Effects of G-CSF and 5-FU as conditioning regimens in platelet-targeted gene therapy for hemophilia B.

Abstract

Platelet-targeted gene therapy for hemophilia entails modifying a patient's hematopoietic stem cells (HSCs) ex vivo to produce platelets containing coagulation factors, offering a potential cure by localized factor release at injury sites. However, the associated bone marrow transplantation carries significant risks, underscoring the critical need for low-toxicity conditioning regimens to enable clinical translation. This study evaluated G-CSF and 5-FU as conditioning regimens to facilitate the engraftment of HSCs expressing platelet-targeted FIX Padua. Hemophilia B (HB) mice were conditioned with G-CSF, 5-FU alone, or a combination of G-CSF, plerixafor, and 5-FU. Bone marrow mononuclear cells (BM-MNCs) from transgenic donors (2bF9-R338L) expressing platelet-stored FIX Padua were transplanted. G-CSF conditioning enabled long-term engraftment with 1 × 10BM-MNCs; however, the efficacy declined significantly with lower cell doses. A single-dose of 5-FU (150 mg/kg) administered one day pre-transplant achieved optimal FIX expression and minimal toxicity. Critically, a combined regimen of G-CSF/plerixafor plus timed 5-FU (day -1) yielded stable, long-term platelet FIX expression and phenotypic rescue. The success of 5-FU underscores the importance of creating a transient niche vacancy for donor HSC engraftment. These results demonstrate that this mobilization-based, non-myeloablative conditioning strategy is a promising approach for platelet-targeted gene therapy for hemophilia.