Expression Of Hemophilia Research Articles

Ultrasound-mediated gene delivery specifically targets liver sinusoidal endothelial cells for sustained FVIII expression in hemophilia A mice

The ability to target the native production site of factor VIII (FVIII)— liver sinusoidal endothelial cells (LSECs)— can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150μs PD) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 μs PD) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of ∼10% over 84 days post-treatment, however, half of the HE-treated mice developed low-titer inhibitors, while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group compared to the HE group. These results indicate that UMGD can safely target LSECs with a lower energy condition to achieve persistent FVIII gene expression. It demonstrates that this novel technology is highly promising for therapeutic correction of HA.

Dexamethasone Transiently Enhances Transgene Expression in the Liver When Administered at Late-Phase Post Long-Term Adeno-Associated Virus Transduction.

Glucocorticoids have anti-inflammatory and immunosuppressive functions and have commonly been used for preventing liver toxicity after the systemic application of a high dose of adeno-associated virus (AAV) vector for gene therapy. Clinical studies have reported that glucocorticoids have rescued factor IX (FIX) expression in patients with hemophilia B who showed a reduced FIX expression at 6 to 10 weeks post-AAV vector administration. In this study, we explored whether glucocorticoids could affect transgene expression in AAV targeted livers in animal models. When dexamethasone was applied before AAV9/FIX vector administration in the wild-type C57BL/6 mice, FIX expression was much higher than that of the control mice at any time point. More importantly, FIX expression transiently increased after dexamethasone was administered at week 6 or later post-AAV injection regardless of the various dexamethasone treatments applied. The transient enhancement in transgene expression was observed once there were one to several consecutive dexamethasone treatments completed. A similar result was also achieved in other wild-type BALB/c and hemophilia B mice that were treated with AAV9/FIX and dexamethasone. This mechanism study demonstrated that the administration of dexamethasone did not change either AAV genome copy number or transgene expression at the transcription level but transiently decreased interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) expression in the livers of mice at a later time after AAV injection. Next, we studied the effect of dexamethasone on late transgene expression in hemophilia B dogs. Dexamethasone was administered 1 year after AAV9/FIX injection. Inconsistent with the results in mice, no significant change of FIX expression was observed in hemophilia B dogs. In summary, the results from this study indicate that dexamethasone may have various effects on transgene expression in AAV-transduced livers in different species, which provides valuable information about the rational application of dexamethasone in future clinical studies.

Activity of transgene-produced B-domain–deleted factor VIII in human plasma following AAV5 gene therapy

AbstractAdeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain–deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

Cross Priming of Transgene Product-Specific CD8+ T Cells in Hepatic AAV Gene Transfer Depends on IL-1 Receptor and XCR1+ Dendritic Cells but Not TLR9

Introduction: Adeno-associated virus (AAV) mediated gene transfer is currently evaluated in multiple Phase I/II and Phase III studies for the treatment of hemophilia. However, immune responses to both the AAV capsid and encoded transgene remain major impediments to clinical translation. Several studies have implicated innate immune sensors such as Toll-like receptors (TLR) and their downstream adaptor molecule MyD88 in sensing viral structures. TLR9-MyD88 signaling has been linked to cross-priming of CD8+ T cell responses to capsid and also to transgene product-specific CD8+ T cell responses. However, little is known about other signaling pathways that may lead to immune activation. Previously, our lab has shown that while liver gene transfer is capable of inducing immunological tolerance to AAV encoded transgene products, vector dose and design play a critical role. For instance, low hepatic gene expression levels may elicit a CD8+ T cell response to the AAV encoded transgene, resulting in loss of the model antigen ovalbumin (OVA) in C57BL/6 mice or of FIX expression in hemophilia B mice. We investigated innate immune sensing pathways that may play a role in initiating transgene specific CD8+ T cell response in the hepatic microenvironment. Further, we determined the contribution of hepatic antigen presenting cells (APC) by selectively depleting/neutralizing APCs and evaluating their effect on presentation of transgene product-derived antigen following AAV8-OVA liver gene delivery. Methods: Wild-type (WT) C57BL6 and specific innate sensing knockout mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8-OVA vector (Mol Ther 25:880, 2017). PBMCs were quantified at 4 weeks for OVA-specific CD8+ T cells using a class I MHC tetramer. Hepatic APC types [Kupffer cells, neutrophils, CD103+ dendritic cell (DC), CD11c+ DC, XCR1+ DC] involved in transgene specific CD8+ T cell activation were selectively depleted/inactivated by pre-treatment with gadolinium chloride (GdCl3), Ly6G, CD103 antibody respectively, or by administering diphtheria toxin (DT) to CD11c-DTR and XCR1-DTR mice. This was followed by intravenous administration of AAV8-OVA and CellTrace violet labeled OT-1 cells. Results: Similar to WT mice, TLR9-/-, TLR2-/-, TRIF-/-, IFNaR-/- and MDA5-/- mice developed a CD8+ T cell response indicating that these sensors do not play a role in transgene specific CD8+ T cells response. Interestingly, adaptor protein MyD88-/- mice did not elicit CD8+ T cell response to OVA, implying a MyD88 dependent but TLR9 independent response. Since MyD88 is an essential adaptor protein not only for TLR but also for interleukin-1 (IL-1) signaling pathways, we next analyzed IL-1R-/- mice. Similar to MyD88-/- mice, IL-1R-/- mice did not show OVA specific CD8+ T cells (p=0.006, 0.007 respectively), indicating that transgene-specific adaptive responses are mediated by IL-1R/MyD88 signaling. Kupffer cells and DCs are principal APCs in liver and infiltrating neutrophils could also act as APCs under inflammatory conditions in liver microenvironment. Using proliferation of OT-I cells as readout we tested if any of these cell types are required for presentation to transgene specific CD8+ T cells. In naïve control, GdCl3 treated and a-Ly6G antibody treated mice, OT-I cell proliferation reached 60%, 65% and 48% on average, respectively. Depletion of CD11c DCs substantially reduced the proliferation of OT-I cells to ~6% (p<0.0001) indicating a critical role for DCs in mediating transgene specific CD8+ T cell responses. Since XCR1+ DCs are the major cross-presenting DCs and hepatic resident CD103+ DCs are shown to have intrinsically enhanced capacity to process and present antigen to naïve CD8+ T cells, we further sought to assess if any of these DCs plays a role in activation of transgene specific CD8+ T cells. Neutralization of CD103+ DCs reduced OT-I proliferation to 39% (p=0.01) whereas depletion of XCR1+ DCs reduced the proliferation to ~20% (p<0.0001) indicating a major role for XCR1+ DCs. Conclusions: In summary, we uncovered a novel-signaling pathway that can activate CD8+ T cell responses during AAV gene transfer independent of TLR9 sensing. The IL-1R/MyD88 pathway drives activation of transgene specific CD8+ T cell, and XCR1+ DCs are critically involved in cross-presenting transgene product-derived antigen to CD8+ T cells. Disclosures Herzog: Takeda Pharmaceuticals: Patents & Royalties.

In vivo enrichment of genetically manipulated platelets for murine hemophilia B gene therapy.

Our previous studies have demonstrated that platelet-targeted factor IX (FIX) gene therapy can introduce sustained platelet-FIX expression in hemophilia B (FIXnull ) mice. In this study, we aimed to enhance platelet-FIX expression in FIXnull mice with O6 -methylguanine-DNA-methyltransferase (MGMT)-mediated in vivo drug selection of transduced cells under nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT lentivirus vector), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the MGMT P140K gene under the murine stem cell virus promoter. Platelet-FIX expression in FIXnull mice was introduced by 2bF9/MGMT-mediated hematopoietic stem cell transduction and transplantation. The 2bF9/MGMT-transduced cells were effectively enriched after drug selection by O6 -benzylguanine/1,3-bis-2-chloroethyl-1-nitrosourea. There were a 2.9-fold higher FIX antigen and a 3.7-fold higher FIX activity in platelets, respectively, posttreatment compared with pretreatment. When a 6-hr tail bleeding test was used to grade the bleeding phenotype, the clotting time in treated animals was 2.6 ± 0.5 hr. In contrast, none of the FIXnull control mice were able to clot within 6 hr. Notably, none of the recipients developed anti-FIX antibodies after gene therapy. One of four recipients developed a low titer of inhibitors when challenged with rhF9 together with adjuvant. In contrast, all FIXnull controls developed inhibitors after the same challenge. Anti-FIX immunoglobulin G were barely detectable in recipients (1.08 ± 0.54 µg/ml), an 875-fold lower level than in the FIXnull controls. Our data demonstrate that using the MGMT-mediated drug selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune tolerance in FIXnull mice.

AAV-Mediated Gene Delivery to the Liver: Overview of Current Technologies and Methods.

Adeno-associated virus (AAV) vectors to treat liver-specific genetic diseases are the focus of several ongoing clinical trials. The ability to give a peripheral injection of virus that will successfully target the liver is one of many attractive features of this technology. Although initial studies of AAV liver gene transfer revealed some limitations, extensive animal modeling and further clinical development have helped solve some of these issues, resulting in several successful clinical trials that have reached curative levels of clotting factor expression in hemophilia. Looking beyond gene replacement, recent technologies offer the possibility for AAV liver gene transfer to directly repair deficient genes and potentially treat autoimmune disease.

Functions of the Heterologous Intron-Derived Fragments Intra and Extra Factor IX-cDNA Coding Region on the Human Factor IX Expression in HepG2 and Hek-293T Cells.

BackgroundHuman FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3′-end formation of mRNAs are required.ObjectivesWe aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells.Materials and MethodsHepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells.ResultsOur data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compared to other constructs in Hek-293 cells. In HepG2 cells, the rABE in combination with CMVp in context of intron-less plasmid induced an increase in total expression of hFIX protein dramatically; ranging from 2.3 to 40 folds increase compared to other constructs. The rABE in combination with CMVp in the hBG intron-I, hBG intron-II, and hBG intron-I,II containing plasmids induced 3.7, 2, and 1.6-fold increase in the total expression of hFIX protein, respectively. The presence of both hBG intronic sequences within the hFIX-cDNA induced a higher secretion level of hFIX than either intron-I or II alone and provided correctly spliced hFIX transcripts in HepG2 and kidney cell lines. The intron-less construct with or without rABE induced the highest hFIX mRNA levels in HepG2 and Hek-293T cells respectively compared to other constructs.ConclusionsThe embryonic kidney cells in addition to the differentiated hepatic cell lines could be successfully targeted by plasmid vectors. The intron-less and hBG intron-I containing plasmids represent a particular interest in producing recombinant hFIX in Hek-293T cells. The synergistic function on the hFIX expression that was achieved by combining the CMVp with the liver-specific rABE would be a useful approach for future designing of the expression cassettes for hepatocyte-mediated gene expression in hemophilia B.

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A

Essentials Factor (F) VIII is an inefficiently expressed protein.Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays.These minimally modified novel FVIII variants have enhanced function.These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. SummaryBackgroundThe major challenge for developing gene‐based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B‐domain‐deleted (BDD) canine FVIII and hFVIII‐R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function.ObjectiveWe hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function.MethodsA series of recombinant hFVIII‐furin deletion variants were introduced into hFVIII‐BDD [Δ1645, 1645‐46(Δ2), 1645‐47(Δ3), 1645‐48(Δ4), or Δ1648] and characterized.Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2‐fold higher procoagulant activity compared with FVIII‐BDD. In vivo, the variants also have improved hemostatic function. After adeno‐associated viral (AAV) vector delivery, the expression of these variants is 2–4‐fold higher than hFVIII‐BDD. Protein challenges of each variant in mice tolerant to hFVIII‐BDD showed no anti‐FVIII immune response.ConclusionsThese data suggest that the furin deletion hFVIII variants are superior to hFVIII‐BDD without increased immunogenicity. In the setting of gene‐based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

Lentivirus-Mediated Platelet Gene Therapy Corrects Bleeding Diathesis and Induces Immune Tolerance in Murine Hemophilia B Mice

Abstract 1101While data from the clinical trials using AAV vector expression FIX in hemophilia B gene therapy in humans are very encouraging, for individuals with severe liver disease or neutralizing antibodies to AAV, an alternative gene therapy approach might be desired. Our previous studies have demonstrated that lentivirus-mediated platelet gene therapy can correct murine hemophilia A phenotype, but this approach has not been explored for hemophilia B. In the current study, we developed a clinical translatable approach for platelet gene therapy of hemophilia B. Platelet-FIX (2bF9) expression in hemophilia B (FIXnull) mice was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). The recipients were analyzed beginning at 3 weeks after bone marrow (BM) transplantation. Expression of the 2bF9 product was detected by PCR in all recipients that received 2bF9 LV-transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF9 LV transfer vector. The expression of the hFIX transgene protein in the transduced platelets was confirmed by immunofluorescent confocal microscopy. Flow cytometry showed that there were 20.8 ± 12.1% (n = 7) and 14.8 ± 10.7% (n = 6) 2bF9 LV-transduced platelets respectively in the recipients preconditioned with 1100 cGy or 660 cGy. The antigen levels of FIX (FIX:Ag) were 2.89 ± 1.75 mU/108 platelets (n = 9) in the recipients preconditioned with 1100 cGy and 1.87 ± 1.30 mU/108 platelets (n = 7) in the 660 cGy group, while the activity (FIX:C) levels were 1.67 ± 1.15 and 1.13 ± 0.85 mU/108 platelets respectively. There was a small amount of FIX detected in the 2bF9 LV-transduced recipient plasma with the average levels of 2.22 mU/ml in 1100 cGy group and 1.44 mU/ml in 660 cGy group. To analyze the distribution of the FIX between platelets and plasma, we normalized FIX levels to total whole blood FIX content. The results demonstrated that 90% to 95% of whole blood FIX was stored in platelets. The tail clip survival test demonstrated that 15 out of 16 mice that received 2bF9 LV-transduced HSCs survived the tail clip challenge, while 8 out of 10 FIXnull control mice died after tail clipping. Nine months after transplantation, sequential transplantation was performed on some of the primary recipients. Platelet-hFIX expression in the secondary recipients was sustained, leading to phenotypic correction and confirming that long-term engrafting HSCs were successfully transduced with 2bF9 LV. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. To investigate whether immune tolerance was induced in 2bF9 LV-transduced recipients, we challenged the recipients with recombinant human FIX (rhFIX) in the presence of adjuvant. Only 1 out of 9 2bF9 LV-transduced recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) as measured by a modified Bethesda assay. In contrast, all of the FIXnull controls developed inhibitory antibodies ranging from 17 – 37 BU/ml after the same challenge (n = 5). To ensure that the immune system was not defective in the 2bF9 LV-transduced recipients and that the tolerance induction is FIX antigen-specific, we further challenged the animals with ovalbumin (OVA) absorbed on Alum. Both the 2bF9 LV-transduced and FIXnull control mice developed high-titer of anti-OVA antibodies. The levels of anti-OVA IgG in the 2bF9 transduced recipients were not significantly different from FIXnull mice after the OVA immunization, confirming that tolerance induction in 2bF9 LV-transduced mice is FIX-specific. Taken together, our data suggest that lentivirus-mediated bone marrow transduction and transplantation can not only provide sustained phenotypic correction, but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans. Disclosures:No relevant conflicts of interest to declare.

In Vivo Expansion of Regulatory T cells With IL-2/IL-2 mAb Complexes Prevents Anti-factor VIII Immune Responses in Hemophilia A Mice Treated With Factor VIII Plasmid-mediated Gene Therapy

Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

Hepatic control elements promote long‐term expression of human coagulation factor IX gene in hydrodynamically transfected mice

Long-term expression of the delivered target gene is critical for successful gene therapy. Recently, hepatic control region I (HCR I) originating from the apolipoprotein (apo)C-I pseudogene was shown to be a critical element for long-term gene expression in the liver of mice. HCR II is another hepatic control region of apoC-I. HCR I, HCR II and HCR I/II-containing plasmids encoding factor IX were prepared and hydrodynamically transferred into the liver of normal and hemophilia B mice. Factor IX expression, clotting activity and formation of antibodies against the expressed gene product were compared. HCR I-, HCR II- and HCR I/II-containing plasmids all induced long-term gene expression in both normal and hemophilia B mice. Post-transfection factor IX expression in the hemophilia B mice remained above 500 ng/ml for 210 days. Antibodies against human factor IX were detected at a low level in the serum, although they had no effect on the levels and clotting activity of the expressed factor IX. We have shown in mouse models that hydrodynamic transfection of pBS-HCRII-HP-FIXA and pBS-HCRI/II-HP-FIXA was able to induce and maintain the expression and clotting activity of human factor IX for a long period of time at a potentially therapeutic level. With an appropriate delivery system, this type of plasmid vector could be clinically useful for the hepatic expression of therapeutic genes including human factor IX.

Hepatic AAV Gene Transfer and the Immune System: Friends or Foes?

Two ongoing clinical trials utilize different adeno-associated viral (AAV) vectors for liver-directed factor IX (F.IX) gene transfer with the goal of sustained therapy in patients with severe hemophilia B. Although preclinical studies have documented immune tolerance and long-term expression of F.IX in animals, the single prior clinical trial of this approach achieved only transient therapeutic gene expression and exposed preexisting immunity to the AAV vector as a major obstacle for therapy.1 Although accumulating preclinical data continue to fuel a debate over the potential impact of immune responses on hepatic AAV gene transfer, these data do not allow us to predict whether the human immune system will reject or tolerate therapy. This commentary dissects the relevant human and animal data, some of which are contradictory or allow us to draw only limited conclusions. More clinical trial data are critically needed and should ultimately help us develop better protocols.

A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice

A longstanding goal for the treatment of hemophilia B is the development of a gene transfer strategy that can maintain sustained production of clotting factor IX (F.IX) in the absence of an immune response. To this end, we have sought to use lentiviral vectors (LVs) as a means for systemic gene transfer. Unfortunately, initial evaluation of LVs expressing F.IX from hepatocyte-specific promoters failed to achieve sustained F.IX expression in hemophilia B mice due to the induction of an anti-F.IX cellular immune response. Further analysis suggested that this may be a result of off-target transgene expression in hematopoietic-lineage cells of the spleen. In order to overcome this problem, we modified our vector to contain a target sequence for the hematopoietic-specific microRNA, miR-142-3p. This eliminated off-target expression in hematopoietic cells, and enabled sustained gene transfer in hemophilia B mice for more than 280 days after injection. Treated mice had more than 10% normal F.IX activity, no detectable anti-F.IX antibodies, and were unresponsive to F.IX immunization. Importantly, the mice survived tail-clip challenge, thus demonstrating phenotypic correction of their bleeding diathesis. This work, which is among the first applications to exploit the microRNA regulatory pathway, provides the basis for a promising new therapy for the treatment of hemophilia B.

The Effect of von Willebrand Factor (VWF) on Site-Specific Factor VIII (FVIII) Expression in Hemophilia A Mice.

von Willebrand factor (VWF) is a carrier protein for factor VIII (FVIII) and protects plasma FVIII from protease degradation. Our laboratory has had a longstanding interest in the association of FVIII with VWF both in vitro and in vivo. Our in vitro studies have demonstrated that FVIII stores together with VWF in both endothelial cells and megakaryocytes if FVIII is made in these cells. Furthermore, we demonstrated that FVIII and VWF are both releasable by agonist stimulation. To investigate the association of VWF and FVIII in vivo, we generated two lines of transgenic mice that express FVIII either in endothelial cells or in platelets using either the endothelial cell-specific Tie2 promoter or the platelet-specific αIIb promoter, respectively. When the platelet-specific FVIII (2bF8) transgene is bred into the FVIIInull mouse, FVIII can only be detected in platelets, with a level of 0.76 ± 0.27 mU/108 platelets in heterozygous and 1.53 ± 0.14 mU/108 platelets in homozygous 2bF8 mice. When the endothelial cell-specific FVIII (Tie2F8) transgene is bred into the FVIIInull mouse, homozygous Tie2F8 mice maintained normal plasma FVIII levels (1.15 ± 0.16 U/ml) and 50% levels in heterozygous mice (0.56 ± 0.16 U/ml). Both 2bF8trans and Tie2F8trans phenotypes effectively abrogate the bleeding diathesis in hemophilic mice. When 2bF8 transgene was bred into a FVIII and VWF double knockout background, the level of platelet-FVIII significantly decreased, but this platelet-derived FVIII was still stored in a-granules and still maintained clinical efficacy. In contrast, when the Tie2F8 transgene was bred into the double knockout background, plasma FVIII dropped to undetectable levels. This is in contrast to the situation in VWFnull mice in which normal endogenous murine FVIII is synthesized with about 10% of normal FVIII activity persisting in plasma. This could be due to a difference in survival between human FVIII and murine FVIII. All Tie2F8trans/FVIIInullVWFnull mice (n=15) survived tail clipping even though there is no FVIII:C detected in the plasma. To investigate the effect of murine VWF on the levels of plasma FVIII, plasma from FVIIInull mice was infused into Tie2F8trans/FVIIInullVWFnull mice to restore VWF levels to 25% of normal. As expected, the endothelial cell-derived plasma FVIII was stabilized by the infused VWF and was detected within 1 hour after infusion, with a peak (25% level) at 4 hours. The level of plasma FVIII at 24 hours was still about 20% of normal while the level of remaining VWF was only 5% of normal. These results demonstrate that VWF is important for site-specific FVIII expression. Co-expression with VWF in platelets is important for optimal platelet-specific FVIII expression and endothelial cell-derived plasma FVIII is VWF-dependent.

1055. High Levels of Human Factor IX Expression in Hemophilia B Mice after Adult Stem Cell-Based Gene Therapy Combined with Non- Myeloablative Conditioning

Top of pageAbstract Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroid- specific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0 |[ndash]| 100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring long-term hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.

Persistent expression of factor VIII in vivo following nonprimate lentiviral gene transfer

AbstractHemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)–based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human α1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain–deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti–human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

601. Persistent Expression of Factor VIII In Vivo Following FIV Lentiviral Gene Transfer

Top of pageAbstract Hemophilia A is a common X-linked coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)-based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudotyped FIV efficiently and showed excellent hepatocyte tropism. The GP64 pseudotyped FIV was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human 1-antitrypsin promoter), further enhanced transgene expression in hepatoctyes. We generated a GP64-pseudotyped FIV vector encoding the B domain-deleted human FVIII coding region driven by the liver-specific promoter, with two beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti-human FVIII antibodies, and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64 pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

Factor IX variants improve gene therapy efficacy for hemophilia B

AbstractIntramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Gene therapy of the hemophilias

Development of hemophilia gene therapy depends on testing gene transfer vectors in hemophilic and nonhemophilic animals. Available animal models include factor VIII or factor IX knockout mice as well as dogs with spontaneous hemophilia A or B. Large animals (particularly dogs) more closely replicate the requirements for correction of human hemophilia than do mice. Small animals are more convenient to maintain and require significantly less vector for testing than do large animals. Nonhemophilic animals (mice or nonhuman primates), whose endogenous factor VIII and factor IX complicate analysis of the human proteins, have utility for safety testing of vectors; some assays can discriminate between human coagulation factors and the endogenous coagulation factors. Most animal models suffer the limitations imposed by the immune response to human factor VIII or IX protein. Clinical trials have failed to achieve significant factor VIII expression in hemophilia A patients, while one clinical trial in hemophilia B patients showed only transient therapeutic increments of factor IX expression. Gene therapy remains an investigational method with many obstacles to overcome before it can be widely used as treatment for hemophilia.

34. Retroviral Vector-Mediated Neonatal Gene Therapy Results in Long-Term and Therapeutic Levels of Canine Factor VIII Expression in Hemophilia A Mice and Dogs

Hemophilia A is a common severe bleeding disorder due to a deficiency of clotting Factor VIII (FVIII). Affected individuals are at risk for spontaneous bleeding, which can be life-threatening. It has been difficult to achieve fully therapeutic levels of expression of FVIII by gene therapy in large animal models. The hypothesis of this study is that neonatal gene therapy with a retroviral vector (RV) could produce a sustained correction of the hemophilic coagulopathy in hemophilia A, and might prevent the development of inhibitory antibodies. A Moloney murine leukemia (MLV)-based RV that contains the liver-specific human 1-antitrypsin promoter, the canine B-domain deleted Factor VIII (cFVIII) cDNA, and the Woodchuck hepatitis virus post-transcriptional regulatory element was generated. Neonatal hemophilia A mice were injected with 11010transducing units (TU)/kg of RV via the temporal vein at birth (N=10). Plasma cFVIII activity was determined by chromogenic substrate assay (COATEST FVIII) using normal canine plasma as the standards. In RV-treated hemophilia A mice, cFVIII activity was 92.5+/-20.1% of normal at 1 month after transduction, and remained high at 109.4+/-10.8% of normal at 11 months thus far. The cFVIII was also functional in an aPTT assay with 23.5% of normal cFVIII levels, and allowed all treated mice to survive after tail-clip. A previous study showed that 50% of mice produced inhibitors after neonatal transduction of a vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped RV expressing human FVIII. However, in our study, none of 12 mice that were transduced as neonates produced cFVIII inhibitors, which was determined by Bethesda assay. To verify the capability of making inhibitors in hemophilia A mice, a single dose of RV was given IV into 6-week old mice at 3.3109 TU/kg (N=3). All three mice made cFVIII inhibitors at 2-3 weeks after transduction. We conclude that completely therapeutic levels of cFVIII can be achieved in mice after neonatal transduction, and neonatal gene therapy with an amphotropic RV expressing cFVIII is not immunogenic. Based upon the above results, we performed neonatal RV transduction in two Chapel Hill hemophilia A dogs. RV was given IV at 3 days after birth at 9109 TU/kg and 7109 TU/kg, respectively. The whole blood clotting time (WBCT) in both dogs has been normalized since day 4 after RV transduction, and remained in the normal range for 4 months thus far. The plasma cFVIII activity in one RV-treated dog was 417% of normal by COATEST assay and 430% of normal by aPTT assay, and the aPTT time was normalized in this dog. The plasma cFVIII activity in another dog was 353% of normal by COATEST assay and 145% of normal by aPTT assay, and the aPTT time was shortened, but not normalized, in this dog. The plasma cFVIII activities have remained stable in both dogs for 4 months thus far. No cFVIII inhibitors were detected. No bleeding has occurred. We conclude that stable expression of therapeutic levels of cFVIII have been achieved in hemophilia A mice and dogs with neonatal transduction of an MLV-based RV.