Bagirath Gangadharan

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Comparison of Factor VIII Transgenes Bioengineered for Improved Expression in Gene Therapy of Hemophilia A

Successful gene therapy of hemophilia A depends on the sustained expression of therapeutic levels of factor VIII (fVIII). Because of mRNA instability, interactions with resident endoplasmic reticulum (ER) chaperones, and the requirement for carbohydrate-facilitated transport from the ER to the Golgi apparatus, fVIII is expressed at much lower levels from mammalian cells than other proteins of similar size and complexity. A number of bioengineered forms of B domain-deleted (BDD) human fVIII have been generated and shown to have enhanced expression. Previously, we demonstrated that recombinant BDD porcine fVIII exhibits high-level expression due to specific sequence elements that increase biosynthesis via enhanced posttranslational transit through the secretory pathway. In the current study, high-expression recombinant fVIII constructs were compared directly in order to determine the relative expression of the various bioengineered fVIII transgenes. The data demonstrate that BDD porcine fVIII expression is superior to that of any of the human fVIII variant constructs tested. Mean fVIII expression of 18 units/10(6) cells/24 hr was observed from HEK-293 cells expressing a single copy of the porcine fVIII transgene, which was 36- to 225-fold greater than that of any human fVIII transgene tested. Furthermore, greater than 10-fold higher expression was observed in human cells transduced with BDD porcine fVIII versus BDD human fVIII-encoding lentiviral vectors, even at low proviral copy numbers, supporting its use over other human fVIII variants in future hemophilia A gene therapy clinical trials.

Functional aspects of factor VIII expression after transplantation of genetically-modified hematopoietic stem cells for hemophilia A

Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti‐fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically‐modified hematopoietic stem cells (HSCs) encoding a B‐domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response.

Enhanced biosynthesis of coagulation factor VIII through diminished engagement of the unfolded protein response.

Human and porcine coagulation factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast, a significant proportion of porcine fVIII was localized to the Golgi indicating efficient transit through the secretory pathway. Overexpression of BiP, an integral UPR protein, reduced the secretion of human fVIII by 50%, but had no effect on porcine fVIII biosynthesis. In contrast, expression of BiP shRNA increased human fVIII expression levels. The current data support the model of differential engagement of UPR by human and porcine fVIII as a non-traditional mechanism for regulation of gene product biosynthesis.

Potentiation of Thrombin Generation in Hemophilia A Plasma by Coagulation Factor VIII and Characterization of Antibody-Specific Inhibition

Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20–30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as “type I”) kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as “type II”) inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.

Development and Characterization of Recombinant Ovine Coagulation Factor VIII

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the worlds population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.

Hematopoietic Stem Cell Function in a Murine Model of Sickle Cell Disease

Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS). We examined the oxidative effects of sickle cell disease on hematopoietic stem cell function in a sickle mouse model. In vitro colony-forming assays showed a significant decrease in progenitor colony formation derived from sickle compared to control bone marrow (BM). Sickle BM possessed a significant decrease in the KSL (c-kit+, Sca-1+, Lineage−) progenitor population, and cell cycle analysis showed that there were fewer KSL cells in the G0 phase of the cell cycle compared to controls. We found a significant increase in both lipid peroxidation and ROS in sickle-derived KSL cells. In vivo analysis demonstrated that normal bone marrow cells engraft with increased frequency into sickle mice compared to control mice. Hematopoietic progenitor cells derived from sickle mice, however, demonstrated significant impairment in engraftment potential. We observed partial restoration of engraftment by n-acetyl cysteine (NAC) treatment of KSL cells prior to transplantation. Increased intracellular ROS and lipid peroxidation combined with improvement in engraftment following NAC treatment suggests that an altered redox environment in sickle mice affects hematopoietic progenitor and stem cell function.

Enforced expression of cytosolic 5'-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection

Purpose: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells. Method: In this study, we investigate retroviral transfer of cytosolic 5′-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2′-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment. Results: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression. Conclusion: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker

912. Strategies using retroviral coexpression of indoleamine 2,3 dioxygenase and fas ligand for allogeneic bone marrow transplantation

Regulation of allogenic immune response could play a very significant role in establishing stable mixed chimerism for the treatment of several hematological diseases. Indoleamine 2, 3 dioxygenase (IDO) is a rate-limiting enzyme in tryptophan degradation along the kynurenine catabolic pathway and has been shown to regulate allogenic immune responses across the placental barrier. Also, engagement of CTLA4 with CD80/86 on antigen presenting cells has been shown to up-regulate IDO expression and play a significant role in immune regulation. Further, IDO responsive T-cells have been shown to be more susceptible to Fas (CD95) induced apoptosis. Fas ligand (FasL) is another protein that plays an important role in immune homeostasis, inducing apoptosis through interaction with its receptor, Fas. In this context, we generated a novel chimeric protein linking the carboxy terminus of IDO to the N-terminus of FasL with a (Gly) 4 (Ser) 3 repeat linker sequence using standard PCR cloning techniques. The cDNA encoding the chimeric protein was cloned into an MSCV retroviral vector co-expressing either eGFP or N-acetyl transferase for selection with puromycin. Control vectors encoding only IDO or FasL were also generated. The retroviral constructs were confirmed by sequencing of the entire transgene and by in vitro analysis of transfected NIH3T3 cells. Recombinant MSCV retrovirus pseudotyped with an ecotropic envelope was generated by transient transfection of GP2-293T producer cells. NIH3T3 cells were transduced with concentrated virus at an MOI of 1, and harvested after 48 hours for FasL and IDO expression. FasL and IDO-FasL modified fibroblasts showed a comparable marked reduction in their growth rate compared to IDO transduced or unmodified NIH 3T3 cells. Flow cytometric analysis of cells modified with IDO-FasL/GFP showed that all GFP expressing NIH3T3 cells were FasL positive. The slower growth rate of the fibroblasts was shown to be the result of Fas expression on NIH3T3 cells. Using the IDO-FasL/puro construct, murine bone marrow cells were transduced and plated in methylcellulose. Colony counts of the IDO-FasL gene modified cells were comparable with that of nontransduced or IDO only transduced cells, indicating expression of the chimeric protein does not change the normal growth of hematopoietic progenitor cells. IDO activity assays, determined spectrophotometrically for kynurenine production, showed similar activities between cells transduced with the IDO-FasL construct and IDO only construct, further indicating the chimeric protein is functional. To study the application of these constructs in an allotransplant setting, non-lethally irradiated mice have been transplanted with bone marrow cells transduced to express IDO, FasL, or the chimeric protein and are being followed for alloengraftment.

253. Regression of Large Solid Tumors Using Engineered Drug Resistant Immunocompetent Cells and Selective Chemotherapy

Chemotherapy remains a central approach for the treatment of cancer but is limited by severe toxic side effects, particularly on the hematopoietic system. Various immunotherapy strategies are less toxic, such as tumor vaccines and cytokine therapy, and are emerging areas of cancer treatment. Although promising, these methods have limited effectiveness on established tumors. Combining chemotherapy agents with immunotherapy has proven difficult because development of an immune response can be significantly impaired in a chemotherapy-induced immunosuppressive environment. Therefore, we are investigating strategies that will allow cytotoxic chemotherapy and immunotherapy treatments to be used concurrently. Our aim is to generate drug resistant immunocompetent T-lymphocytes using retroviral gene transfer of cDNA sequences that confer drug resistance. Our drug resistant immunotherapy approach entails generating tumor-specific genetically engineered immunocompetent cells, such as T and NK cells, by transducing hematopoietic stem cells (HSC) with a retroviral vector encoding the L22Y variant of dihydrofolate reductase (L22YDHFR). This construct confers >10-fold resistance to the cytotoxic agent trimetrexate (TMTX) compared to non-modified hematopoietic cells. Drug resistant lymphocytes are generated by transplanting drug resistant bone marrow into irradiated recipient mice. Upon engraftment immunocompetent cells encoding the drug resistant marker are generated. To examine the effectiveness of drug resistance immunotherapy, C3H mice reconstituted with drug resistant bone marrow were inoculated with AG104 sarcoma cells and treated with either an agonistic anti-4-1BB antibody alone, TMTX alone, or the combination. Approximately 60% of animals transplanted with gene-modified cells and treated with anti-4-1BB, which induces CD8+ and NK anti-tumor responses, achieved complete tumor regression within 30 days. Although TMTX treatment alone regressed tumor size transiently, there was no effect on tumor clearance. However, animals treated with the combination of TMTX and anti-4-1BB antibody completely eliminated tumors in 100% of mice. Also important, compared to animals transplanted with non-modified marrow, hematopoiesis of animals transplanted with drug resistant marrow is protected against chemotherapy-induced toxicities. These results show that drug resistant immunocompetent cells can be conserved after a chemotherapy challenge and appear to retain their anti-tumor activity. This study supports our hypothesis that, in the context of drug resistant immunocompentent cells, chemotherapy can be used to control tumor growth and provide the critical time needed for the establishment of an effective anti-tumor immune response.