Patent ID: 11891614
Assignee: CELLECTIS
Field: Pharmaceuticals (Chemistry)
Classification: CPC C  A | IPC A  C

Claim 0:
1. A method of preparing engineered T-cells for immunotherapy comprising
(a) inactivating PDCD1 in the T-cells comprising introducing into the T-cells by electroporation of RNAs encoding at least two half Transcription activator-like effector nucleases (TALE-nucleases) able to selectively inactivate by DNA cleavage a gene encoding PDCD1, to produce engineered T-cells; and
(b) expanding said engineered T-cells,, wherein said at least two half TALE-nucleases recognize and cleave a target sequence selected from SEQ ID NO:77 and SEQ ID NO:78;
wherein introducing said at least two half Transcription activator-like effector nucleases (TALE-nucleases) into said T-cells comprises contacting said engineered T-cells with RNAs encoding said at least two half TALE-nucleases; and
i. applying an agile pulse sequence consisting of: one electrical pulse with a voltage range from 2250 to 3000 V per centimeter, a pulse width of 0.1 ms and a pulse interval of 0.2 to 10 ms between the electrical pulses of steps (i) and (ii);
ii. one electrical pulse with a voltage range from 2250 to 3000 V with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (ii) and the first electrical pulse of step (iii); and
iii. 4 electrical pulses with a voltage of 325 V with a pulse width of 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses.