In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection.

BACKGROUND: Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A.
OBJECTIVE: In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities.
METHODS: A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMT(P140K) cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O(6)-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis.
RESULTS: Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 10(8) platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge.
CONCLUSION: we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy.