CRISPR-Mediated Base Conversion Allows Discriminatory Depletion of Endogenous T Cell Receptors for Enhanced Synthetic Immunity

• Main Authors: Roland Preece, Andrea Pavesi, Soragia Athina Gkazi, Kerstin A. Stegmann, Christos Georgiadis, Zhi Ming Tan, Jia Ying Joey Aw, Mala K. Maini, Antonio Bertoletti, Waseem Qasim
• Format: Article
• Language: English
• Published: Elsevier 2020-12-01
• Series: Molecular Therapy: Methods & Clinical Development
• Subjects: CRISPR/Cas9Base editing; Recombinant TCR; Gene therapy; Cell therapy; Hepatocellular carcinoma (HCC); Hepatitis B virus (HBV)
• Online Access: http://www.sciencedirect.com/science/article/pii/S232905012030187X

Emerging base editing technology exploits CRISPR RNA-guided DNA modification effects for highly specific C > T conversion, which has been used to efficiently disrupt gene expression. These tools can enhance synthetic T cell immunity by restricting specificity, addressing histocompatibility leukocyte antigen (HLA) barriers, and promoting persistence. We report lentiviral delivery of a hepatitis B-virus (HBV)-specific recombinant T cell receptor (rTCR) and a linked CRISPR single-guide RNA for simultaneous disruption of endogenous TCRs (eTCRs) when combined with transient cytosine deamination. Discriminatory depletion of eTCR and coupled expression of rTCR resulted in enrichment of HBV-specific populations from 55% (SEM, ±2.4%) to 95% (SEM, ±0.5%). Intensity of rTCR expression increased 1.8- to 2.9-fold compared to that in cells retaining their competing eTCR, and increased cytokine production and killing of HBV antigen-expressing hepatoma cells in a 3D microfluidic model were exhibited. Molecular signatures confirmed that seamless conversion of C > T (G > A) had created a premature stop codon in TCR beta constant 1/2 loci, with no notable activity at predicted off-target sites. Thus, targeted disruption of eTCR by cytosine deamination and discriminatory enrichment of antigen-specific T cells offers the prospect of enhanced, more specific T cell therapies against HBV-associated hepatocellular carcinoma (HCC) as well as other viral and tumor antigens.