Signal-sensor polynucleotides for the alteration of cellular phenotypes

Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of signal-sensor polynucleotides, primary transcripts and mmRNA molecules.

We claim: 
     
         1 . An isolated synthetic signal-sensor polynucleotide, wherein said isolated synthetic signal-sensor polynucleotide comprises an mRNA which encodes an oncology-related polypeptide of interest and one or more sensor sequences selected from the group consisting of any of SEQ ID NOs: 3529-4549, SEQ ID NOs: 5571-6591 and functional variants thereof. 
     
     
         2 . The isolated synthetic signal-sensor polynucleotide of  claim 1  wherein the oncology-related polypeptide of interest is selected from the group consisting of SEQ ID NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516 and 7517. 
     
     
         3 . The isolated synthetic signal-sensor polynucleotide of  claim 1  wherein the mRNA comprises at least an open reading frame of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2488-2496, 6617-6621, 7348-7354, 7362-7489, 7491, 7494, 7506, 7511 and 7513. 
     
     
         4 . The isolated synthetic signal-sensor polynucleotide of  claim 3 , wherein the open reading frame is codon optimized. 
     
     
         5 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , wherein the mRNA comprises two stop codons. 
     
     
         6 . The isolated synthetic signal-sensor isolated polynucleotide of  claim 1 , wherein the mRNA comprises a first stop codon “TGA” and a second stop codon selected from the group consisting of “TAA,” “TGA” and “TAG.” 
     
     
         7 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , wherein the mRNA has a 3′ tailing sequence of linked nucleosides selected from the group consisting of a poly-A tail of at least 140 nucleotides, a triple helix, and a poly A-G quartet. 
     
     
         8 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , wherein the mRNA comprises at least one 5′terminal cap selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. 
     
     
         9 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , where the isolated synthetic signal-sensor polynucleotide is substantially purified. 
     
     
         10 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , wherein the isolated synthetic signal-sensor polynucleotide comprises at least one chemical modification. 
     
     
         11 . The isolated synthetic signal-sensor polynucleotide of  claim 10 , wherein the at least one chemical modification is 1-methylpseudouridine. 
     
     
         12 . The isolated synthetic signal-sensor polynucleotide of  claim 11 , further comprising the chemical modification 5-methylcytidine. 
     
     
         13 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , where the isolated synthetic signal-sensor polynucleotide comprises at least two chemical modifications. 
     
     
         14 . The isolated synthetic signal-sensor polynucleotide of  claim 13 , wherein the modifications are located on one or more of a nucleoside and/or the backbone of said nucleotides. 
     
     
         15 . The isolated synthetic signal-sensor polynucleotide of  claim 13 , where the modifications are located on both a nucleoside and a backbone linkage. 
     
     
         16 . The isolated synthetic signal-sensor polynucleotide of  claim 13 , where the modifications are located on the backbone linkage. 
     
     
         17 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , wherein the isolated signal-sensor polynucleotide is codon optimized. 
     
     
         18 . The isolated synthetic signal-sensor polynucleotide of  claim 1 , wherein the isolated signal-sensor polynucleotide is formulated. 
     
     
         19 . The isolated synthetic signal-sensor polynucleotide of  claim 1  wherein the polypeptide of interest is a factor modulating the affinity between HIF subunits and/or HIF-dependent gene expression. 
     
     
         20 . The isolated synthetic signal-sensor polynucleotide of  claim 19  wherein the HIF subunits are selected from the group consisting of SEQ ID NO: 6611-6616. 
     
     
         21 . The isolated synthetic signal-sensor polynucleotide of  claim 1  wherein the isolated synthetic signal-sensor polynucleotide comprises at least one translation enhancer element. 
     
     
         22 . An isolated synthetic signal-sensor polynucleotide comprising:
 (a) a first region of linked nucleosides, said first region encoding an oncology-related polypeptide of interest selected from the group consisting of SEQ ID NOs: 1321-2487, 6611-6616 and 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516 and 7517;   (b) a first flanking region located 5′ relative to said first region comprising;
 (i) a sequence of linked nucleosides selected from the group consisting of the native 5′ untranslated region (UTR) of any of the nucleic acids that encode any of SEQ ID NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516, 7517, SEQ ID NO: 1-4 and functional variants thereof; 
   (c) a second flanking region located 3′ relative to said first region comprising;
 (i′) a sequence of linked nucleosides selected from the group consisting of the native 3′ UTR of any of the nucleic acids that encode any of SEQ ID NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516, 7517, SEQ ID NO: 5-21 and functional variants thereof; 
 (ii′) one or more sensor sequences located selected from the group consisting of the any of SEQ ID NOs: 3529-4549, SEQ ID NOs: 5571-6591 and functional variants thereof; and 
 (iii′) a 3′ tailing sequence of linked nucleosides. 
   
     
     
         23 . The isolated synthetic signal-sensor polynucleotide of  claim 22  wherein the first region of linked nucleosides comprises at least an open reading frame of a nucleic acid sequence, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 2488-2496, 6617-6621, 7348-7354, 7362-7489, 7491, 7494, 7506, 7511 and 7513. 
     
     
         24 . The isolated synthetic signal-sensor polynucleotide of  claim 23 , wherein the open reading frame is codon optimized. 
     
     
         25 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , wherein the first region comprises two stop codons. 
     
     
         26 . The isolated synthetic signal-sensor isolated polynucleotide of  claim 22 , wherein the first region comprises a first stop codon “TGA” and a second stop codon selected from the group consisting of “TAA,” “TGA” and “TAG.” 
     
     
         27 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , wherein the 3′ tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail of at least 140 nucleotides, a triple helix, and a poly A-G quartet. 
     
     
         28 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , wherein the first flanking region further comprises at least one 5′terminal cap. 
     
     
         29 . The isolated synthetic signal-sensor polynucleotide of  claim 28 , wherein the at least one 5′ terminal cap is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. 
     
     
         30 . The isolated synthetic signal-sensor polynucleotide of  claim 28  where the isolated signal-sensor polynucleotide is substantially purified. 
     
     
         31 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , wherein the isolated synthetic signal-sensor polynucleotide comprises at least one chemical modification. 
     
     
         32 . The isolated synthetic signal-sensor polynucleotide of  claim 31 , wherein the at least one chemical modification is 1-methylpseudouridine. 
     
     
         33 . The isolated synthetic signal-sensor polynucleotide of  claim 32 , further comprising the chemical modification 5-methylcytidine. 
     
     
         34 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , comprising at least two chemical modifications in the first region. 
     
     
         35 . The isolated synthetic signal-sensor polynucleotide of  claim 34 , wherein the modifications are located on one or more of a nucleoside and/or the backbone of said nucleotides. 
     
     
         36 . The isolated synthetic signal-sensor polynucleotide of  claim 34 , where the modifications are located on both a nucleoside and a backbone linkage. 
     
     
         37 . The isolated synthetic signal-sensor polynucleotide of  claim 34 , where the modifications are located on the backbone linkage. 
     
     
         38 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , where the isolated signal-sensor polynucleotide is codon optimized. 
     
     
         39 . The isolated synthetic signal-sensor polynucleotide of  claim 38 , wherein the first region of linked nucleosides is codon optimized. 
     
     
         40 . The isolated synthetic signal-sensor polynucleotide of  claim 22 , wherein the isolated signal-sensor polynucleotide is formulated. 
     
     
         41 . A method of treating a disease, disorder and/or condition in a subject in need thereof by increasing the level of an oncology-related polypeptide of interest comprising administering to said subject an isolated synthetic signal-sensor polynucleotide encoding said oncology-related polypeptide. 
     
     
         42 . A method of reducing, eliminating or preventing tumor growth in a subject in need thereof by increasing the level of an oncology-related polypeptide of interest comprising administering to said subject an isolated synthetic signal-sensor polynucleotide encoding said oncology-related polypeptide. 
     
     
         43 . A method of reducing and/or ameliorating at least one symptom of cancer in a subject need thereof by increasing the level of an oncology-related polypeptide of interest comprising administering to said subject an isolated synthetic signal-sensor polynucleotide encoding said oncology-related polypeptide. 
     
     
         44 . The method of any of  claims 41 - 43  wherein the disease, disorder and/or condition is selected from the group consisting of adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment. 
     
     
         45 . The method of  claim 44  wherein the tumor growth is results from a disease, disorder and/or condition selected from the group consisting of adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment. 
     
     
         46 . The method of any of  claims 41 - 43  wherein the administration of the isolated synthetic signal-sensor polynucleotide reduces the number of cancer cells, eliminates cancer cells, prevents an increase in cancer cells and/or alleviates the symptoms of cancer in a subject. 
     
     
         47 . The method of  claim 43  wherein the at least one symptom of cancer is selected from the group consisting of weakness, aches and pains, fever, fatigue, weight loss, blood clots, increased blood calcium levels, low white blood cell count, short of breath, dizziness, headaches, hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth, change in bowel habits, change in bladder function, long-lasting sores, white patches inside the mouth, white spots on the tongue, unusual bleeding or discharge, thickening or lump on parts of the body, indigestion, trouble swallowing, changes in warts or moles, change in new skin and nagging cough and hoarseness. 
     
     
         48 . The methods of any of  claims 41 - 43 , wherein the isolated synthetic signal-sensor polynucleotide is formulated. 
     
     
         49 . The method of  claim 48 , wherein the isolated synthetic signal-sensor polynucleotide is administered at a total daily dose of between 0.001 ug and 150 ug. 
     
     
         50 . The method of  claim 49 , wherein administration is by injection, topical administration, ophthalmic administration or intranasal administration. 
     
     
         51 . The method of  claim 50 , wherein administration is by injection and said injection is selected from the group consisting of intradermal, subcutaneous and intramuscular. 
     
     
         52 . The method of  claim 50 , wherein administration is topical administration and said topical administration is selected from the group consisting of cream, lotion, ointment, gel, spray, solution and the like. 
     
     
         53 . A method of preferentially inducing cell death in cancer cells in a tissue or organ, comprising
 (a) contacting said tissue or organ with an isolated synthetic signal-sensor polynucleotide, wherein said isolated synthetic signal-sensor polynucleotide encodes
 (i) an oncology-related polypeptide whose expression triggers apoptosis or cell death, and 
 (ii) at least one microRNA binding site of a microRNA, where the expression of said microRNA in the cancer cell is lower than the expression of said microRNA in normal, non cancerous cells.