Abstract
Fanconi anemia (FA) is a congenital multisystem disorder characterized by early-onset bone marrow failure (BMF) and cancer susceptibility. While ex vivo gene addition and repair therapies are being considered as treatment options, depleted hematopoietic stem cell (HSC) pools, poor HSC mobilization, compromised survival during ex vivo transduction, and increased sensitivity to conventional conditioning strategies limit eligibility for FA patients to receive gene therapies. As an alternative approach, we explored in vivo protein replacement by mRNA delivery via lipid nanoparticles (LNPs). Our study aims to address several key obstacles to current mRNA-LNP treatment: access to the HSC niche, effective expression half-life, and potential mRNA LNP immunogenicity. Results demonstrate efficient in vivo LNP transfection of murine BM via intravenous or intrafemoral injections, yielding reporter expression across hematopoietic and non-hematopoietic BM niche populations. Functionally, LNP delivery of modified Fancc mRNA restored ex vivo expansion. In a proof of principle approach, LNP-treated murine Fancc -/- HSPCs engrafted with restored alkylator resistance up to 120 h post-treatment using circularized mRNA constructs. In vitro delivery of mRNA LNPs resulted in modest differences in innate immune target gene expression in both FA and wild-type HSPCs. Our results suggest that mRNA-LNP-based protein replacement therapy holds promise for clinical translation.