US2013102034A1PendingUtilityA1
Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
Individually held — no corporate assignee on recordPriority: Oct 1, 2010Filed: May 25, 2012Published: Apr 25, 2013
Est. expiryOct 1, 2030(~4.2 yrs left)· nominal 20-yr term from priority
Inventors:Jason P. Schrum
C12N 2310/335C07H 21/02C12N 15/11C12N 15/67C12N 5/0602C12N 15/1138A61K 48/0066C07K 2317/24C07H 19/10C12N 15/102C12P 21/00G01N 33/559C12N 15/1136C07K 16/2887C07K 16/00C12N 2310/3341
66
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Claims
Abstract
The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using thereof.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A compound comprising a nucleotide that disrupts binding of a major groove binding partner with a nucleic acid comprising the nucleotide, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
2 . The compound of claim 1 , wherein the nucleotide comprises a chemical modification located on the major groove face of a nucleobase portion of the nucleotide.
3 . The compound of claim 2 , wherein the nucleobase portion comprises a pyrimidine nucleobase, and wherein the chemical modification comprises replacing or substituting an atom of the major groove face of the pyrimidine nucleobase with an amine, an SH, a methyl, an ethyl, a chloro or a fluoro group.
4 . The compound of claim 2 , wherein the chemical modification is located on a sugar portion of the nucleotide.
5 . The compound of claim 2 , wherein the chemical modification is located on a phosphate backbone of the nucleotide.
6 . The compound of claim 1 , having Formula I:
wherein:
Z is O or S;
each of Y 1 is independently selected from —OR a1 , —NR a1 R b1 and —SR a1 ;
each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
each of Y 3 is independently selected from O and S;
Y 4 is selected from H, —OR a , —SR a , and —NHR a ;
n is 0, 1, 2, or 3;
m is 0, 1, 2 or 3;
B is a nucleobase;
R a is H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, or C 6-20 aryl;
R a1 and R b1 are each independently H or a counterion; and
—Y 3 —R c1 is OH or SH at a pH of about 1 or —Y 3 —R c1 is O − or S − at physiological pH;
or —Y 3 —R c1 is C 1-20 alkoxy, C 2-20 —O-alkenyl, or C 1-20 —O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH.
7 . The compound of claim 6 , wherein B is a nucleobase of Formula II-a, II-b, or II-c:
wherein:
denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or —OR c ,
wherein C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl are each optionally substituted with —OH, —NR a R b , —SH, —C(O)R D , —C(O)OR S , —NHC(O)R c , or —NHC(O)OR c ;
and wherein when V is N then R 1 is absent;
R 2 is H, —OW, —SR S , —NR a R b , or halo;
or when V is C then R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, —OH, —SH, —NR a R b , C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, or C 1-6 thioalkyl;
R 3 is H or C 1-6 alkyl;
R 4 is H or C 1-6 alkyl; wherein when denotes a double bond then R 4 is absent, or N—R 4 , taken together, forms a positively charged N substituted with C 1-6 alkyl;
R a and R b are each independently H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, or C 6-10 aryl; and
R c is H, C 1-6 alkyl, C 1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
8 . The compound of claim 7 , wherein B is a nucleobase of Formula II-al, II-a2, II-a3, II-a4, or II-a5:
9 . The compound of claim 6 , wherein B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
10 . The compound of claim 6 , having Formula I-a:
11 . The compound of claim 6 , having Formula I-b:
12 . The compound of claim 6 , having Formula I-c:
13 . The compound of claim 6 , selected from the group consisting of:
14 . The compound of claim 6 , selected from the group consisting of:
15 . A nucleic acid sequence comprising at least two nucleotides, the nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
16 . The nucleic acid sequence of claim 15 , comprising a compound of Formula I-d:
wherein:
Z is O or S;
each of Y 1 is independently selected from —OR al , —NR a1 R b1 , and —SR a1 ;
each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
B is a nucleobase;
R a is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, or C 6-10 aryl; and
R a1 and R b1 are each independently H or a counterion; and
—OR c1 is OH at a pH of about 1 or —OR c1 is O − at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH.
17 . The nucleic acid sequence of claim 16 , wherein B is a nucleobase of Formula II-a, II-b, or II-c:
wherein:
denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or —OR c , wherein C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl are each optionally substituted with —OH, —NR a R b , —SH, —C(O)R D , —C(O)OR c , —NHC(O)R c , or —NHC(O)OR c ;
and wherein when V is N then R 1 is absent;
R 2 is H, —OR c , —SR c , —NR a R b , or halo;
or when V is C then R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, —OH, —SH, —NR a R b , C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, or C 1-6 thioalkyl;
R 3 is H or C 1-6 alkyl;
R 4 is H or C 1-6 alkyl; wherein when denotes a double bond then R 4 is absent, or N—R 4 , taken together, forms a positively charged N substituted with C 1-6 alkyl;
R a and R b are each independently H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, or C 6-10 aryl; and
R c is H, C 1-6 alkyl, C 1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
18 . The nucleic acid sequence of claim 17 , wherein B is a nucleobase of Formula II-al, II-a2, II-a3, II-a4, or II-a5:
19 . The nucleic acid sequence of claim 16 , wherein B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
20 . The nucleic acid sequence of claim 16 , wherein the nucleic acid sequence contains a plurality of structurally unique compounds of Formula I-d.
21 . The nucleic acid sequence of claim 16 , wherein at least 25% of the cytosines are replaced by a compound of Formula I-d and/or wherein at least 25% of the uracils are replaced by a compound of Formula I-d.
22 . The compound of claim 1 , wherein the major groove interacting partners are selected from the group consisting of: TLRs (Toll-like Receptors) 3, 7, and 8; RIG-I (retinoic acid-inducible gene I); MDA5 (melanoma differentiation-associated gene 5); laboratory of genetics and physiology 2 (LGP2); HIN-200 domain containing proteins; and Helicase-domain containing proteins.
23 . A non-naturally occurring nucleotide comprising one or more chemical modifications of a naturally occurring nucleotide, wherein the nucleotide reduces the induction of the cellular innate immune response of a cell to a modified nucleic acid comprising the non-naturally occurring nucleotide when the modified nucleic acid is introduced into the cell, as compared to the induction of the cellular innate immune in a cell induced by a corresponding unmodified nucleic acid.
24 . The compound of claim 23 , wherein the nucleotide reduces the innate immune response or the secretion of pro-inflammatory cytokines or both by at least about 10%.
25 . The compound of claim 23 , wherein the nucleotide reduces the innate immune response or the secretion of pro-inflammatory cytokines or both by about 75%.
26 . The compound of claim 23 , wherein the nucleotide reduces the innate immune response or the secretion of pro-inflammatory cytokines or both by at least 90%.
27 . The modified nucleic acid comprising the non-naturally occurring nucleotide of claim 23 , further comprising a translateable region encoding a protein of interest.
28 . A composition comprising the modified nucleic acid of claim 27 , in an amount sufficient to increase the production of the protein of interest when introduced into a target cell, as compared to the amount of protein produced in a cell containing a corresponding unmodified nucleic acid encoding the protein of interest.
29 . The composition of claim 28 , wherein the increase is at least about 10%.
30 . The composition of claim 28 , wherein the increase is at least about 50%.
31 . The composition of claim 28 , wherein the increase is at least about 100%.
32 . The modified nucleic acid of claim 27 , wherein at least 25% of the cytosines in the nucleic acid are replaced by a compound of Formula I-d.
33 . The modified nucleic acid of claim 27 , wherein at least 90% of the cytosines in the nucleic acid are replaced by a compound of Formula I-d.
34 . The modified nucleic acid of claim 27 , wherein about 100% of the cytosines in the nucleic acid are replaced by a compound of Formula I-d.
35 . The modified nucleic acid of claim 27 , wherein at least 25% of the uracils in the nucleic acid are replaced by a compound of Formula I-d.
36 . The modified nucleic acid of claim 27 , wherein at least 90% of the uracils in the nucleic acid are replaced by a compound of Formula I-d.
37 . The modified nucleic acid of claim 27 , wherein about 100% of the uracils in the nucleic acid are replaced by a compound of Formula I-d.
38 . The modified nucleic acid of claim 27 , wherein at least 25% of the cytosines in the nucleic acid are replaced by a first compound of Formula I-d and wherein at least 25% of the uracils in the nucleic acid are replaced by a second compound of Formula I-d.
39 . The modified nucleic acid of claim 27 , wherein about 100% of the cytosines in the nucleic acid are replaced by a first compound of Formula I-d and wherein about 100% of the uracils in the nucleic acid are replaced by a second compound of Formula I-d.
40 . The composition of claim 28 , further comprising an RNA polymerase, a cDNA template, or a combination thereof.
41 . The composition of claim 40 , further comprising a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
42 . A method of preparing a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, wherein the nucleic acid sequence comprises a compound of Formula I-d:
wherein:
the nucleotide has decreased binding affinity to the major groove binding partner;
Z is O or S;
each of Y 1 is independently selected from —OR al , —NR a1 R b1 , and —SR a1 ;
each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
B is a nucleobase; and
R a1 and R b1 are each independently H or a counterion; and
—OR c1 is OH at a pH of about 1 or —OR c1 is O − at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH;
the method comprising:
reacting a compound of Formula I-c:
with an RNA polymerase, and a cDNA template.
43 . The method of claim 42 , wherein the reaction is repeated from 1 to about 7,000 times.
44 . The method of claim 42 , wherein B is a nucleobase of Formula II-a, II-b, or II-c:
wherein:
denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or —OR c ,
wherein C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl are each optionally substituted with —OH, —NR a R b , —SH, —C(O)R D , —C(O)OR c , —NHC(O)R c , or —NHC(O)OR c ;
and wherein when V is N then R 1 is absent;
R 2 is H, —OR c , —SR c , —NR a R b , or halo;
or when V is C then R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, —OH, —SH, —NR a R b , C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, or C 1-6 thioalkyl;
R 3 is H or C 1-6 alkyl;
R 4 is H or C 1-6 alkyl; wherein when denotes a double bond then R 4 is absent, or N—R 4 , taken together, forms a positively charged N substituted with C 1-6 alkyl;
R a and R b are each independently H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, or C 6-10 aryl; and
R c is H, C 1-6 alkyl, C 1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
45 . The method of claim 44 , wherein B is a nucleobase of Formula II-al, II-a2, II-a3, II-a4, or II-a5:
46 . A method of amplifying a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, the method comprising:
reacting a compound of Formula I-c:
wherein:
the nucleotide has decreased binding affinity to the major groove binding partner;
Z is O or S;
each of Y 1 is independently selected from —OR al , —NR a1 R b1 , and —SR a1 ;
each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
B is a nucleobase; and
R a1 and R b1 are each independently H or a counterion; and
—OR c1 is OH at a pH of about 1 or —OR c1 is O − at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH;
with a primer, a cDNA template, and an RNA polymerase.
47 . The method of claim 46 , wherein B is a nucleobase of Formula II-a, II-b, or II-c:
wherein:
denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or —OR c ,
wherein C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl are each optionally substituted with —OH, —NR a R b , —SH, —C(O)R D , —C(O)OR c , —NHC(O)R c , or —NHC(O)OR c ;
and wherein when V is N then R 1 is absent;
R 2 is H, —OR c , —SR c , —NR a R b , or halo;
or when V is C then R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, —OH, —SH, —NR a R b , C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, or C 1-6 thioalkyl;
R 3 is H or C 1-6 alkyl;
R 4 is H or C 1-6 alkyl; wherein when denotes a double bond then R 4 is absent, or N—R 4 , taken together, forms a positively charged N substituted with C 1-6 alkyl;
R a and R b are each independently H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, or C 6-10 aryl; and
R c is H, C 1-6 alkyl, C 1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
48 . The method of claim 46 , wherein B is a nucleobase of Formula II-al, II-a2, II-a3, II-a4, or II-a5:
48 . A method of synthesizing a pharmaceutical nucleic acid, comprising the steps of:
a) providing a complementary deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein of interest; b) selecting a nucleotide that is known to disrupt a binding of a major groove binding partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to the major groove binding partner; and c) contacting the provided cDNA and the selected nucleotide with an RNA polymerase, under conditions such that the pharmaceutical nucleic is synthesized.
49 . The method of claim 48 , wherein the pharmaceutical nucleic acid is a ribonucleic acid (RNA).
50 . A method of inducing a physiological change in a target cell population, comprising the steps of:
a) providing a first nucleic acid comprising i) a translatable region encoding a protein of interest and ii) a nucleic acid modification, wherein the first nucleic acid is substantially resistant to cellular degradation; and b) contacting an effective amount of the first nucleic acid to a producer cell under conditions such that the protein of interest is produced in the producer cell and secreted therefrom, wherein the secreted protein of interest contacts the target cell population and induces a physiological change therein.
51 . The method of claim 50 , wherein the protein of interest is capable of binding to a receptor on the surface of at least one cell present in the target cell population.
52 . The method of claim 50 , wherein the secreted protein is capable of interacting with a receptor on the surface of at least one cell present in the target cell population.
53 . The method of claim 50 , wherein the secreted protein is Granulocyte-Colony Stimulating Factor (G-CSF).
53 . The method of claim 51 , wherein the target cell population comprises one or more cells that express the G-CSF receptor.
54 . A compound comprising a nucleic acid comprising one or more nucleotides having Formula I:
wherein:
Z is O or S;
each of Y 1 is independently selected from —OR a1 , —NR a1 R b1 , and —SR a1 ;
each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
n is 0, 1, 2, or 3;
m is 0, 1, 2 or 3;
B is a nucleobase;
R a is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, or C 6-10 aryl;
R a1 and R b1 are each independently H or a counterion; and
—OR a1 is OH at a pH of about 1 or —OR c1 is O − at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH, and
wherein a cell comprising the nucleic acid is characterized by:
i) Decreased cellular secretion of s pro-inflammatory cytokine;
ii) decreased activation of a cellular innate immune responder;
iii) decreased suspectibility to a cellular nuclease;
iv) decreased binding to a negative regulator of gene expression;
v) decreased binding to a nucleic acid;
vi) increased protein translation efficiency;
vii) increased half-life;
viii) or a combination thereof.Join the waitlist — get patent alerts
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