To facilitate genetic editing of T cells without electroporation, Hamilton and Tsuchida et al. used virus-like particles to deliver Cas9 ribonucleoprotein complexes (Cas9-VLPs). Cas9-VLPs targeting B2M effectively knocked out B2M expression on murine T cell lines and primary human T cells, and could also co-deliver a CAR transgene, enabling one-step production of CAR T cells with additional gene editing. The transduction efficiency and tropism depended on the VLP surface glycoprotein, and Cas9-VLPs engineered with an HIV-1 envelope glycoprotein could preferentially transduce CD4+ T cells in the presence of CD8+ T cells.
Contributed by Alex Najibi
ABSTRACT: As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4(+) cells while sparing co-cultured CD8(+) cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.
Author Info: (1) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkel
Author Info: (1) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (2) Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. (3) Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA. (4) Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA. (5) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (6) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (7) Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA. (8) Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA. (9) Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94158, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. (10) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.
