POLYNUCLEOTIDES ENCODING METHYLMALONYL-CoA MUTASE
The disclosure relates to polynucleotides comprising an open reading frame of linked nucleosides encoding human methylmalonyl-CoA mutase precursor, human methylmalonyl-CoA mutase (MCM) mature form, or functional fragments thereof. In some embodiments, the disclosure includes methods of treating methylmalonic acidemia in a subject in need thereof comprising administering an mRNA encoding an MCM polypeptide.
1 - 15 . (canceled)
16 . A messenger RNA (mRNA) comprising:
(i) a 5′-terminal cap;
(ii) a 5′ untranslated region (UTR);
(iii) an open reading frame (ORF) encoding the human methylmalonyl-CoA mutase (MCM) polypeptide of SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, or SEQ ID NO:213, wherein at least 95% of uracils in the ORF are 5-methoxyuracils, and wherein the uracil content in the ORF is below 150% of the theoretical minimum;
(iv) a 3′ UTR; and
(v) a poly-A tail.
17 . The mRNA of claim 16 , wherein the uracil content in the ORF is above 100% of the theoretical minimum.
18 . The mRNA of claim 16 , wherein the uracil content in the ORF is between 123% and 125%, between 122% and 126%, between 121% and 127%, between 120% and 128%, between 119% and 129%, between 118% and 130%, between 117% and 131%, between 116% and 132%, between 115% and 133%, between 114% and 134%, between 113% and 135%, between 112% and 136%, or between 111% and 137% of the theoretical minimum.
19 . The mRNA of claim 16 , wherein the uracil content in the ORF is between 118% and 129% of the theoretical minimum.
20 . The mRNA of claim 16 , wherein the ORF encodes the human MCM polypeptide of SEQ ID NO:213.
21 . The mRNA of claim 20 , wherein the ORF is at least 80% identical to the nucleotide sequence of SEQ ID NO:732.
22 . The mRNA of claim 20 , wherein the ORF is at least 85% identical to the nucleotide sequence of SEQ ID NO:732.
23 . The mRNA of claim 20 , wherein the ORF is at least 90% identical to the nucleotide sequence of SEQ ID NO:732.
24 . The mRNA of claim 20 , wherein the ORF is at least 95% identical to the nucleotide sequence of SEQ ID NO:732.
25 . The mRNA of claim 20 , wherein the ORF is at least 98% identical to the nucleotide sequence of SEQ ID NO:732.
26 . The mRNA of claim 20 , wherein the ORF is at least 99% identical to the nucleotide sequence of SEQ ID NO:732.
27 . The mRNA of claim 20 , wherein the ORF is 100% identical to the nucleotide sequence of SEQ ID NO:732.
28 . The mRNA of claim 21 , wherein the 5′ UTR comprises the nucleotide sequence of SEQ ID NO:215.
29 . The mRNA of claim 21 , wherein the mRNA comprises the miR-142-3p binding site depicted in SEQ ID NO:722.
30 . The mRNA of claim 21 , wherein the 5′ terminal cap is Cap1.
31 . The mRNA of claim 21 , wherein the poly-A tail is 100 residues in length.
32 . The mRNA of claim 27 , wherein the 5′ UTR comprises the nucleotide sequence of SEQ ID NO:215.
33 . The mRNA of claim 27 , wherein the mRNA comprises the miR-142-3p binding site depicted in SEQ ID NO:722.
34 . The mRNA of claim 27 , wherein the 5′ terminal cap is Cap1.
35 . The mRNA of claim 27 , wherein the poly-A tail is 100 residues in length.
36 . The mRNA of claim 27 , wherein the 5′ UTR comprises the nucleotide sequence of SEQ ID NO:215, wherein the mRNA comprises the miR-142-3p binding site depicted in SEQ ID NO:722, wherein the 5′ terminal cap is Cap1, and wherein the poly-A tail is 100 residues in length.
37 . A pharmaceutical composition comprising the mRNA of claim 16 and a pharmaceutically acceptable excipient.
38 . A pharmaceutical composition comprising the mRNA of claim 27 and a pharmaceutically acceptable excipient.
39 . A pharmaceutical composition comprising the mRNA of claim 36 and a pharmaceutically acceptable excipient.
40 . A lipid nanoparticle comprising the mRNA of claim 16 .
41 . A lipid nanoparticle comprising the mRNA of claim 27 .
42 . A lipid nanoparticle comprising the mRNA of claim 36 .
43 . A method of treating methylmalonic acidemia in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the pharmaceutical composition of claim 37 .
44 . A method of treating methylmalonic acidemia in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the pharmaceutical composition of claim 38 .
45 . A method of treating methylmalonic acidemia in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the pharmaceutical composition of claim 39 .