Targeted Lipid Nanoparticle Delivery of an RNA Gene Writer In Vivo Enables Generation of CAR-T Cells in a Humanized Mouse Model

The FDA approval of autologous ex vivo Chimeric Antigen Receptor (CAR) T-cell therapies have added a powerful tool to treat relapsed and refractory leukemias and lymphomas. However, significant challenges limit access to these therapies including long vein-to-vein times for drug product delivery, viral vector supply chain limitations, cumbersome manufacturing processes and high costs. Allogeneic CAR-T therapies have not achieved the same level of therapeutic efficacy as conventional autologous T cell therapies highlighting the critical unmet need for same-day in vivo treatment options. RNA Gene Writers leverage target-primed reverse transcription (TPRT) biochemistry that evolved from non-LTR retrotransposons to modify the genome of cells using RNA templates. Moreover, RNA Gene Writers can be engineered to catalyze a variety of editing reactions, such as writing or introducing gene-length DNA sequences, as well as rewriting or making single nucleotide substitutions or small insertions or deletions to the genome. These edits can be achieved by delivering all-RNA compositions to primary cells in vitro or in vivo, eliminating the need for viral vectors and DNA template-based genome editing. Initial testing of T cell specific LNPs containing a Gene Writer with a GFP or CAR template in vitro led to an observed >70% GFP+ and ~40% CAR+ T cells, respectively, in primary human T cells t. Moreover, these CAR+ T cells can clear antigen specific tumors in vivo in human xenograft mice mediating robust cytotoxic response and CAR-T cell expansion. For in vivo applications, targeting and editing of resting T cells is a critical milestone to overcome. Optimization of our T cell specific LNPs and delivery to freshly isolated human primary T cells in vitro, without pre-activation, resulted in an average of 21% CAR expression. CAR+ cells generated in this manner are potent in their ability to drive cytotoxicity, cytokine production and CAR-T cell expansion in response to multiple tumor types. In proof-of-concept studies, we achieved transient delivery of GFP mRNA to approximately 80% and 40% of T cells in humanized mouse and non-human primate (NHP) models, with minimal delivery to B cells. We next validated our in vitro CAR-T results in various in vivo models. Studies in humanized NSG mice achieved an average of 6% CAR writing in implanted human T cells when using T cell targeted LNPs. CAR writing in other relevant tissues, such as B cells and liver, was not observed. The ability to generate in vivo CAR-T cells using Gene Writers and our proprietary LNP delivery platform provides a unique opportunity to potentially eliminate the challenges associated with conventional ex vivo CAR-T cell therapies and viral based in vivo CAR-T gene therapies.