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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a/ Y0 h: ?5 k( Q' {7 \, j$ A
promising approach to treat cancer, such as B-cell malignancy- s  ?4 r8 V" H4 N  \' t
[1]. However, the current standard treatment
% ^+ Q( {& v, @6 t1 Nrequires autologous adoptive cell transfer, which is expensive
3 p7 @6 p+ ~1 _. C" l! z) Z& H5 land time-consuming. For newborn and elder
+ d6 \. c4 V6 e9 c4 r6 {+ Apatients, it is often difficult to obtain enough T cells with
  f7 J" t  e/ N* a* Y) U( xgood quality to generate patient-specific CAR-T cells.3 M3 L0 W+ l* w8 g$ q1 j' c+ n
To make CAR-T therapy more accessible, it is highly
. l( c- p# H6 B, O8 \' y& ]desirable to develop an allogeneic adoptive transfer2 {  L) Y: a: H! U9 K; v
strategy, in which universal CAR-T cells derived from T8 m1 J! W( M3 T: e; A
cells from healthy donors can be applied to treat multiple
* R- X% ?3 l7 ~patients. For this strategy to work, the αβ T-cell receptor( n. N2 l! l# u3 [
(TCR) on allogeneic CAR-T cells needs to be eliminated
. H# M+ l: g- u4 pto avoid graft-versus-host-disease (GVHD), and human
8 b( X! y. ^  y: z# l1 \7 dleukocyte antigens class I (HLA-Is) on CAR-T cells need
( o+ a7 \2 h- x/ d( ]: T5 S$ ito be removed to minimize their immunogenicity. Previous: }: {" i, @0 {. m7 i# P
studies have shown that mutation in TCRα subunit( z2 U( z: i7 H0 K
constant (TRAC) leads to loss of αβ TCR on T-cell surface' Q- X6 o$ m5 o: h/ t
[2], and beta-2 microglobulin (B2M) is essential for
/ v" u6 t+ R7 V4 ^8 Rcell-surface expression of HLA-I heterodimers [3]. Thus,
8 s1 K$ `$ g2 o2 p7 B  ewe attempted to target TRAC and B2M genes in CAR-T
8 n, t  i1 u! Ycells. Considering blocking programmed death-1 (PD-
2 r7 C$ u5 z. m: f1) signaling can effectively treat cancers via reversing
' P0 o0 O* R. B1 a* Q5 d' F. Wimmunosuppression, we also targeted PD-1 in CAR-T
/ O. `  ^4 u5 {) V3 ^0 fcells to render them nonresponsive to PD-1 signaling