Assessment of Transduction of CD34+ Human Hematopoietic Stem Cells from Patients with Severe Hemophilia-Α with Lentiviral Vector Carrying a High Expression FVIII Transgene (CD68-ET3-LV)

Background: Alternative strategies are needed for the large number of people with hemophilia who are ineligible for AAV based gene therapy due to age or high levels of anti-AAV neutralizing antibodies. High inter-individual variability of expression and ill sustained factor levels are also challenges with AAV based gene therapy for hemophilia A, in particular. We have developed a third-generation lentiviral vector mediated hematopoietic stem cell-based gene therapy for hemophilia A. (Doering et al Human Gene Therapy 2018; 29: 1183-1201) This vector (CD68-ET3-LV) has a high expression FVIII transgene with a CD68 promoter targeting expression in monocytic cells predominantly. We report here successful transduction of severe hemophilia A patient derived human hematopoietic stem cells with this vector. Methods: Mobilized peripheral blood stem cells were collected by apheresis from three patients with severe hemophilia A without inhibitors who also received prophylactic clotting factor replacement therapy during this period. CD34+ hematopoietic stem cells (HSCs) were enriched on the CliniMACS Plus® system (Miltenyi Biotec, Bergish Gladbach, Germany) using the CliniMACS CD34® reagent system. Purified HSC were then transduced with this CD68-ET3-LV as follows - HSCs cultured on retronectin coated surfaces in xenofree media with cytokines were exposed to clinical grade CD68-ET3-LV vector in two ways - a double transduction protocol without any enhancer and a single transduction with an enhancer. After completion of this step, the product was assessed for viability and vector copy number (VCN) by trypan blue labelling and Q-PCR performed on genomic DNA from CFU cells, respectively. Transduced HSCs were also assessed for their engraftment potential. CD68-ET3-LV vector transduced HSC (1x10 6) were transplanted into NBSGW mice via tail vein injection. Engraftment was assessed at 16 weeks after transplantation. This protocol was approved by the Institutional Review Board of the Christian Medical College, Vellore, India. Results: G-CSF (10 ug/kg/day) based peripheral blood stem cell mobilization was well tolerated by all participants. The apheresis procedures which were done with plasma FVIII levels in the normal range after prophylactic replacement therapy were unremarkable. The total collection of mobilized CD34+ cells from the three donors was 268±80.5x10 6 CD34+ cells. An aliquot of 2x10 6 CD34+ cells/ml was used in the transduction experiments. The data on viability and vector copy number post transduction shown in the tables 1 and 2 confirm that viability was not affected by the manipulation of these HSCs in both double and single transduction methods. The vector copy number (VCN) in the genomic DNA obtained from CFU cells collected from CD34+ HSCs cultured in MethoCult H4434™ (Stem Cell Technologies™, Vancouver, Canada) varied from 0.62 to 1.21 in double transduction (n=3) and nearly doubled in the single transduction method with an enhancer to VCNs of 1.5 and 2.4 in two samples tested. Transplantation studies in NBSGW mice showed successful engraftment of human CD34+ HSCs with a mean engraftment of 80.99 ± 4.2% of CD45+ multilineage hematopoietic cells in the bone marrow 16 weeks after transplantation. Conclusion: To the best of our knowledge, this is the first report of transduction of mobilized peripheral blood CD34+ HSCs from patients with severe hemophilia A using a clinical grade lentiviral vector with a high expression FVIII transgene. These data establish feasibility and safety of the procedure. The transduction protocol showed good efficiency with very high viability, significant VCN and good engraftment of these gene modified human HSCs in a mouse model. These protocols are currently being evaluated in a first in human phase 1 clinical trial of gene therapy for severe hemophilia A without inhibitors.