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. 2015 Mar 10;23(4):617–626. doi: 10.1038/mt.2015.20

Intraosseous Delivery of Lentiviral Vectors Targeting Factor VIII Expression in Platelets Corrects Murine Hemophilia A

Xuefeng Wang 1, Simon C Shin 1, Andy F J Chiang 1, Iram Khan 1, Dao Pan 2, David J Rawlings 1,3, Carol H Miao 1,3,*
PMCID: PMC4395783  PMID: 25655313

Abstract

Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage-Sca1+c-Kit+ hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

Introduction

Hemophilia A (HemA) is a serious bleeding disorder caused by defects in factor VIII (FVIII) gene. HemA patients can be treated with acute or prophylactic FVIII replacement.1 However, ~20–30% of the patients develop inhibitory antibodies (Abs) against FVIII. An effective, single treatment gene therapy protocol that can achieve sustained and therapeutic levels of FVIII comprises a key goal for treating severe HemA patients.

Several gene therapy phase 1 clinical trials for HemA patients have been performed previously.2,3,4 However, only transient, low-level FVIII protein expression has been achieved due to inefficient gene transfer and development of immune responses against FVIII and/or associated gene transfer vectors. The self-renewal potential of hematopoietic stem cells (HSCs) in bone marrow (BM) suggests that this population might comprise an ideal target for stable genomic integration of therapeutic genes capable of correcting genetic diseases. Transplantation of retrovirally transduced HSCs carrying FVIII gene produced low levels of FVIII protein in mouse circulation.5,6 Recent improvement using a vector encoding a porcine FVIII and immunosuppressive agents achieved therapeutic levels of FVIII long-term in HemA mice with or without pre-existing anti-FVIII antibodies.7,8 In these studies, a nonmyeloblative conditioning regimen involving busulfan was used to establish stable mixed chimerism with transduced cells. However, the requirement for preconditioning of hemophilia subjects is undesirable.

The feasibility of in vivo gene transfer by direct intraosseous (IO) injection of adeno-, retro-, and lenti-viral vectors into mice has recently been demonstrated.9,10 Efficient transduction of HSCs can be achieved with GFP expression detected in progenitors and differentiated cell lineages. It was also demonstrated that virally transduced HSCs retain their potential to differentiate into all blood cell lineages and maintain their reconstitution ability,10,11 leading to long-term correction of BM defects such as Fanconi anemia in mice.11 This in vivo approach would avoid the difficulties encountered by ex vivo HSC gene transfer, including maintenance of stem cell properties, the loss of engraftment potential, and potential cytokine stimulation.10,12,13 In addition, in vitro manipulation of stem cells and preconditioning of the subject is not required to achieve a therapeutic benefit. Therefore, this approach may provide a novel treatment for HemA.

Platelets are released by BM megakaryocyte precursor cells into the circulation, where they play a crucial role in the maintenance of hemostasis.14 Platelets may comprise an ideal vehicle for ectopic FVIII expression due to multiple reasons. First, circulating platelets are recruited to, and activated through contact with collagen or von Willebrand factor (vWF) at sites of damaged vessel walls where they release vesicular contents potentially including ectopic FVIII to promote clot formation. Second, circulating platelets are produced daily from megakaryocytes in BM, and could potentially provide FVIII whenever needed. Third, FVIII stored in platelet α-granules is protected from neutralizing inhibitory Abs.15,16 Previous studies have shown that platelet-restricted expression of FVIII using megakaryocyte-specific promoters (glycoprotein (Gp) αIIb,16,17 Gp1bα18 and platelet factor 4 (ref. 19)) can partially correct hemophilia phenotype in transgenic mice or in lethally irradiated HemA mice treated with ex vivo gene therapy. Thus, FVIII ectopic expression in platelets may be a way to locally deliver protein and concomitantly evade inhibitory antibody responses in treating HemA.

In order to avoid specific challenges posed by ex vivo gene delivery and limit transgene expression to platelets in HemA mice, we treated HemA mice with IO infusions of LVs containing a B-domain variant of human FVIII (hFVIII/N6)20 gene under the control of the strictly megakaryocytic lineage-specific Gp1bα promoter.21 We demonstrate that a single IO infusion of LVs can produce long-term stable expression of hFVIII in platelets and correct hemophilia phenotype for at least 5 months after treatment.

Results

GFP expression in HSCs after IO delivery of M-GFP-LV in mice

In order to evaluate if IO delivery of lentiviral vectors can efficiently transduce BM cells, we first constructed a SIN-LV vector encoding GFP driven by the retroviral-derived, ubiquitous MND promoter (M-GFP-LV, Figure 1a). The M-GFP-LV vector (1.1 × 108 ifu/animal, n = 6) was delivered IO into the tibia of mice at 10 µl/minute using a syringe pump with the goal of retaining LVs in BM for in situ transduction (Figure 1b and Supplementary Figure S1a). Seven days after infusion, a significant GFP signal was observed in the BM of the treated leg using immunofluorescence microscopy (Figure 1c). Flow cytometry demonstrated 15.43 ± 3.97% GFP+ BM cells (Figure 1d,f). In HSCs, defined as Lin-Sca1+c-Kit+ cells, 14.33 ± 4.82% cells expressed GFP (Figure 1e,g). Viral copy number, as assessed using real-time quantitative PCR (qPCR), was 0.48 ± 0.30 copies per BM cell. In a separate experiment, we found that GFP expression levels in BM HSCs, B220+ cells, and CD11c+ cells of the treated leg correlated with the vector doses used (Supplementary Figure S1b). Furthermore, transgene expression levels in HSCs of the treated legs were higher than that in the untreated legs on day 7 after treatment (Supplementary Figure S2a). However, in the long-term (124 days after infusion) follow-up, we found that the percentage of GFP+ HSCs cells in untreated legs was almost equivalent to that in treated legs (P = 0.92) (Supplementary Figure S2b). These results confirmed that direct IO LV infusion can efficiently transduce BM cells including primitive HSCs in vivo.

Figure 1.

Figure 1

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GFP expression in BM cells following IO infusion of M-GFP-LV. C57BL/6 mice were intraosseously delivered with M-GFP-LV (1.1 × 108 ifu/animal, n = 6). (a) Schematic of self-inactivating LV genome encoding GFP under the control of the modified myeloid proliferative sarcoma virus promoter (MND) (M-GFP-LV). (b) Schematic of IO infusion of vectors into the mice with an infusion speed of 10 µl/minute, which was precisely controlled by a programmable microfluidics syringe pump. (c) GFP expression in BM cells of LV-treated mice detected by a fluorescent microscope. Top panel, bright field; bottom panel, green fluorescent signals. (d,f) Flow cytometry analysis of total BM cells isolated from naive or LV-treated mice. Top panel, naive mice; bottom panel, mice treated with LVs. (e,g) GFP expression in Lin-Sca1+c-Kit+ HSCs. Top panels, Lin- cells were gated; middle panels, Lin-Scal+c-Kit+ HSCs were gated; bottom panels, GFP+Lin-Sca1+c-Kit+ HSCs were gated. d and e shown were representative plots of control and treated mice. Data were expressed as mean ± SD. Differences were considered significant at P < 0.01 (**).

IO in vivo transduction and transgene expression of G-GFP-LV

Next, for restricting transgene expression in megakaryocytes, G-GFP-LV containing the platelet-specific Gp1bα promoter was constructed (Figure 2a). Four weeks after IO infusion of G-GFP-LV (5 × 107 ifu/animal, n = 3) or PBS (20 µl/animal, n = 3), BM cells were collected and GFP expression levels in HSCs, B220+ and CD11c+ cells were examined by flow cytometry (Figure 2b). In G-GFP-LV treated mice, no GFP positive HSCs or B220+ or CD11c+ cells were detected, whereas, in mice previously treated with comparable titers of M-GFP-LV, 8.89 ± 6.92% HSCs, 6.12 ± 3.52% B220+, and 8.82 ± 4.15% CD11c+ BM cells expressed GFP (Supplementary Figure S1b, bottom panel). These results indicated that the platelet-specific Gp1bα promoter in G-GFP-LV was inactive in these cell types including the early myeloid progenitors, while the ubiquitous MND promoter in M-GFP-LV directed GFP expression in all three types of cells. This is consistent with the in vitro experimental results that Gp1bα promoter restricted transgene expression specifically in megakaryocyte cell line Dami (Supplementary Figure S3) and the previous observations that Gp1bα promoter is only active in the late differentiated stage of megakaryocytes.21

Figure 2.

Figure 2

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In vivo GFP expression in transduced BM cells and platelets following IO infusion of G-GFP-LV. (a) Schematic of self-inactivating LV genomes encoding GFP under the control of platelet-specific glycoprotein 1bα promoter (Gp1bα) (G-GFP-LV). (b) C57BL/6 mice were intraosseously infused with G-GFP-LV (5 × 107 ifu/animal, n = 3) or PBS (20 µl/animal, mock, n = 3) on day 0. The experimental mice were subsequently sacrificed, their BM cells were isolated, and GFP expression levels in Lin-Sca1+c-Kit+ HSCs (left panel), B220+ (middle panel) and CD11c+ (right panel) cells were detected by flow cytometry on day 31. (c) For long-term follow up, C57/BL6 mice were treated with G-GFP-LV (5 × 107 ifu/animal, n = 5) or PBS (20 µl/animal, mock, n = 3) on day 0. Platelets were isolated from peripheral blood and marked with CD42d+, and their GFP expression levels over 5 months were measured by flow cytometry. Left panel, representative flow data at 5 months after infusion; right panel, summary plot over time. Figures shown were representative of two independent experiments.

Although GFP as an intracellular protein is not stored in α-granules,22 minor amounts of GFP remained in platelets after fragmentation of megakaryocytes. We evaluated GFP expression in platelets of LV-treated mice overtime (Figure 2c). After 161 days, 0.1% persistent GFP expression was observed in CD42d+ platelets in G-GFP-LV treated mice (Figure 2c, left panel). GFP containing platelets were also evaluated after infusion in G-GFP-LV treated (n = 5) or mock (n = 3) mice over time (Figure 2c, right panel). The percentage of GFP containing platelets declined during the first 5 weeks, and then remained at a stable level afterwards. Both HSCs and pre- and pro-megakaryocytes were probably transduced by lentiviral vectors initially and contributed to the higher percentage of GFP-containing platelets during the first 5 weeks. However, only transduced HSCs will contribute to GFP-containing platelets at later time points and maintain the GFP-containing platelets at stable levels. These results indicated that Gp1bα promoter directed restricted and sustained GFP transgene expression in megakaryocytes.

FVIII activity directed by ubiquitous expression of E-F8-LV vectors in vivo

For treating HemA mice, we first constructed a LV encoding hFVIII/N6 cDNA20 capable of limiting anti-FVIII antibody titers23,24 driven by a ubiquitous human elongation factor 1α (EF-1α) promoter (E-F8-LV, Figure 3a). EF-1α promoter, derived from a human housekeeping gene, was chosen to reduce promoter inactivation or silencing in hematopoietic cells. In mice following IO infusion of E-F8-LV (1 × 108 ifu/animal), we detected FVIII expression in HSCs (1.34 ± 1.17%, n = 3) by flow cytometry (Figure 3b and Supplementary Figure S4a,b) and in mouse plasma by Western blot25 (Supplementary Figure S4c), which is consistent with 3–20% (n = 4) circulatory FVIII activity in plasma detected by a modified activated partial thromboplastin time assay (aPTT) (Figure 3c, left panel). FVIII activity subsequently decreased to undetectable levels and this decrease correlated with the appearance of anti-FVIII inhibitory antibodies in most animals detected by hFVIII Bethesda inhibitor assay (Figure 3c, right panel).

Figure 3.

Figure 3

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Circulatory FVIII in plasma of HemA mice following IO infusion of E-F8-LV. (a) Schematic of a self-inactivating LV genome encoding hFVIII variant with the proximal 226 amino acid region of the B-domain (FVIII/N6) under the control of EF-1α promoter (E-F8-LV). (b) E-F8-LV (1 × 108 ifu/animal, n = 3) or PBS (20 µl/animal, mock, n = 2) was intraosseously infused into HemA mice on day 0. hFVIII expression in Lin-Sca1+c-Kit+ HSCs was checked by flow cytometry on day 12 after infusion. Figures shown were representative of two independent experiments. (c) HemA mice were intraosseously infused with E-F8-LV (5 × 107 ifu/animal, n = 4) or PBS (20 µl/animal, mock, n = 3) on day 0. Plasma samples were collected and hFVIII activity and anti-FVIII antibodies were measured by aPTT and Bethesda assay, respectively. In mock mice, the level of hFVIIII activity was corrected as 0% of Normal and the level of anti-FVIII antibodies was 0 Bethesda Unit. Each line represents an individual LV-treated animal. Data shown were representative of four independent experiments.

IO infusion of G-F8-LV into HemA mice mediates platelet-specific FVIII expression

To bypass immune responses against FVIII, we next constructed a LV carrying a hFVIII/N6 cDNA under the control of platelet-specific promoter Gp1bα (G-F8-LV; Figure 4a). G-F8-LV (2.2 × 107 ifu/animal, n = 6) or PBS (20 µl/animal, n = 3) were infused IO into HemA mice. BM and peripheral blood were collected from treated mice, and single cell suspensions were stained intracellularly with anti-FVIII antibody and analyzed by flow cytometry to evaluate FVIII expression. Compared with control mice, there was no detectable hFVIII expression in HSCs (0.28 ± 0.21% versus 0.32 ± 0.02% in controls) on day 8 after treatment (Figure 4b) or in blood CD3ɛ+ and CD11c+ (0.12 ± 0.06% versus 0.14 ± 0.04%), B220+ (0.09 ± 0.04% versus 0.09 ± 0.05%), and CD11b+ (0.08 ± 0.07% versus 0.12 ± 0.04%) cells on day 35 after treatment (Figure 4c) in G-F8-LV treated mice. Furthermore, no FVIII gene expression was observed in HSCs and WBCs at later time points (representative figures on day 91 were shown in Supplementary Figure S5). However, 1.80 ± 1.27% CD42d+ platelets contained hFVIII in HemA mice on day 91 after infusion of G-F8-LV (Figure 4d), and correlated with viral integration number of 0.49 ± 0.42 in total BM cells. These results comply that G-F8-LV transduced HSCs, and over time, these transduced HSCs differentiated into megakaryocytes where the Gp1bα promoter became activated and directed hFVIII gene expression. Subsequently, hFVIII was sorted and stored in α-granules via a regulated secretary pathway.16

Figure 4.

Figure 4

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hFVIII specific expression in CD42d+ platelets. (a) Schematic of a self-inactivating LV genome encoding hFVIII variant with the proximal 226 amino acid region of the B-domain (FVIII/N6) under the control of Gp1bα promoter (G-F8-LV). HemA mice were intraosseously infused with G-F8-LV (2.2 × 107 ifu/animal, n = 6) or PBS (20 µl/animal, mock, n = 3) on day 0. (b) BM cells, (c) white blood cells and (d) platelets from peripheral blood were isolated. hFVIII expression was detected in Lin-Sca1+c-Kit+ HSCs on day 8 (b), in CD3ɛ+, CD11c+, B220+, CD11b+ blood cells on day 35 (c), and in CD42d+ platelets on day 91 (d) by flow cytometry. Representative flow data of two independent experiments were shown in the figures.

Stable, long-term hFVIII expression in platelets of HemA mice treated with IO infusion

To test whether hFVIII stored in platelets enhanced clot formation during bleeding, a single IO infusion of G-F8-LV (2.2 × 107 ifu/animal and 2.2 × 106 ifu/animal) or PBS (20 µl/animal, mock) was given to HemA mice. In high-titer G-F8-LV recipients (n = 8), the average percentage of CD42d+ platelets containing hFVIII was 2% on day 27, expression declined slightly on day 84, and stabilized at ~2% by day 160 (Figure 5a). The average percentage of hFVIII containing CD42d+ platelets in the recipients of low-titer LV (n = 5) was about half of that in high-titer LV treated mice and followed a similar expression profile. In contrast, negligible levels of CD42d+hFVIII+ platelets (<0.2% due to nonspecific staining) was observed in mock treated HemA mice. Next, hFVIII antigen level in platelets on day 112 was measured by ELISA (Figure 5b). For the mice treated with high-titer LV (n = 5), the average hFVIII antigen level was 1 mU (for hFVIII, 1 mU = 0.1 ng/ml) per 1 × 108 platelets which is comparable to platelet FVIII:C described in 2bF8trans HemA mice evaluated by chromogenic assay.16 The average hFVIII antigen in low-titer LV treated mice (n = 5) was 0.3 mU per 1 × 108 platelets, slightly lower than the high-titer LV recipients (P = 0.17).

Figure 5.

Figure 5

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Long-term stable hFVIII levels in platelets were obtained in HemA mice after a single IO infusion of G-F8-LVs and their phenotype was corrected. HemA mice were given IO infusion of G-F8-LV (2.2 × 107 ifu/animal or 2.2 × 106 ifu/animal) or PBS (20 µl/animal, mock) on day 0. (a) Platelets were isolated from peripheral blood of high- (n = 8) or low- (n = 5) titer G-F8-LV treated or mock (n = 3) mice. hFVIII expression levels in CD42d+ platelets were evaluated by flow cytometry on day 27, 62, 84, 112 and 160. (b) hFVIII levels in platelet lysates of high (n = 5) or low (n = 5) titer LV-treated mice or mock (n = 3) were measured by ELISA on day 112. (c) HemA phenotype correction of G-F8-LV treated mice was monitored by tail clip assay on day 35, 118, and 160 (n = 4–7/group). The average blood loss of untreated HemA mice was set as 100%. Wild-type C57BL/6 mice were used as positive controls. (d) Plasma samples were collected from high-titer G-F8-LV treated (n = 10) or mock (n = 3) mice, and hFVIII activity and anti-FVIII antibodies were measured by aPTT and Bethesda assay, respectively. For mock mice, the level of hFVIIII activity was corrected as 0% of normal and the level of anti-FVIII antibodies was 0 Bethesda unit. Each symbol represented an individual animal. Data were expressed as mean ± SD. Differences were considered significant at P < 0.05 (*) and P < 0.01 (**). n.s. was an abbreviation for non-significant. Data shown were from two independent experiments.

LV-treated HemA mice were subsequently examined for phenotypic correction of bleeding diathesis by tail clip assay (Figure 5c). In the high-titer group, the average blood loss was 41% (n = 7), 48% (n = 5), and 33% (n = 5) compared with mocked treated HemA (~100%), and wild-type (2.5%) mice on days 35, 118, and 160, respectively. In the low-titer group, the average percentage of the blood loss was 43% (n = 7), 54% (n = 4), and 42% (n = 4) on days 35, 118, and 160, respectively. Overall, blood loss did not differ significantly between the high versus low-titer recipients (P = 0.08 on day 35, P = 0.27 on day 118, and P = 0.24 on day 160). Blood loss for both groups was significantly different from the HemA controls, indicating that treatment with G-F8-LV significantly enhanced clot formation during bleeding.

Plasma hFVIII activity and anti-hFVIII antibodies in mice treated with high-titer G-F8-LV (n = 10) were examined on day 160 (Figure 5d). There was neither detectable hFVIII activity nor anti-hFVIII antibodies in blood, indicating that hFVIII was not expressed in lineages other than megakaryocytes and that hFVIII was not released systemically from storage in platelets, thereby limiting the immune response. Thus, single IO infusion of G-F8-LV is sufficient to achieve long-term, partial correction of the HemA phenotype in mice.

Platelet FVIII expression mediates phenotypic correction of HemA despite pre-existing inhibitors

We next examined whether hFVIII stored in platelets could overcome anti-hFVIII antibodies and correct the hemophilia phenotype following IO infusion of G-F8-LV. “Inhibitor” mice were established by repeated intraperitoneal injections of 3U recombinant hFVIII (Figure 6a). Average antibody titers in these animals were 83 ± 56 Bethesda Unit (BU). A single dose of G-F8-LV (2.2 × 107 ifu/animal or 2.2 × 106 ifu/animal) was delivered IO to inhibitor mice. hFVIII antigen levels were measured by ELISA on day 27 after infusion. In high-titer LV recipients, the average hFVIII antigen level was 0.74 mU per 108 platelets (n = 5) (Figure 6b), significantly greater than levels in lower-titer recipients (0.14 mU per 108 platelets, n = 5; data not shown). Phenotypic correction of inhibitor mice was assessed by tail clip assay on day 160 (Figure 6c). While anti-hFVIII antibody levels did not change after IO treatment (112 ± 77 BU), average blood loss significantly improved to 39% (n = 7) in high-titer group mice compared with control HemA (100%, n = 10) and wild-type mice (12%, n = 8). Average blood loss was 80% (n = 3) in low-titer group mice (data not shown). Average viral copy number was 0.66 in the BM cells derived from high-titer recipient mice. These results indicate that platelet-derived FVIII can correct the HemA phenotype despite of the presence of inhibitory antibodies.

Figure 6.

Figure 6

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hFVIII expression in platelets of G-F8-LV treated inhibitor HemA mice corrected their hemophilia A phenotype. (a) Inhibitor HemA mice were established by repeated intraperitoneal injection (3x/week for 2 weeks) of 3U rhFVIII into 10- to 12-week-old HemA mice. These inhibitor HemA mice were then intraosseously infused with G-F8-LV (2.2 × 107 ifu/animal) or PBS (20 µl/animal, mock) on day 0. (b) Platelets were isolated from peripheral blood of LV-treated (n = 5) and mock (n = 3) mice and lysed. The resulting lysate was examined for hFVIII expression level by ELISA on day 27 after infusion. (c) The phenotypic correction of G-F8-LV treated HemA inhibitor mice (n = 7) was examined by tail clip assays on day 160 after infusion. The average blood loss of untreated HemA (n = 10) mice was set as 100%. Wild-type C57BL/6 mice (n = 8) were used as positive controls. Data shown were from two independent experiments. Data were expressed as mean ± SD. Differences were considered significant at P < 0.05 (*) and P < 0.01 (**).

Discussion

Hemophilia is an attractive target for gene therapy since there is a wide therapeutic window wherein a small rise (1% of normal) in circulating clotting factors can provide a significant clinical benefit.26 Recent clinical trial using adenovirus-associated virus (AAV) vector mediated gene transfer of human factor IX under the control of a liver-specific promoter resulted in encouraging results in HemB patients.27 Furthermore, single dose intravenous administration of liver-directed AAV vector encoding a codon-optimized hFVIII variant in mice or macaques generated therapeutic levels of hFVIII.28 The duration of clinical benefit using this approach in a clinical setting remains to be determined. Compared with AAVs that persist as episomal, concatemerized vectors in vivo, LV transduction results in integration of the therapeutic gene into the host cell genome, leading to persistent expression in a range of genetic disorders. If successful, repeated treatment may not be needed. Unlike gamma retroviral vectors that only target dividing cells, LVs can transduce both nondividing and dividing cells, leading to more efficient targeting of stem cells. In addition, self-inactivating (SIN)-LVs offer improved safety by incorporating SIN LTR elements that exhibit reduced transactivation capacity29 and permit incorporation of enhancer-less internal promoters.30 Two recent clinical trials in Wiskott-Aldrich Syndrome (WAS)31 and Metachromatic leukodystrophy (MLD)32 patients showed significant improvement in their clinical scores following ex vivo LV gene therapy. Importantly, integration analyses showed no evidence of aberrant clonal expansion.

Although ex vivo HSC transduction/transplantation protocols can successfully deliver FVIII into HemA mice,7,17 the procedure requires preconditioning using potentially toxic, myelosuppressive agents. In situ transduction of HSCs is more feasible for hemophilia treatment in clinics because it doesn't require this step. Furthermore, no manipulation of stem cells would be needed. Moreover, in contrast to intravenous administration that distributes LVs into circulating blood, IO infusion focused delivery to BM microenvironment and increases the efficiency of HSC LV transduction. Previous results of clony-forming unit assay and a duplex real-time PCR assay confirmed successful LV-mediated gene transfer and gene expression in hematopoietic stem/progenitor cells using a GFP-LV vector driven by a ubiquitous EF1α promoter in primary LV treated mice and secondary BMT recipients following IO infusion.10 LV transduction is observed mainly in HSCs with minor transduction in other tissues. The observed marking in the liver, spleen and lung at 4 months after transduction may not necessarily correspond to transduced parachymal cells but could be from gene-marked myeloid or lymphoid cells that homed or trapped into these tissues. Our qRT-PCR data obtained from mice treated with IO infusion of G-F8-LV showed that very little expression of FVIII was detected in these tissues (Supplementary Figure S6) due to the use of a megakaryocyte-specific promoter. Moreover, IO infusion resulted in phenotypic correction of Fanconi anemia in a preclinical animal model in the absence of preconditioning.11 In the current study, we used a pump to infuse LV into the BM with the goal of more efficiently targeting marrow cells. Our results showed that following IO infusion of M-GFP-LVs, average of 30% HSCs were transduced and expressed GFP up to 160 days (Supplementary Figure S2c), indicating that IO LV infusion can efficiently transduce primitive progenitor cells in BM. In addition, as shown in Supplementary Figure S2a,b, transduced cells in the control untreated leg were initially significantly lower than the treated leg, whereas at later time points, comparable numbers of transduced cells were observed at both sites; findings consistent with transit of transduced HSCs from treated to untreated BM sites over time. Taken together, these results further support the conclusion that IO infusion of LVs mediates sustained transduction of hematopoietic stem/progenitor cells.

We compared IO delivery of LVs expressing FVIII utilizing two different promoters, a ubiquitous EF-1α promoter and a human megakaryocytic-specific GP1bα promoter. EF-1α promoter from a house keeping gene was used to reduce promoter silencing. We chose to incorporate GP1bα promoter in the LV as this element is specifically active late during megakaryocyte differentiation. Following LV transduction, the GP1bα promoter was megakaryocyte-specific, whereas the alternative platelet active GPIIb promoter is more leaky with activity in immature megakaryocyte progenitor cells.21 Of note we also incorporated a cDNA coding for hFVIII/N6 (ref. 20) as this construct limits anti-FVIII antibody titers.23,24 Despite efficient transduction of HSCs following E-F8-LV IO delivery, robust anti-FVIII immune responses were induced that eliminated functional FVIII in the circulation. In contrast, persistent platelet-specific expression of FVIII was obtained following a single IO delivery of G-F8-LV, leading to long-term, partial correction of HemA phenotype. Interestingly, neither hFVIII activity in plasma nor anti-hFVIII antibodies was detected up to 160 days after LV infusion, implying that this construct led to little or no systemic hFVIII expression. Our results are also consistent with previous work in the HemA model using the Gp1bα promoter including transgenic animals expressing human B-domain deleted FVIII18 and animals treated with ex vivo gene therapy using simian immunodeficiency virus (SIV)-based LVs expressing hFVIII.33 The latter study also demonstrated that FVIII colocalized with vWF in platelets. Our data confirmed that Gp1bα also specifically drives FVIII expression in platelets following IO infusion and that FVIII stored in platelets corrects the HemA phenotype.

Following IO delivery, FVIII driven by platelet-specific promoters is synthesized and stored in up to 3% of platelets. Notably, during thrombopoiesis, α-granule proteins including hFVIII are not evenly distributed in platelets34 and flow cytometry may underestimate hFVIII levels. Although the timing or kinetics of clot formation mediated via platelet delivered FVIII is temporally and spatially different from plasma FVIII, both delivery routes were effective in clot response to laser injury in cremaster arterioles and venules in mice.35 Notably, phenotypic correction of HemA mice treated with platelet-targeted gene therapy has previously been assessed using a range of injury models including tail bleeding, digital cuticular bleeding, ferric chloride-induced arterial injury, and survival assay.15,16,17,18 Our results clearly show that even low levels of FVIII released from platelets can partially correct the HemA phenotype. We anticipate that increasing transgene expression using variant FVIII cDNA with higher expression levels including codon optimized FVIII28,36,37 is likely to further enhance the therapeutic effect of platelet delivery following IO treatment. In addition, while the results from bleeding assays suggest that the platelet-stored FVIII can restore hemostasis, there are limitations to the mouse models. Ultimately, clinical trials in human need to demonstrate a reduction in incidence of spontaneous bleeds (such as joint bleeds).

Inhibitory antibody production remains a challenging problem following FVIII protein or gene therapy for hemophilia. Extensive previous work has focused on developing immunomodulation protocols38 or in tailoring vectors (including promoter and envelope) and delivery route39,40 to achieve long-term tolerance to FVIII in this setting. Shi et al.15,41 showed that transplantation of transgenic or gene modified HSCs that express FVIII in platelets can improve hemostasis in mice with pre-existing inhibitory antibodies. However this approach required either myeloablative or reduced intensity marrow conditioning regimens for successful application. In this study, we demonstrate that a single G-F8-LV IO in fusion led to significant functional FVIII activity despite of pre-existing high-titer anti-FVIII antibodies in the circulation. Thus, FVIII ectopically expressed and stored in platelet α-granules and released at the sites of injury represents a promising potential strategy to HemA patients with high-titer inhibitor antibodies, including individuals who have previously been excluded from clinical gene therapy trials. The IO infusion technique is already performed in humans for delivery of drugs. The technology can be readily translated to large animal models and eventually to humans.

In conclusion, we achieved effective platelet-specific hFVIII expression in HemA mice following IO infusion of LVs containing FVIII under the control of the Gp1bα promoter. Platelets generated from transduced HSC exhibited sustained FVIII expression, leading to partial phenotypic correction in animals both with and without pre-existing inhibitory antibodies. This approach may provide a novel and readily translatable treatment for hemophilia.

Materials and Methods

Animals. Mice were housed according to the guidelines of National Institutes of Health and Seattle Children's Research Institute (SCRI). The protocols were approved by the Institutional Animal Care and Use Committee at SCRI. HemA mice with C57BL/6 genetic background were generated as previously described.42

Lentiviral vector construction and production. pRRLSIN·cPPT·MCS·WPRE and pRRLSIN·MND·eGFP·WPRE (M-GFP construct) were obtained from the SCRI viral core. Promoters (the human Gp1bα promoter (from -274 to 54) amplified from genomic DNA and a ubiquitous EF-1α-short promoter) and transgenes (hFVIII/N6 (ref. 20) kindly provided by Steven Pipe at University of Michigan and eGFP) were inserted into pRRLSIN·cPPT·MCS·WPRE at NheI site, and between XhoI and SpeI sites, respectively, to generate pRRLSIN·Gp1bα·hFVIII/N6·WPRE (G-F8 construct), pRRLSIN·EF-1α·hFVIII/N6·WPRE (E-F8 construct), and pRRLSIN·Gp1bα·eGFP·WPRE (G-GFP construct). All reagents for cloning were purchased from Qiagen (Valencia, CA). The LVs (M-GFP-LV, G-GFP-LV, E-F8-LV and G-F8-LV) were produced by transient transfection of three plasmids using polyethylenimine in 293T cells and concentrated by ultracentrifugation.43 Viral titers were determined by infection in Nalm-6 cells, followed by qPCR and expressed as ifu/ml44 (see details in Supplementary Materials and Methods).

Mammalian cell transduction. A human megakaryocyte cell line (Dami),45 a human T cell lymphoblast-like cell line (Jurkat) and a human pre-B cell line (Nalm-6) were transduced with LVs and transgene expression was evaluated using a flow cytometer (LSRII, BD Biosciences, San Jose, CA) (see details in Supplementary Materials and Methods).

IO infusion of LVs into mice. IO infusion procedure was modified from a previously reported protocol.10 Briefly, a 27-gauge needle was twisted into the tibia through the joint of an anesthetized mouse. LVs loaded in a Microliter syringe (Hamilton, Reno, NV) connected to the needle via short sterilized ultra-thin tubing were infused into the mice. The infusion speed was precisely controlled by a syringe pump (NE-1010, New Era Pump Systems, Farmingdale, NY). The optimal infusion volume (20 µl) was determined by a titration experiment using trypan blue (see details in Supplementary Materials and Methods). Viral copy numbers in BM cells of treated mice were determined by the qPCR method as previously described.46

Immunostaining and flow cytometric analysis. For M-GFP-LV or G-GFP-LV treated mice, BM cells were stained for HSC surface markers (Lineage (Lin), Sca1 and c-Kit), B220 and CD11c. Platelets isolated from peripheral blood were stained for surface marker CD42d. For E-F8-LV or G-F8-LV treated mice, after surface staining, samples were fixed and stained with mouse anti-hFVIII monoclonal antibody (ESH8, American Diagnostica, Seabrook, TX) or isotype control (puried mouse IgG2α, BD Biosciences), and secondary antibody goat anti-mouse Ig FITC (BD Biosciences). All surface marker antibodies were purchased from eBioscience (San Diego, CA). Flow cytometric analysis was conducted using LSRII or Calibur (BD Bioscience) and the data were analyzed using FlowJo 8.8.1 (Ashland, OR).

Assays for measuring hFVIII activities and anti-hFVIII antibodies. hFVIII activities in mouse plasma were analyzed using aPTT and anti-hFVIII antibodies were measured by hFVIII Bethesda inhibitor assay as previously described.42,47 The aPTT values were confirmed by a chromogenic assay (Chromogenix Coatest SP Factor VIII; Diapharma, West Chester, OH) as shown in Supplementary Figure S4d. hFVIII expression in mouse plasma after E-F8-LV treatment was also detected by Western blot assay (see the detailed method in Supplementary Materials and Methods).

Assessment of hFVIII levels in platelets. An enzyme-linked immunosorbent assay (ELISA) was used to determine hFVIII:Ag levels in platelets (see the detailed method in Supplementary Materials and Methods).

Tail clip assay. Correction of the bleeding phenotype of HemA mice was measured using a modified tail clip assay. Briefly, the tail of the anesthetized mouse was first incubated in 0.9% saline in a 15 ml tube at 37 °C for 2 minute. The distal part of the tail at a 2- to 3-mm diameter was then cut and placed back immediately into the warm saline and allowed to bleed without disturbance for 10 minute. The tail was then cauterized to prevent further bleeding. The collected blood cells were lysed with ACK buffer and centrifuged. The hemoglobin content in the resulting supernatant was measured using a Victor spectrophotometer (PerkinElmer, Santa Clara, CA) at 560 nm.

Statistical analysis. Data were expressed as mean ± SD. The statistical significance of the data between different groups of animals was evaluated by a two-tailed Student's t test. Differences were considered significant at P < 0.05 (*) and P < 0.01 (**).

SUPPLEMENTARY MATERIAL Figure S1. In vivo GFP expression in transduced bone marrow cells following IO infusion of M-GFP-LV. Figure S2. In vivo GFP expression in BM HSCs after IO infusion of M-GFP-LV. Figure S3. Comparison of LVs encoding GFP driven by two different promoters. Figure S4. In vitro and in vivo hFVIII expression in E-F8-LV transduced 293T cells or E-F8-LV infused HemA mice. Figure S5. Long-term expression of hFVIII in bone marrow HSCs and blood cells in G-F8-LV treated HemA mice. Figure S6. hFVIII mRNA expression in liver, spleen and lung in G-F8-LV or pBS-HCRHPI-FVIIIA treated HemA mice. Materials and Methods.

Acknowledgments

Research reported in this publication was supported by the NHLBI of the National Institutes of Health under award numbers: R21 HL112148 (C.H.M.) and 5PL1HL092557 (D.J.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Steven Pipe for providing us the hFVIII/N6 cDNA and Liping Chen, Joseph Pangallo and Meghan Lyle for their technical help. The authors declare no conflict of interest. X.W. designed and performed research, collected and analyzed data and wrote the paper. S.C.S., A.C., and I.K. performed research. D.P. provided suggestions. D.J.R. provided suggestions in project design, reviewed data, and revised the paper. C.H.M. designed the project and research, analyzed data and wrote the paper.

Supplementary Material

Supplementary Figures
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Supplementary Materials
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References

  1. Gringeri A, Mantovani LG, Scalone L, Mannucci PM. COCIS Study Group Cost of care and quality of life for patients with hemophilia complicated by inhibitors: the COCIS Study Group. Blood. 2003;102:2358–2363. doi: 10.1182/blood-2003-03-0941. [DOI] [PubMed] [Google Scholar]
  2. 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;344:1735–1742. doi: 10.1056/NEJM200106073442301. [DOI] [PubMed] [Google Scholar]
  3. Powell JS, Ragni MV, White GC, 2nd, Lusher JM, Hillman-Wiseman C, Moon TE, et al. Phase 1 trial of FVIII gene transfer for severe hemophilia A using a retroviral construct administered by peripheral intravenous infusion. Blood. 2003;102:2038–2045. doi: 10.1182/blood-2003-01-0167. [DOI] [PubMed] [Google Scholar]
  4. White GC., 2nd Gene therapy in hemophilia: clinical trials update. Thromb Haemost. 2001;86:172–177. [PubMed] [Google Scholar]
  5. Evans GL, Morgan RA. Genetic induction of immune tolerance to human clotting factor VIII in a mouse model for hemophilia A. Proc Natl Acad Sci USA. 1998;95:5734–5739. doi: 10.1073/pnas.95.10.5734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Moayeri M, Hawley TS, Hawley RG. Correction of murine hemophilia A by hematopoietic stem cell gene therapy. Mol Ther. 2005;12:1034–1042. doi: 10.1016/j.ymthe.2005.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ide LM, Gangadharan B, Chiang KY, Doering CB, Spencer HT. Hematopoietic stem-cell gene therapy of hemophilia A incorporating a porcine factor VIII transgene and nonmyeloablative conditioning regimens. Blood. 2007;110:2855–2863. doi: 10.1182/blood-2007-04-082602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doering CB, Gangadharan B, Dukart HZ, Spencer HT. Hematopoietic stem cells encoding porcine factor VIII induce pro-coagulant activity in hemophilia A mice with pre-existing factor VIII immunity. Mol Ther. 2007;15:1093–1099. doi: 10.1038/sj.mt.6300146. [DOI] [PubMed] [Google Scholar]
  9. McCauslin CS, Wine J, Cheng L, Klarmann KD, Candotti F, Clausen PA, et al. In vivo retroviral gene transfer by direct intrafemoral injection results in correction of the SCID phenotype in Jak3 knock-out animals. Blood. 2003;102:843–848. doi: 10.1182/blood-2002-12-3859. [DOI] [PubMed] [Google Scholar]
  10. Worsham DN, Schuesler T, von Kalle C, Pan D. In vivo gene transfer into adult stem cells in unconditioned mice by in situ delivery of a lentiviral vector. Mol Ther. 2006;14:514–524. doi: 10.1016/j.ymthe.2006.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Habi O, Girard J, Bourdages V, Delisle MC, Carreau M. Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer. Anemia. 2010;2010:pii: 947816. doi: 10.1155/2010/947816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Baum C, Düllmann J, Li Z, Fehse B, Meyer J, Williams DA, et al. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood. 2003;101:2099–2114. doi: 10.1182/blood-2002-07-2314. [DOI] [PubMed] [Google Scholar]
  13. Pan D, Gunther R, Duan W, Wendell S, Kaemmerer W, Kafri T, et al. Biodistribution and toxicity studies of VSVG-pseudotyped lentiviral vector after intravenous administration in mice with the observation of in vivo transduction of bone marrow. Mol Ther. 2002;6:19–29. doi: 10.1006/mthe.2002.0630. [DOI] [PubMed] [Google Scholar]
  14. Michelson AD. How platelets work: platelet function and dysfunction. J Thromb Thrombolysis. 2003;16:7–12. doi: 10.1023/B:THRO.0000014586.77684.82. [DOI] [PubMed] [Google Scholar]
  15. Kuether EL, Schroeder JA, Fahs SA, Cooley BC, Chen Y, Montgomery RR, et al. Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity. J Thromb Haemost. 2012;10:1570–1580. doi: 10.1111/j.1538-7836.2012.04791.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shi Q, Wilcox DA, Fahs SA, Weiler H, Wells CW, Cooley BC, et al. Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies. J Clin Invest. 2006;116:1974–1982. doi: 10.1172/JCI28416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shi Q, Wilcox DA, Fahs SA, Fang J, Johnson BD, DU LM, et al. Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A. J Thromb Haemost. 2007;5:352–361. doi: 10.1111/j.1538-7836.2007.02346.x. [DOI] [PubMed] [Google Scholar]
  18. Yarovoi HV, Kufrin D, Eslin DE, Thornton MA, Haberichter SL, Shi Q, et al. Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment. Blood. 2003;102:4006–4013. doi: 10.1182/blood-2003-05-1519. [DOI] [PubMed] [Google Scholar]
  19. Damon AL, Scudder LE, Gnatenko DV, Sitaraman V, Hearing P, Jesty J, et al. Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice. Thromb Haemost. 2008;100:1111–1122. doi: 10.1160/th08-04-0242. [DOI] [PubMed] [Google Scholar]
  20. Pipe SW. Functional roles of the factor VIII B domain. Haemophilia. 2009;15:1187–1196. doi: 10.1111/j.1365-2516.2009.02026.x. [DOI] [PubMed] [Google Scholar]
  21. Lavenu-Bombled C, Izac B, Legrand F, Cambot M, Vigier A, Massé JM, et al. Glycoprotein Ibalpha promoter drives megakaryocytic lineage-restricted expression after hematopoietic stem cell transduction using a self-inactivating lentiviral vector. Stem Cells. 2007;25:1571–1577. doi: 10.1634/stemcells.2006-0321. [DOI] [PubMed] [Google Scholar]
  22. Briquet-Laugier V, Lavenu-Bombled C, Schmitt A, Leboeuf M, Uzan G, Dubart-Kupperschmitt A, et al. Probing platelet factor 4 alpha-granule targeting. J Thromb Haemost. 2004;2:2231–2240. doi: 10.1111/j.1538-7836.2004.01037.x. [DOI] [PubMed] [Google Scholar]
  23. Cerullo V, Seiler MP, Mane V, Cela R, Clarke C, Kaufman RJ, et al. Correction of murine hemophilia A and immunological differences of factor VIII variants delivered by helper-dependent adenoviral vectors. Mol Ther. 2007;15:2080–2087. doi: 10.1038/sj.mt.6300308. [DOI] [PubMed] [Google Scholar]
  24. Jirovska D, Ye PQ, Pipe SW, Miao CH. Reduction of inhibitory anti-FVIII antibody titer by using a B domain vairant FVIII/N6 cDNA for nonivral gene therapy in hemophilia A mice. Blood. 2008;112:1212. [Google Scholar]
  25. Krishnan S, Kolbe HV, Lepage P, Faure T, Sauerwald R, de la Salle H, et al. Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II. Eur J Biochem. 1991;195:637–644. doi: 10.1111/j.1432-1033.1991.tb15748.x. [DOI] [PubMed] [Google Scholar]
  26. High KA. Gene transfer as an approach to treating hemophilia. Semin Thromb Hemost. 2003;29:107–120. doi: 10.1055/s-2003-37945. [DOI] [PubMed] [Google Scholar]
  27. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011;365:2357–2365. doi: 10.1056/NEJMoa1108046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McIntosh J, Lenting PJ, Rosales C, Lee D, Rabbanian S, Raj D, et al. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood. 2013;121:3335–3344. doi: 10.1182/blood-2012-10-462200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhou S, Mody D, DeRavin SS, Hauer J, Lu T, Ma Z, et al. A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells. Blood. 2010;116:900–908. doi: 10.1182/blood-2009-10-250209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Knight S, Bokhoven M, Collins M, Takeuchi Y. Effect of the internal promoter on insertional gene activation by lentiviral vectors with an intact HIV long terminal repeat. J Virol. 2010;84:4856–4859. doi: 10.1128/JVI.02476-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Aiuti A, Biasco L, Scaramuzza S, Ferrua F, Cicalese MP, Baricordi C, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science. 2013;341:1233151. doi: 10.1126/science.1233151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science. 2013;341:1233158. doi: 10.1126/science.1233158. [DOI] [PubMed] [Google Scholar]
  33. Ohmori T, Mimuro J, Takano K, Madoiwa S, Kashiwakura Y, Ishiwata A, et al. Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibalpha promoter: in vivo model for platelet-targeting gene therapy. FASEB J. 2006;20:1522–1524. doi: 10.1096/fj.05-5161fje. [DOI] [PubMed] [Google Scholar]
  34. Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical correlates. Blood Rev. 2009;23:177–189. doi: 10.1016/j.blre.2009.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Neyman M, Gewirtz J, Poncz M. Analysis of the spatial and temporal characteristics of platelet-delivered factor VIII-based clots. Blood. 2008;112:1101–1108. doi: 10.1182/blood-2008-04-152959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ward NJ, Buckley SM, Waddington SN, Vandendriessche T, Chuah MK, Nathwani AC, et al. Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood. 2011;117:798–807. doi: 10.1182/blood-2010-05-282707. [DOI] [PubMed] [Google Scholar]
  37. Siner JI, Iacobelli NP, Sabatino DE, Ivanciu L, Zhou S, Poncz M, et al. Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype. Blood. 2013;121:4396–4403. doi: 10.1182/blood-2012-10-464164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Scott DW, Pratt KP, Miao CH. Progress toward inducing immunologic tolerance to factor VIII. Blood. 2013;121:4449–4456. doi: 10.1182/blood-2013-01-478669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Matsui H, Shibata M, Brown B, Labelle A, Hegadorn C, Andrews C, et al. A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells. Blood. 2009;114:677–685. doi: 10.1182/blood-2009-03-202267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sack BK, Merchant S, Markusic DM, Nathwani AC, Davidoff AM, Byrne BJ, et al. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy. PLoS ONE. 2012;7:e37671. doi: 10.1371/journal.pone.0037671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shi Q, Fahs SA, Wilcox DA, Kuether EL, Morateck PA, Mareno N, 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;112:2713–2721. doi: 10.1182/blood-2008-02-138214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Miao CH, Harmeling BR, Ziegler SF, Yen BC, Torgerson T, Chen L, et al. CD4+FOXP3+ regulatory T cells confer long-term regulation of factor VIII-specific immune responses in plasmid-mediated gene therapy-treated hemophilia mice. Blood. 2009;114:4034–4044. doi: 10.1182/blood-2009-06-228155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Toledo JR, Prieto Y, Oramas N, Sánchez O. Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors. Appl Biochem Biotechnol. 2009;157:538–544. doi: 10.1007/s12010-008-8381-2. [DOI] [PubMed] [Google Scholar]
  44. Kerns HM, Ryu BY, Stirling BV, Sather BD, Astrakhan A, Humblet-Baron S, et al. B cell-specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia. Blood. 2010;115:2146–2155. doi: 10.1182/blood-2009-09-241869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Greenberg SM, Rosenthal DS, Greeley TA, Tantravahi R, Handin RI. Characterization of a new megakaryocytic cell line: the Dami cell. Blood. 1988;72:1968–1977. [PubMed] [Google Scholar]
  46. Sather BD, Ryu BY, Stirling BV, Garibov M, Kerns HM, Humblet-Baron S, et al. Development of B-lineage predominant lentiviral vectors for use in genetic therapies for B cell disorders. Mol Ther. 2011;19:515–525. doi: 10.1038/mt.2010.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Kasper CK, Aledort L, Aronson D, Counts R, Edson JR, van Eys J, et al. Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. 1975;34:612. [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary Figures
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Supplementary Materials
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