Primary cell gene editing

A polynucleotide comprising:

• a. first and second homology arms homologous to first and second target nucleic acid sequences;
• b. a TCR gene sequence positioned between the first and second homology arms;
• and
• c. a first 2A-coding sequence positioned upstream of the TCR gene sequence and a second 2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second 2A-coding sequences code for the same amino acid sequence but are codon-diverged relative to each other.

A polypeptide comprising, in order from N-terminus to C-terminus:

• a. a human growth hormone signal sequence;
• b. a first 2A sequence;
• c. a TCR polypeptide sequence;
• and
• d. a second 2A sequence.

A method of treating cancer in a human patient comprising:

• a. modifying a patient-derived T cell by introducing a polynucleotide into the T cell, wherein the polynucleotide comprises:
• i. first and second homology arms homologous to first and second endogenous sequences of the cell;
• ii. a TCR gene sequence positioned between the first and second homology arms;
• and
• iii. a first 2A-coding sequence positioned upstream of the TCR gene sequence and a secondP2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second 2A-coding sequences code for the same amino acid sequence but codon-diverged relative to each other;
• b. recombining the polynucleotide into an endogenous locus of the T cell, wherein the endogenous locus comprises the first and second endogenous sequences homologous to the first and second homology arms of the polynucleotide;
• and
• c. culturing the modified T cell to produce a population of T cells;
• and
• d. administering a therapeutically effective number of the modified T cells to the human patient to thereby treat the cancer.