PRODUCTION OF ENGINEERED T-CELLS BY SLEEPING BEAUTY TRANSPOSON COUPLED WITH METHOTREXATE SELECTION
Aspects of the invention described herein include methods of treating, inhibiting, ameliorating and/or eliminating a virus or cancer cells in a subject utilizing genetically engineered human T-cells having receptors for a molecule presented by the virus or the cancer cells, wherein the genetically engineered T cells are isolated utilizing a two-stage MTX selection that employs increasing concentrations of MTX.
1 . A gene delivery polynucleotide for stable insertion of a nucleic acid into an oligonucleotide, wherein the nucleic acid for insertion is flanked by inverted terminal repeat gene sequences in the gene delivery polynucleotide and, wherein the gene delivery polynucleotide is selectable, the gene delivery polynucleotide comprising:
a first sequence, wherein the first sequence comprises a first inverted terminal repeat gene sequence;
a second sequence, wherein the second sequence comprises a second inverted terminal repeat gene sequence;
a third sequence, wherein the third sequence comprises a promoter region sequence;
a fourth sequence, wherein the fourth sequence comprises at least one gene, wherein the at least one gene encodes a protein or encodes a sequence for mRNA transcription, and wherein the fourth sequence is optimized;
a fifth sequence, wherein the fifth sequence comprises at least one selectable marker cassette encoding a double mutant of dihydrofolate reductase, wherein the double mutant of dihydrofolate reductase has a 15,000 fold or about 15,000 fold reduced affinity for methotrexate, wherein the methotrexate can be used to select for cells transduced with the gene delivery polynucleotide to enhance the ratio of cells expressing the at least one gene and wherein the fifth sequence is optimized;
a sixth sequence, wherein the sixth sequence comprises a first attachment site (attP); and
a seventh sequence, wherein the seventh sequence comprises a second attachment site (attB); wherein each of the first sequence, second sequence, third sequence, fourth sequence, fifth sequence, sixth sequence, and seventh sequence have a 5′ terminus and a 3′ terminus, and wherein the 3′ terminus of the first sequence comprising the first inverted terminal repeat gene sequence is adjacent to the 5′ terminus of the third sequence, the 3′ terminus of the third sequence is adjacent to the 5′ terminus of the fourth sequence, the 3′ terminus of the fourth sequence is adjacent to the 5′ terminus of the fifth sequence and the 3′ terminus of the fifth sequence is adjacent to the 5′ terminus of the second sequence comprising a second inverted terminal repeat.
2 .- 64 . (canceled)