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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a2 r5 O, [, Y( u! Y& K2 c& b
promising approach to treat cancer, such as B-cell malignancy0 x; ?" _1 l9 ^7 N
[1]. However, the current standard treatment
- g6 l$ Y1 o0 D: ]1 q$ wrequires autologous adoptive cell transfer, which is expensive
/ h# x- `4 ^6 G! s/ r* z" \and time-consuming. For newborn and elder# G, b7 L' Q6 i0 a2 A
patients, it is often difficult to obtain enough T cells with
8 T& o7 o$ D" C7 fgood quality to generate patient-specific CAR-T cells.
  b" L7 k- Z9 W* @1 ]) n" BTo make CAR-T therapy more accessible, it is highly3 t5 k9 q" z6 F$ ?: l$ K
desirable to develop an allogeneic adoptive transfer# v( X8 `4 b+ ^, ?1 l& R/ g
strategy, in which universal CAR-T cells derived from T
- @1 a7 e. M- zcells from healthy donors can be applied to treat multiple
: W( N' [* Y# i+ epatients. For this strategy to work, the αβ T-cell receptor+ T8 m8 l* Z! C3 V; i% X5 f# C
(TCR) on allogeneic CAR-T cells needs to be eliminated
" Z: R6 e+ d1 f  kto avoid graft-versus-host-disease (GVHD), and human0 {& f, A* @/ W
leukocyte antigens class I (HLA-Is) on CAR-T cells need4 G7 `" Y+ K$ B
to be removed to minimize their immunogenicity. Previous- p. Q- A, Y, Z5 }
studies have shown that mutation in TCRα subunit
; ], ^8 A$ ?1 k* Bconstant (TRAC) leads to loss of αβ TCR on T-cell surface8 E8 b. i) U2 N: j6 I+ n/ ?
[2], and beta-2 microglobulin (B2M) is essential for) M! {" {" n$ t( s
cell-surface expression of HLA-I heterodimers [3]. Thus,( a/ ^3 E5 e; `/ f! D/ b
we attempted to target TRAC and B2M genes in CAR-T
+ G9 a* c5 O: g( Scells. Considering blocking programmed death-1 (PD-1 }  M  P# J( v% F( X
1) signaling can effectively treat cancers via reversing( o9 x0 q. ~: ]3 N1 f; A" t1 @
immunosuppression, we also targeted PD-1 in CAR-T7 F7 \# }) s7 w
cells to render them nonresponsive to PD-1 signaling