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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a
3 P+ z) C* X; Y' r% U2 H. tpromising approach to treat cancer, such as B-cell malignancy3 }7 l7 b; n2 l% u9 x" X: _- ^
[1]. However, the current standard treatment
" h/ K9 w' R% V+ A# \9 P* f; qrequires autologous adoptive cell transfer, which is expensive0 D6 G4 |" L) o- ?0 U+ Q
and time-consuming. For newborn and elder( G- H) }# N1 c) {5 }6 H+ |
patients, it is often difficult to obtain enough T cells with
1 m. {- P3 ]9 W: y6 o4 G* h6 A4 n1 q! ~good quality to generate patient-specific CAR-T cells.
0 r, C, ^+ |3 E( |7 x( TTo make CAR-T therapy more accessible, it is highly
8 n* a, J5 q" |# u* s+ Mdesirable to develop an allogeneic adoptive transfer! N; Y" m" K# p/ a* Q+ ]6 Q, |
strategy, in which universal CAR-T cells derived from T
2 a3 y! y# L4 D, lcells from healthy donors can be applied to treat multiple
; ^6 K4 R* {8 U3 D6 J7 bpatients. For this strategy to work, the αβ T-cell receptor
) V; Y; I( ]" E! D' \(TCR) on allogeneic CAR-T cells needs to be eliminated
+ h, x5 c- Y  O+ X# G+ f# N/ Ito avoid graft-versus-host-disease (GVHD), and human
6 S) F! v# B$ Cleukocyte antigens class I (HLA-Is) on CAR-T cells need
) I" n5 Z! c1 E; C7 L) V: gto be removed to minimize their immunogenicity. Previous& \4 s/ ^$ Y& X$ P7 @4 {$ \) @4 o
studies have shown that mutation in TCRα subunit( x% i3 s+ S. h0 C- V
constant (TRAC) leads to loss of αβ TCR on T-cell surface
( d+ u+ s5 A8 \) q/ |* b[2], and beta-2 microglobulin (B2M) is essential for2 E2 P3 O9 l* p+ h$ m. j
cell-surface expression of HLA-I heterodimers [3]. Thus,) N% B: S" t% T9 Y' r/ Z7 q
we attempted to target TRAC and B2M genes in CAR-T/ v( p3 G6 f* l# o% V2 W2 i
cells. Considering blocking programmed death-1 (PD-
% e$ f' Q6 d, V1) signaling can effectively treat cancers via reversing
7 o8 K# H% a2 s* \# |immunosuppression, we also targeted PD-1 in CAR-T3 m$ J$ V+ P+ }
cells to render them nonresponsive to PD-1 signaling