Preclinical Development and Validation of a Hematopoietic Stem Cell-Based Lentivector Treatment for Hemophilia a

Hemophilia A is a monogenic bleeding disorder currently treated by lifelong infusions of factor VIII (fVIII). The development of gene-based treatments for hemophilia A has been hampered by several biological hurdles including the low level expression of fVIII from genetically engineered target cells, the large transgene size, and transgene product immunogenicity. We now show that these major hurdles are overcome by i) using a bioengineered FVIII transgene (ET3) that expresses at 10 - 100-fold higher levels compared to other human FVIII sequences, ii) targeting hematopoietic stem cells (HSC) ex vivo using a self-inactivating (SIN) lentiviral vector (LV) encoding ET3, and iii) using a non-myeloablative conditioning regimen prior to HSC transplantation that results in transgene product associated immune tolerance. The clinical trial design and preclinical data were favorably reviewed by the RAC, Institutional Biosafety Committee, and FDA. However upon full-scale clinical production of the SIN-ET3-LV, an additional hurdle was discovered that resulted in low LV manufacturing titer, which we showed is due to promoter competition between the CMV/HIV-LTR and the internal promoter driving ET3 production (eg. EF1a). We show that high-titer recombinant LV encoding ET-3 can be achieved by replacing the EF1a internal promoter with one that has low activity in the 293T producer cells. CD68 is a monocyte-restricted promoter that is not active in 293T producer cell lines and thus minimizes promoter interference. Using a SIN-CD68-ET3-LV expression vector, high titer virus was generated using a laboratory scale production strategy as well as a 6 L scalable clinical LV production platform. Our scalable manufacturing process generated titers >2 x 10⁹/ml, which is sufficient to support an ex vivo HSC transduction/transplantation pilot/phase I clinical trial. Late stage preclinical testing using clinically processed vector showed that the high titer virus efficiently transduces hematopoietic cell lines, as determined by quantitative PCR, and Southern analysis, which confirmed integrated transgene stability over 8 weeks. Efficient and stable gene transfer into murine Sca-1+ cells harvested from hemophilia A mice was also achieved. A dose response of LV addition to Sca-1 cells and FVIII expression was observed in transplanted mice. Expression of >40% normal FVIII levels was achieved at multiplicity of infections (MOIs) as low 20, and long term FVIII expression was observed post HSCT. No difference in the engraftment of genetically modified cells as compared to mock-modified cells was observed. In addition, efficient, stable and dose responsive gene marking (up to 0.6 VCN/cell) of human mobilized peripheral blood(mPB)-derived CD34+ cells was reproducibly achieved. Transduction of mPB-CD34+ cells at MOIs ranging from 1 to 125 had no effect on methylcellulose colony forming unit growth when comparing SIN-CD68-ET3-LV to non-modified cells. In addition, there was no effect on mPB-CD34+ cell engraftment of NSG mice when comparing modified and mock modified cells. Furthermore, next generation sequencing-based insertion site analysis showed integration of SIN-CD68-ET3-LV to be similar, with respect to integration events near oncogenes, to that of other vectors in current clinical trials. Collectively, this comprehensive, preclinical data set provides support for regulatory submissions to conduct a pilot/phase I clinical trial of a SIN-CD68-ET3-LV transduced-HSC transplantation product for the treatment of hemophilia A.