Preece, R;
Pavesi, A;
Gkazi, SA;
Stegmann, KA;
Georgiadis, C;
Tan, ZM;
Joey Aw, JY;
... Qasim, W; + view all
(2020)
CRISPR mediated base conversion allows discriminatory depletion of endogenous T cell receptors for enhanced synthetic immunity.
Molecular Therapy: Methods & Clinical Development
10.1016/j.omtm.2020.09.002.
(In press).
Preview |
Text
1-s2.0-S232905012030187X-main.pdf - Accepted Version Download (5MB) | Preview |
Abstract
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 HLA barriers and promoting persistence. We report lentiviral delivery of a Hepatitis B virus (HBV) specific recombinant TCR (rTCR) and a linked CRISPR single-guide RNA for simultaneous disruption of endogenous TCRs (eTCR) 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 1.8-2.9 fold compared to cells retaining their competing eTCR and increased cytokine production and killing of HBV antigen-expressing hepatoma cells in a 3D microfluidic model was exhibited. Molecular signatures confirmed 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 tumour antigens.
Archive Staff Only
 |
View Item |
