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

peliang12

Chimeric antigen receptor (CAR) T cell therapy is a6 D4 D# L" R* w, B' x4 y
promising approach to treat cancer, such as B-cell malignancy
: ~/ t  S0 T# O; Y[1]. However, the current standard treatment
2 W, r6 z1 W$ grequires autologous adoptive cell transfer, which is expensive/ Y: f$ B5 f2 @  r$ B
and time-consuming. For newborn and elder3 E  d7 j$ M- ]9 `6 ]* v
patients, it is often difficult to obtain enough T cells with
9 n7 w6 F7 k) m5 _( vgood quality to generate patient-specific CAR-T cells.
9 G2 E3 [3 k  U" d- y6 MTo make CAR-T therapy more accessible, it is highly
: f! b2 f5 Q# d$ X# ~3 Mdesirable to develop an allogeneic adoptive transfer/ X4 k* h: ?. ^: ]
strategy, in which universal CAR-T cells derived from T- |; i8 p2 N* B3 X5 `
cells from healthy donors can be applied to treat multiple7 P4 X! a( {" A! F5 R: I4 x8 C
patients. For this strategy to work, the αβ T-cell receptor
: K" i7 B6 F  i. D& R! B% c$ a+ P(TCR) on allogeneic CAR-T cells needs to be eliminated( T9 f; W' V! C* A6 o
to avoid graft-versus-host-disease (GVHD), and human
& k( I- v" s: ^+ H$ ileukocyte antigens class I (HLA-Is) on CAR-T cells need
) T2 g# |) b( q/ Nto be removed to minimize their immunogenicity. Previous5 n. l$ ?2 d/ L  _; h% Q
studies have shown that mutation in TCRα subunit" e' S' E: J  C9 ^6 J
constant (TRAC) leads to loss of αβ TCR on T-cell surface
# [) M4 g& u! g. K. }$ Q& U; O[2], and beta-2 microglobulin (B2M) is essential for
- f* Y+ i& g( l4 @' `' Ycell-surface expression of HLA-I heterodimers [3]. Thus,
9 k- y6 d$ V7 x3 c% W/ m( twe attempted to target TRAC and B2M genes in CAR-T! y5 N  }0 P! l! ~3 i# f
cells. Considering blocking programmed death-1 (PD-( u7 O, b! H8 I* i1 l8 {
1) signaling can effectively treat cancers via reversing7 t" W( J" X" Y$ K7 n8 V9 z
immunosuppression, we also targeted PD-1 in CAR-T3 l" w5 [% ]4 h9 Y2 o  c3 W
cells to render them nonresponsive to PD-1 signaling