Modified messenger RNA comprising functional RNA elements
The present disclosure provides messenger RNAs (mRNAs) having chemical and/or structural modifications, including RNA elements and/or modified nucleotides, which provide a desired translational regulatory activity to the mRNA.
1. A messenger RNA (mRNA) comprising
(i) a 5′ untranslated region (UTR) comprising at least one RNA element that provides a translational regulatory activity;
(ii) a full open reading frame comprising an initiation codon and encoding a polypeptide; and
(iii) a 3′ UTR,
wherein the at least one RNA element is a GC-rich RNA element comprising guanine (G) and cytosine (C) nucleobases and, optionally, adenine (A) and uracil (U) nucleobases, or derivatives or analogs thereof, wherein the GC-rich RNA element is at least 50% or greater cytosine (C) nucleobases and is at least 6 and up to 30 nucleotides in length, wherein the GC-rich RNA element is located about 15-20, about 10-15, about 5-10 or less than 5 nucleotides upstream a Kozak consensus sequence in the 5′ UTR, and wherein the mRNA comprises one or more chemically modified nucleotides.
2. The mRNA of claim 1 , wherein the GC-rich RNA element is about 50%-55% cytosine, about 55%-60% cytosine, about 60%-65% cytosine, about 65%-70% cytosine, about 70%-75% cytosine or about 75%-80% cytosine.
3. The mRNA of claim 1 , wherein the GC-rich RNA element does not comprise adenine (A) or uracil (U) nucleobases.
4. The mRNA of claim 1 , wherein the GC-rich RNA element comprises a nucleotide sequence of about 6-30 guanine (G) and cytosine (C) nucleotides, or derivatives or analogues thereof, wherein the sequence is >50% cytosine, >60% cytosine or >70% cytosine nucleobases, and wherein the GC-rich RNA element comprises a repeating sequence motif.
5. The mRNA of claim 4 , wherein the repeating sequence motif is (i) [CCG] n , wherein n=2 to 10, 2 to 5, 4, 3 or 2; or (ii) [GCC] n , wherein n=2 to 10, 2 to 5, 4, 3 or 2.
6. The mRNA of claim 1 , comprising a second RNA element that provides a translational regulatory activity, wherein the second RNA element comprises a stable RNA secondary structure.
7. The mRNA of claim 1 , wherein the initiation codon comprises at least one modified nucleotide, and wherein the at least one modified nucleotide is selected from the group consisting of 2-thiouridine, 2′-O-methyl-2-thiouridine, 2-selenouridine, 2′-O-methyl ribose, a modified nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon, inosine, 2-methylguanosine, 6-methyl-adenosine, a deoxyribonucleotide.
8. The mRNA of claim 7 , wherein the mRNA comprises:
a first polynucleotide, wherein the first polynucleotide is chemically synthesized, wherein the first polynucleotide comprises a 5′ UTR; and
(ii) a second polynucleotide, wherein the second polynucleotide is synthesized by in vitro transcription, and wherein the second polynucleotide comprises a full open reading frame encoding a polypeptide, and a 3′ UTR, and wherein (i) and (ii) are chemically cross-linked or enzymatically ligated.
9. The mRNA of claim 1 , wherein the mRNA comprises a poly A tail, optionally wherein the poly A tail is about 100 nucleotides in length.
10. The mRNA of claim 1 , wherein the mRNA comprises a 5′ Cap 1 structure.
11. The mRNA of claim 1 , wherein the one or more chemically modified nucleotides is selected from the group consisting of pseudouridine or a pseudouridine analog.
12. The mRNA of claim 1 , wherein the one or more chemically modified nucleotides is N1-methylpseudouridine.
13. The mRNA of claim 12 , wherein the mRNA is fully modified with N1-methylpseudouridine.
14. A composition comprising the mRNA of claim 1 and a pharmaceutically acceptable carrier.
15. A lipid nanoparticle comprising the mRNA of claim 1 .
16. A pharmaceutical composition comprising the lipid nanoparticle of claim 15 , and a pharmaceutically acceptable carrier.
17. The mRNA of claim 1 , wherein the GC-rich RNA element is located about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of the Kozak consensus sequence in the 5′ UTR.
18. The mRNA of claim 1 , wherein the translational regulatory activity is selected from the group consisting of:
(a) inhibits or reduces leaky scanning of the mRNA by the PIC or ribosome;
(b) increases an amount of a polypeptide translated from the full open reading frame;
(c) increases initiation of polypeptide synthesis at or from the initiation codon;
(d) inhibits or reduces initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon;
(e) inhibits or reduces an amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame;
inhibits or reduces translation of truncated or aberrant translation products from the mRNA; and
(g) a combination of any of (a)-(f).
19. A messenger RNA (mRNA) comprising
(i) a 5′ untranslated region (UTR) comprising at least one RNA element that provides a translational regulatory activity;
(ii) a full open reading frame comprising an initiation codon and encoding a polypeptide; and
(iii) a 3′ UTR,
wherein the at least one RNA element is a GC-rich RNA element comprising guanine (G) and cytosine (C) nucleobases and, optionally, adenine (A) and uracil (U) nucleobases, or derivatives or analogs thereof, wherein the GC-rich RNA element is at least 50% or greater cytosine (C) nucleobases and is at least 6 and up to 30 nucleotides in length, wherein the GC-rich RNA element is located upstream of and immediately adjacent to a Kozak consensus sequence in the 5′ UTR, and wherein the mRNA comprises one or more chemically modified nucleotides.
20. The mRNA of claim 19 , wherein the GC-rich RNA element is about 50%-55% cytosine, about 55%-60% cytosine, about 60%-65% cytosine, about 65%-70% cytosine, about 70%-75% cytosine or about 75%-80% cytosine.
21. The mRNA of claim 19 , wherein the GC-rich RNA element does not comprise adenine (A) or uracil (U) nucleobases.
22. The mRNA of claim 19 , wherein the GC-rich RNA element comprises a nucleotide sequence of about 6-30 guanine (G) and cytosine (C) nucleotides, or derivatives or analogues thereof, wherein the sequence is >50% cytosine, >60% cytosine or >70% cytosine nucleobases, and wherein the GC-rich RNA element comprises a repeating sequence motif.
23. The mRNA of claim 22 , wherein the repeating sequence motif is (i) [CCG] n , wherein n=2 to 10, 2 to 5, 4, 3 or 2; or (ii) [GCC] n , wherein n=2 to 10, 2 to 5, 4, 3 or 2.
24. The mRNA of claim 19 , wherein the mRNA comprises a poly A tail, optionally wherein the poly A tail is about 100 nucleotides in length.
25. The mRNA of claim 19 , wherein the mRNA comprises a 5′ Cap 1 structure.
26. The mRNA of claim 19 , wherein the one or more chemically modified nucleotides is N1-methylpseudouridine, optionally wherein the mRNA is fully modified with N1-methylpseudouridine.
27. The mRNA of claim 19 , wherein the translational regulatory activity is selected from the group consisting of:
(a) inhibits or reduces leaky scanning of the mRNA by the PIC or ribosome;
(b) increases an amount of a polypeptide translated from the full open reading frame;
(c) increases initiation of polypeptide synthesis at or from the initiation codon;
(d) inhibits or reduces initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon;
(e) inhibits or reduces an amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame;
(f) inhibits or reduces translation of truncated or aberrant translation products from the mRNA; and
(g) a combination of any of (a)-(f).
28. A composition comprising the mRNA of claim 19 and a pharmaceutically acceptable carrier.
29. A lipid nanoparticle comprising the mRNA of claim 19 .
30. A pharmaceutical composition comprising the lipid nanoparticle of claim 29 , and a pharmaceutically acceptable carrier.