Circular polynucleotides
The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of circular polynucleotides.
1. A single-stranded circular polynucleotide encoding a polypeptide, wherein said circular polynucleotide comprises a sequence of Formula II:
[A n ]-L 1 -[B o ] Formula II
wherein each A and each B independently comprises any nucleoside;
n and o are independently 15 to 1000; and
L 1 has the structure of Formula III:
wherein a, b, c, d, e, and f are each, independently, 0 or 1;
each of R 1 , R 3 , R 5 , and R 7 , is, independently, selected from optionally substituted C 1 -C 6 alkylene, optionally substituted C 1 -C 6 heteroalkylene, O, S, and NR 8 ;
R 2 and R 6 are each, independently, selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl;
R 4 is optionally substituted C 1 -C 10 alkylene, optionally substituted C 2 -C 10 alkenylene, optionally substituted C 2 -C 10 alkynylene, optionally substituted C 2 -C 9 heterocyclylene, optionally substituted C 6 -C 12 arylene, optionally substituted C 2 -C 100 polyethylene glycolene, or optionally substituted C 1 -C 10 heteroalkylene, or a bond linking (R 1 ) a —(R 2 ) b —(R 3 ) c to (R 5 ) d —(R 6 ) e —(R 7 ) f ,
wherein if c, d, e, and f are 0, R 4 is not a bond; and
R 8 is hydrogen, optionally substituted C 1 -C 4 alkyl, optionally substituted C 2 -C 4 alkenyl, optionally substituted C 2 -C 4 alkynyl, optionally substituted C 2 -C 6 heterocyclyl, optionally substituted C 6 -C 12 aryl, or optionally substituted C 1 -C 7 heteroalkyl;
wherein L 1 is attached to [A n ] and [B o ] at the sugar of one of said nucleosides; and
wherein said circular polynucleotide comprises a coding region, a 5′ untranslated region (UTR) and a 3′UTR.
2. A single-stranded circular polynucleotide encoding a polypeptide, wherein said circular polynucleotide comprises a sequence of Formula II:
[A n ]-L 1 -[B o ] Formula II
wherein each A and B independently comprises any nucleoside;
n and o are, independently 15 to 1000; and
L 1 is a bond or has the structure of Formula III:
wherein a, b, c, d, e, and f are each, independently, 0 or 1;
each of R 1 , R 3 , R 5 , and R 7 , is, independently, selected from optionally substituted C 1 -C 6 alkylene, optionally substituted C 1 -C 6 heteroalkylene, O, S, and NR 8 ;
R 2 and R 6 are each, independently, selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl;
R 4 is optionally substituted C 1 -C 10 alkylene, optionally substituted C 2 -C 10 alkenylene, optionally substituted C 2 -C 10 alkynylene, optionally substituted C 2 -C 9 heterocyclylene, optionally substituted C 6 -C 12 arylene, optionally substituted C 2 -C 100 polyethylene glycolene, or optionally substituted C 1 -C 10 heteroalkylene, or a bond linking (R 1 ) a —(R 2 ) b —(R 3 ) c to (R 5 ) d —(R 6 ) e —(R 7 ) f ; and
R 8 is hydrogen, optionally substituted C 1 -C 4 alkyl, optionally substituted C 2 -C 4 alkenyl, optionally substituted C 2 -C 4 alkynyl, optionally substituted C 2 -C 6 heterocyclyl, optionally substituted C 6 -C 12 aryl, or optionally substituted C 1 -C 7 heteroalkyl;
wherein L 1 is attached to [A n ] and [B o ] at the sugar of one of the nucleosides;
wherein at least one of [A n ] and [B o ] includes the structure of Formula IV or Formula XVIII:
wherein each of N 1 and N 2 is independently a nucleobase;
each of R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 16 is, independently, H, halo, hydroxy, thiol, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted C 2 -C 6 heteroalkenyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted amino, azido, or optionally substituted C 6 -C 10 aryl;
each of g and h is, independently, 0 or 1;
each X 1 and X 4 is, independently, O, NH, or S; and
each X 2 is independently O, NH, or S; and
each X 3 is OH or SH, or a salt thereof;
wherein, for Formula IV, at least one of X 1 , X 2 , or X 4 is NH or S; and
wherein said circular polynucleotide comprises a coding region, a 5′ untranslated region (UTR) and a 3′ UTR.
3. The circular polynucleotide of claim 2 , wherein X 1 is NH.
4. The circular polynucleotide of claim 2 , wherein X 4 is NH.
5. The circular polynucleotide of claim 2 , wherein X 2 is S.
6. The circular polynucleotide of claim 2 , further comprising at least one 5′ cap structure.
7. The circular polynucleotide of claim 6 , further comprising a poly-A tail region.
8. The circular polynucleotide of claim 7 , wherein the one of the coding region, the 5′ UTR, the 3′ UTR, the 5′ cap structure, or the poly-A tail comprises [A n ]-L1-[B o ].
9. The circular polynucleotide of claim 7 , wherein one of the coding region, the 5′ UTR, the 3′ UTR, the 5′ cap structure, or the poly-A tail comprises [A n ] and another of the coding region, the 5′ UTR, the 3′ UTR, the 5′ cap structure, or the poly-A tail comprises [B o ].
10. The circular polynucleotide claim 2 , wherein said 5′ UTR comprises at least one Kozak sequence.
11. The circular polynucleotide of claim 2 wherein the circular polynucleotide comprises at least one modified nucleoside.
12. The circular polynucleotide of claim 11 , wherein the modified nucleoside is 1 methyl-pseudouridine.
13. The circular polynucleotide of claim 2 , wherein R 4 is optionally substituted C 2-9 heterocyclylene.
14. The circular polynucleotide of claim 13 , wherein the optionally substituted C 2-9 heterocyclyl has the structure:
15. The circular polynucleotide of claim 2 , wherein L 1 comprises the structure:
16. The circular polynucleotide of claim 2 , wherein L 1 is attached to [A n ] at the 3′ or 4′ position of the sugar of one of the nucleosides and to [B o ] at the 5′ or 6′ position of the sugar of one of the nucleosides.
17. A method of producing a composition comprising the circular polynucleotide of claim 2 encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula Va or Vb:
the method comprising reacting a compound having the structure of Formula VIa or VIb:
with a compound having the structure of Formula VII:
wherein each of N 1 and N 2 is independently a nucleobase;
each of R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 16 is, independently, H, halo, hydroxy, thiol, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted C 2 -C 6 heteroalkenyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted amino, azido, or optionally substituted C 6 -C 10 aryl;
each of g and h is, independently, 0 or 1;
each X 1 and X 4 is, independently, O, NH, or S; and
each X 2 is O or S; and
each X 3 is independently OH or SH, or a salt thereof;
each of R 17 and R 19 is, independently, a region of linked nucleosides; and
R 18 is a halogen;
to produce a composition comprising a circular polynucleotide encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula Va or Vb.
18. A method of producing a composition comprising the circular polynucleotide of claim 2 encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula VIIIa or VIIIb:
the method comprising reacting a compound having the structure of Formula IXa or IXb:
with a compound having the structure of Formula Xa or Xb:
wherein each of N 1 and N 2 is independently a nucleobase;
each of R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 16 is, independently, H, halo, hydroxy, thiol, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted C 2 -C 6 heteroalkenyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted amino, azido, or optionally substituted C 6 -C 10 aryl;
each of g and h is, independently, 0 or 1;
each X 4 is, independently, O, NH, or S; and
each X 1 and X 2 is independently O or S;
each X 3 is independently OH, SH, or a salt thereof;
each of R 20 and R 23 is, independently, a region of linked nucleosides; and
each of R 21 and R 22 is, independently, optionally substituted C 1 -C 6 alkoxy;
to produce a composition comprising a circular polynucleotide encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula VIIIa or VIIIb.
19. A method of producing a composition comprising the circular polynucleotide of claim 2 encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula XIa, XIb, XIIa, or XIIb:
the method comprising reacting a compound having the structure of Formula XIIIa, XIIIb, XIVa, or XIVb:
with a compound having the structure of Formula XVa or XVb:
wherein each of N 1 and N 2 is independently a nucleobase;
each of R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 16 is, independently, H, halo, hydroxy, thiol, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted C 2 -C 6 heteroalkenyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted amino, azido, or optionally substituted C 6 -C 10 aryl;
each of g and h is, independently, 0 or 1;
each X 1 and X 4 is, independently, absent, O, NH, or S; or a salt thereof;
each of R 24 and R 27 is, independently, a region of linked nucleosides; and
each of R 25 , R 25′ , R 26 , and R 26′ is, independently, absent, optionally substituted C 1 -C 6 alkylene or optionally substituted C 1 -C 6 heteroalkylene or R 25 or R 26′ and the alkynyl group together form optionally substituted cycloalkynyl;
to produce a composition comprising a circular polynucleotide encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula XIa, XIb, XIIa, or XIIb.
20. A method of producing a composition comprising the circular polynucleotide of claim 2 encoding a polypeptide, wherein the circular polynucleotide has a sequence comprising Formula II:
[A n ]-L 1 -[B o ] Formula II
the method comprising reacting a compound having the structure of Formula XVI:
[A n ]-(R 1 ) a —(R 2 ) b —(R 3 ) c —N 3 Formula XVI
with a compound having the structure of Formula XVII:
R 27 —(R 5 ) d —(R 6 ) e —(R 7 ) f —[B o ] Formula XVII
wherein each A and B is independently any nucleoside;
n and o are, independently 15 to 1000; and
L 1 has the structure of Formula III:
wherein a, b, c, d, e, and f are each, independently, 0 or 1;
each of R 1 , R 3 , R 5 , and R 7 , is, independently, selected from optionally substituted C 1 -C 6 alkylene, optionally substituted C 1 -C 6 heteroalkylene, O, S, and NR 8 ;
R 2 and R 6 are each, independently, selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl;
R 4 is an optionally substituted triazolene; and
R 8 is hydrogen, optionally substituted C 1 -C 4 alkyl, optionally substituted C 3 -C 4 alkenyl, optionally substituted C 2 -C 4 alkynyl, optionally substituted C 2 -C 6 heterocyclyl, optionally substituted C 6 -C 12 aryl, or optionally substituted C 1 -C 7 heteroalkyl; and
R 27 is an optionally substituted C 2 -C 3 alkynyl or an optionally substituted C 8 -C 12 cycloalkynyl, wherein L 1 is attached to [A n ] and [B o ] at the sugar of one of the nucleosides;
to produce a composition comprising a circular polynucleotide encoding a polypeptide, wherein the circular polynucleotide has a sequence comprising Formula II.
21. A method of producing a composition comprising the circular polynucleotide claim 2 encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula XVIII:
the method comprising reacting a compound having the structure of Formula XIX:
with a compound having the structure of Formula XX:
wherein each of N 1 and N 2 is, independently, a nucleobase;
each of R 13 , R 14 , R 15 , and R 16 is, independently, H, halo, hydroxyl, thiol, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted C 2 -C 6 heteroalkenyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted amino, azido, or optionally substituted C 6 -C 10 aryl;
h is 0 or 1; and
X 4 is O, NH, or S;
to produce a composition comprising a circular polynucleotide encoding a polypeptide, wherein the circular polynucleotide comprises the structure of Formula XVIII.