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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a+ `. y) j' Z2 g7 t
promising approach to treat cancer, such as B-cell malignancy
' E1 }. d" ^' R, F* @8 }+ A" u( j[1]. However, the current standard treatment
; |4 R% `: Q8 M6 e* Y) o1 Hrequires autologous adoptive cell transfer, which is expensive
; W5 [  M, l; o# K6 T7 c( Iand time-consuming. For newborn and elder
& X" L8 v* N& d- }patients, it is often difficult to obtain enough T cells with2 c# D! E* X9 P6 A- M/ b- P
good quality to generate patient-specific CAR-T cells.1 p! E$ f" |# V# L2 ^" l! h! g+ {8 e# w( N
To make CAR-T therapy more accessible, it is highly
0 l, J$ Z, P2 g+ P8 S5 b3 Mdesirable to develop an allogeneic adoptive transfer/ k1 F3 ?" i# N  W- {/ J/ b# W
strategy, in which universal CAR-T cells derived from T
/ _) m5 {) f6 Ucells from healthy donors can be applied to treat multiple
2 A. z' d! a, r. _' O5 Ypatients. For this strategy to work, the αβ T-cell receptor
: p" L) H5 ^6 \* x* q4 L6 Q(TCR) on allogeneic CAR-T cells needs to be eliminated
; M; P7 U1 W' Y9 ~4 f7 Gto avoid graft-versus-host-disease (GVHD), and human
+ [( c0 i; W. A0 b( i3 B! y" z( Hleukocyte antigens class I (HLA-Is) on CAR-T cells need
4 \8 l/ i( w; d, p4 ~to be removed to minimize their immunogenicity. Previous
/ g7 F8 @0 u. X4 A* gstudies have shown that mutation in TCRα subunit  f7 P. Y" O# j/ R0 I% v* p3 y
constant (TRAC) leads to loss of αβ TCR on T-cell surface
* h& U9 H5 `* Q; o5 O4 E+ R' n[2], and beta-2 microglobulin (B2M) is essential for5 s: `$ m' D$ X! m
cell-surface expression of HLA-I heterodimers [3]. Thus,) M% ^# C* \+ X; l$ a1 ]
we attempted to target TRAC and B2M genes in CAR-T$ M/ Y" M# z2 c/ }, g1 d" C
cells. Considering blocking programmed death-1 (PD-6 ?  ^* ]- w, G( T
1) signaling can effectively treat cancers via reversing
8 j' `( v1 \+ {- T4 K! aimmunosuppression, we also targeted PD-1 in CAR-T) @" C3 ]) b1 G' E4 }8 J1 x* l9 i# V. k
cells to render them nonresponsive to PD-1 signaling