In Vivo Selection Of Genetically Manipulated Hematopoietic Stem Cells For Platelet Gene Therapy Of Hemophilia A

Our previous studies have demonstrated that targeting FVIII expression to platelets (2bF8) by lentiviral (LV) gene delivery to hematopoietic stem cells (HSCs) corrects bleeding diathesis in hemophilia A mice with or without inhibitors. Although the bleeding diathesis is improved in transduced recipients, the transduction efficiency using our current 2bF8 LV, is only about 10%, resulting a median level of platelet-FVIII (Plt-F8) of 1.5 mU/108 platelets even thought a myeloablative conditioning regimen was employed. It has been shown in clinical trials that efficient stem cell gene transfer and myeloablation is not required when there is a powerful selective advantage to the genetically modified cells. We hypothesize that incorporating a drug-resistance gene into the 2bF8 LV construct will allow for in vivo selection of 2bF8 LV-transduced cells which will result in the increase of therapeutic levels of Plt-F8 for hemophilia A gene therapy and reduce the potential for genotoxicity. To address our hypothesis, we constructed a new lentiviral vector, pWPT-2bF8/MGMT, which harbors dual genes, the 2bF8 gene and a drug-resistance gene, the MGMTP140K cassette. To explore the feasibility of the MGMT-based in vivo selection system, HSCs from FVIIInull mice were transduced with 2bF8/MGMT LV at an MOI (multiplicity of infectious) of 1, which is 1/10 of the MOI used for our regular 2bF8 LV transduction, and transplanted into littermates pre-conditioned with a non-myeloablative regimen, 660 cGy total body irradiation (TBI). We chose a low MOI because one of the goals of using the MGMT selection system is to reduce the potential for genotoxicity. After bone marrow (BM) reconstitution, the recipients were treated with O6-benzylguanine (BG) followed by 1, 3-bis-2 chloroethyl-1-nitrosourea (BCNU) monthly for 3 or 4 times. As determined by a chromogenic assay on platelet lysates, functional Plt-F8 expression in recipients was only 0.22 ± 0.15 mU/108 platelets before the drug treatment, but remarkably increased to 4.33 ± 5.48 mU/108 platelets (n = 16) after BG/BCNU drug-selective treatments. The levels of Plt-F8 in the untreated transduced control group remained low over the study period. FVIII activity was not detected in the plasma in any of the recipients even with Plt-F8 as high as 22 mU/108 platelets. The average copy number of 2bF8/MGMT proviral DNA per cell was determined by quantitative real-time PCR. 2bF8 proviral DNA was barely detectable (0.01 ± 0.02 copies/cell) in recipients before drug-selective treatment, but it increased to 0.42 ± 0.15 copies/cell after BG/BCNU treatments, confirming that 2bF8/MGMT genetically modified cells were effectively enriched in vivo after drug-selective treatment. When the tail clip survival test was used to assess phenotypic correction of the FVIIInull coagulation defect, 15 of 16 treated animals survived the tail clip challenge; in contrast, none of the untransduced FVIIInull control mice survived. When ROTEM analysis was used to determine the whole blood clotting time (CT), the CT was shortened from 3043 ± 728 seconds (n = 7) to 931± 273 seconds (n = 6) (P < 0.0001) in treated transduced recipients when compared to FVIIInull mice. There was no significant difference between wild type (722 ± 270 seconds, n = 7) and treated recipients. To ensure sustained Plt-F8 expression in BG/BCNU treated transduced recipients, some primary recipients were sacrificed 9 months after transplantation and BM mononuclear cells were transplanted into secondary recipients. Platelet lysate FVIII activity assays showed that the levels of Plt-F8 in secondary recipients were similar to those in primary recipients, confirming that long-term repopulating HSCs were successfully genetically modified by 2bF8/MGMT LV. When a low intensity pre-conditioning regimen of 440 cGy TBI was used, the levels of Plt-F8 increased from 0.06 ± 0.12 mU/108 platelets to 1.86 ± 2.06 mU/108 platelets after BG/BCNU drug-selective treatment. It is notable that no anti-FVIII inhibitory antibodies were detected in the treated recipients even after rhFVIII challenge, indicating that immune tolerance was induced in the treated animals. In contrast, all FVIIInull mice under the same challenge developed various levels of inhibitors. Taken together, we have established a powerful in vivo selective system that allows us to enrich 2bF8 LV-transduced cells and to enhance platelet-FVIII expression for hemophilia A gene therapy. Disclosures: No relevant conflicts of interest to declare.