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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a
2 u9 e. O# Z9 l5 w0 H) hpromising approach to treat cancer, such as B-cell malignancy
6 l2 y. i4 v$ ^+ H8 U[1]. However, the current standard treatment
/ s* p9 N- w8 z1 C4 O3 Urequires autologous adoptive cell transfer, which is expensive: ]5 u9 r  j- J. ~) a
and time-consuming. For newborn and elder- l4 a. _6 e' _) S1 V
patients, it is often difficult to obtain enough T cells with3 [5 G( A  O' o7 ^/ `! r
good quality to generate patient-specific CAR-T cells.; ?4 o9 i- c* m! Y) K$ C
To make CAR-T therapy more accessible, it is highly6 `; t. ^4 `5 e8 r$ \5 f
desirable to develop an allogeneic adoptive transfer
8 D1 u3 B' G4 }& i6 estrategy, in which universal CAR-T cells derived from T
' Y8 Y4 P+ l- c4 c' I% q6 @4 {$ Bcells from healthy donors can be applied to treat multiple
9 N* e# r8 n) z0 N' G. V7 ppatients. For this strategy to work, the αβ T-cell receptor/ `5 s/ L; G1 S6 W# X+ e% d) T
(TCR) on allogeneic CAR-T cells needs to be eliminated% \- N/ G) ]+ }- d5 B
to avoid graft-versus-host-disease (GVHD), and human
/ o- j  y* V; ]' f7 ]# sleukocyte antigens class I (HLA-Is) on CAR-T cells need" ?. f, c7 X3 J+ P' G9 \) X& ]
to be removed to minimize their immunogenicity. Previous/ ^3 V9 W7 S) B4 D
studies have shown that mutation in TCRα subunit
" [# G# v; u/ c0 p* R/ e7 Q4 lconstant (TRAC) leads to loss of αβ TCR on T-cell surface
; n0 U7 @7 s. r+ w[2], and beta-2 microglobulin (B2M) is essential for9 ?2 F8 n3 A- D& E
cell-surface expression of HLA-I heterodimers [3]. Thus,1 x& M+ f( A8 H6 q
we attempted to target TRAC and B2M genes in CAR-T
4 L3 V; L( x% m  Mcells. Considering blocking programmed death-1 (PD-
: I" f1 ~% h( `- U3 H( k2 c1) signaling can effectively treat cancers via reversing
8 {8 H, V2 D  timmunosuppression, we also targeted PD-1 in CAR-T1 h2 b" W" q# Q
cells to render them nonresponsive to PD-1 signaling