CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a
* u4 X) S% |5 u0 M. ^8 ?" J! {promising approach to treat cancer, such as B-cell malignancy8 L: P& r1 X1 O$ f6 l; o! h! {
[1]. However, the current standard treatment. r3 I6 h& y3 ~. s) M; ^
requires autologous adoptive cell transfer, which is expensive# [" T4 I) u  e: @+ ~
and time-consuming. For newborn and elder
# y0 N& i! J. G( U! kpatients, it is often difficult to obtain enough T cells with
) |! x/ Z- |4 q) f* wgood quality to generate patient-specific CAR-T cells.
( k. }- v/ T1 [% c# z% cTo make CAR-T therapy more accessible, it is highly
0 c: \" r' I. z* R/ E+ ^* tdesirable to develop an allogeneic adoptive transfer
# [1 E5 B1 c& X# pstrategy, in which universal CAR-T cells derived from T
& w3 f, b# S! t4 }: W7 ncells from healthy donors can be applied to treat multiple
8 Q' i8 i$ T2 L2 T( Gpatients. For this strategy to work, the αβ T-cell receptor
0 \) }+ ]* Y0 c5 {) Z# Z(TCR) on allogeneic CAR-T cells needs to be eliminated3 l( g0 q/ Z+ T6 d4 ?: k
to avoid graft-versus-host-disease (GVHD), and human, q/ O0 L$ P8 c/ L+ u
leukocyte antigens class I (HLA-Is) on CAR-T cells need
/ }) w, u0 @/ L# t) jto be removed to minimize their immunogenicity. Previous1 Q. w) }6 `8 u3 T4 j6 ]
studies have shown that mutation in TCRα subunit
9 b+ ]$ Z$ \3 h# Hconstant (TRAC) leads to loss of αβ TCR on T-cell surface$ J3 F! [3 E7 X' V
[2], and beta-2 microglobulin (B2M) is essential for
% n9 {! m) a. A! ~$ O* E7 \( Acell-surface expression of HLA-I heterodimers [3]. Thus,
" _0 L$ z' ^% R: ~we attempted to target TRAC and B2M genes in CAR-T3 J$ N- N: T' ~
cells. Considering blocking programmed death-1 (PD-; \1 `. P+ o" Z
1) signaling can effectively treat cancers via reversing
# H% }9 Q/ u& X9 I1 vimmunosuppression, we also targeted PD-1 in CAR-T0 V2 b4 b* B# z9 d( F
cells to render them nonresponsive to PD-1 signaling