Phenotypic HSPC Rescue By RNA Lipid Nanoparticles in a Murine Model of Fanconi Anemia

Background: Fanconi Anemia (FA) is a recessively heritable multisystem disorder that manifests with bone marrow failure in early childhood. Current gene therapy trials for FA rely on transplantation of ex vivo transduced autologous hematopoietic stem cells (HSCs). This approach is limited by poor mobilization efficiency and a depleted HSC pool in FA patients. The recent use of mRNA encapsulated within lipid nanoparticles (LNP^mRNA) for cancer immunotherapy and vaccines has created an interest in adapting mRNA delivery for protein replacement therapies. Phase 1/2 trials have demonstrated both safety and efficacy for in vivo treatment of multiple monogenic metabolic disorders. We hypothesized that LNP^Fancc could be developed as an in vivo protein replacement therapy for FA.

Methods: Custom mRNA transcripts modified with N1-methylpseudouridine and packaged into LNPs (diameter 80 ± 5nm; PDI 0.01), their performance was tested ex vivo and in vivo. LNP^mRNA were delivered either intrafemorally or by tail vein injection into Fancc^-/- mice, a mouse model of FA or Ai14 mice, a Cre-sensitive reporter mouse model. We also tested LNP performance using ex vivo expanded hematopoietic stem and progenitor cell (HSPC) populations and fresh CD150⁺CD48^-Lin^-Sca1⁺ckit⁺ (long-term hematopoietic stem cells; LT-HSC) cells obtained from wildtype (WT, Fancc^+/+) and knockout (KO, Fancc^-/-) mice. LNP formulations delivered mRNA transcripts of the fluorescent reporter mCherry (LNP^mCherry), cre-recombinase (LNP^Cre), luciferase bioluminescent reporter gene (LNP^Luc) or a truncated murine FANCC gene (LNP^Fancc).

Results: To characterize access to the bone marrow compartment in vivo, we delivered LNP^mCherry or LNP^Cre via systemic (intravenous) and direct (intraosseous) routes to healthy WT and Ai14 mice. Using LNP^Cre, we achieve up to 92.6% ± 5% tdTomato reporter expression in Lin^-Sca1⁺ckit⁺ (LSK) cells 5 days following intraosseous delivery. Consistent with reports by others, we demonstrate lower average transfection rate of 24.8% ± 15.9% fluorescent positivity rates in LSK cells after intraosseous LNP^mCherry delivery, and 9.5% ± 6.7% after intravenous delivery. mCherry fluorescent signal was detectable by FACS for up to 72 hours following LNP^mCherry injection. We reasoned that meaningful FA HSC correction would require more extended protein expression. Circular mRNA shows improved biostability and durability of expression. Comparing both linear and circular mRNA performance, we observed that circularized LNP^Luc is expressed for up to 7 days post ex vivo exposure in LT-HSC populations as compared to 3 days when using linear LNP^Luc. Functionally, ex vivo LNP^Fancc treatment improves proliferation rates and Mitomycin C (MMC) resistance in colony forming unit (CFU) assays of Fancc^-/- HSPC. Here, LNP^Fancc treated HSPC demonstrate improved MMC survival rates reaching 37.2% ± 10.5% up from 16.1% ± 8.2%. Ex vivo expanded Fancc^-/- HSPC demonstrate an even greater benefit from LNP^Fancc treatment, with CFU survival rates increasing to 62.7% from a baseline of 3.3%. Finally, we demonstrate improved repopulation of LNP^Fancc treated HSPC 2 weeks after intravenous delivery to myeloablated recipients, measuring up to 73.9% donor chimerism after treatment with circular LNP^Fancc compared to 50% among untreated (no LNP) controls.

Conclusion: Our studies show that LNP^Fancc can rescue Fancc^-/- HSPC in vitro, and ongoing experiments reveal improved repopulation in vivo. Along with evidence for efficient in vivo delivery to the bone marrow, our data support the use of LNP^mRNA as a potential in vivo treatment of bone marrow failure in FA patients.