SIGNAL-SENSOR POLYNUCLEOTIDES FOR THE ALTERATION OF CELLULAR PHENOTYPES
The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of signal-sensor polynucleotides, primary transcripts and mmRNA molecules.
1 .- 53 . (canceled)
54 . A method of dampening expression of a polypeptide in a hepatocyte, comprising administering a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding the polypeptide and a 3′untranslated region (3′UTR) comprising at least one microRNA (miR)-122 binding site.
55 . The method of claim 54 , wherein the mRNA is fully modified with chemically-modified uridines.
56 . The method of claim 55 , wherein the chemically-modified uridines are pseudouridine analogs.
57 . The method of claim 56 , wherein the pseudouridine analogs are 1-methyl pseudouridines.
58 . The method of claim 54 , wherein the mRNA is fully modified with 5-methyl-cytidine nucleosides.
59 . The method of claim 54 , wherein the mRNA comprises at least two miR-122 binding sites or at least three miR-122 binding sites.
60 . The method of claim 54 , wherein the miR-122 binding site is a miR-122-3p binding site.
61 . The method of claim 60 , wherein the miR-122-3p binding site comprises the sequence set forth in SEQ ID NO: 3587.
62 . The method of claim 54 , wherein the miR-122 binding site is a miR-122-5p binding site.
63 . The method of claim 62 , wherein the miR-122-5p binding site comprises the sequence set forth in SEQ ID NO: 3592.
64 . The method of claim 54 , wherein the mRNA comprises a 5′UTR.
65 . The method of claim 64 , wherein the 5′UTR comprises a translation initiation sequence selected from the group consisting of a Kozak sequence and an internal ribosome entry site (IRES).
66 . The method of claim 54 , wherein the mRNA comprises at least one 5′ cap structure.
67 . The method of claim 66 , wherein the at least one 5′cap structure is selected from the group consisting 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2).
68 . The method of claim 54 , wherein the mRNA comprises a poly A tail.
69 . The method of claim 68 , wherein the poly A tail comprises at least 100, at least 120, or at least 140 nucleosides.
70 . The method of claim 54 , wherein the mRNA is formulated with a liposome, lipoplex or lipid nanoparticle.
71 . The method of claim 54 , wherein the mRNA is formulated with a lipid nanoparticle.
72 . The method of claim 71 , wherein the lipid nanoparticle comprises a cationic or ionizable lipid.
73 . The method of claim 72 , wherein the cationic lipid is selected from the group consisting of DLin-MC3-DMA, DLin-DMA, C 12 -200 and DLin-KC2-DMA.
74 . The method of claim 54 , wherein the polypeptide is an oncology-related polypeptide.
75 . A method of treating a disease, disorder or condition in a subject, comprising administering an mRNA comprising an ORF encoding a polypeptide of interest and a 3′UTR comprising at least one miR-122 binding site, wherein the mRNA is fully modified with chemically-modified uridines, and wherein expression of the polypeptide from the mRNA is dampened in the liver of the subject.
76 . The method of claim 75 , wherein the disease, disorder or condition is cancer.
77 . The method of claim 76 , wherein the mRNA is administered intratumorally.
78 . The method of claim 76 , wherein the polypeptide of interest is an oncology-related polypeptide.