Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

Hyatt Balke-Want; Vimal Keerthi; Nikolaos Gkitsas; Andrew G Mancini; Gavin L Kurgan; Carley Fowler; Peng Xu; Xikun Liu; Kyle Asano; Sunny Patel; Christopher J Fisher; Annie K Brown; Ramya H Tunuguntla; Shabnum Patel; Elena Sotillo; Crystal L Mackall; Steven A Feldman

doi:10.1186/s12943-023-01799-7

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

Mol Cancer. 2023 Jun 26;22(1):100. doi: 10.1186/s12943-023-01799-7.

Authors

Hyatt Balke-Want¹, Vimal Keerthi¹, Nikolaos Gkitsas¹, Andrew G Mancini², Gavin L Kurgan³, Carley Fowler¹, Peng Xu¹, Xikun Liu¹, Kyle Asano¹, Sunny Patel¹, Christopher J Fisher¹, Annie K Brown¹, Ramya H Tunuguntla¹, Shabnum Patel¹, Elena Sotillo¹, Crystal L Mackall⁴, Steven A Feldman⁵

Affiliations

¹ Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
² MaxCyte, Inc, Rockville, MD, USA.
³ Integrated DNA Technologies, Inc, Coralville, IA, 52241, USA.
⁴ Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA. cmackall@stanford.edu.
⁵ Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA. feldmans@stanford.edu.

Abstract

Background: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA).

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 in our system. 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 high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 10⁸-3.6 × 10⁹ 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.

Keywords: CRISPR/Cas9; GMP; Genomic safety; Homology-independent targeted insertion (HITI); Non-viral CAR-T cell; Targeted insertion.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

DNA*
Humans
Immunotherapy, Adoptive
Recombinational DNA Repair
T-Lymphocytes*

Substances

DNA