Platelet-targeted Gene Therapy Research Articles

Gene Therapy Expressing Platelet-Derived Factor VIII for Correction of Hemophilia Α with a History of Inhibitors

Introduction: Platelet targeted gene therapy with human factor VIII has established hemostasis in murine and canine models with severe hemophilia A withouteliciting inhibitory antibodies. Here-in we describe feasibility, safety and efficacy of the first subject treated on a first-in-human phase 1 trial that targets factor FVIII synthesis and storage within platelet α-granules for delivery at the site of vascular injury for hemophilia A (NCT03818763). Method: Eligible are severe hemophilia A patients ≥18 years with a history of inhibitors to factor VIII (≥0.6 Bethesda Units [BU]/ml). Following mobilization, autologous CD 34+ cells (≥6 x 10 6/kg) are collected and transduced with a lentiviral vector encoding the ITGA2B gene promoter for ectopic expression of human B-domain-deleted factor VIII within the megakaryocyte lineage. The transduced cells are infused after reduced intensity cytoreduction with fludarabine (120 mg/m 2) and melphalan (120 mg/m 2) followed by a washout period of ≥24 hours. The primary endpoints of feasibility and safety are defined as: 1) feasibility of the cell manufacturing procedure by the availability of ≥4 x 10 6/kg transduced clinical grade CD34+cells, cell viability ≥70% and undetectable microbial contamination and 2) safety defined as hematopoietic recovery ≤28 days of infusion and absence of ≥ grade 3 toxicity (CTCAE version 5.0). Outcome: Subject 1 is a 29-year-old male with severe hemophilia A who developed inhibitors to factor VIII (2.6 BU/ml) in his first year of life. Immune tolerance to factor VIII had been established with a non-detectable inhibitor titer at enrollment. Hemostatic prophylaxis was maintained with emicizumab weekly and recombinant factor VIII for breakthrough bleeding. His annualized bleeding rate was 16 in the 12-month period preceding infusion. The cell product was the result of 1 day of collection yielding 6.73 x 10 6 /kg total viable CD34+ cells post-transduction and vector copy number by qPCR of 1.16 copies/cell. Megakaryocyte factor VIII:C levels were 0.00 mU/10 6 before transduction and 101.32 mU/10 6 after transduction. The cell product satisfied release criteria. Neutrophil recovery (≥0.5 x 10 9/L) and platelet transfusion independence (≥50 x 10 9/L) was achieved 15 days post-infusion and sustained for >12 months. The duration of hospitalization was 21 days with no re-admissions. There was no breakthrough bleeding during collection and hospitalization. During and post-infusion there was no unexpected toxicity ≥ grade 1. He has not required immune suppression. Emicizumab was discontinued 3.6 months post-infusion after demonstration of whole blood vector copy number at 1 and 3-months. There has been no spontaneous bleeding or a need for “on demand” factor VIII after discontinuing emicizumab. Secondary outcomes are summarized in Table 1. No replication competent lentivirus was detected through month 12 post-infusion. Integration site analysis was carried out, and none of the samples tested through 12 months contained cell clones which exceeded 20% relative abundance. Transduced cells were highly polyclonal. Integration site analysis showed no enrichment of integration near cancer-associated genes in the cell product nor in whole blood cell lineages post-infusion. Conclusion: We report feasibility, safety and efficacy in the first subject with severe hemophilia A and a history of inhibitors who received lentiviral vector gene therapy directed to induce megakaryocytes to synthesize and store factor VIII within platelets. These findings extend the potential to treat severe hemophilia A patients who are not eligible for valoctcogene roxaoarvovec (AAV5-hFVIII-SQ).

Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

Abstract The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model. FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period. We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway. In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. Disclosures No relevant conflicts of interest to declare.

Platelet-Targeted Gene Transfer Induces Immune Tolerance in a Primed Model

Abstract Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) is effective in inducing immune tolerance not only to the coagulation factor VIII and IX, but also to the non-coagulant protein ovalbumin (OVA) in a model without pre-existing immunity (the non-primed model). In the present study, we explored whether platelet-specific gene transfer could induce immune tolerance in the primed model. To address this question, we first used C57BL6 wild type (WT) mouse models. Both donors and recipients were immunized with OVA to induce anti-OVA antibody development. OVA expression was introduced by 2bOVA lentivirus (LV) transduction of hematopoietic stem cells (HSCs) from WT/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with a sub-lethal 6.6Gy total body irradiation (TBI). 2bGFP was used as a control vector in parallel. Animals were analyzed after at least 4 weeks of bone marrow (BM) reconstitution. The viability of OVA genetically modified HSCs was confirmed by PCR in all 2bOVA-transduced recipients. After full bone marrow constitution, which takes about 12 weeks, the engraftment in the 2bOVA group (87.4±4.4%, n=5) was similar to that in the 2bGFP group (87.8±3.9%, n=5) as determined by flow cytometry. The OVA protein level was 6.7±2.8 ng/108platelets (n=5) in 2bOVA transduced recipients. Anti-OVA antibody titers declined with time in both groups. Six months after transplantation, recipients were rechallenged with OVA. All of the anti-OVA titers in 2bGFP-transduced recipients (n=5) increased dramatically, while only one in the 2bOVA group slightly increased. Normalized anti-OVA antibody titers in the 2bOVA group (90.8±152.7) were significantly lower than those in the 2bGFP group (1278±2101) after OVA rechallenge, demonstrating that platelet-specific OVA gene delivery to HSCs can suppress anti-OVA immune responses in OVA-primed WT mice. To explore how immune suppression is established after platelet-specific gene transfer in the OVA-primed model, we transduced HSCs from OVA-specific TCR transgenic mice (OTII/CD45.2, in which greater than 97% CD4 T+ cells are OVA-specific) with 2bOVALV and transplanted into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy TBI. Untransduced transplanted mice were used as a control. After BM reconstitution, the OVA protein level was 32.3±6.3 ng/108 platelets (n=5) in the 2bOVA-transduced group. Anti-OVA antibody titers in both groups declined with time. When all the recipients were rechallenged with OVA, 2 of 3 anti-OVA titers in the untransduced group increased, while none of the titers in the 2bOVA group increased. Normalized anti-OVA antibody titers in the 2bOVA group (4.1±4.9) were significantly lower than those in the un-transduced group (152.3±214.5), demonstrating that platelet-specific OVA gene delivery to HSCs can also suppress the anti-OVA immune response in the OVA-primed WT mice that received 2bOVA-transduced HSCs from OTII mice (the OTII/WT model). After BM reconstitution, the engraftments in the 2 groups were similar (81.4±2.6%, and 80.9±2.5%, respectively), but donor-derived CD45.2+CD4+ (OTII/CD4) T cells in the 2bOVA (0.2±0.1%) group were consistently significantly lower than in the untransduced group (4.2±1.1%) in peripheral blood during the entire study course. Also, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA group compared to the untransduced group. Interestingly, there were no differences in either percentage or total cell number of CD45.2+CD4+ T cells in the thymus between the 2 groups, indicating that central tolerance may not play a role in the immune tolerance induced after platelet-targeted gene therapy. Meanwhile, the frequency and total number of endogenous CD4+ T cells were similar in the 2 groups. Of note, the percentages of donor-derived regulatory T (Treg) cells in the 2bOVA group were significantly higher than those in the untransduced group in peripheral blood, spleen, and lymph nodes, while there were no significant differences in the total numbers of donor-derived Treg cells between the 2 groups. Taken together, our studies demonstrate that platelet-specific gene therapy can induce immune tolerance in a primed system through two distinct pathways, peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. Disclosures No relevant conflicts of interest to declare.

Platelet-Targeted Gene Therapy of Hemophilia a Using Hematopoietic Stem Cells Derived from Genome-Edited Induced Pluripotent Stem Cells

Platelet-Targeted Gene Therapy of Hemophilia a Using Hematopoietic Stem Cells Derived from Genome-Edited Induced Pluripotent Stem Cells

Treatment of a Hemophilia B Mouse Model with Platelet-Targeted Expression of Factor IX Padua.

Targeting the coagulation factor IX (FIX) expression in platelets has been shown to be effective in ameliorating bleeding in hemophilia B (HB) mice. To improve the therapeutic effects and evaluate the safety of this gene therapy strategy, we generated a transgenic mouse model on an HB background with FIX Padua target expressed in platelets. The transgenic mice exhibited stable expression and storage of FIX Padua in platelets. The platelet-stored FIX Padua could be released with the activation of platelets, and the proportion of platelet-stored FIX Padua in whole blood was the same as that of platelet-stored wild-type human FIX. The platelet-derived FIX Padua showed substantially increased specific activity compared with wild-type FIX. Reduced bleeding volume in the FIX Padua transgenic mice demonstrated that bleeding in the mice was improved. Levels of thrombin-antithrombin complex, fibrinogen, D-Dimer, and blood cell counts were normal in the transgenic mice, suggesting that thrombotic risk was not increased in this mouse model. However, the leakage and failure to overcome the presence of inhibitor to wild-type FIX is also observed with FIX Padua, as expected. Taken together, our results support the conclusion that targeting FIX Padua expression in platelets may be an effective and safe gene therapy strategy for HB, and could provide an ideal model to evaluate the safety of platelet-targeted gene therapy for treating hemophilia.

Platelet gene therapy induces robust immune tolerance even in a primed model via peripheral clonal deletion of antigen-specific T cells

While platelet-specific gene therapy is effective in inducing immune tolerance to a targeted protein, how the reactivity of pre-existing immunity affects the efficacy, and whether CD8 T cells were involved in tolerization, is unclear. In this study, ovalbumin (OVA) was used as a surrogate protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either wild-type (WT)/CD45.2 or OT-II/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6 Gy of irradiation. Sustained platelet-OVA expression was achieved in >85% of OVA-primed recipients but abolished in animals with high-reactive pre-existing immunity. As confirmed by OVA rechallenge and skin graft transplantation, immune tolerance was achieved in 2bOVA-transduced recipients. We found that there is a negative correlation between platelet-OVA expression and the percentage of OVA-specific CD4 T cells and a positive correlation with the OVA-specific regulatory T (Treg) cells. Using the OT-I/WT model, we showed that antigen-specific CD8 T cells were partially deleted in recipients after platelet-targeted gene transfer. Taken together, our studies demonstrate that robust antigen-specific immune tolerance can be achieved through platelet-specific gene therapy via peripheral clonal deletion of antigen-specific CD4 and CD8 T effector cells and induction of antigen-specific Treg cells. There is an antagonistic dynamic process between immune responses and immune tolerance after platelet-targeted gene therapy.

Enhancing therapeutic efficacy of in vivo platelet-targeted gene therapy in hemophilia A mice

Our previous studies demonstrated that intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a modified B domain-deleted factor VIII (FVIII) transgene driven by a megakaryocyte-specific promoter (GP1Bα promoter; G-F8/N6-LV) successfully transduced hematopoietic stem cells (HSCs) to produce FVIII stored in the platelet α-granules. Platelet FVIII corrected the bleeding phenotype with limited efficacy in hemophilia A (HemA) mice with and without preexisting anti-FVIII inhibitors. The present study sought to further enhance the therapeutic efficacy of this treatment protocol by increasing both the efficiency of LV transduction and the functional activity of platelet FVIII. A combined drug regimen of dexamethasone and anti-CD8α monoclonal antibody enhanced the percentage of transduced bone marrow and HSCs over time. In G-F8/N6-LV-treated HemA mice, significant improvement in phenotypic correction was observed on day 84. To improve platelet FVIII functionality, genes encoding FVIII variant F8X10K12 with increased expression or F8N6K12RH with increased functional activity compared with F8/N6 were incorporated into LVs. Treatment with G-F8X10K12-LV in HemA mice produced a higher level of platelet FVIII but induced anti-FVIII inhibitors. After treatment with combined drugs and IO infusion of G-F8/N6K12RH-LV, HemA mice showed significant phenotypic correction without anti-FVIII inhibitor formation. These results indicate that new human FVIII variant F8/N6K12RH combined with immune suppression could significantly enhance the therapeutic efficacy of in vivo platelet-targeted gene therapy for murine HemA via IO delivery. This protocol provides a safe and effective treatment for hemophilia that may be translatable to and particularly beneficial for patients with preexisting inhibitory antibodies to FVIII.

Platelet-Targeted Gene Therapy Induces Robust Immune Tolerance Even in a Primed Model Via Peripheral Clonal Deletion of CD4 and CD8 T Cells and Expansion of Treg Cells

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) results in effective immune tolerance induction to FVIII and FIX as well as the non-coagulant protein ovalbumin (OVA) in the unprimed model. Here we explored how the reactivity of immune responses affects the efficacy of transgene expression and the potential immune tolerance mechanisms in platelet-specific gene transfer in the primed model. We used OVA as a surrogate protein for this study. Recipient wild-type (WT) CD45.1 mice were primed with OVA protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either WT/CD45.2 or OTII/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy irradiation. We found that pre-existing high-reactive immune responses devastate platelet-OVA expression in recipients in both the WT/WT and OTII/WT models. Using the WT/WT mouse model, 6 of 7 2bOVA-transduced recipients had sustained platelet-OVA expression with levels ranging from 4.53-8.84 ng/108 platelets. Anti-OVA total IgG titers declined with time in 2bOVA-transduced OVA-primed recipients even when rechallenged with OVA. In contrast, recall memory immune responses were elicited in 2bGFP and untransduced transplanted controls. Furthermore, full-thickness tail skin grafts from CAG-OVATg mice were successfully engrafted onto 2bOVA-transduced recipients and survived throughout the remaining lifetimes of the animals. In contrast, skin grafts were completely rejected in control 2bGFP-transduced and untransduced transplanted recipients. To ensure that the immune system was not inactive in 2bOVA-transduced recipients, animals were immunized with unrelated antigen recombinant human FVIII (rhF8) using a protocol known to induce anti-F8 immune responses even in WT mice. All 2bOVA-transduced recipients developed anti-F8 inhibitory antibodies after rhF8 immunization with no differences between the 2bOVA group and the 2bGFP and untransduced control groups. Using the OVA-specific CD4 TCR transgenic model (OTII/WT), we showed that sustained platelet-OVA expression was achieved in 90% of 2bOVA-transduced recipients. OVA-specific CD4 T cells were deleted and Treg cells were expanded in peripheral lymphoid organs in 2bOVA-transduced OVA-primed recipients. The levels of platelet-OVA expression negatively correlates with the percentages of OVA-specific CD4 T cells, but positively correlates with Treg cells. Immune tolerance was also achieved in the OTII/WT model even though all donor-derived CD4 T cells are OVA-specific. However, our studies showed that high-reactive immune responses could influence the neoprotein expression in platelet-targeted gene therapy. Using the OVA-specific CD8 TCR transgenic model (OTI/WT), in which all donor-derived CD8 T cells are OVA-specific, we found that low levels of platelet-OVA expression were obtained at week 5 after transplantation (0.72±0.65 ng/108 platelets) and then dropped to barely detectable at subsequent time points. Flow cytometry analysis showed that there were 33.9±12.9% platelets positive with GFP in 2bGFP LV-transduced recipients, demonstrating that high transduction efficiency and successful transplantation were achieved. Of note, OVA-specific CD8 T cells in the 2bOVA group were 48% lower than in the 2bGFP control group, demonstrating that antigen-specific CD8 T cells were partially deleted in the OTI/WT model after platelet-targeted OVA gene transfer. Interestingly, we found that the percentage of the antigen-specific CD8+Foxp3+ cells in the 2bOVA group was significantly higher than in the 2bGFP group (0.19±0.14% vs. 0.05±0.01%, respectively). These results suggest that there is a counterpace process between immune reaction and immune tolerance after platelet-targeted gene therapy. In conclusion, our studies demonstrate that platelet-targeted gene therapy can induce antigen-specific immune tolerance even when the immune response has been mounted via peripheral clonal deletion of antigen-specific CD4 and CD8 T cells and expansion of Tregs, suggesting that platelet-targeted gene therapy is a promising approach to induce immune tolerance for treatment of diseases with undesired immune responses or even with pre-existing immune responses, such as hemophilia A with inhibitors or autoimmune diseases. Disclosures No relevant conflicts of interest to declare.

Platelet-Targeted Gene Transfer Induces Immune Tolerance through Two Distinct Pathways

Abstract Our previous studies using hemophilia A and B mouse models have demonstrated that targeting FVIII or FIX expression to platelets under control of the aIIb promoter through lentivirus-mediated delivery to hematopoietic stem cells (HSCs) results in transgene protein expression and storage in platelet a-granules and that platelet-derived FVIII or FIX not only restores hemostasis but also induces immune tolerance in transduced recipients. In the current studies, we explored how immune tolerance is induced after platelet-specific gene therapy and whether this approach can be applied to induce immune tolerance to a non-coagulant protein. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). Since VWF propeptide (Vp) can reroute secreting proteins to a storage pathway, we designed another vector, 2bVpOVA, which contains Vp to secure OVA storage in platelet granules. We first confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to HSCs can induce anti-OVA immune tolerance in wild-type (WT) C57BL6 mice. 2bOVA or 2bVpOVA-transduced HSCs (CD45.2/B6) were transplanted into CD45.1/B6 recipients pre-conditioned with 6.6Gy total body irradiation (TBI). We found that 95% and 98% of OVA protein in whole blood was stored in platelets with an OVA protein level of 24.22±8.72 ng/108 platelets (n=10) and 1.41±0.73 ng/108 platelets (n=10) in 2bOVA and 2bVpOVA transduced recipients, respectively. Electronic microscope analysis demonstrated that the OVA transgene protein using both vectors was stored in transduced platelet a-granules. When the transduced recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA group (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in untransduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. Of note, the titer of anti-OVA total IgG titer in 2bF8 (an unrelated control vector) transduced FVIIInull/B6 recipients without OVA immunization was 413±61 (n=12), which was not significantly different compared to the 2bOVA or 2bVpOVA group even after OVA immunization. In another unrelated control group, 2bGFP, anti-OVA titer was 84±17 (n=9), which was significantly higher than the data obtained from untransduced WT animals without immunization (33±7, n=24). Why there were various levels of anti-OVA antibody titers in unrelated vectors transduced recipients is still unclear and needed further investigation. To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP (a control vector) and transplanted into CD45.1/B6 recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (86.4±2.3%, 86.2±2.2%, and 87.4±2.0%, respectively), but donor-derived CD45.2+ CD4+ T cells in the 2bOVA (0.2±0.1%, n=5) and 2bVPOVA groups (0.9±0.4%, n=6) were consistently significantly lower than in the 2bGFP group (3.1±0.9%, n=6) in peripheral blood during the entire study course. Similarly, donor-derived CD45.2+ CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in either percentage or total cell number of CD45.2+ CD4+ T cells in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy. Notably, the frequency and total number of endogenous CD4 T cells were similar in the 3 groups. Annexin-V staining revealed that the percentage of apoptotic CD45.2+ CD4+ T cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in both spleen and lymph nodes, but not in the thymus. The frequency of donor-derived regulatory T cells cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in peripheral blood, spleen, and lymph nodes, but not in the thymus. Taken together, our studies demonstrate that platelet-specific gene therapy induces immune tolerance through peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. Thus, platelet gene therapy can be a promising approach for immune tolerance induction. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

De Novo Synthesis & Storage of Human Factor VIII In Platelets Reduces Bleeding In Canine Hemophilia A

Abstract Abstract 2198 Introduction: The goal of our study was to develop a clinically relevant strategy for platelet-targeted gene therapy of the inherited bleeding disorder, Hemophilia A. We hypothesized that adult dogs (25 kg) affected with Hemophilia A could serve as a relevant “large animal” model to test if Factor VIII (FVIII) could be synthesized and sequestered within platelets derived from lentivirus-transduced hematopoietic stem cells. This approach is novel because it should permit the regulated release of FVIII from activated platelet progeny directly at a site of vascular injury as a physiological hemostatic response to a severe intermittent bleeding challenge. Methods: cG-CSF/cSCF mobilized peripheral blood stem cells (PBSC) were immuno-selected for CD34 antigen from an apheresis product, transduced with a lentivirus vector under the transcriptional control of platelet-specific integrin αIIb gene promoter driving expression of human BDD-FVIII. The PBSC (2 × 106/kg) were then autologously transplanted (Tx) into animals that were preconditioned with Bulsulfan (5-10 mg/kg i.v.). After transplant, the dogs received oral cyclosporine to maintain levels at 200 – 400 ng/ml for 90 days and MMF at 8 mg/kg for 35 days. Results: Three Tx recipients underwent periodic testing for incorporation and expression of the FVIII transgene as well as immune tolerance and phenotypic correction of Hemophilia A. LAM PCR analysis detected and localized the lentivirus vector within genomic DNA isolated from circulating peripheral blood leukocytes from each animal. Immunofluorescence confocal microscopy detected FVIII within a subset of circulating peripheral blood platelets. Immune electron microscopic analysis revealed that the FVIII had been trafficked and stored within the platelet secretory α-granules. Chromogenic analysis of platelets isolated from dogs demonstrated that FVIII was present in its biologically active form (FVIII:C) at approximately 5 mU/ml/1×108 platelets from 20 weeks through greater than two years after PBSC Tx. In contrast, FVIII:C was not detected within the plasma of these animals. This result coupled with the use immunomodulation drugs may help to explain why the dogs remained tolerant of human FVIII as indicated by our inability to detect inhibitory antibodies to FVIII. Following successful gene transfer and PBSC engraftment, all animals showed signs of clinical improvement of Hemophilia A: the longest Tx recipient had one bleed per year for 2 years (vs expected 5 – 6/year), the second dog has had no bleeds for twenty months following PBSC Tx and the third animal had no bleeding episodes for first seven months post Tx. In addition, the first Tx dog showed significant recovery from prolonged (months) history of chronic gastrointestinal bleeding. Remarkably, all three animals displayed improved whole blood clotting times. These data are consistent with our previous results demonstrating synthesis, trafficking, storage and regulated release of FVIII from α-granules of human megakaryoyctes in vitro and platelets of the murine “small animal” model for Hemophilia A. Conclusions: The outcome of this work demonstrates a feasible strategy for controlling severe bleeding episodes within patients with Hemophilia A by providing a locally inducible secretory pool of FVIII in platelets derived from an autologous Tx of FVIII-transduced PBSC. Disclosures: No relevant conflicts of interest to declare.

Platelet-Targeted Expression of Human BDD-FVIII Reduces Bleeding in Canine Hemophilia A.

Abstract Abstract 691 Introduction: The goal of our study was to develop a clinically relevant strategy for platelet-targeted gene therapy of the commonly inherited bleeding disorder, Hemophilia A. We hypothesized that adult dogs (25 kg) affected with Hemophilia A could serve as a relevant “large animal” model to test if FVIII could be synthesized and sequestered within platelets derived from lentivirus-transduced hematopoietic stem cells. This approach is novel because it should permit the regulated release of FVIII from activated platelet progeny directly at the wound site as a physiological hemostatic response to bleeding challenges. Methods: cG-CSF/cSCF mobilized peripheral blood stem cells (PBSC) were immuno-selected for CD34 antigen from an apheresis product, transduced with a lentivirus vector under the transcriptional control of platelet-specific integrin αaIIb gene promoter driving expression of human BDD-FVIII. The PBSC (3 × 10 6/kg) were then autologously transplanted into animals that were preconditioned with Bulsulfan (5-10 mg/kg i.v.). After transplant, the dogs received oral cyclosporine to maintain levels at 200–400 ng/ml for 90 days and MMF at 8mg/kg for 35 days. Results: Two transplant recipients underwent periodic testing for incorporation and expression of the FVIII transgene as well as immune tolerance and phenotypic correction of Hemophilia A. PCR analysis detected the lentivirus vector within genomic DNA isolated from circulating peripheral blood leukocytes for more than one year after transplant. Immunofluorescence confocal microscopy showed synthesis of FVIII within tissue cultured canine megakaryocytes and circulating peripheral blood platelets. Chromogenic analysis of platelets isolated from transplanted dogs demonstrated the presence of a biologically active form of FVIII (FVIII:C) at approximately 5 mU/ml/1×108 platelets from 20 weeks through greater than one year after PBSC transplant. In contrast, FVIII:C was not detected within the plasma of these animals. This result coupled with the immunomodulation drugs may help to explain why the dogs failed to develop inhibitory antibodies to human FVIII. Following successful gene transfer and engraftment, both animals showed signs of clinical improvement of Hemophilia A: one dog has had one bleed per year for 2 years (vs expected 5–6/year) and the second dog has had no bleeds for 8 months following transplantation. In addition, one dog showed significant recovery from prolonged (months) history of gastrointestinal bleeding. These data are consistent with our previous results demonstrating synthesis, trafficking and storage of FVIII within αa-granules of human megakaryoyctes in vitro and platelets of the murine “small animal” model for Hemophilia A. Conclusions: This outcome raises the possibility of developing a protocol for delivering a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Wilcox:Amgen, INC: Research Reagents, canine growth factors: cG-CSF and cSCF.

Platelet-Targeted Expression of Coagulation Factor VIII (FVIII) Shows Efficacy for Using the Dog as a Large Animal Model for Gene Therapy of Hemophilia a

Abstract In order to develop a large animal model for platelet-targeted gene therapy of Hemophilia A, we transduced normal canine bone marrow with a lentivirus vector under the transcriptional control of integrin αIIb gene promoter driving expression of human BDD-FVIII. Bone marrow was collected from normal dogs and transduced in the presence of recombinant canine c-SCF, c-IL-6, human h-TPO, and h-flt-3/flk-2 ligand differentiation factor. Immunofluorescence analysis using antibodies against human FVIII, and the megakaryocyte-specific marker integrin αIIb revealed synthesis of FVIII within tissue cultured megakaryocytes derived from lentivirus transduced bone marrow cells. This result appeared similar to synthesis, trafficking and storage of FVIII trafficked to α-granules of the murine “small animal” model for hemophilia A and human megakaryoyctes in vitro. To examine the effect of FVIII expression in platelets, in vivo, FVIII-transduced canine bone marrow was xenotransplanted into immune-compromised “NOD-SCID” mice treated with a sublethal dose (350 cGy) of irradiation. Flow cytometric analysis using antibodies specific for canine glycoproteins Ibα and integrin αIibβ3 revealed that circulating canine platelets comprised approximately 20–30% of the total platelet population in whole blood isolated from the mice within four weeks after xenotransplant. Immunofluorescence confocal microscopy detected a punctuate staining for FVIII that co-localized with a platelet α–granule protein, P-selectin, within a subpopulation of canine platelets isolated from murine whole blood. In contrast, FVIII was not detected in platelets from control samples. In addition, chromogenic analysis of platelets isolated from mice transplanted with FVIII-transduced bone marrow demonstrated the presence of a biologically active form of FVIII, FVIII:C at 2 mU/ml/lysate of 1×108 platelets. These results indicate that canine megakaryocytes are able to synthesize and store a biologically active form of FVIII that can be retained within progeny platelets. We speculate that dogs affected with hemophilia A should serve as an ideal “large animal” model to test if FVIII can undergo regulated release from platelets following physiologic hemostatic response to vessel injury. This raises the possibility of developing a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells.