Download 1 Platelet gene therapy corrects the hemophilic

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Blood First Edition Paper, prepublished online November 22, 2013; DOI 10.1182/blood-2013-08-520478
Platelet gene therapy corrects the hemophilic phenotype in immunocompromized
hemophilia A mice transplanted with genetically manipulated human cord blood
stem cells
Qizhen Shi1,2,3,4, Erin L. Kuether1,2,3, Yingyu Chen2,5, Jocelyn A. Schroeder1,2,3, Scot A.
Fahs2, and Robert R. Montgomery1,2,3, 4
1Department
of Pediatrics, Medical College of Wisconsin; 2Blood Research Institute,
BloodCenter of Wisconsin; 3Children’s Research Institute, Children’s Hospital of
Wisconsin; 4MACC Fund Research Center, Milwaukee, WI 53226, USA; 5Fujian
Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian
Medical University Union Hospital, Fuzhou, Fujian, China.
Running title: Human platelet gene therapy of hemophilia A
Address correspondence: Qizhen Shi, MD, PhD. Department of Pediatrics, Medical
College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Tel: 414937-3853; Fax: 414-937-6811; e-mail: [email protected]
1
Copyright © 2013 American Society of Hematology
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Key Points
•
Platelet-specific lentiviral gene delivery to human HSCs can efficiently introduce
therapeutic levels of FVIII expression in human platelets
•
Human platelet-derived FVIII can ameliorate the hemophilic phenotype in an
immunocompromised hemophilia A mouse model.
2
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Abstract
Our previous studies have demonstrated that platelet-FVIII (2bF8) gene therapy can
improve hemostasis in hemophilia A mice even in the presence of inhibitory antibodies,
but none of our studies has targeted human cells, only mouse cells. Here we evaluated
the feasibility for lentivirus (LV)-mediated human platelet gene therapy of hemophilia A.
Human platelet-FVIII expression was introduced by 2bF8LV-mediated transduction of
human cord blood (hCB) CD34+ cells followed by xeno-transplantation into
immunocompromised NSG mice or NSG mice in a FVIIInull background (NSGF8KO).
Platelet-FVIII was detected in all recipients that received 2bF8LV-transduced hCB cells
as long as human platelet chimerism persists. Electron microscopy demonstrated that
FVIII was colocalized with VWF in 2bF8LV-transduced human platelets. All NSGF8KO
recipients (n = 7) that received 2bF8LV-transduced hCB cells survived tail clipping if
animals had greater than 2% of platelets derived from 2bF8LV-transduced hCB cells,
while 5 of 7 survived when human platelets were 0.3-2%. Whole blood clotting time
analysis confirmed that hemostasis was improved in NSGF8KO mice that received
2bF8LV-transduced hCB cells. We demonstrate for the first time the feasibility of
2bF8LV gene delivery to human HSCs to introduce FVIII expression in human platelets
and that human platelet-derived FVIII can improve hemostasis in hemophilia A.
Key words: Platelet; Gene therapy; hemophilia A; Human cells
3
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Introduction
Hemophilia A is a congenital bleeding disorder caused by a deficiency of factor VIII
(FVIII). Protein replacement therapy using either plasma derived or recombinant FVIII is
effective for the treatment of hemophilia A patients. However, it is expensive and
requires frequent infusions owing to the short half-life of the protein. Furthermore, up to
35% of patients will develop anti-FVIII inhibitory antibodies, referred to as inhibitors,
after exogenous FVIII replacement therapy.1-3 The clinical hallmark of inhibitor
development in hemophilia A patients is failure to respond to routine replacement therapy
for bleeding episodes.3-6
Gene therapy is an attractive strategy for hemophilia A treatment. The goal of gene
therapy is to introduce long-term expression of therapeutic levels of FVIII in vivo by
genetically modifying the target cells resulting in a cure of the disease. Although
substantial progress has been achieved in the past decade, potential development of an
immune response to transgene product or vector remains a significant concern in genetic
therapy.7-9 We have developed a novel clinically translatable platelet-targeted gene
therapy approach using lentiviral gene delivery to hematopoietic stem cells (HSCs), in
which FVIII expression is under control of the platelet-specific glycoprotein (GP) IIb
promoter (2bF8).10 Our previous studies have demonstrated that 2bF8 lentivirus
(2bF8LV)-mediated platelet-specific gene therapy can efficiently introduce therapeutic
levels of platelet-FVIII in hemophilia A mice with neither inhibitory nor non-inhibitory
antibody development.10 Further studies have demonstrated that sustained therapeutic
levels of platelet-FVIII are obtained while inhibitor titers decline with time after 2bF8
4
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gene therapy in hemophilia A mice with pre-existing anti-FVIII immunity.11 However,
this approach has not been studied in human cells.
Since our ultimate goal is to express FVIII in hemophilia A patient’s platelets, the
questions we addressed in the current study include: 1) whether human hematopoietic
stem cells (hHSCs) can be transduced by 2bF8LV; 2) whether 2bF8LV-transduced
hHSCs can normally give rise to blood cells including the platelet lineage; 3) whether
2bF8LV-mediated gene transfer can efficiently introduce FVIII expression in human
platelets; and 4) whether human platelet-derived FVIII can correct the hemophilic
bleeding diathesis. We demonstrate for the first time the feasibility of 2bF8 lentiviral
gene delivery to hHSCs to introduce FVIII expression in human platelets and that human
platelet-derived FVIII can improve hemostasis in hemophilia A.
5
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Materials and methods
Mice. Immunocompromised NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which
combine the features of the NOD background, the severe combined immune deficiency
mutation (scid) and IL2 receptor gamma chain (IL2rg) deficiency,12 were purchased
from the Jackson Laboratory (Bar Harbor, ME). The NSG mice have been shown to
support greater engraftment of hHSCs than all other strains.12;13 The
immunocompromised hemophilia A mouse model (NSGF8KO) was generated by our
laboratory by crossing NSG mice into the FVIIInull background. The FVIIInull mice (in
C57BL/6 x 129/SV background) were generated by targeted disruption of exon 17 of the
F8 gene.14 All animals were kept in non-specific pathogen free microisolator cages at the
animal facilities operated by the Medical College of Wisconsin. Animal studies were
performed according to a protocol approved by the Institutional Animal Care and Use
Committee of the Medical College of Wisconsin.
Virus production, purification, and titering. The lentiviral construct, pWPT-2bF8,
was generated as previously described.10 Recombinant lentivirus was generated from
HEK 293T cells by transient transfection. The procedures for virus production and
purification were described in previous reports.10;15
Lentivirus-mediated transduction of human cord blood (hCB) CD34+ cells. Human
cord blood (hCB) CD34+ cells were purchased from ALLCELLs (Emeryville, CA) and
transduced with 2bF8LV using a protocol similar to that described in previous
reports.10;15 The details are provided in the supplemental materials.
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Xeno-transplantation. Six to seven week old NSG or NSGF8KO mice were conditioned
for xeno-transplantation with busulfan treatment. Busulfan (6 mg/mL) (DSM
Pharmacenticals, Inc. Greenville, NA) was diluted 1: 4 in PBS and administered
intravenously at 25 mg/kg per day on days -2 and -1. Twenty-four hours after busulfan
treatment, 1-2 x 106 2bF8 LV-transduced hCB CD34+ cells were transplanted in a volume
of 300 μL of QBSF-60 media per mouse by retro-orbital vein injection. Untransduced
cells were transplanted into controls. The animals that received hCB CD34+ cell
transplantation (hCBT) are defined as a humanized model.
Human cell chimerism analysis. Blood samples from animals were collected by tail
bleeds and blood counts were performed using the Scil Vet ABC Plus blood counter (Scil
Animal Care Company, Gurnee, IL). The details are provided in the supplemental
materials.
PCR and quantitative real-time PCR (qPCR) analysis. PCR and qPCR were
performed as previously described.10;11 The details are provided in the supplemental
materials.
Immunogold electron microscopy (EM). EM studies were performed by Clive W.
Wells in The Electron Microscopy Facility at the Medical College of Wisconsin. Isolated
platelets were processed and stained for FVIII protein as previously described.16 The
details are provided in the supplemental materials.
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FVIII activity (FVIII:C) assays. Platelets and plasma were isolated as previously
described.17 Functional FVIII:C in platelet lysates or plasma was quantitated by a
modified FVIII chromogenic assay as previously described.16;17 Recombinant human Bdomain deleted FVIII (rhFVIII) (Xyntha, Wyeth Pharmaceuticals, Collegeville, PA) was
used as the standard. Platelets and plasma from NSG mice, C57BL6 mice, and humans
were used as controls.
Assessment of phenotypic correction. To evaluate whether 2bF8LV-transduced human
platelets can improve hemostasis in hemophilic mice, the tail clip survival test and
rotational thromboelastometry (ROTEM) analysis were used. The tail clip tests were
performed between 4-6 weeks after transplantation as previously reported.11;16;17
ROTEM analysis of whole blood clotting time (CT) was performed between 6-8 weeks
after transplantation as described in our previous report.18
2bF8LV integration site analysis. 2bF8LV integration site analysis was performed on
genomic DNA extracted from peripheral blood using non-restrictive LAM-PCR (nrLAMPCR), which is faster and more cost-efficient than LAM-PCR as reported.19 The details
are provided in the supplemental materials.
Statistical analysis. Data are presented as mean ± SD, and the significance of
differences was evaluated by 2-tailed Student t-test. A value of P < .05 was considered
statistically significant.
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Results
Human cell chimerism. Using our culture conditions for in vitro transduction of hCBderived hHSCs, we were able to maintain both a similar cell number and the “stemness”
(as indicated by CD34 expression) compared to the initial hCB sample. Greater than 90%
of the cells were still CD34+ after 2bF8LV-transduction. To determine whether 2bF8LVtransduced hCB CD34+ cells can differentiate into human platelets in our xenograft
model, flow cytometry was used to analyze peripheral blood cells from transplanted
recipients. No human cells could be detected 1 week after transplantation, but both
human platelets and leukocytes were detected in NSG mice that received 2bF8LVtransduced hCB CD34+ cells at later time points. Three weeks after transplantation, 48.9
± 13.5% (n = 10) of total platelets and 73.2 ± 9.6% (n = 6) of total leukocytes in
peripheral blood were of human origin (Figure 1 A-D). Human platelet chimerism
dropped to 7.4 ± 4.3% (n = 6) at week 5 while leukocyte chimerism was still maintained
at greater than 47% (Figure 1C and D).
Total human platelet number was calculated based on the whole blood cell count and the
percent of chimerism determined by flow cytometry analysis. The peak levels of human
platelet chimerism in recipients occurred 2-3 weeks after transplantation, while the
animals exhibited murine cell thrombocytopenia at that time point (Figure 1E). There
were 56.3 ± 27.0 x 103/ μL human platelets in the blood at 2 weeks, but dropped to 12.4
± 11.9 x 103/ μL at 5 weeks and 1.9 ± 0.6 x 103/ μL at 12 weeks after transplantation.
Mouse platelet number was 9.3 ± 5.5 x 103/ μL at 2 weeks after transplantation, which is
only about 1% of the platelet number of NSG mice. The mouse platelet number started
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to increase at 3 weeks after transplantation, but never recovered to pre-transplantation
levels during the study course (Figure 1E).
In contrast to platelets, the peak levels of human leukocyte chimerism occurred at 7
weeks after transplantation and were maintained around that level for the rest of the study
course. When we looked at murine leukocytes, the animals had developed severe murine
leukopenia at 3 to 5 weeks after transplantation. Compared to untreated NSG mice, there
were only about 3% of mouse leukocytes remaining at 3 weeks after transplantation.
Mouse leukocyte levels started to recover after 7 weeks. These levels were approximately
21% of normal in NSG mice and were maintained around that level onward during the
study course (Figure 1F).
Analysis of 2bF8LV transduction in engrafted human cells. To investigate the
transduction of viably engrafted 2bF8LV-transduced hCB-derived HSCs, PCR was used
to detect the 2bF8 transgene in peripheral blood cell-derived DNA from NSG recipients.
Representative results from recipients at least 3 weeks after transplantation are shown in
Figure 2A. The 2bF8 transgene was detected in all NSG mice that received 2bF8LV
transduced human cord blood CD34+ cells. To determine the average copy number of
2bF8 proviral DNA in human cells, qPCR was used to quantify the 2bF8 expression
cassette, normalized to the human GAPDH gene. There were 1.88 ± 0.52 copies of the
2bF8 transgene per human cell in NSG mice that received 2bF8LV-transduced hCB
CD34+ cells (Figure 2B). As expected, no 2bF8 proviral DNA was detected in NSG
control mice.
10
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Analysis of FVIII expression in 2bF8 LV-transduced human platelets. The levels of
platelet-FVIII expression were determined by a chromogenic assay of platelet lysates
from recipients at least 3 weeks after transplantation. Functional FVIII activity was
detected in the NSG recipients that received 2bF8LV-transduced hCB CD34+ cells, at an
average of 0.76 ± 0.43 mU/108 total platelets (n = 6) (Figure 3A). This level of plateletFVIII corresponds to about 17.77 ± 10.36 mU/108 human platelets when calculated
according to chimerism levels measured by flow cytometry (Figure 3B). As expected, no
FVIII was detected in platelet lysates from control mice (NSG and C57BL6) and human
control platelets.
Localization of human FVIII transgene protein expression was determined by
immunogold electron microscopy. Transgene hFVIII protein was labeled with small
colloidal gold particles and endogenous VWF was labeled with large colloidal gold
particles. Transgene hFVIII protein was detected in 2bF8LV-transduced human platelets
recovered from transplanted NSG mice and was co-localized with endogenous VWF in
platelet α-granules (Figure 4 A-C). No FVIII was detected in platelets from
untransplanted NSG mice or normal human individuals although a normal distribution of
endogenous human VWF (Figure 4 D-F) or murine VWF (Figure 4 G-I) was observed.
Insertion site analysis. To investigate the chromosomal insertion sites of 2bF8LV in
human cells, we used non-restrictive LAM-PCR. We identified 19 insertion sites in 5 of
the NSG recipients and found 13 insertion sites in the introns of genes, 2 in the exons,
and 4 in intergenic regions (Table 1). Similar to what we have found in transduced mouse
cell studies,11 the integration of 2bF8LV into the first intron of human genes appears to
11
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have occurred at a higher frequency (27%) than other areas within genes. The frequency
of sense versus anti-sense orientation of 2bF8 transgene within human genes appears to
be approximately, equal at 47% and 53%, respectively. No identified integration sites
were located within known proto-oncogenes in human hematopoiesis.
Evaluation of clinical efficacy of human platelet-derived FVIII. Using the NSG
mouse model, we have examined the feasibility of targeting FVIII expression to human
platelets by 2bF8 lentiviral gene transfer to hCB-derived hHSCs. To explore whether
2bF8LV-transduced human platelets can improve the bleeding diathesis in hemophilia A,
we generated an immunocompromised hemophilia A mouse model by crossing NSG and
FVIIInull to produce NSGF8KO mice in which the genetic background is a mixture of
NOD, 129/SV, and C57BL/6. 2bF8LV-transduced hCB-derived CD34+ cells were
transplanted into NSGF8KO mice pre-conditioned with busulfan.
Because the IL2rg gene is relatively near the F8 locus (the distance between the two
genes is 7.5 centimorgan) on the X-chromosome, there was some difficulty with crossing
to achieve linkage of the defective IL2rg gene with the FVIII knockout gene, but after
multiple matings, we successfully identified a cross-over between these two genes and
obtained NSGF8KO mice for our studies. We found that human platelet chimerism was
markedly decreased in recipients that had lost the NOD phenotype during breeding, while
leukocyte chimerism was less affected. Three weeks after transplantation, 20.9 ± 23.9%
(n = 14) of total platelets in NSGF8KO recipients were of human origin and only 2.1 ±
1.7% (n = 12) at week 5, significantly lower than in NSG recipients (P < .01) (Figure
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1C). In contrast, human leukocyte chimerism in NSGF8KO recipients was not
significantly different from the NSG recipients (Figure 1D).
As expected, no FVIII:C was detected in plasmas from NSGF8KO recipients (data not
shown). Functional platelet-FVIII activity was detected in NSGF8KO recipients at an
average the level of 0.56 ± 0.42 mU/108 total mouse plus human platelets (Figure 3A),
which corresponded to 17.72 ± 8.65 mU/108 human platelets (Figure 3B) when calculated
based on human platelet chimerism as determined by flow cytometry. There was no
significant difference in the level of platelet-FVIII expression between NSG and
NSGF8KO groups after xeno-transplantation of 2bF8LV-transduced hCB (Figure 3A and
B).
Tail clip survival tests were used to assess phenotypic correction of the FVIIInull
coagulation defect in NSGF8KO mice. Four to 6 weeks after transplantation, all
recipients survived tail clipping if greater than 2% of platelets were derived from
2bF8LV-transduced hCB CD34+ cells; 5 of 7 survived when human platelets were
between 0.3% and 2%. In contrast, none of the untransplanted NSGF8KO mice or
NSGF8KO mice that received untransduced hCB CD34+ cells survived tail clipping
(Figure 5A). To further assess whether 2bF8LV-transduced human platelets improve
hemostasis in hemophilia A mice, ROTEM analysis was used to analyze whole blood CT
(Figure 5B and C). Six to 8 weeks after transplantation, the whole blood CT was 1296 ±
234 seconds (n = 7) in NSGF8KO/2bF8LV-hCBT recipients, in which human platelet
chimerism was 0.4-5.5% at the time of the ROTEM analysis. This whole blood CT was
significantly shorter than that obtained from NSGF8KO control mice [2792 ± 220
13
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seconds (n = 10)], confirming that hemostasis was improved in NSGF8KO mice that
received 2bF8 LV-transduced hCB CD34+ cells.
Taken together, our results show that 2bF8 lentiviral gene delivery to hHSCs can
efficiently introduce FVIII expression in human platelets and that 2bF8LV-transduced
human platelets can improve hemostasis in a hemophilia A mouse model.
14
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Discussion
Previous studies by our group10;11;16;17 and others20-23 using mouse models have
demonstrated that platelet-targeted expression of FVIII may be a promising approach for
gene therapy of hemophilia A and hemophilia A with inhibitors. In the current study, we
established a novel immunocompromised hemophilia A mouse model and used
humanized mouse models to explore the feasibility and efficacy of targeting FVIII
expression to human platelets for hemophilia A gene therapy. FVIII expression was
introduced by ex vivo transduction of hCB-derived hHSCs followed by xenotransplantation into immunocompromised hemophilia A mice. We conclude that 2bF8
lentiviral gene delivery to hHSCs can efficiently introduce functional FVIII expression in
human platelets and that human platelet-derived FVIII can ameliorate the bleeding
diathesis in hemophilia A mice.
Human thrombocytopoiesis in the 2bF8LV-genetically modified humanized NSG
model.
Our study demonstrated that 2bF8LV can efficiently transduce hCB CD34+ cells,
resulting in successfully genetically manipulated hHSCs. In preliminary experiments, we
transplanted the transduced hCB CD34+ cells into NSG mice preconditioned with a
sublethal irradiation dose of 200 cGy or 300 cGy total body irradiation, but the mortality
was extremely high. It has been reported that busulfan chemotherapy is an efficient
preconditioning regimen to achieve successful engraftments in humanized models for
white blood cell related studies.24-27 Thus, we for the first time used busulfan
chemotherapy as a preconditioning regimen for xeno-transplants to study the feasibility
of 2bF8 gene delivery to hHSCs and the clinical efficacy of human platelet-derived FVIII
15
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in gene therapy of hemophilia A. We found that the transduced repopulating hHSCs were
able to successfully engraft into immunocompromised animals preconditioned with
busulfan and give rise to human blood cells, including platelets that contain functional
FVIII activity.
Since the final target for FVIII expression in our gene therapy approach is platelets, it is
important to investigate whether 2bF8LV-transdcued hHSCs can differentiate into human
platelets. Human platelet chimerism peaked at 2-3 weeks after xeno-transplantation, when
up to 93% of platelets were of human origin, but by week 5 they had dropped to 3-18%
although leukocyte chimerism was still maintained at greater than 47%. The rapid
reduction of human platelet chimerism in NSG recipients also occurs in non-transduced
transplants (data not shown), so it is not due to 2bF8 genetic modification. This is a
common problem in studies of human thrombocytopoiesis in immunocompromised
mouse models and the cause is still unknown.28-31 There was a small population that was
positive for both human and mouse platelet markers, which could be due to low level
platelet activation during sample processing resulting in human and mouse platelet
association.
We had monitored some animals up to 7 months after transplantation and found that
leukocyte chimerism was sustained, but the reduction of human platelet chimerism
between weeks 3 and 7 was remarkable, decreasing by 70 to 95%. 2bF8 proviral DNA
was consistently detected in peripheral blood cell-derived DNA at all time points during
the study course, indicating that long-term repopulating hHSCs were successfully
engrafted and were genetically modified by 2bF8LV. Although the temporal window for
16
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studying human platelets derived from 2bF8LV-transduced hCBT in the NSG mouse
model was narrow, there were up to 133 x 103 human platelets/μL circulating in the
peripheral blood in xeno-transplanted recipients with the highest chimerism between
weeks 2 and 3, nearly reaching the range of normal platelet number in humans (150 - 450
x 103/μL).
Since high levels of white blood cell chimerism were sustained in 2bF8LV-transduced
recipients, we suspect the quick reduction of human platelets in xeno-transplant recipients
may be due to incompatibility of murine molecular signals involving thrombocytopoiesis
or maintenance of the proper hematopoietic microenvironment, for human platelet
production. While we did infuse rhTPO into 3 of the NSG mice that received 2bF8LVtransduced hCB CD34+ cells, human platelet chimerism did not significantly increase
(data not shown). Recent studies done by Hu et al have demonstrated that poor human
platelet reconstitution in their xeno-transplantion models is mainly caused by
macrophage-mediated rejection.32 The macrophage rejection mechanism cannot explain
the phenotype of blood cells in our xeno-transplanted recipients, in which human platelets
peak in the early phase (2 weeks) when there were 12% murine endogenous leukocytes
including macrophages and decrease dramatically at 5 weeks when murine endogenous
leukocytes are barely detectable (0.9%). These results indicate that there are other
mechanisms, which are still unknown, playing roles in human thrombocytopoiesis in
immunocompromised mouse models.
17
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Human thrombocytopoiesis in the 2bF8LV-genetically modified humanized
NSGF8KO model.
While we obtained high levels of human platelet chimerism in NSG mice that received
2bF8LV-transduced hCB CD34+ cell, the human platelet chimerism in NSGF8KO
recipients was dramatically lower. This might be owing to loss of the NOD background
during crossbreeding of NSG mice to the FVIIInull background as well as scid
“leakiness”.26 It is known that the NOD background contributes to the absence of
hemolytic complement, defective macrophages, and enhanced human HSC
engraftment.33-35 For genotyping of NSGF8KO mice, we used PCR to detect the
FVIIInull allele, the scid mutation, and the IL-2Rγ(null) gene. However, there is currently
no genotyping approach available to track the NOD background. Thus, the genetic
background of NOD was likely diluted during crossbreeding of NSG into FVIIInull, so
macrophages from resulting NSGF8KO mice might function normally and phagocytize
human platelets in xeno-transplanted recipients.
The scid mutation prevents maturation of T and B cells,36 but scid leakiness can result in
the production of some functional B and T cells as the mice age,37 causing a rejection of
xeno-grafts. It has been reported that such leakiness is higher in mice housed under nonspecific pathogen free conditions, which is the type of facility in which we house our
mice, and leakiness is generally higher on the C57BL/6J genetic background.38-40 The
FVIIInull mice crossed with NSG mice were on a mixed genetic background of C57BL/6
and 129/SV, so NSGF8KO mice inherited a mixed genetic background of C57BL/6,
129/SV, and NOD. Backcrossing NSGF8KO mice to a pure NOD background will need
to be pursued in future studies.
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FVIII expressed in human platelets has functional activity.
Although human platelet reconstitution was not sustained in 2bF8LV-transduced hCBT
recipients, platelets produced by 2bF8LV-transduced hCB CD34+ cells contained
functional FVIII activity. When the levels of platelet-FVIII expression were converted to
average per human platelet values according to chimerism levels determined by flow
cytometry, the amount of FVIII in 2bF8LV-transduced human platelets (17.77 mU/108
platelets) is significantly greater than those obtained from transduced or transgenic mouse
platelets (1.56 mU/108 platelets or 1.41 mU/108 platelets, respectively) in our previous
studies.11;16 This phenomena was consistent for both NSG and NSGF8KO mice that
received 2bF8LV-transduced hCB CD34+ cells.
While it is still unclear why the average amount of FVIII in human platelets derived from
2bF8 genetically engineered hHSCs is greater than in mouse HSCs, there are three
potential explanations. First, human platelets are larger than mouse platelets. Human
platelets are approximately 2.0-4.0 μm in diameter with a mean volume of 7.8 fL,
whereas mouse platelets are 1.0-2.0 μm in diameter with a mean volume of 5.5 fL.41;42
Thus, each 2bF8LV-transduced human platelet has a higher capacity to carry cargo
including transgene FVIII protein than mouse platelets. Secondly, 2bF8LV may
transduce human cord blood-derived HSCs more efficiently than mouse bone marrowderived HSCs. The vector that we use to deliver 2bF8 transgene to HSCs is a second
generation human immunodeficiency virus type 1-based vector, which has been reported
to transduce human cells more efficiently than their mouse counterparts.43-45 Indeed, the
average copy number of 2bF8 proviral DNA per human cell was significantly higher than
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that obtained from the transduced mouse cells in our previous reports.10;11 A third reason
for potentially higher expression (not infection) is that human platelets contain human
VWF which works better with human FVIII.
Our previous studies have demonstrated that hFVIII expressed in mouse platelets can
rescue the bleeding diathesis in hemophilia A mice.10;11;16;46 In the current study using a
humanized NSGF8KO model we demonstrated that 2bF8LV can efficiently introduce
FVIII expression in human platelets and that human platelet-derived FVIII can restore
hemostasis in a hemophilia A mouse model as long as human platelet persist. The
bleeding diathesis was significantly improved even with a small proportion of platelets
(0.3% - 2%) derived from 2bF8LV-transduced hCB CD34+ cells. Our data suggest that
sufficiently high transduction efficiencies can be practically achieved to ensure adequate
transgene delivery to hHSCs to produce therapeutic levels of platelet-FVIII using 2bF8
lentiviral gene transfer.
In conclusion, we have demonstrated for the first time that 2bF8 lentiviral gene delivery
to hHSCs can efficiently introduce therapeutic levels of FVIII expression in human
platelets and that this can ameliorate the hemophilic phenotype in an
immunocompromised hemophilia A mouse model. Our data strongly suggest that 2bF8
lentiviral gene delivery to hHSCs can be a promising approach for gene therapy of
hemophilia A. In the scenario of a hemophilia A patient, autologous cells from cord
blood or mobilized HSCs will be transduced with 2bF8LV ex vivo followed by autotransplantation of the transduced cells.
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Acknowledgments
We thank H. Kazazian at the University of Pensylvania School of Medicine for FVIIInull
mice. This work was supported by National Institutes of Health grants HL-102035 (QS)
and HL-44612 (RRM), Hemophilia Association of New York grant (QS), and generous
gifts from the Children’s Hospital Foundation (QS) and MACC Fund (QS).
Authorship
Contribution: Q. S. designed and performed research, analyzed data, and wrote the
manuscript; E. L. K. and Y. C. performed research and analyzed data; J. A. S., and S. A.
F. performed research, analyzed data, and made comments to the manuscript; R. R. M.
helped in the design of the project and made comments to the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
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Reference List
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engraftment of human hematopoietic stem cells. Nat Immunol 2007;8(12):13131323.
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(42) Bath PM, Carney C, Markandu ND, MacGregor GA. Platelet volume is not
increased in essential hypertension. J Hum Hypertens 1994;8(6):457-459.
(43) Ricks DM, Kutner R, Zhang XY, Welsh DA, Reiser J. Optimized lentiviral
transduction of mouse bone marrow-derived mesenchymal stem cells. Stem Cells
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(44) Siapati EK, Bigger BW, Miskin J et al. Comparison of HIV- and EIAV-based
vectors on their efficiency in transducing murine and human hematopoietic
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(45) Davis BM, Humeau L, Slepushkin V et al. ABC transporter inhibitors that are
substrates enhance lentiviral vector transduction into primitive hematopoietic
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(46) Shi Q, Fahs SA, Kuether EL, Cooley BC, Weiler H, Montgomery RR. Targeting
FVIII expression to endothelial cells regenerates a releasable pool of FVIII and
restores hemostasis in a mouse model of hemophilia A. Blood
2010;116(16):3049-3057.
25
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Table 1. Non-restrictive linear amplification-mediated PCR (nrLAM-PCR) determined
insertion sites of 2bF8 transgene
#
Chr.
1
2
3
4
5
1
1
1
3
6
Dir. of
T.g.
↑
↑
↑
↑
↑
6
7
7
7
↓
↑
8
9
7
8
↓
↑
10
11
12
13
11
11
11
11
↓
↓
↓
↓
14
12
↓
15
16
17
18
19
14
14
16
16
17
↑
↓
↑
↑
↓
Gene
↓ DDI2 (DNA Damage Inducible Protein 2) Intron 1
↑ ZSWIM5 (Zinc Finger, SWIM Domain Containing 5) Intron 1
↓ AHS1L (Probable histone-lysine N-methyltransferase ASH1L) Intron 14
↓ CLASP2 (Cytoplasmic Linker Associated Protein 2) Intron 12
Intergenic [Located downstream of Hypothetical Protein LOC100288169 and
upstream of DUSP22 (Dual Specificity Phosphatase 22)]
↑ DGKB (Diacylglyceral kinase beta isoform 1, 2) Intron 2
↑ GTF2IRD2 (General transcription factor II-I repeat domain-containing protein 2A)
Intron 2
↓ PILRB (Paired immunoglobulin-like type 2 receptor beta precursor) Intron 3
Intergenic [Located downstream of UNC5D (Netrin receptor UNC5D precursor) and
upstream of KCNUI (potassium channel subfamily U member)]
↓ RBM14-RBM41 (RBM14-RBM4 protein isoform 1, 2) Intron 1
↑ ARRB1 (beta-arrestin-1 isoform A and B) Intron 1
↑ BSCL2 (Bernadinelli-Seip Congenital Lipidystrophy 2 [Seipin]) Intron 5
Intergenic [Located downstream of LTBP3 (latent transforming growth factor beta
binding protein 3) and upstream of SSSCA1 (Sjogren syndrome / scleroderma
autoantigen 1)]
Intergenic [Located downstream of ATP2B1 (plasma membrane calcium-transporting
ATPase 1isoform 1a, 1b) and upstream of C12orf12 (hypothetical protein
LOC196477)]
↓ SOCS4 (Suppressor of Cytokine Signaling 4) Exon 4 in 3’UT
↑ GPR132 (G protein-coupled receptor 132) Intron 1
↑ CREBBP (Creb Binding Protein) Exon 30
↑ KIAA0430 (Limkain b-1 isoform 1, 2, 3) Intron 6
↓ SUZ12 (Supressor of Zeste 12 Homolog [Drosophila]) Intron 15
“Chr.” means chromosome; Arrows indicate the directions of the transgene (Tg) or gene
relative to the standard chromosomeal numbering. The insertion sites listed in this table
were from 5 NSG recipients.
26
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Figure legends
Figure 1. Human cell chimerism in NSG and NSGF8KO mice that received 2bF8
LV-transduced hCB CD34+ cells. Blood samples were collected from tail bleeds.
Human platelets were identified by a mouse anti-human GPIbα antibody, AP1. Mouse
platelets were stained with a rat anti-mouse CD41 (GPIIb) antibody. Human leukocytes
were identified by mouse anti-human CD45 antibody and mouse leukocytes were
identified by rat anti-mouse CD45 antibody. (A) Representative flow cytometric analysis
of platelets in peripheral blood of NSG and NSGF8KO recipients at 3 weeks after
transplantation. (B) Representative flow cytometric analysis of leukocytes in peripheral
blood of NSG and NSGF8KO recipients between 5-7 weeks after transplantation. (C)
Human platelet chimerism in NSG and NSGF8KO recipients. Samples from NSG mice
and normal human individuals were used as controls. Data are summarized from 10 trials
of xeno-transplants. D) Human leukocyte chimerism in NSG and NSGF8KO recipients.
Samples from NSG mice and normal human individuals were used as controls. Data are
summarized from 10 trials of xeno-transplants. (E) Time course of human platelet
production in NSG recipients. Data are summarized from 2 trials of xeno-transplants. n =
4 to 9 mice for each group. (F) Time course of human leukocyte production in NSG
recipients. Data are summarized from 2 trials of xeno-transplants. n= 4 to 7 mice for each
group.
Figure 2. Genetic analysis of 2bF8 LV-transduced hCB transplantation recipients.
(A) PCR analysis of the 2bF8 transgene. Genomic DNA was purified from peripheral
leukocytes. Leukocytes from NSG mice and a normal human individual were used as
controls. 2bF8 transgenic mouse DNA was used as a positive control for the 2bF8
27
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transgene. Shown is one representative experiment that was performed three times. Wildtype (WT) mouse FVIII and human GAPDH were used as internal controls. (B) Realtime quantitative PCR quantified the average copy number of the 2bF8 transgene per cell
in 2bF8 LV-transduced hCB transplantation recipients. Peripheral leukocyte-derived
genomic DNA was used to analyze 2bF8 proviral DNA with normalization to human
GAPDH. Bars represent mean ± SD. For individual mice analyzed more than once over
the course of study, the average copy number was calculated. Genomic DNA from NSG
mice was used as a control. Data are summarized from 2 trials of xeno-transplants.
Figure 3. Analysis of platelet FVIII expression in 2bF8 LV-transduced xenotransplanted recipients. Platelets were lysed in 0.5% CHAPS. The levels of plateletderived FVIII expression were determined by chromogenic FVIII activity (FVIII:C)
assay of platelet lysates from recipients at least three weeks after transplantation. Bars
represent mean ± SD. For individual mice analyzed more than once over the course of
study, the average FVIII level was calculated. Platelets from NSG mice, C57BL6 mice,
and normal human individuals were used as controls. (A) Quantitative evaluation of
FVIII activity levels in platelet lysates from 2bF8 LV-transduced hCBT recipients
(humanized) NSG or NSGF8KO recipients. (B) Quantitative evaluation of FVIII activity
levels in hCB-derived human platelets based on human platelet numbers as determined
by flow cytometry. Data are summarized from 10 trials of xeno-transplants.
Figure 4. Electron microscopy determines cellular location of 2bF8 transgene
protein. Isolated platelets from 2bF8LV-transduced xeno-transplanted NSG recipients
(A-C), normal human individuals (D-F), and untransplanted NSG control mice (G-I)
28
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were immunostained for hFVIII and either endogenous human or mouse VWF. hFVIII
was stained with mouse anti-human FVIII monoclonal antibody 103.3 and probed with
goat anti-mouse colloidal gold probe (6 nm, some indicated by arrow). VWF was stained
with rabbit anti-human VWF polyclonal antibody (Dako), which cross reacts with mouse
VWF, and probed with goat anti-rabbit colloidal gold probe (10 nm, some indicated by
arrowhead). The results show hFVIII is stored together with human VWF in platelet αgranules of 2bF8LV-transduced human platelets (A-C). Scale bar is 100 nm.
Figure 5. Phenotypic correction assessment. (A) Tail clip survival test assessing the
phenotypic correction in 2bF8LV-transduced humanized NSGF8KO mice. The tail
clipping test was performed at least 4 weeks after transplantation. Mice surviving beyond
24 hours were considered to have achieved phenotypic correction. Untransduced hCBtransplanted and untransplanted NSGF8KO recipients were tested as controls. Data are
summarized from 10 trials of xeno-transplants. (B) ROTEM analysis. The ROTEM
analysis was performed between 6 and 8 weeks after transplantation when human platelet
chimerism was 0.4-5.5%. Representative TEMograms from untransplanted NSG,
untransplanted NSGF8KO, and 2bF8LV-transduced humanized NSGF8KO (with 2%
human platelet chimerism) mice are shown. (C) Whole blood clotting time determined by
ROTEM analysis. Untransplanted NSG and NSGF8KO mice were used as controls. Data
are summarized from 5 trials of xeno-transplants.
29
Figure 1
Platelets
Human control
NSG/2bF8LV
NSG/2bF8LVhCBT recipient
0.00
0.00
98.11
0.05
56.49
0.78
0.95
99.05
1.28
0.56
15.67
27.07
NSGF8KO/2bF8LV- NSGF8KO/2bF8LVNSGF8KO/2bF8LV
NSGF8KO/2bF8LV
hCBT recipient
hCBT recipient
58.9
0.49
18.4
0.44
10.8
29.8
6.48
74.4
Mouse GPIIb
Leukocytes
y
Huma
an CD45
NSG control
Human control
NSG/2bF8LVNSG/2bF8LV
hCBT recipient
0.03
0.25
98.4
1.03
81.4
0.56
0.72
99.0
0.53
0.002
8.12
9.93
Mouse CD45
NSGF8KO/2bF8LV- NSGF8KO/2bF8LVNSGF8KO/2bF8LV
NSGF8KO/2bF8LV
hCBT recipient
hCBT recipient
Hum
man CD45
B
Mouse GPIIb
91.1
0.39
86.1
0.16
2.48
6.02
7.56
6.18
Mouse CD45
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Human
n GPIb
NSG control
Human GPIb
A
P < .01
60
P < .01
40
20
n=10 n=6 n=14 n=12 n=10 n=9
5w NSG Human
40
20
0
n=6
n=6 n=14 n=12
n=9
3w
5w
NSG Human
3w
5w
n=9
80
1400
1200
1000
60
800
40
600
400
20
200
0
0
0 1 2 3 4 5 7 10 12
Time after transplantation (weeks)
ytes (x 103/μL)
Human leukocy
F
Human platelets
Murine platelets
Mouse plateletts (x 103/μL)
Human plateletts (x103/μL)
E
100
60
NSG/
N
hCBT
2bF8LV-h
recipients
3w
Co
ontrols
5w
NSG/
V-hCBT
2bF8LV
reciipients
3w
GF8KO/
NSG
2bF8LV
V-hCBT
reciipients
0
80
1.75
1.50
Human leukocytes
Murine leukocytes
1.75
1.50
1.25
1.25
1
00
1.00
1
00
1.00
0.75
0.75
0.50
0.50
0.25
0.25
0.00
0.00
0 1 2 3
4 5 7 10 12
Time after transplantation (weeks)
ytes (x103/μL)
Mouse leukocy
80
P = .15
100
Controls
Human leu
ukocyte chimerrism (%)
100
P = .12
D
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platelet chimeris
sm (%)
Human p
C
NSGF8
8KO/
hCBT
2bF8LV-h
recipients
Figure 1
2bF8tg+ contrrol
dH2O
4
3
2
1
DNA Marker
NSG/2bF8LVhCBT recipients
2bF8 (650 bp)
WT mFVIII (233 bp)
Average c
copy number of 2
2bF8
transgene
e per human cell
hGAPDH (100 bp)
B
4
3
2
1
0
n=7
n=3
NSG/2bF8LVNSG
hCBT recipients control
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NSG control
A
Human control
Figure 2
Figure 3
n=4
n=4
C
57
B
L6
H
um
an
n=4
SG
n = 14
N
0.00
n=6
2bF8LV-hCBT
2bF8LV
hCBT Untransduced and
untransplanted
recipients
controls
25
20
15
10
5
0
n=6
n = 14
NA
n=4
NA
n = 12
n=4
C
57
B
L6
H
um
an
0.25
P = 0.99
N
SG
0.50
30
N
SG
N
SG
F8
K
O
0.75
Platelet FVIII:C
elets
(mU//108 human plate
1.00
1 00
N
SG
N
SG
F8
K
O
Platelet FVIII:C
U/108 total platele
ets)
(mU
1.25
B
P = .33
Untransduced
t
d
d and
d
2bF8LV-hCBT U
untransplanted
recipients
controls
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A
uman platelets
s
Hu
untransduced))
(u
2bF
F8LV-transduc
ced
hu
uman platelets
s
A
B
C
D
E
F
G
H
I
100 nm
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ouse platelets
s
Mo
(untransduced )
Figure 4
Figure 5
B
C
mm NSG
60
40
20
20
40
60 CT
% Surrvival
80
60
40
10
20
N/A
n=3
mm
60
40
20
20
40
60
20 30 40 50 min
NSGF8KO control
CT
10
NSGF8KO
control
n=4
Un
ntransduced
recipients 20 - 60
0.3
3-2
2.1 - 15
n=7
2bF8 LVtransduced
recipients
0
Human platelet
chimerism (%)
(at tail clipping)
n=7
mm
60
40
20
20
40
60
control
20 30 40 50 min
NSGF8KO/2bF8LVhCBT recipient
CT
10
20
30
40
50 min
P < .001
3600
3000
2400
1800
1200
600
0
NSG
control
NSGF8KO
control
NSGF8KO/
2bF8LV-hCBT
recipients
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100
CT (time to 2 mm
m in second)
A
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Prepublished online November 22, 2013;
doi:10.1182/blood-2013-08-520478
Platelet gene therapy corrects the hemophilic phenotype in
immunocompromized hemophilia A mice transplanted with genetically
manipulated human cord blood stem cells
Qizhen Shi, Erin L. Kuether, Yingyu Chen, Jocelyn A. Schroeder, Scot A. Fahs and Robert R.
Montgomery
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