Direct Delivery of mRNA-Loaded Lipid Nanoparticles to the Hematopoietic Stem Cell Niche

Therapeutic access to sufficient hematopoietic stem cells and progenitors for ex vivo gene therapy applications continues to pose significant challenges in patients with bone marrow failure. The advent of lipid nanoparticles (LNPs) has broadened prospects of in vivo delivery as an alternative to mobilization strategies for conventional ex vivo correction of autologous transplants using viral platforms. A continued shortcoming for LNP in vivo delivery to the hematopoietic stem cell (HSC) niche are off-target losses in lungs, liver, spleen, and other organs of high blood perfusion. Here, we hypothesized that delivery to hematopoietic and non-hematopoietic target cells in the HSC niche can be improved by direct delivery. To this end, we are performing comparative studies between intrafemorally (IF) and intravenously (IV) delivered reporter mRNA-LNPs.

Up to 12.5μg of custom mCherry mRNA (5’ CleanCap, 101 poly-A tail, N1-methylpseudouridine modified) packaged into LNPs (diameter 80 ± 5nm; PDI 0.01) was delivered by either IF (injected into one femur at distal end between condyles) or IV (tail vein) injection in C57BL/6 mice aged 6-9 weeks. Injected animals were sacrificed at set intervals post-injection. Red blood cell lysed cell fractions were harvested from flushed ipsilateral and contralateral femurs and from spleens before immunolabeling (Lin, Sca-1, c-kit, CD48, CD150) to identify short-term and long-term HSCs and multipotent progenitors by FACS. Expression of mCherry was measurable as early as 6 hours and was detected up to 72 hours post-injection. When compared to IV injections, robust increases in IF bone marrow transfection rates were measured across several metrics of delivery and expression. At 24 hours post-injection, we measured over 15.8 ± 7.3% mCherry positive cells across all live cells, and LSK progenitor subpopulations were on average 21.1 ± 11.1% positive, reaching over two-fold increase in average reporter positivity rates in IF injected femurs compared to those in IV injected animals. Within mCherry positive populations, over two-fold increase in mean fluorescence intensity (MFI) was detected for all populations measured, suggesting increased levels of mRNA delivery and gene product per cell. RNA extracted from processed whole bone marrow cells revealed increased levels of mCherry mRNA present in injected femur cell fractions and was measured at 24 hours post-injection with relative concentrations on average 196.7 ± 108.4 fold higher than tail vein. Despite the elevated mCherry expression in the injected femur between IF and IV delivery, positivity rates and intensities in other measured tissues were similar. Given evidence of niche cell dysfunction in several bone marrow failure syndromes, studies looking at delivery to other bone marrow niche cells, including endothelial cells, mesenchymal stem cells, and osteoprogenitors are currently underway. For comparison, we performed in vivo (IF or IV) delivery of VSV-G pseudotyped lentivirus with reporter transgene cassettes under constitutive promoters. Yet, despite achieving copy numbers of up to 0.2 copies per genome in injected femurs, we did not observe measurable amounts of fluorescence within the same time frame as with LNPs or up to two weeks post-injection.

Overall, we believe that direct delivery of LNPs to the bone marrow niche warrants further study to reverse hematopoietic manifestations of bone marrow failure disorders.

Alameh:AexeRNA: Other: Co-founder; McKinsey and Co.: Membership on an entity's Board of Directors or advisory committees. Weissman:AexeRNA: Other: Co-founder; BioNTech: Research Funding. Rivella:Sanofi Aventis U.S.: Consultancy; Protagonist Therapeutics: Consultancy; Noble insight: Consultancy; Keros Therapeutics: Consultancy; Slingshot Insight: Consultancy; Ghost Tree Capital: Consultancy; FORMA Therapeutics: Consultancy; First Manhattan Co.: Consultancy; Catenion: Consultancy; Celgene Corporation: Consultancy; Cambridge Healthcare Res: Consultancy; Disc Medicine: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Techspert.io: Consultancy; BVF Partners L.P.: Consultancy; Rallybio, LLC: Consultancy; venBio Select LLC: Consultancy.