In Vivo RNA delivery by targeted lipid nanoparticles enable gene editing in hematopoietic stem cells and T cells

Abstract Non-viral in vivo genetic medicines promise novel and potentially curative therapies with broader patient access than ex vivo gene therapy approaches. Effective in vivo genetic medicines require safe and efficient delivery of gene modifying payloads to intended cells and tissues. While clinical validation of gene therapy products formulated with lipid nanoparticles (LNPs) aimed at the treatment of liver diseases is underway, the use of LNPs for in vivo extrahepatic gene therapy remains a challenge. By optimizing LNPs for extrahepatic delivery and subsequent pairing with Tessera's novel RNA-based Gene Writing platform, we aim to enable in vivo gene editing of hematopoietic stem cells (HSCs) and T cells. Tessera's Gene Writers use target-primed reverse transcription to facilitate a range of genetic edits, from single nucleotide changes for gene correction to transgene insertions for gain of function. Multiple strategies are used to optimize our LNPs for extrahepatic delivery, including tailoring the ionizable lipid, altering the ratios of LNP components and incorporating cell-specific targeting ligands to enhance cell-specific uptake. Our HSC- LNPs achieved transient GFP mRNA reporter delivery to 90% of long-term HSCs (LT-HSCs, defined as Lin-CD34+CD38-CD90+CD45RA- cells) in both humanized mice and NHPs. In B6 mice, a single intravenous dose of ourHSC-LNP formulation reduced GFP reporter expression in the liver by 11-fold compared to a standard liver-directed LNP formulation. Furthermore, the addition of HSC-targeting elements increased GFP delivery by 55-fold in the bone marrow while maintaining low GFP expression in the liver. By pairing our HSC-targeted LNPs with an all-RNA composition consisting of Gene Writer mRNA and a beta-2 microglobulin (B2M) targeting template, we achieved approximately 75% B2M editing in LT-HSCs in humanized mice and cynomolgus macaques. In NHP, whole tissue analysis revealed 3-fold higher B2M gene editing in the bone marrow versus the liver. Our Gene Writer platform can be applied to correct the sickle cell disease (SCD)-causing mutation (E6V) to wild-type in HSCs. We first evaluated whether SCD impacted HSC delivery efficiency of our LNP. with similar GFP expression. Next, we explored the editing efficiency of our Gene Writer with a beta-globin template in mice and NHP. In humanized mice (n=6 human donors), a single dose of our LNP formulated with Gene Writer enabled an average of 50% Makassar (E6A) installation in LT-HSCs. In cynomolgus macaques, we achieved an average of 24% Makassar editing in LT-HSCs with stable multi-lineage editing observed in HSC progeny over 9 months. Together, these promising results demonstrate our ability to introduce a specific mutation in beta-globin in vivo usingour HSC-targeted Gene Writer LNPs. Separately, towards the pursuit of in vivo CAR-T therapies, we are developing T cell-targeted LNPs to enable CAR writing using our Gene Writers. A single dose of T cell-targeted LNPs with a Gene Writer and CD20 CAR template in a non-activated humanized mouse model resulted in 20% CAR-expressing T cells and peripheral B-cell aplasia. Flow analysis of peripheral T cells at peak CAR levels demonstrated an expansion of effector memory T cells, with the majority of CAR positive cells being CD8+ vs untreated control. In addition to the peripheral B cell aplasia, immunohistochemistry confirmed eradication of B cells in the spleen. Collectively, the optimized LNPs drove a potent CD8+ CAR-T response that achieved rapid and systemic B cell clearance in humanized mice. Overall, these results demonstrate the versatility of our LNP delivery and Gene Writer platforms for in vivo gene editing of HSCs and T cells. We believe our platform holds considerable promise towards the development of in vivo genomic medicines for sickle cell disease and CAR-T therapies for immune oncology and autoimmune disease.