RNA ENGINEERED T CELLS FOR THE TREATMENT OF CANCER

1 - 62 . (canceled)

63 . A method of generating a population of RNA-engineered T cells transiently expressing an exogenous RNA, the method comprising:

(a) introducing into a population of T cells an in vitro transcribed (IVT) RNA or a synthetic RNA comprising a chimeric antigen receptor (CAR) nucleic acid,

wherein the IVT RNA or the synthetic RNA is transcribed from an in vitro transcription vector or a DNA plasmid comprising a poly(A) tail comprising about 150 adenosine bases, a 3′UTR comprising at least one repeat of a 3′UTR derived from human beta-globulin, and a 5′UTR comprising a 5′ cap; and

wherein the CAR comprises an extracellular domain comprising an antigen binding domain that binds a tumor antigen, a transmembrane domain, a costimulatory signaling region, and a signaling domain of CD3-zeta; and

(b) culturing the CAR expressing population of T cells in a culture media.

64 . The method of claim 63 , wherein prior to introducing the IVT RNA or the synthetic RNA, the population of T cells is isolated from peripheral blood lymphocytes, and enriched for CD3 + T cells, CD28 + T cells, CD4 + T cells, CD8 + T cells, CD45RA + T cells, CD45RO + T cells, and any combination thereof.

65 . The method of claim 64 , wherein, the population of T cells is enriched for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

66 . The method of claim 64 , wherein the population of T cells is enriched for at least about 10 hours to about 24 hours.

67 . The method of claim 63 , wherein prior to introducing the IVT RNA or the synthetic RNA, the population of T cells is enriched for CD4 + T cells, CD25 + T cells, CD62L hi T cells, GITR + T cells, and FoxP3 + T cells.

68 . The method of claim 63 , wherein prior to introducing the IVT RNA or the synthetic RNA, the population of T cells is depleted of CD4 + T cells, CD25 + T cells, CD62L hi T cells, GITR + T cells, FoxP3 + T cells, or any combination thereof.

69 . The method of claim 68 , wherein the population of T cells is depleted of CD25 + T cells.

70 . The method of claim 63 , wherein introducing comprises electroporating the IVT RNA or the synthetic RNA into the population of T cells.

71 . The method of claim 63 , wherein the in vitro transcription vector or the DNA plasmid comprises two repeats of the 3′UTR derived from human beta-globulin (2bgUTR).

72 . The method of claim 63 , wherein the IVT RNA or the synthetic RNA further comprises an internal ribosome entry site (IRES) sequence.

73 . The method of claim 63 , wherein:

(a) the tumor antigen is an antigen associated with a cancer selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, and any combination thereof;

(b) the tumor antigen is selected from the group consisting of CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, and GD-2;

(c) the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof, and/or

(d) the transmembrane domain is selected from transmembrane region of a molecule selected from the group consisting of alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

74 . The method of claim 63 , wherein the DNA plasmid or the in vitro transcription vector comprises a sequence selected from the group consisting of SEQ ID NO: 6-24.

75 . The method of claim 63 , wherein the DNA plasmid or the in vitro transcription vector comprises a sequence selected from the group consisting of:

(a) SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24;

(b) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; or

(c) SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

76 . The method of claim 63 , wherein the in vitro transcription vector comprises the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 28, or SEQ ID NO: 27.

77 . The method of claim 63 , wherein the CAR nucleic acid is:

(a) modified to remove internal open reading frames;

(b) codon optimized for use in humans; and/or

(c) modified to remove dileucine motifs.

78 . The method of claim 63 , wherein the costimulatory signaling region comprises a CD28, a CD2, or a 4-1BB costimulatory domain.

79 . A population of RNA-engineered T cells generated by the method of claim 63 .

80 . A method of treating a cancer, the method comprising administering to a patient in need thereof the population of RNA-engineered T cells (RNA CAR T cells) generated by the method of claim 63 .

81 . The method of claim 80 further comprising repeating the administration of the RNA CAR T cells for at least two, three, or four times.

82 . The method of claim 81 further comprising administering a chemotherapeutic agent to the patient.

83 . The method of claim 82 , wherein the chemotherapeutic agent is Cyclophosphamide (Cytoxan).

84 . The method of claim 82 , wherein the chemotherapeutic agent is administered at least seven days after a first RNA CAR T cells administration.

85 . The method of claim 82 , wherein the chemotherapeutic agent is administered at least one day before a second, a third, or a fourth RNA CAR T cell administration.