.
Exp Hematol. 2003;31(12):1357-1362.
57. Sabatino DE, Lange AM, Altynova ES, et al.
Efficacy and safety of long-term prophylaxis in
severe hemophilia A dogs following liver gene
therapy using AAV vectors. Mol Ther. 2011;19(3):
442-449.
41. Wiegering V, Sauer K, Winkler B, Eyrich M,
Schlegel PG. Indication for allogeneic stem cell
transplantation in Glanzmann’s thrombasthenia.
Hamostaseologie. 2013;33(4):305-312.
42. Wiegering V, Winkler B, Langhammer F, et al.
Allogeneic hematopoietic stem cell
transplantation in Glanzmann thrombasthenia
complicated by platelet alloimmunization. Klin
Padiatr. 2011;223(3):173-175.
43. Wilcox DA, Olsen JC, Ishizawa L, et al.
Megakaryocyte-targeted synthesis of the integrin
b(3)-subunit results in the phenotypic correction of
Glanzmann thrombasthenia. Blood. 2000;95(12):
3645-3651.
44. Fang J, Hodivala-Dilke K, Johnson BD, et al.
Therapeutic expression of the platelet-specific
integrin, alphaIIbbeta3, in a murine model for
Glanzmann thrombasthenia. Blood. 2005;106(8):
2671-2679.
45. Fang J, Jensen ES, Boudreaux MK, et al. Platelet
gene therapy improves hemostatic function for
integrin alphaIIbbeta3-deficient dogs. Proc Natl
Acad Sci USA. 2011;108(23):9583-9588.
46. Shi Q, Wilcox DA, Morateck PA, Fahs SA, Kenny
D, Montgomery RR. Targeting platelet GPIbalpha
transgene expression to human megakaryocytes
and forming a complete complex with
endogenous GPIbbeta and GPIX. J Thromb
Haemost. 2004;2(11):1989-1997.
47. Kanaji S, Kuether EL, Fahs SA, et al. Correction
of murine Bernard-Soulier syndrome by lentivirusmediated gene therapy. Mol Ther. 2012;20(3):
625-632.
48. Kufrin D, Eslin DE, Bdeir K, et al. Antithrombotic
thrombocytes: ectopic expression of urokinasetype plasminogen activator in platelets. Blood.
2003;102(3):926-933.
49. Stonebraker JS, Bolton-Maggs PH, Soucie JM,
Walker I, Brooker M. A study of variations in the
reported haemophilia A prevalence around the
world. Haemophilia. 2010;16(1):20-32.
50. Berntorp E, Shapiro AD. Modern haemophilia
care. Lancet. 2012;379(9824):1447-1456.
51. High KH, Nathwani A, Spencer T, Lillicrap D.
Current status of haemophilia gene therapy.
Haemophilia. 2014;20(suppl 4):43-49.
52. Roth DA, Tawa NE Jr, O’Brien JM, Treco DA,
Selden RF; Factor VIII Transkaryotic Therapy
Study Group. Nonviral transfer of the gene
encoding coagulation factor VIII in patients with
severe hemophilia A. N Engl J Med. 2001;
344(23):1735-1742.
36. Nurden AT, Nurden P. Inherited disorders of
platelet function: selected updates. J Thromb
Haemost. 2015;13(suppl 1):S2-S9.
53. Powell JS, Ragni MV, White GC II, et al. Phase 1
trial of FVIII gene transfer for severe hemophilia
A using a retroviral construct administered by
peripheral intravenous infusion. Blood. 2003;
102(6):2038-2045.
37. Franchini M, Lippi G. NovoSeven (recombinant
factor VIIa) for the treatment of bleeding episodes
54. Nathwani AC, Tuddenham EG, Rangarajan S,
et al. Adenovirus-associated virus vector-
58. Kitazawa T, Igawa T, Sampei Z, et al. A bispecific
antibody to factors IXa and X restores factor VIII
hemostatic activity in a hemophilia A model. Nat
Med. 2012;18(10):1570-1574.
59. McIntosh J, Lenting PJ, Rosales C, et al.
Therapeutic levels of FVIII following a single
peripheral vein administration of rAAV vector
encoding a novel human factor VIII variant. Blood.
2013;121(17):3335-3344.
60. Chuah MK, Nair N, VandenDriessche T. Recent
progress in gene therapy for hemophilia. Hum
Gene Ther. 2012;23(6):557-565.
61. Siner JI, Iacobelli NP, Sabatino DE, et al. Minimal
modification in the factor VIII B-domain sequence
ameliorates the murine hemophilia A phenotype.
Blood. 2013;121(21):4396-4403.
62. Doering CB, Spencer HT. Replacing bad (F)
actors: hemophilia. Hematology Am Soc Hematol
Educ Program. 2014;2014(1):461-467.
63. Johnston JM, Denning G, Doering CB, Spencer
HT. Generation of an optimized lentiviral vector
encoding a high-expression factor VIII transgene
for gene therapy of hemophilia A. Gene Ther.
2013;20(6):607-615.
64. Wilcox DA, Shi Q, Nurden P, et al. Induction
of megakaryocytes to synthesize and store a
releasable pool of human factor VIII. J Thromb
Haemost. 2003;1(12):2477-2489.
65. Shi Q, Wilcox DA, Fahs SA, et al. Factor VIII
ectopically targeted to platelets is therapeutic in
hemophilia A with high-titer inhibitory antibodies.
J Clin Invest. 2006;116(7):1974-1982.
66. Shi Q, Wilcox DA, Fahs SA, et al. Lentivirusmediated platelet-derived factor VIII gene therapy
in murine haemophilia A. J Thromb Haemost.
2007;5(2):352-361.
67. Yarovoi HV, Kufrin D, Eslin DE, et al. Factor VIII
ectopically expressed in platelets: efficacy in
hemophilia A treatment. Blood. 2003;102(12):
4006-4013.
68. Gould J. Gene therapy: genie in a vector. Nature.
2014;515(7528):S160-S161.
69. Haberichter SL, Jozwiak MA, Rosenberg JB,
Christopherson PA, Montgomery RR. The von
Willebrand factor propeptide (VWFpp) traffics an
unrelated protein to storage. Arterioscler Thromb
Vasc Biol. 2002;22(6):921-926.
70. Haberichter SL, Jacobi P, Montgomery RR.
Critical independent regions in the VWF
propeptide and mature VWF that enable normal
VWF storage. Blood. 2003;101(4):1384-1391.
71. Yarovoi H, Nurden AT, Montgomery RR, Nurden
P, Poncz M. Intracellular interaction of von
Willebrand factor and factor VIII depends on
cellular context: lessons from platelet-expressed
factor VIII. Blood. 2005;105(12):4674-4676.
72. Wang X, Shin SC, Chiang AF, et al. Intraosseous
delivery of lentiviral vectors targeting factor VIII
expression in platelets corrects murine hemophilia
A. Mol Ther. 2015;23(4):617-626.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1268
WILCOX
BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10
73. Sharma D, Brummel-Ziedins KE, Bouchard BA,
Holmes CE. Platelets in tumor progression: a host
factor that offers multiple potential targets in the
treatment of cancer. J Cell Physiol. 2014;229(8):
1005-1015.
79. Wang Y, Hayes V, Jarocha D, et al. Comparative
analysis of human ex vivo-generated platelets vs
megakaryocyte-generated platelets in mice: a
cautionary tale. Blood. 2015;125(23):
3627-3636.
74. Montecinos VP, Morales CH, Fischer TH, et al.
Selective targeting of bioengineered platelets to
prostate cancer vasculature: new paradigm for
therapeutic modalities. J Cell Mol Med. 2015;
19(7):1530-1537.
80. Greene TK, Lyde RB, Bailey SC, et al. Apoptotic
effects of platelet factor VIII on megakaryopoiesis:
implications for a modified human FVIII for
platelet-based gene therapy. J Thromb Haemost.
2014;12(12):2102-2112.
75. Li J, Luo J, Luo YQ, et al. Overexpression of
tumstatin in genetically modified megakaryocytes
changes the proangiogenic effect of platelets.
Transfusion. 2014;54(8):2106-2117.
81. Hacein-Bey-Abina S, Le Deist F, Carlier F,
et al. Sustained correction of X-linked severe
combined immunodeficiency by ex vivo gene
therapy. N Engl J Med. 2002;346(16):
1185-1193.
76. Fang J, Yao M, Jing W, Sun B, Johnson BD,
Wilcox DA. Platelets engineered to store
interleukin-24 inhibited melanoma growth in
mice [abstract]. J Thromb Haemost. 2015;
13(suppl S2):225. Abstract OR342.
77. Feng Q, Shabrani N, Thon JN, et al. Scalable
generation of universal platelets from human
induced pluripotent stem cells. Stem Cell Rep.
2014;3(5):817-831.
78. Lambert MP, Sullivan SK, Fuentes R, French DL,
Poncz M. Challenges and promises for the
development of donor-independent platelet
transfusions. Blood. 2013;121(17):3319-3324.
82. Cavazzana-Calvo M, Fischer A. Efficacy of gene
therapy for SCID is being confirmed. Lancet.
2004;364(9452):2155-2156.
83. Cavazzana-Calvo M, Hacein-Bey S, de Saint
Basile G, et al. Gene therapy of human severe
combined immunodeficiency (SCID)-X1 disease.
Science. 2000;288(5466):669-672.
84. Kaiser J. Gene therapy. Panel urges limits on XSCID trials. Science. 2005;307(5715):1544-1545.
85. Hacein-Bey-Abina S, von Kalle C, Schmidt M,
et al. A serious adverse event after successful
gene therapy for X-linked severe combined
immunodeficiency. N Engl J Med. 2003;348(3):
255-256.
86. Kaiser J. Gene therapy. Seeking the cause of
induced leukemias in X-SCID trial. Science. 2003;
299(5606):495.
87. Bradford GB, Williams B, Rossi R, Bertoncello I.
Quiescence, cycling, and turnover in the primitive
hematopoietic stem cell compartment. Exp
Hematol. 1997;25(5):445-453.
88. Biffi A, Bartolomae CC, Cesana D, et al. Lentiviral
vector common integration sites in preclinical
models and a clinical trial reflect a benign
integration bias and not oncogenic selection.
Blood. 2011;117(20):5332-5339.
89. Shi Q, Fahs SA, Wilcox DA, et al. Syngeneic
transplantation of hematopoietic stem cells that
are genetically modified to express factor VIII in
platelets restores hemostasis to hemophilia A
mice with preexisting FVIII immunity. Blood. 2008;
112(7):2713-2721.
90. Neyman M, Gewirtz J, Poncz M. Analysis of the
spatial and temporal characteristics of plateletdelivered factor VIII-based clots. Blood. 2008;
112(4):1101-1108.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2016 127: 1260-1268
doi:10.1182/blood-2015-07-607937 originally published
online January 19, 2016
Megakaryocyte- and megakaryocyte precursor−related gene therapies
David A. Wilcox
Updated information and services can be found at:
http://www.bloodjournal.org/content/127/10/1260.full.html
Articles on similar topics can be found in the following Blood collections
Free Research Articles (4545 articles)
Gene Therapy (584 articles)
Platelets and Thrombopoiesis (732 articles)
Review Articles (710 articles)
Review Series (155 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.