Busulfan combined with immunosuppression allows efficient engraftment of gene-modified cells in a rhesus macaque model

Abstract Busulfanconditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppressionis required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene therapy model. We transduced half of rhesus CD34 + cells with an enhanced green fluorescent protein (GFP)-encoding vector ( immunogenic) and the other half with a γ- globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfanconditioning (5.5mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months post-transplant and stable engraftment of γ- globin-transduced cells in two animals, demonstrating that ablative busulfanconditioning is sufficient for engraftment of gene-modified cells producing non- immunogenicproteins, but insufficient to permit engraftment of immunogenicproteins. We then added immunosuppressionwith abataceptand sirolimusto busulfanconditioning and observed engraftment of both GFP- and γ- globin-transduced cells in two animals, demonstrating that additional immunosuppressionallows for engraftment of gene-modified cells expressing immunogenicproteins. In conclusion, myeloablative busulfanconditioning should permit engraftment of gene-modified cells producing non- immunogenicproteins, while additional immunosuppressionis required to prevent immunological rejection of a neoantigen.