Transposase polypeptides and uses thereof
View Patent ↗Transposase polypeptides and polynucleotides are provided, which have a high activity in mammalian cells. Methods for engineering cells, such as chimeric antigen T-cells, with the transposes are also provided.
1. A method of providing an immune response in a human subject in need thereof comprising:
(a) transfecting an immune cell with
(i) a nucleic acid encoding a recombinant polypeptide having transposase activity, wherein said polypeptide comprises an amino acid sequence at least 90% identical to the sequence of SEQ ID NO: 1, wherein the polypeptide comprises:
(i) an Arg at the position corresponding to position 14;
(ii) an Ala at the position corresponding to position 33;
(iii) a His at the position corresponding to position 115;
(iv) an Arg at the position corresponding to position 136;
(v) an Asp at the position corresponding to position 214;
(vi) an Ala at the position corresponding to position 215;
(vii) a Val at the position corresponding to position 216;
(viii) a Gln at the position corresponding to position 217;
(ix) a His at the position corresponding to position 243;
(x) a His at the position corresponding to position 253; and
(xi) an Arg at the position corresponding to position 255; and
(ii) a DNA encoding a CAR or TCR flanked by transposon repeats; and
(b) administering the transfected immune cells expressing a CAR or TCR to the subject to provide a T-cell response.
2. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 1.
3. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 1.
4. The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
5. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 3.
6. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 3.
7. The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3.
8. The method of claim 1 , wherein the CAR specific to a tumor antigen.
9. The method of claim 8 , wherein the tumor antigen is CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2.
10. The method of claim 1 , further comprising transfecting the immune cells with a nucleic acid encoding a membrane-bound cytokine.
11. The method of claim 10 , wherein the membrane-bound cytokine is membrane-bound IL-15.
12. The method of claim 1 , wherein the immune cell is a natural killer (NK) cell, a T-cell, a precursor of a NK cell or a precursor of a T-cell.
13. The method of claim 1 , wherein the nucleic acid is mRNA.
14. A method of treating a human subject having a disease cancer comprising:
(a) transfecting an immune cell with
(i) a nucleic acid encoding a recombinant polypeptide having transposase activity, wherein said polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, wherein the polypeptide comprises:
(i) an Arg at the position corresponding to position 14;
(ii) an Ala at the position corresponding to position 33;
(iii) a His at the position corresponding to position 115;
(iv) an Arg at the position corresponding to position 136;
(v) an Asp at the position corresponding to position 214;
(vi) an Ala at the position corresponding to position 215;
(vii) a Val at the position corresponding to position 216;
(viii) a Gln at the position corresponding to position 217;
(ix) a His at the position corresponding to position 243;
(x) a His at the position corresponding to position 253; and
(xi) an Arg at the position corresponding to position 255; and
(ii) a DNA encoding a CAR or TCR flanked by transposon repeats; and
(b) administering a therapeutically effective amount of the transfected immune cells expressing a CAR or TCR to the subject, thereby effecting an anti-tumor response to reduce or eliminate tumors in the subject.
15. The method of claim 14 , wherein the cancer is cancer of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
16. The method of claim 14 , wherein the cancer is a lymphoma, leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or B cell-associated autoimmune disease.
17. The method of claim 14 , further comprising transfecting the immune cells with a nucleic acid encoding a membrane-bound cytokine.
18. The method of claim 17 , wherein the membrane-bound cytokine is membrane-bound IL-15.
19. The method of claim 14 , wherein the immune cell is a natural killer (NK) cell, a T-cell, a precursor of a NK cell or a precursor of a T-cell.