Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering

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.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

“…Virus-like particles (VLPs), assemblies of viral proteins that can infect cells but lack viral genetic material, have emerged as potentially promising vehicles for delivering gene editing agents as RNPs ( Campbell et al., 2019 ; Choi et al., 2016 ; Gee et al., 2020 ; Hamilton et al., 2021 ; Indikova and Indik, 2020 ; Lyu et al., 2019 , 2021 ; Mangeot et al., 2019 ; Yao et al., 2021 ). VLPs that deliver RNP cargoes exploit the efficiency and tissue targeting advantages of viral delivery but avoid the risks associated with viral genome integration and prolonged expression of the editing agent.…”

Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41

“…For instance, to avoid electroporation-induced cell death, the usage of LV particles for Cas9 RNP delivery looks very attractive. To date, several Cas9-VLP technologies have been developed that utilize retroviral ( 63 ) or lentiviral ( 64 , 65 ) Gag assembly. Although they demonstrated good efficiency for gene knockout, VLPs specifically recruiting repair template have not been devised.…”

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

“…Although VLPs can be developed to deliver Cas9 RNPs [20-27], delivering Cas9 mRNA may achieve short-term yet strong Cas9 expression to increase genome editing efficiency. This is especially relevant when targeting loci in heterochromatin regions.…”

“…Delivering Cas9 ribonucleoprotein (Cas9 RNP) [18] or Cas9 mRNA [19] enabled short-term expression of CRISPR/Cas9 and greatly improved safety. Based on these observations, viral vectors have been modified for delivering Cas9 RNPs [20-27] or Cas9 mRNAs [28-34] to improve safety.…”