GENOME EDITED IMMUNE EFFECTOR CELLS

1 .- 162 . (canceled)

163 . A method of editing a TCRα allele in a population of T cells comprising:

(a) activating a population of T cells and stimulating the population of T cells to proliferate;

(b) introducing an mRNA encoding megaTAL into the population of T cells;

(c) transducing the population of T cells with one or more viral vectors comprising a donor repair template;

wherein expression of the engineered nuclease creates a double strand break at a target site in the TCRα allele, and the donor repair template is incorporated into the TCRα allele by homology directed repair (HDR) at the site of the double-strand break (DSB).

164 . The method of claim 163 , wherein the donor repair template comprises a 5′ homology arm homologous to the TCRα sequence 5′ of the DSB; a polynucleotide encoding an immunopotency enhancer, an immunosuppressive signal damper, or an engineered antigen receptor; and a 3′ homology arm homologous to the TCRα sequence 3′ of the DSB.

165 . The method of claim 164 , wherein the polynucleotide further comprises an RNA polymerase II promoter operably linked to the polynucleotide encoding the immunopotency enhancer, immunosuppressive signal damper, or engineered antigen receptor.

166 . The method of claim 164 , wherein:

(a) the lengths of the 5′ and 3′ homology arms are independently selected from about 100 bp to about 2500 bp;

(b) the lengths of the 5′ and 3′ homology arms are independently selected from about 600 bp to about 1500 bp;

(c) the 5′homology arm is about 1500 bp and the 3′ homology arm is about 1000 bp;

or

(d) the 5′homology arm is about 600 bp and the 3′ homology arm is about 600 bp.

167 . The method of claim 163 , wherein the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.

168 . The method of claim 167 , wherein:

(a) the rAAV has one or more ITRs from AAV2;

(b) the rAAV has a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10;

(c) the rAAV has an AAV6 serotype;

(d) the retrovirus is a lentivirus; or

(e) the retrovirus is an integrase deficient lentivirus.

169 . The method of claim 163 , wherein the megaTAL comprises a TALE DNA binding domain and an engineered meganuclease.

170 . The method of claim 169 , wherein the TALE binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

171 . The method of claim 169 , wherein:

(a) the meganuclease is engineered from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-NcrI, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdil41I;

(b) the meganuclease is engineered from an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI; or

(c) the meganuclease is engineered from an I-OnuI LHE.

172 . The method of claim 163 , wherein a DSB is generated in both TCRα alleles; and the donor template is inserted into one or both modified TCRα alleles; and the cell is further transduced with a lentiviral vector comprising an engineered antigen receptor.

173 . The method of claim 163 , wherein the T cells are cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.

174 . The method of claim 163 , wherein the mRNA encoding the engineered nuclease further encodes a viral self-cleaving 2A peptide and an end-processing enzyme.

175 . The method of claim 163 , wherein the method further comprises introducing an mRNA encoding an end-processing enzyme into the T cell.

176 . The method of claim 174 or claim 175 , wherein the end-processing enzyme exhibits 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerases activity.

177 . The method of claim 176 , wherein the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.

178 . The method of claim 163 , wherein the T cell is activated and stimulated in the presence of a PI3K inhibitor.

179 . A population of T cells modified by the method according to claim 163 .

180 . A composition comprising the population of T cells of claim 179 .

181 . A composition comprising the population of T cells of claim 179 and a physiologically acceptable carrier, diluent, or excipient.