In Utero mRNA Lipid Nanoparticle Delivery in a Murine Model of Fanconi Anemia

Fanconi anemia (FA) is a rare multisystem genetic disorder characterized by defects in DNA repair, cancer predisposition and early failure of the hematopoietic system. Hematopoietic stem cell (HSC) transplant is curative, but eligibility is limited by donor availability. Alternatively, early phase clinical trials of lentiviral HSC gene transfer show great promise but remain constrained by a depleted HSC pool in older patients, ineffective mobilization, and HSC sensitivity to ex vivo manipulation. Recent studies revealed the fetal onset of HSC depletion in FA before the later manifestation of peripheral cytopenia, suggesting that prenatal gene therapy offers a minimally invasive, potentially preventive approach during fetal immune tolerance. Building on the successful use of lipid nanoparticles (LNP) for mRNA vaccines and for treatment of monogenic metabolic disorders; we hypothesize that LNP delivery of modified Fancc mRNA for protein replacement may rescue fetal HSC pool expansion and delay postnatal HSC attrition in Fancc^-/- (KO) mice.

To demonstrate feasibility, we used 5‘ Clean Cap, 101 poly-A tail methyl-pseudouridine modified luciferase mRNA packaged into LNPs (diameter 80 ± 5nm; PDI 0.01) and compared the half-life of linear vs. circular constructs. C57BL/6 mouse bone marrow derived hematopoietic stem and progenitor cells (HSPC) treated with circularized luciferase mRNA had detectable luminescence for up to 7 days as compared to 4 days when using linear mRNA. To test FA phenotype rescue, we used polyvinyl-EtOH supplemented ex vivo expansion of Fancc^-/- CD150⁺ CD48^- Lin^- Sca1⁺ c-kit⁺ long term (LT- HSC) as a novel assay to test for HSPC proliferation. We showed that a single dose of circular LNP^Fancc (LNP^CFancc) improved HSPC tolerance to mitomycin (MMC) up to 5 days, compared with 3 days upon treatment with Linear LNP^Fancc. To further verify functionality on ex vivo expanded LT-HSC, serial treatment with LNP^CFancc days 0, -7 and -14 resulted in significantly improved Fancc^-/- doubling time, reaching wildtype (WT) equivalence by day 14. This was accompanied by significantly improved progenitor colony formation. For an in vivo assessment of LNP^CFancc on prenatal HSC expansion, C57BL/6 Fancc^+/− heterozygote pregnancies were used to generate Fancc^-/- and Fancc^+/+ (WT) littermate fetuses. Baseline analysis of Fancc^-/- embryos at gestational age day E15.5 showed higher numbers of KO fetuses, 26% as compared to 14% observed P21 postnatally, suggesting a late gestational lethality. Compared to their wildtype WT littermates, KO fetus’ weights and total fetal liver cell count were 87% and 72% of WT, respectively. In the fetal HSPC compartment, single vitelline vein injections of LNP^CFancc to embryos at E14.5 resulted in small improvements in short term (ST-HSC) and LT-HSC without significant effect on myeloid or lymphoid progenitor cell population. However, LNP^CFancc treatment improved colony formation of sorted KSL cells (c-Kit⁺ Sca^-1+ Lin^-), reaching 81% of WT level (from a 69% lower baseline) and conferred a significant improvement in MMC tolerance by 2-fold over untreated controls.

In conclusion, our data show that protein replacement with LNP^CFancc can support FA HSPC function and proliferation. While transient, this method for protein replacement to HSC during rapid fetal liver expansion may rescue expansion of the FA HSC pool and delay the onset of postnatal bone marrow failure. Our study provides further evidence that in utero LNP delivery of mRNA can ameliorate fetal phenotype in FA and sets the stage for gene editing in FA as a curative approach.

Rivella:Techspert.io: Consultancy; Slingshot Insight: Consultancy; Protagonist Therapeutics: Consultancy; BMS: Consultancy; BioMarin: Consultancy; Disc Medicine: Membership on an entity's Board of Directors or advisory committees, Research Funding; Vifor: Membership on an entity's Board of Directors or advisory committees; Meira GTx: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BVF Partners L.P.: Consultancy; venBio Select LLC: Consultancy; LifeSci Capital: Consultancy.