Homology-independent targeted dna insertion in human t cells
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsCrystal L. Mackall, Steven A. Feldman, Hyatt Balke-Want, Andrew Guy Mancini
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Abstract
The present disclosure provides methods and compositions for site directed CAR integration in primary human T cells using DNA delivered via homology-independent targeted insertion (HITI). The methods provide higher cell yield compared to homology-directed repair (HDR) mediated gene insertion. Post-HITI CRISPR EnrichMENT (CEMENT) using GMP grade reagents enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of CAR+ T cells.
Claims56
A method for enriching for T cells that express a chimeric antigen receptor (CAR) inserted at a target genomic locus following homology-independent targeted insertion (HITI), comprising:
- transfecting a plurality of T cells with
- (a) a linear nucleic acid molecule comprising:
- (i) an expression cassette comprising a promoter operably linked to a sequence encoding a CAR and a sequence encoding a selectable marker;
- and
- (ii) a single protospacer adjacent motif (PAM) sequence that binds a guide RNA-CRISPR/Cas9 ribonucleoprotein (RNP), wherein the PAM is located 5′ or 3′ of the expression cassette of (i);
- and
- (b) a guide RNA-CRISPR/Cas9 ribonucleoprotein (RNP);
- wherein the nucleic acid molecule is inserted into a target site in the genome of the T cell via non-homologous end joining (NHEJ);
- and
- isolating T cells that express the selectable marker, wherein the number of CAR+ T cells is increased compared to T cells that do not express the selectable marker.
A method for treating a tumor in a subject, comprising administering to the subject an effective amount of a genetically modified T cell comprising:
- a nucleic acid molecule comprising:
- (i) an expression cassette comprising a promoter operably linked to a sequence encoding a chimeric antigen receptor (CAR) and a sequence encoding a selectable marker;
- and
- (ii) a single protospacer adjacent motif (PAM) sequence that binds a guide RNA-CRISPR/Cas9 ribonucleoprotein (RNP), wherein the PAM is located 5′ or 3′ of the expression cassette of (i);
- wherein the nucleic acid molecule is inserted into a target site in the genome of the T cell via non-homologous end joining (NHEJ), thereby treating the tumor.
A genetically modified T cell comprising:
- a nucleic acid molecule comprising:
- (i) an expression cassette comprising a promoter operably linked to a sequence encoding a CAR and a sequence encoding DHFR-FS;
- and
- (ii) a single protospacer adjacent motif (PAM) sequence that binds a guide RNA-CRISPR/Cas9 ribonucleoprotein (RNP), wherein the PAM is located 5′ or 3′ of the expression cassette of (i).
Dependencies are inferred from the claim text.