Restoring hematopoietic stem and progenitor cell function in Fancc mice by in situ delivery of RNA lipid nanoparticles

Fanconi anemia (FA) is a congenital multisystem disorder characterized by early-onset bone marrow failure (BMF) and cancer susceptibility. While <i>ex vivo</i> gene addition and repair therapies are being considered as treatment options, depleted hematopoietic stem cell (HSC) pools, poor HSC mobilization, compromised survival during <i>ex vivo</i> transduction, and increased sensitivity to conventional conditioning strategies limit eligibility for FA patients to receive gene therapies. As an alternative approach, we explored <i>in vivo</i> 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 <i>in vivo</i> 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 <i>Fancc</i> mRNA restored <i>ex vivo</i> expansion. In a proof of principle approach, LNP-treated murine <i>Fancc</i> ^<i>-/-</i> HSPCs engrafted with restored alkylator resistance up to 120 h post-treatment using circularized mRNA constructs. <i>In vitro</i> 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.