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H - Clinical Translation of Gene and Cell Therapies -> Cell Therapy Product Engineering, Development and Manufacturing

319: Efficient and Minimally Perturbative CAR-T Cell Engineering Using Peptide-Enabled CRISPR RNP Delivery

Type: Oral Abstract Session

Presentation Details
Session Title: Cell Therapy Product Manufacturing
Location: Petree Hall D
Start Time: 5/20/2023 8:00
End Time: 5/20/2023 8:15

Precision genome editing of primary human T cells offers the potential for sophisticated and durable cell therapies, but the use of electroporation for delivering genome-editing reagents is a source of cell toxicity and requires costly, cumbersome hardware and reagents. This manufacturing burden vastly limits the access and wide implementation of genome-edited T cell therapies. We developed a method called peptide-enabled RNP delivery for CRISPR engineering (PERC) to conduct precise editing without the need for electroporation. In this process, an RNP enzyme such as Cas9/gRNA or Cas12a/gRNA is mixed with a particular amphiphilic peptide and then applied to cells in culture, resulting in efficient gene knockout (up to 90%). Compared to electroporation, PERC produces significantly higher yields of edited cells, preserves the naïve and memory phenotype, and is less perturbative at the transcriptomic level. PERC can be paired with adeno-associated virus (AAV) bearing a homology-directed repair template for making efficient electroporation-free knock-ins, such as of a CAR transgene to the TCR alpha constant (TRAC) locus under endogenous promoter control, which has been shown to improve anti-tumor potency compared to virus-mediated pseudorandom integration. With PERC and AAV, we achieved up to 75% TRAC-CAR knock-in efficiency. As PERC is minimally perturbative, it can be used to edit multiple loci sequentially and thereby avoid chromosomal translocations, which arise when multiple RNPs are delivered simultaneously. Remarkably, we achieved above 25% dual knock-in at TRAC and B2M without detectable chromosomal rearrangement. Functionally, TRAC CAR-T cells produced using PERC maintained the advantage of their electroporation counterpart over pseudorandomly engineered CAR-T cells in targeting antigen-expressing tumor cells. Furthermore, multi-locus-edited CAR-T cells produced using PERC demonstrated therapeutic potency matching that of electroporation in an in vivo model of B-ALL. In summary, PERC paired with AAV enables efficient, convenient, non-toxic, minimally perturbative, and multi-locus editing. Looking forward, this approach has the potential to decrease the cell handling steps and infrastructure involved in making precisely engineered T cell therapies and could lower the barrier to point-of-care manufacturing.

Joseph J. Muldoon1,2, Dana V. Foss3,4, David N. Nguyen1,2,3, Daniel Carr1,2,3, Srishti U. Sahu3,4, Alexander Marson1,2,3, Justin Eyquem1,2, Ross C. Wilson3,4

1Department of Medicine, UCSF, San Francisco, CA,2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA,3Innovative Genomics Institute, UC Berkeley, Berkeley, CA,4California Institute for Quantitative Biosciences at UC Berkeley, Berkeley, CA
 J.J. Muldoon: None.

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