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.