RNA CAP ANALOGS AND METHODS OF USE
Provided herein are compounds that are cap analogs for polynucleotides, e.g., RNA molecules, such as mRNA molecules. Also provided are capped polynucleotides, e.g., capped RNA molecules, such as capped miRNA molecules, wherein the 5′ end of the RNA molecule comprises a cap analog disclosed herein, drug products comprising the capped RNA molecules, methods for making capped polynucleotides disclosed herein, and kits for making the capped polynucleotides.
1 . A compound of Formula (I), or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof:
wherein
ring G is guanine or a modified guanine;
each of R 1 , R 2 , R 4 , and R 6 is independently H, C 1-6 alkyl, halo, OH, CN, or OR x , where R x is C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl, and R x is optionally substituted with one or more substituents independently selected from halo, OH, C 1-6 alkoxy, and OC(O)—C 1-6 alkyl;
R 1A is H, or R 1A and R 1 together form —O—C 1-6 alkylene-;
R 4A is H, or R 4A and R 4 together form —O—C 1-6 alkylene-;
X is O, S, SO, SO 2 , or Se;
L is a linker;
R 3 is H, C 1-6 alkyl, C 1-6 alkoxy, OH, halo, or a polynucleotide;
R 5 is OH, SH, C 1-6 alkyl, C 1-6 alkoxy, or N(R N5 ) 2 and each R N5 is independently H or C 1-6 alkyl;
W is O, S, or NS(O) 2 C 1-6 alkyl; and
each of ring A and ring B is independently a nucleobase.
2 . The compound, stereoisomer, tautomer, or salt of claim 1 , wherein
L is —Y 0 —(P(O)(R P )Y 1 )—(P(O)(R P )Y 2 ) m —(P(O)(R P )Y 3 ) n —,
each R P is independently H, halo, C 1-6 alkyl, OH, SH, SeH, or BH 3 − ;
each of Y 0 ,Y 1 , Y 2 , and Y 3 is independently O, S, NH, N(C 1-6 alkyl), or CH 2 ; and
m+n is 1 to 5.
3 . The compound, stereoisomer, tautomer, or salt of claim 1 or 2 , wherein m+n is 1 or 2.
4 . The compound, stereoisomer, tautomer, or salt of claim 3 , wherein L is —Y 0 —(P(O)(R P )Y 1 )—(P(O)(R P )Y 2 )—(P(O)(R P )Y 3 )—.
5 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 4 , wherein each R P is OH.
6 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 4 , wherein at least one R P is SH.
7 . The compound, stereoisomer, tautomer, or salt of claim 6 , wherein each R P is SH.
8 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 7 , wherein Y 0 is 0.
9 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is 0.
10 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is CH 2 .
11 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is NH.
12 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is S.
13 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is 0.
14 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is CH 2 .
15 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is NH.
16 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is S.
17 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 16 , wherein Y 3 is 0.
18 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 16 , wherein Y 3 is CH 2 .
19 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 16 , wherein Y 3 is NH.
20 . The compound, stereoisomer, tautomer, or salt of claim 4 , wherein L is —O—(P(O)(OH)O)—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)O)—(P(O)(OH)O)—(P(O)(SH)O)—, —O—(P(O)(OH)NH)—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)S)—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)CH 2 )—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)S)—(P(O)(OH)S)—(P(O)(OH)O)—, or —O—(P(O)(SH)NH)—(P(O)(SH)O)—(P(O)(SH)O)—.
21 . The compound, stereoisomer, tautomer, or salt of claim 20 , wherein L is —O—(P(O)(OH)O)—(P(O)(OH)O)—(P(O)(OH)O—.
22 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 21 , wherein R 1A is H.
23 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 22 , wherein R 1 is OH.
24 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 21 , wherein R 1A and R 1 together form —O—C 1-6 alkylene-.
25 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 24 , wherein R 2 is OH.
26 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 25 , wherein R 4A is H.
27 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 26 , wherein R 4 is OH.
28 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 25 , wherein R 4A and R 4 together form —O—C 1-6 alkylene-.
29 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 28 , wherein R 6 is OR x .
30 . The compound, stereoisomer, tautomer, or salt of claim 29 , wherein R 6 is OC 1-6 alkyl.
31 . The compound, stereoisomer, tautomer, or salt of claim 29 or 30 , wherein R 6 is methoxy.
32 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 31 , wherein X is S.
33 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 31 , wherein X is 0.
34 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 33 , wherein ring G is guanine.
35 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 33 , wherein ring G is modified guanine.
36 . The compound, stereoisomer, tautomer, or salt of claim 35 ,
wherein
ring G is
R N1 is C 1-6 alkyl, C 1-6 alkylene-aryl, or C 1-6 alklyene-O-aryl; and
each R N2 is independently H, C 1-6 alkyl, or benzyl.
37 . The compound, stereoisomer, tautomer, or salt of claim 36 , wherein R N 1 is Me or Et.
38 . The compound, stereoisomer, tautomer, or salt of claim 36 , wherein R N 1 is benzyl or benzyloxy.
39 . The compound, stereoisomer, tautomer, or salt of any one of claims 36 to 38 , wherein at least one R N2 is C 1-6 alkyl.
40 . The compound, stereoisomer, tautomer, or salt of any one of claims 36 to 38 , wherein at least one R N2 is benzyl.
41 . The compound, stereoisomer, tautomer, or salt of any one of claims 36 to 40 , wherein at least one R N2 is H.
42 . The compound, stereoisomer, tautomer, or salt of claim 41 , wherein each R N2 is H.
43 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 42 , wherein W is O.
44 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 42 , wherein W is NS(O) 2 C 1-6 alkyl.
45 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 44 , wherein R 5 is OH.
46 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 44 , wherein R 5 is SH.
47 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 44 , wherein R 5 is C 1-6 alkyl.
48 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 47 , wherein R 3 is OH or C 1-6 alkoxy.
49 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 47 , wherein R 3 is H or halo.
50 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 47 , wherein R 3 is a polynucleotide.
51 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 50 , wherein ring A is adenine.
52 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 50 , wherein ring A is N 6 -methyladenine.
53 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 52 , wherein ring B is guanine.
54 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 52 , wherein ring B is uracil.
55 . A compound having a structure as shown in Table 1, or a stereoisomer, tautomer, or salt thereof.
56 . An RNA molecule, wherein the 5′ end of the RNA molecule comprises the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 55 .
57 . The RNA molecule of claim 56 , wherein the RNA molecule is mRNA.
58 . The RNA molecule of claim 57 , wherein the mRNA encodes for one or more immunomodulatory agents (e.g., a checkpoint inhibitor such as an anti-PD-1 antibody, anti-PDL-1 antibody, anti-CTLA4, etc., an immunosuppression antagonist, a pro-inflammatory agent/cytokine), therapeutic proteins, antibodies, or antigens (e.g., a tumor specific antigen).
59 . The RNA molecule of any one of claims 56 to 58 , wherein the RNA molecule has a half-life that is more than that of a corresponding natural RNA molecule in a cellular environment.
60 . A drug product comprising the RNA molecule of any one of claims 56 to 59 and one or more pharmaceutically acceptable excipients.
61 . A kit for capping an RNA molecule comprising the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 55 , and an RNA polymerase.
62 . The kit of claim 61 , wherein the RNA molecule is mRNA.
63 . A method for making a capped RNA molecule from a polynucleotide template by in vitro transcription, comprising:
(a) combining a plurality of nucleotides, the polynucleotide template and an RNA polymerase to produce a reaction mix;
(b) incubating the reaction mix; and
(c) adding the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 55 to the mix in order to produce the capped RNA molecule.
64 . The method of claim 63 , wherein the RNA molecule is mRNA.
65 . The method of claim 63 or claim 64 , wherein at least a portion of the plurality of nucleotides are unmodified.
66 . The method of any one of claims 63 to 65 , wherein the at least a portion of the plurality of nucleotides are modified.
67 . The method of any one of claims 63 to 66 , wherein the polynucleotide template is a DNA template.
68 . The method of any one of claims 63 to 67 , wherein the capping efficiency is greater than about 95%.
69 . The method of any one of claims 63 to 68 , wherein the translation yield is greater than about 75%.