In Vivo Delivery of RNA By Lipid Nanoparticles (LNP) to Hematopoietic Stem Cells and T Cells in Humanized Mice and Non-Human Primates

Genomic medicines have significant therapeutic potential to cure rare genetic diseases and cancer, but currently, most treatments rely on ex vivo cell editing, which is time consuming, costly, and suffers from logistic and manufacturing challenges. In contrast, in vivo delivery of genomic medicines would unlock new therapeutic areas and provide broader patient access. However, the success of in vivo delivery is dependent on the development of safe delivery vehicles capable of transporting the genomic medicines to their intended target tissues and cells. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used for in vivo delivery beyond liver and muscle tissue. Challenges in extrahepatic delivery of LNPs include adequate tissue-specific targeting to achieve therapeutic levels of genome editing while avoiding anti-drug immune responses. We have developed novel LNPs designed to deliver RNA payloads to either T cells or hematopoietic stem cells (HSCs) in vivo.

Using our novel T-cell LNPs, we have demonstrated single-dose in vivo delivery of GFP mRNA in two humanized mouse models. In the spleen of humanized NSG mice (pre-engrafted with human HSCs 12-weeks prior to study), we achieved GFP expression in 24% of human CD3⁺ T cells. In another model, NSG mice infused with human T cells 14-days prior to study, GFP expression was observed in 76% of T cells in the spleen. Single-dose administration of this T-cell LNP in rhesus macaque resulted in 40% GFP expression in peripheral blood T cells. Furthermore, we did not observe any detectable GFP expression in human or rhesus B cells, suggesting this LNP system may be suitable for the development of in vivo CAR-T therapies for B cell malignancies.

Separately, we treated humanized NBSGW mice (pre-engrafted with human HSCs for 12 weeks) with a single dose of our HSC LNPs delivering GFP mRNA. Expression of GFP was observed in approximately 90% of bone marrow hematopoietic stem and progenitor cells (HSPCs defined as Lin^-CD34⁺CD38^- cells) and approximately 95% of long-term bone marrow HSC-enriched population (HSCs defined as Lin^-CD34⁺CD38^-CD90⁺CD45RA^- cells). Single-dose intravenous administration of the same LNP in cynomolgus macaques led to GFP expression in 61% of HSPCs and 62% of HSCs in the bone marrow.

Gene editing presents a greater challenge than reporter mRNA delivery due to the increased size and complexity of the RNA cargos. Toward this goal, we went on to demonstrate that our HSC LNP platform can deliver Cas9 mRNA, with a beta-2 microglobulin (B2M) sgRNA, to human HSCs in humanized NBSGW mice resulting in the knockout (KO) of the B2M gene through the introduction of insertion or deletion mutations. Likewise, following administration of a B2M editing T-cell LNP in humanized NSG mice, we demonstrated B2M KO in splenic human CD3⁺ T cells.

Together, these in vivo delivery results indicate our LNP delivery platform is capable of efficiently delivering RNA payloads to different hematopoietic cell types in blood and tissues, providing a potential delivery platform for in vivo genome editing.