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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

232: Cell Type-Programmable CRISPR-Cas9 Delivery for Human T Cell Engineering

Type: Oral Abstract Session

Presentation Details
Session Title: Gene Targeting and Gene Correction: New Technologies
Location: Room 515 AB
Start Time: 5/18/2023 16:45
End Time: 5/18/2023 17:00

The lack of approaches for delivering genome editors to specific cell types limits the therapeutic application of CRISPR-Cas9. Enveloped delivery vehicles (EDVs), such as virus-like particles, enable targeted genome editing by leveraging the cell binding specificity of viral glycoproteins for the transient delivery of CRISPR-Cas9 tools. However, the repertoire of naturally occurring viral glycoproteins with cell-specific tropisms (ex. HIV-1 envelope glycoprotein to target CD4+ T cells) is limited. Inspired by chimeric antigen receptors and their ability to link programmable targeting to cellular function, we hypothesized that displaying antibody fragments on Cas9-guide RNA complex-packaging enveloped delivery vehicles (Cas9-EDVs) would allow for the delivery of genome editors to specific cells. Working with engineered 293T cells, we found that antibody-targeted Cas9-EDVs direct genome editor delivery to cognate ligand-expressing cells, leaving bystander cells largely unmodified. We next screened a panel of antibody-based molecules for genome editing primary human T cells. We identified Cas9-EDV targeting molecules for specifically editing T cell subpopulations (anti-CD4) and T cells broadly (anti-CD3). CD3-targeted Cas9-EDVs mediate genome editing levels comparable to Cas9-EDVs pseudotyped with the broadly-transducing VSV-G viral glycoprotein. Additionally, this antibody-directed targeting approach can be applied to lentiviruses for precisely delivering transgenes to T cells, both ex vivo and in vivo. Retro-orbital administration of CD3-targeted lentiviral EDVs leads to the generation of chimeric antigen receptor (CAR) T cells in humanized mice, demonstrating targeted delivery in vivo. Together, antibody-targeted EDVs are a programmable approach for the transient, cell-selective delivery of transgenes and CRISPR-Cas9 genome editor complexes.

Jennifer R. Hamilton1,2, Evelyn Chen1,2, Barbara S. Perez1,2, Cindy R. Sandoval Espinoza3, Jennifer A. Doudna1,2,4,5,6

1UC Berkeley, Berkeley, CA,2Innovative Genomics Institute, Berkeley, CA,3Stanford University, Palo Alto, CA,4Howard Hughes Medical Institute, Berkeley, CA,5Gladstone Institutes, San Francisco, CA,6Lawrence Berkeley National Laboratory, Berkeley, CA
 J.R. Hamilton: None.

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