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.

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.

Hemophilia A is an X-linked gene disorder that results in a deficiency of circulating coagulation factor VIII (fVIII) and may be ameliorated by only modest amounts of circulating protein, which makes it a logical candidate for gene therapy. Due to the potential risk of insertional mutagenesis from oncoretroviral-mediated gene therapy, cell-specific expression of transgenes using self-inactivating viral vectors may provide a safer gene therapy approach for use in humans. Therefore, we constructed simian immunodeficiency virus (SIV)-based lentiviral vectors containing a 5′ long-terminal repeat (LTR) and 3′ LTR with self-inactivating U3 deletion, the bovine growth hormone polyA signal, a packaging signal (ψ), and a single internal ankyrin-1 or β-globin promoter, designated SIV-Ank and SIV-Bg, respectively. The minimal 314-bp ankyrin-1 promoter and 180-bp β-globin promoter flanked upstream by enhancing sequences, HS2, HS3, and HS4 (Hanawa et al., Hum Gene Ther, 2002) from the locus control region were cloned into the SIV vector backbone upstream from either enhanced green fluorescent protein (eGFP) or B-domain deleted porcine factor VIII (BDDpfVIII). The erythroid-specificity of each promoter was evaluated in vitro by measurement of either eGFP or fVIII expression following transduction of SIV-Ank and SIV-Bg constructs into both K562 myelogenous leukemic cells and 293T human embryonic kidney cells. GFP expression, as measured by flow cytometry, in transduced cells revealed that the ankyrin-1 and β-globin promoters are more active in K562 cells as compared to 293T cells. The β-globin promoter yielded higher mean fluorescent intensity values for GFP compared to the ankyrin-1 promoter at similar MOIs in K562 cells, suggesting stronger β-globin promoter activity in these cells. Transduction of cells with the SIV vector encoding BDDpfVIII driven by the β-globin promoter resulted in a 14-fold higher number of transcripts per DNA copy number in K562 cells compared to 293T cells, while cells transduced with the ankyrin-l promoter had only a 1.4-fold greater number of transcripts per DNA copy number. In addition, SIV-Bg-fVIII-modified K562 cells produced a 5.2-fold greater number of transcripts per DNA copy number than SIV-Ank- fVIII-modified cells. To evaluate the usefulness of these vectors for in vivo expression of BDDpfVIII, hemophilia A mice (exon 16 knockout) were conditioned with 11 Gy total body irradiation and transplanted with gene-modified Sca-1+ cells transduced with either SIV-Ank-fVIII, SIV-Ank-eGFP, SIV-Bg-fVIII, or SIV-Bg-eGFP. The expression of eGFP from donor red blood cells in recipient mice was approximately 8–12% using both the ankyrin-1 and β-globin promoter constructs. Mice that received cells transduced with SIVAnk- fVIII demonstrated therapeutic levels of plasma fVIII up to 0.5 units/mL (i.e. 50% normal human levels). However, fVIII expression decreased over time and real-time PCR analysis of peripheral blood cells confirmed the loss of detectable fVIII transgene by 6 weeks after transplantation, suggesting there was predominantly gene transfer into short-term repopulating hematopoietic cells. Mice transplanted with SIV-Bg-fVIII-modified hematopoietic stem cells demonstrated a similar rise and fall of fVIII expression within the first 4 weeks after transplantation, and showed an increase in fVIII expression by 6 weeks. At 8 weeks post transplantation, fVIII levels greater than 300% normal human levels were observed. Red blood cell count, hemoglobin, and red blood cell morphology were normal despite the high level of expression of fVIII. Overall these data demonstrate the potential for therapeutic expression of factor VIII using a self-inactivating lentiviral vector containing an erythroid-specific internal promoter.