The three-day event, organized by president Izuho Hatada will be held at Tower Hall Funabori in Tokyo, and in person for the first time in three years. The event is intended as a forum to exchange ideas and thoughts on various topics such as CRISPR-Cas9, new various models used in genome editing and new emerging technologies.
Come visit us and discover MaxCyte's cGMP-compliant non-viral cell engineering platform and how it enables the acceleration of therapeutic development from concept to clinic.
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Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing
Date: June 8, 2023
Time: 12:35 - 1:20 PM (JST)
Presenter: Hyatt Balke-Want
Background: Chimeric Antigen Receptor (CAR)-T cell therapies have become an effective therapeutic option for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to viral vector engineered CAR-T cell therapies faces significant challenges to meet clinical needs, in large part due to high cost and long lead times for manufacturing GMP vector . Therefore, moving to a non-viral gene delivery platform for the manufacture of CAR-T cell therapies is a logical next step. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application. We discuss here a targeted but HDR-independent approach for the manufacture of CAR-T cell therapies.
Methods: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach.
Results: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides higher cell yield compared to HDR mediated gene insertion. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5x108-3.6x109 CAR+ T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity.
Conclusions: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.