Targeted lnp delivery of an RNA gene writer In Vivo enables generation of functional CAR-T cells

Abstract Autologous ex vivo Chimeric Antigen Receptor (CAR) T-cell therapies are a powerful treatment option for relapsed and refractory leukemias and lymphomas. However, significant challenges limit access to these therapies, including long vein-to-vein times for drug delivery, viral vector supply chain constraints, and complex manufacturing processes. While allogeneic CAR-T therapies offer an alternative, they have not yet matched the therapeutic efficacy of conventional autologous T-cell therapies, highlighting the need for in vivo treatment solutions. RNA Gene Writers are designed to edit cellular genomes using RNA templates by harnessing the mechanism of target-primed reverse transcription (TPRT). These Gene Writers can be engineered to perform various editing reactions, from introducing gene-length DNA sequences to rewriting genomic regions by making single-nucleotide substitutions to small insertions or deletions. 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 methods. For in vivo applications, targeting and editing of resting T cells remains a critical hurdle to overcome. Using T cell targeted LNPs (tLNPs), we co-delivered Gene Writer mRNA and a CD19 CAR template to human primary T cells in vitro, without pre-activation, and generated an average of ~40% CD19 CAR+ cells. These CAR+ T cells were observed to be functional, driving cytotoxicity, cytokine production, and cell expansion in response to multiple tumor types in vitro. For in vivo proof-of-concept studies, we tested two humanized xenograft mouse models for CAR specific activity. We performed a single intravenous infusion of tLNPs formulated with Gene Writer mRNA and a CD19 CAR template in a tumor-bearing xenograft mouse model. This generated an average of 24% CAR-T cells in vivo that subsequently expanded and eradicated tumor burden. Additionally, tLNP delivery of Gene Writer mRNA with a CD20 CAR template in a CD34+ engrafted humanized mouse model achieved an average of 55% CD20 CAR+ T-cells at peak expansion as well as complete elimination and sustained clearance of human B cells, highlighting its therapeutic potential in autoimmune diseases. Central to demonstrating the therapeutic potential of in vivo CAR-T in humans is the development of proof-of-concept studies in non-human primate (NHP) models. We tested our lead candidates in vitro and found that our Gene Writing system and tLNPs were cross reactive and generated up to 60% CAR writing in NHP T cells. To evaluate the delivery system in vivo in NHP, we used tLNPs formulated with a transient GFP mRNA reporter and observed approximately 40% of T cells expressed GFP, with minimal delivery to B cells. This enables a path towards in vivo NHP editing, either through Gene Writer-mediated permanent T cell editing or transient T cell engineering by CAR mRNA delivery. The ability to generate CAR-T cells in vivo using Gene Writers and our proprietary tLNP 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 for both oncology and autoimmune diseases.