Several hurdles limit the successful gene therapy treatment of hemophilia A. For example,

  • human coagulation factor VIII (fVIII) is inefficiently biosynthesized and has proven difficult to express using recombinant viral gene transfer,

  • target cells have been inefficiently transduced, and

  • fVIII expression can result in inhibitory antibody formation to the transgene product.

Recently, we showed that B-domain-deleted porcine fVIII (BDDpfVIII) is expressed at levels 100-fold higher than BDD human fVIII (BDDhfVIII) in both in vitro and in vivo systems. Ex vivo modification of murine bone marrow cells with recombinant BDDpfVIII-encoding retrovirus followed by transplantation into non-myeloablative hemophilia A murine recipients resulted in long-term fVIII expression at levels considered curative, and the transplanted mice were immunogenically tolerant to the BDDpfVIII expressed from the genetically-modified hematopoietic cells. We now have identified the residues within BDDpfVIII responsible for the high-level expression and incorporated them into the cDNA encoding BDDhfVIII, thus generating a high-expression hybrid human/porcine (HP) construct composed of 90% human and only 10% porcine sequence. High titer recombinant oncoretroviral and lentiviral vectors were generated with the chimeric HP-fVIII sequence and used to transduce human cell lines, murine sca-1+ cells, and primary human hematopoietic cells. The lentiviral vectors efficiently transduced the human cell lines HEK-293, Hela, K562, EU-1, jurkat and U937, the former 4 being hematopoietic cell-derived. In HEK-293 cells, a linear increase in fVIII expression was observed with increasing MOI, and at 7-days after transduction the expression was 28 units/106 cells/24 hr, compare to only 4 units/106 cells/24 hr for BDDhfVIII (MOI = 3). In the hematopoietic cells lines, therapeutically significant, but approximately 10-fold lower expression was observed. Transplantation of oncoretroviral or lentiviral transduced murine hematopoietic stem and progenitor cells into lethally conditioned hemophilia A recipient mice resulted in long-term fVIII expression at therapeutic levels (>10% normal human levels) despite having only 5% or less genetically-modified blood mononuclear cells. No fVIII activity was observed in hemophilia A mice transplanted under identical conditions using recombinant virus encoding BDDhfVIII. Human CD34+ cells were isolated from fresh bone marrow aspirates, cultured overnight, and then transduced with either lentivirus encoding eGFP or the chimeric HP-fVIII (MOI = 5). Similar numbers of progenitor colonies grew in methylcellulose cultures for each construct indicating no increased toxicity resulting from HP-fVIII expression. Real-time PCR using genomic DNA isolated from pooled colonies from methylcellulose plates showed gene-marking levels of approximately 0.3 proviral genomes/diploid genome equivalent, thus indicating a transduction efficiency of approximately 30%. Furthermore, fVIII activity levels were greater in CD34+ cell cultures transduced with vectors encoding chimeric HP-fVIII (0.1 units/106 cells/24 hr) than negative control CD34+ cells transduced with vectors encoding eGFP (<0.01 units106 cells/24 hr). From these studies, it is concluded that humanized high-expression HP-fVIII transgenes can be utilized to significantly increase fVIII expression levels in the context of future clinical gene transfer-based therapies for hemophilia A.

Disclosures: Doering:Expression Therapeutics: Equity Ownership. Denning:Expression Therapeutics: Employment. Kerstann:Expression Therapeutics: Employment. Keefe:Lentigen: Employment. Dropolic:Lentigen: Employment, Equity Ownership. Spencer:Expression Therapeutics: Equity Ownership.

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