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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a) H* e' U! L" @0 k: _, }4 h+ e
promising approach to treat cancer, such as B-cell malignancy
+ r  l  y1 C  j! }# w6 b& `, O. o[1]. However, the current standard treatment
" g/ w1 f+ i, \: j& B! qrequires autologous adoptive cell transfer, which is expensive( o7 h0 Z* a, O2 P" ?, G" ?/ @
and time-consuming. For newborn and elder/ X7 m* E- ~, F' P% m' _! Z
patients, it is often difficult to obtain enough T cells with
& b; L9 E& g: ]$ y2 Egood quality to generate patient-specific CAR-T cells.7 C' j# p* A7 M* c1 l
To make CAR-T therapy more accessible, it is highly
2 [( E8 M% _8 B/ o- R9 Wdesirable to develop an allogeneic adoptive transfer
8 e" K/ H0 J2 a* |" X) ]; X' ustrategy, in which universal CAR-T cells derived from T
0 b, r0 \6 ?  i% {% O8 G/ @: Vcells from healthy donors can be applied to treat multiple) N8 C4 I7 p# W4 o
patients. For this strategy to work, the αβ T-cell receptor
0 r4 \( e1 q) q6 ~, }( q(TCR) on allogeneic CAR-T cells needs to be eliminated5 H, ?  b3 `) p1 y2 F/ y9 N" O
to avoid graft-versus-host-disease (GVHD), and human! p* k' E/ h" t6 t" ^5 u, A( Q
leukocyte antigens class I (HLA-Is) on CAR-T cells need8 e" Z3 L; l# b0 e0 a$ W0 s# \1 A
to be removed to minimize their immunogenicity. Previous& J; ^1 }" _8 `1 C9 R
studies have shown that mutation in TCRα subunit6 a- W' P2 j- _! s7 E
constant (TRAC) leads to loss of αβ TCR on T-cell surface
& ]+ e" G  K. W* x* V; b8 `. J[2], and beta-2 microglobulin (B2M) is essential for- c6 f3 w5 k+ u9 a9 K5 d0 H! H- s' |" c
cell-surface expression of HLA-I heterodimers [3]. Thus,+ G) N8 T7 H. Q% a
we attempted to target TRAC and B2M genes in CAR-T3 k/ d; r# N# e. O- D6 L# ~' M
cells. Considering blocking programmed death-1 (PD-
1 g; W/ Q" T! d1) signaling can effectively treat cancers via reversing
5 \' ~8 Z- X+ q/ o/ E: }immunosuppression, we also targeted PD-1 in CAR-T
' ?% F% ^5 y0 n) y4 ~cells to render them nonresponsive to PD-1 signaling