MODIFIED NUCLEIC ACID MOLECULES AND USES THEREOF
The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using them.
1 . An mRNA encoding a polypeptide, wherein the mRNA comprises:
(i) at least one 5′-cap structure;
(ii) a 5′-UTR;
(iii) an open reading frame encoding the polypeptide wherein at least one nucleotide is 5-methyl-2-thio-uridine; and
(iv) a 3′-UTR.
2 . The mRNA of claim 1 , wherein the open reading frame encoding the polypeptide and consisting of nucleotides including 5-methyl-2-thio-uridine, cytosine, adenine, and guanine
3 . The mRNA of claim 1 , wherein the at least one 5′-cap structure is cap0, cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.
4 . The mRNA of claim 3 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA.
5 . The mRNA of claim 1 , wherein the 3′-UTR is an alpha-globin 3′-UTR.
6 . The mRNA of claim 1 , wherein the mRNA further comprises a poly-A region
7 . The mRNA of claim 6 , wherein the poly-A region is at least 160 nucleotides in length.
8 . The mRNA of claim 1 , wherein the 5′-UTR comprises a Kozak sequence.
9 . The mRNA of claim 1 , wherein the mRNA is purified.
10 . The mRNA of claim 1 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine.
11 . A pharmaceutical composition comprising the mRNA of claim 1 and a pharmaceutically acceptable excipient.
12 . A method of expressing a polypeptide of interest in a mammalian cell, the method comprising:
(i) providing the mRNA of claim 1 ; and
(ii) introducing the mRNA to a mammalian cell under conditions that permit the expression of the polypeptide of interest by the mammalian cell.