Get Help Sign In
ProcessingProcessing

CRISPR-mediated deletion of glucocorticoid receptor protects viral-specific T cells against lymphocytotoxic effects of glucocorticoid treatment

Basar R, Daher M, et al. (2020) Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells. Blood Adv 4(14): 3357–3367.

Immunocompromised patients can be protected from viral infection with an immunotherapy that uses viral-specific T cells (VSTs) to target the infection. However, the same patients are often given glucocorticoids to treat graft-versus-host disease (GVHD). Glucocorticoids can interfere with viral infection treatment by attacking VSTs. Therefore, enhancing the function and survival of VSTs is of great importance for these patients. In a research study, Basar et al. used the CRISPR-Cas9 system to delete the glucocorticoid receptor gene and showed that its deletion protected the VSTs while retaining the glucocorticoid effectiveness against GVHD. Furthermore, use of Alt‑R S.p. HiFi Cas9 Nuclease V3 minimized off-target edits, characterized by the rhAmpSeq amplicon sequencing system.

Background

Immunocompromised patients can be treated for viral infection using immunotherapy by infusing patients with viral-specific T cells (VSTs). VSTs target viral infections, but efficacy can be blocked by glucocorticoids. Glucocorticoids are steroid hormones that reduce inflammation by inducing T cell death. They are used to treat graft-versus-host disease (GVHD), a condition that often affects immunocompromised patients. Enhancing the function and survival of VSTs and protecting them from eradication by glucocorticoids is critical for patient survival.

Here, Basar et al. reduced glucocorticoid activity by editing out the glucocorticoid receptor (GR) in the VSTs before infusion using the CRISPR-Cas9 system, rendering VSTs resistant to the lymphocytotoxic effects of glucocorticoids, while retaining glucocorticoid effectiveness against GVHD. The CRISPR-Cas9 system is a genome editing technique that cuts DNA to modify gene function. Several clinical trials have demonstrated the feasibility, safety, and efficacy of cell and gene therapy via genome editing, allowing CRISPR genome editing to move toward therapeutic applications. This technique can have unintended effects, so it is important to find edit locations and characterize them. Potential editing sites must be detected before they can be characterized using next generation sequencing.

Experiment

VSTs were generated using peripheral blood mononuclear cells isolated from donors. The cells were treated with virus-specific peptide pools and a cytokine cocktail. Exon 2 of the GR was knocked out in VSTs with the ribonucleoprotein (RNP) complex: Alt-R™ guide RNA complexed with Alt-R HiFi Cas9 protein. For small-scale knockout, electroporation was used to transfect CRISPR RNP complexes. For the GMP-compliant, large-scale protocol, a 4D nucleofector (Lonza) was used. Knockout efficiency was tested using PCR gel electrophoresis and GR protein expression was confirmed with western blot. Various flow cytometry assays confirmed VST functionality. The CRISPR-modified VSTs were also tested in vivo using a xenograft immunodeficient mouse model. GUIDE-seq of HEK293 cells revealed off-target editing events which were quantified using the rhAmpSeq™ amplicon sequencing system—a strategy for multiplexed target enrichment by amplification. Amplicons were sequenced on an Illumina MiSeq™ instrument and analyzed with Picard tools.

Results and discussion

VSTs were successfully generated to target cytomegalovirus, BK virus, and adenovirus. CRISPR-mediated silencing of GR did not impact functionality or phenotype of the VSTs and enabled glucocorticoid resistance both in vitro and in vivo. GUIDE-seq revealed low frequencies of off-target events in cells treated with WT S.p. Cas9 protein. Use of high-fidelity Cas9 (Alt-R HiFi Cas9 v3) further reduced off-targeting event incidence down to <0.5%, which was validated with the rhAmpSeq amplicon sequencing system. Sequencing results confirmed on-target editing efficiency using Alt-R HiFi Cas9 v3 was consistently >90% and specificity of editing was >95%, confirming the direct relationship of the phenotype to the effect of the exon 2 editing event. VSTs produced using the large-scale production protocol using GMP-compliant material retained their function. Edited cells responded strongly to viral activity and were able to proliferate and release cytokines at all dose levels tested. Since GMP-compliant scale-up has been demonstrated to be technically feasible, Basar et al. are interested in clinical translation and are looking forward to assessing safety and efficacy of their strategy in a clinical study.

Published Aug 18, 2020

Unless otherwise specified, all IDT products referenced in this article are for Research Use Only and are not approved for use in clinical trials, diagnostic applications, or therapeutic applications.