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C6 - Gene Targeting and Gene Correction New Technologies

12: RNA-Based Gene Writer and Lipid Nanoparticle (LNP) Delivery Enables Generation of Functional Chimeric Antigen Receptor (CAR) T Cells with In Vitro and In Vivo Anti-Tumor Activity and T Cell-Specific Genome Engineering In Vivo

Type: Oral Abstract Session

Presentation Details
Session Title: New Technologies for Gene Targeting and Gene Correction

Autologous Chimeric Antigen Receptor (CAR) T-cell therapies have added an effective tool to the treatment options for relapsed and refractory leukemias and lymphomas. However, significant challenges limit patient access to these therapies, including eligibility, cost, scalability, and manufacturing. These limitations highlight the critical unmet need for effective same day treatment options. We are combining our Gene Writing and delivery platforms to enable T cell specific genome engineering, in an effort to reduce the challenges associated with conventional autologous CAR-T approaches. RNA Gene Writers are designed to introduce a broad range of alterations to the genome, from writing in a transgene, inserting an exon, or introducing single nucleotide changes, providing an attractive toolkit for sophisticated cell engineering. RNA Gene Writers are inspired by the biology of non-LTR retrotransposons, a class of mobile genetic elements that can edit the genome through target-primed reverse transcription, or TPRT. RNA Gene Writers are designed to be delivered as all-RNA composition, which is intended to facilitate in vivo delivery by lipid nanoparticles, or LNP.   LNP-RNA delivery of RNA Gene Writers resulted in the integration and expression of green fluorescent protein (GFP) transgenes in >70% of primary human T cells in vitro. We achieved successful LNP delivery of RNA to T cells in vivo in both mouse and non-human primate (NHP) models, with approximately 80% and 40% GFP reporter expression observed, respectively.   We demonstrated that LNP delivery of one of our RNA Gene Writers achieved the integration and CAR expression in an average of ~21% of primary human T cells in vitro. We did not observe detriment to cell viability or proliferative capacity in these CAR-T generated cells, and robust tumor cell killing was also observed in vitro. Moreover, RNA Gene Writer derived CAR-T cells demonstrated antigen specific tumor clearance in vivo when introduced into mouse xenograft models. We also demonstrated an average of ~6% GFP reporter expression in T cells after in vivo administration of LNP-RNA delivery into a humanized mouse model, potentially opening up the possibility of using RNA Gene Writers for in vivo CAR-T treatment. In addition, the modularity of our RNA Gene Writers delivered in an LNP potentially allows for multiplex editing to co-introduce multiple genetic changes in a single step using all-RNA compositions. In preclinical studies, we achieved >90% knockout of the TRAC and B2M genes, individually, in T cells, that when multiplexed led to ~88% of T cells with knockout of both TRAC and B2M. We also demonstrated T cells containing 3 edits, including TRAC and B2M knockout, and either GFP or CAR writing, showing that rewriting and writing can be multiplexed. Further, we observed approximately 30% B2M knockout by rewriting following in vivo administration in a humanized mouse model. We believe our RNA Gene Writers are uniquely positioned to drive a broad range of genetic alterations, including multiplex editing, and the ability to deliver RNA Gene Writers as all-RNA compositions packaged in a LNP potentially offers greater simplicity and flexibility compared to conventional lentiviral based CAR-T therapies.

Kartika Venugopal, Michael Magee, Alberto De Iaco, Jason Rodriguez, James Rottman, Donghui Li, Giulia Schiroli, Yu Cao, Rebecca Levy, Travis Schute, Mohit Gupta, Christina Ferren, Michael Monte, Jane Wang, Bill Querbes, Cecilia Cotta-Ramusino, Madhusudan V. Peshwa, Michael C. Holmes

Tessera Therapeutics, Somerville, MA"

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