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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a
! O2 E) N6 X4 }" u0 A- _promising approach to treat cancer, such as B-cell malignancy
3 ?9 X/ b  {  t[1]. However, the current standard treatment
. L8 W4 V9 Y8 U+ ^- ]8 d# P$ Zrequires autologous adoptive cell transfer, which is expensive
' x4 l  k, ~& G  ?and time-consuming. For newborn and elder& H7 y( H4 I5 n0 g2 Q. V
patients, it is often difficult to obtain enough T cells with
3 _' U4 h/ x- w. p/ ugood quality to generate patient-specific CAR-T cells.
8 t- p" ~* \: ~. D' q  gTo make CAR-T therapy more accessible, it is highly
, t3 e$ W  [( _- C) |( tdesirable to develop an allogeneic adoptive transfer& T( g( p& h' R! u
strategy, in which universal CAR-T cells derived from T5 ~0 n6 l# d$ P# b
cells from healthy donors can be applied to treat multiple
) _5 ~& a( s& p/ i+ T* f0 D. ~5 W+ tpatients. For this strategy to work, the αβ T-cell receptor
7 b! n# |/ i6 X3 ]2 `% D% x8 [9 l(TCR) on allogeneic CAR-T cells needs to be eliminated/ B& c# S# o* K& z/ u; U' `
to avoid graft-versus-host-disease (GVHD), and human& n% n2 p/ R: ?8 Q' v2 p# k2 o
leukocyte antigens class I (HLA-Is) on CAR-T cells need1 `& f; y$ O, `. v; T1 P
to be removed to minimize their immunogenicity. Previous
/ [7 ?( Y4 s- ~: v$ M7 X& Ystudies have shown that mutation in TCRα subunit
- D1 D7 v, a, X: w- W; ~; j! cconstant (TRAC) leads to loss of αβ TCR on T-cell surface! n8 @1 k1 i; L0 |* \9 K) U
[2], and beta-2 microglobulin (B2M) is essential for5 w0 W$ @% {3 F& x$ D* ?
cell-surface expression of HLA-I heterodimers [3]. Thus,
9 q3 U6 E, g) V$ L# s. l) A: ^we attempted to target TRAC and B2M genes in CAR-T
) b. ^; F" s2 l. d8 Rcells. Considering blocking programmed death-1 (PD-
. [5 C( w6 S4 O" `2 d1) signaling can effectively treat cancers via reversing
4 E9 [9 [* D& ~, g3 Uimmunosuppression, we also targeted PD-1 in CAR-T
; A& \8 B/ k" N( P' g% \3 |cells to render them nonresponsive to PD-1 signaling