Mirna modulation of t cell signaling and uses thereof

2:

• The method of claim 1, wherein the miRNA targets a plurality of T cell mRNAs selected from the group consisting of mRNAs encoding tyrosine-protein phosphatase non-receptor type (PTPN) 11 (PTPN11), PTPN22, dual specificity protein phosphatase (DUSP) 5 (DUSP5), and DUSP6.

3:

• The method of claim 2, wherein the miRNA targets each of the mRNAs encoding PTPN11, PTPN22, DUSP5, and DUSP6.

4:

• The method of claim 1, wherein the RNA transcript comprises a sequence corresponding to a precursor miRNA (pre-miRNA).

5:

• The method of claim 1, wherein the RNA transcript comprises a sequence corresponding to a primary miRNA (pri-miRNA).

6:

• The method of claim 1, wherein the RNA transcript comprises a loop region having the nucleotide sequence of SEQ ID NO:
• 4 or 5, or a variant thereof comprising up to 3 nucleotide substitutions.

7:

• The method of claim 1, wherein the RNA transcript comprises a stem region having the nucleotide sequence of SEQ ID NO:
• 3, or a variant thereof comprising up to 3 nucleotide substitutions.

8:

• The method of claim 4, wherein the sequence corresponding to the pre-miRNA has the nucleotide sequence of SEQ ID NO:
• 6 or 7.

9:

• The method of claim 5, wherein the sequence corresponding to the pri-miRNA has the nucleotide sequence of SEQ ID NO:
• 8 or 9.

10:

• The method of claim 1, further comprising introducing the exogenous nucleic acid molecule into a population of input T cells, thereby generating the population of modified T cells.

11:

• A method of treating cancer in an individual, comprising administering to the individual a population of modified T cells that recognize a cancer-associated antigen in the individual, wherein the modified T cells comprise a modification that increases the expression of endogenous miR-181a, and wherein the modified T cells have a lower T cell receptor (TCR) signaling threshold and/or increased TCR sensitivity to the cancer-associated antigen as compared to T cells not modified to increase the expression of endogenous miR-181a.

12:

• The method of claim 11, wherein the modification comprises introducing a nucleic acid molecule encoding a) a fusion protein comprising a nuclease-deficient sequence-guided endonuclease (SGEN) fused to an activator of RNA polymerase II;
• and b) a guide nucleotide directing the fusion protein to the promoter region of the miR-181a gene.

13:

• The method of claim 11, wherein the modification comprises inserting a nucleic acid sequence that upregulates miR-181a into the genome of the T cells.

14:

• The method of claim 13, wherein the nucleic acid sequence encodes miR-181a.

15:

• The method of claim 13, wherein the nucleic acid sequence comprises a promoter sequence and is inserted such that the promoter sequence is operably linked to the miR-181a gene.

16:

• The method of claim 11, further comprising modifying a population of input T cells, thereby generating the population of modified T cells.

17:

• The method of claim 1, further comprising administering a second therapy or therapeutic agent.

18:

• The method of claim 17, wherein the method further comprises administering a) a conditioning chemotherapy prior to administration of the modified T cells, b) a chemotherapeutic agent, or c) an immunotherapeutic agent.

19:

• The method of claim 1, wherein the modified T cells are autologous to the individual.

20:

• The method of claim 19, wherein the T cells are isolated from a solid tumor in the individual.

21:

• The method of claim 1, wherein the modified T cells are allogeneic to the individual.

22:

• The method of claim 1, wherein the individual is human.

23:

• The method of claim 1, wherein the cancer is a solid tumor.

24:

• A method of producing a population of modified T cells, comprising introducing into a population of input T cells recognizing a cancer-associated antigen an exogenous nucleic acid molecule encoding an miRNA that comprises a seed sequence having the nucleotide sequence of SEQ ID NO:
• 1, wherein the modified T cells have a lower T cell receptor (TCR) signaling threshold and/or increased TCR sensitivity to the cancer-associated antigen.

25:

• The method of claim 24, wherein the miRNA targets a plurality of T cell mRNAs selected from the group consisting of mRNAs encoding tyrosine-protein phosphatase non-receptor type (PTPN) 11 (PTPN11), PTPN22, dual specificity protein phosphatase (DUSP) 5 (DUSP5), and DUSP6.

26:

• The method of claim 25, wherein the miRNA targets each of the mRNAs encoding PTPN11, PTPN22, DUSP5, and DUSP6.

27:

• The method of claim 24, wherein the RNA transcript comprises a sequence corresponding to a precursor miRNA (pre-miRNA).

28:

• The method of claim 24, wherein the RNA transcript comprises a sequence corresponding to a primary miRNA (pri-miRNA).

29:

• The method of claim 24, wherein the RNA transcript comprises a loop region having the nucleotide sequence of SEQ ID NO:
• 4 or 5, or a variant thereof comprising up to 3 nucleotide substitutions.

30:

• The method of claim 24, wherein the RNA transcript comprises a stem region having the nucleotide sequence of SEQ ID NO:
• 3, or a variant thereof comprising up to 3 nucleotide substitutions.

31:

• The method of claim 27, wherein the sequence corresponding to the pre-miRNA has the nucleotide sequence of SEQ ID NO:
• 6 or 7.

32:

• The method of claim 28, wherein the sequence corresponding to the pri-miRNA has the nucleotide sequence of SEQ ID NO:
• 8 or 9.

33:

• A method of producing a population of modified T cells, comprising introducing into a population of input T cells recognizing a cancer-associated antigen a modification that increases the expression of endogenous miR-181a, wherein the modified T cells have a lower TCR signaling threshold and/or increased TCR sensitivity to the cancer-associated antigen.

34:

• A composition comprising a population of modified T cells prepared by the method of claim 24.

35:

• A polyclonal population of modified T cells that recognize two or more cancer-associated antigens in an individual, wherein the modified T cells comprise an exogenous nucleic acid molecule encoding a microRNA (miRNA) that comprises a seed sequence having the nucleotide sequence of SEQ ID NO:
• 1, and wherein the modified T cells have a lower T cell receptor (TCR) signaling threshold and/or increased TCR sensitivity to the cancer-associated antigen.

36:

• A polyclonal population of modified T cells that recognize two or more cancer-associated antigens in an individual, wherein the modified T cells comprise a modification that increases the expression of endogenous miR-181a, and wherein the modified T cells have a lower T cell receptor (TCR) signaling threshold and/or increased TCR sensitivity to the cancer-associated antigen.

37:

• A pharmaceutical composition comprising the polyclonal population of modified T cells of claim 35 and a pharmaceutically acceptable carrier.