Granted Patent B2

US 12,534,724 · App. 17/331,146 · Granted Jan 27, 2026

Inventors: Anastasia Khvorova (Westborough, MA); Loïc Maurice René Jean Roux (Worcester, MA); Ken Yamada (Boston, MA)

Assignee: UNIVERSITY OF MASSACHUSETTS

C12N15/113C07H21/04C12N2310/11C12N2310/141

Abstract

Claims (106)

1 . A modified RNA oligonucleotide comprising a 5′ end, a 3′ end and at least two modified intersubunit linkages of Formula Ia:

wherein:

B is a base moiety;

W is O or O(CH 2 )n 1 , wherein n 1 is an integer between 1 and 10;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z 1 is O or O(CH 2 ) n 2 , wherein n 2 is an integer between 1 and 10;

Z 2 is O or O(CH 2 ) n 3 , wherein n 3 is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof;

wherein at least two of the at least two modified intersubunit linkages are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide, and

wherein:

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O, and Y is O − ;

Z 1 is O, W is O(CH 2 ) n 1 , n 1 is 1, and Y is O − ;

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O, and Y is S − ;

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O(CH 2 ) n , and Y is O − ; or

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O(CH 2 ) n , and Y is S − .

2 . The modified RNA oligonucleotide of claim 1 , wherein the base moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

3 . The modified RNA oligonucleotide of claim 1 , wherein between two and ten of the at least two modified intersubunit linkages are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide.

4 . The modified RNA oligonucleotide of claim 1 , wherein the at least two modified intersubunit linkages are consecutive.

5 . The modified RNA oligonucleotide of claim 1 , wherein the modified RNA oligonucleotide is selected from the group consisting of an siRNA, a miRNA, an antisense oligonucleotide, a ribozyme, and a mRNA.

6 . A modified RNA oligonucleotide comprising a 5′ end, a 3′ end and at least two modified intersubunit linkages of one or more of:

Formula IIa:

wherein:

B is a base moiety;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S, R 2 , and SH;

Z is O or O(CH 2 ) n , wherein n is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof;

Formula IIIa:

wherein:

B is a base moiety;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z is O or O(CH 2 ) n , wherein n is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof;

Formula Va:

wherein:

B is a base moiety;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z is O or O(CH 2 ) n , wherein n is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof;

Formula III:

wherein:

B is a base moiety;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z is O or O(CH 2 ) n , wherein n is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and/or

Formula V:

wherein:

B is a base moiety;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z is O or O(CH 2 ) n , wherein n is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

wherein at least two of the at least two modified intersubunit linkages of any one or more of Formula (IIa), (IIIa), (Va), (III), and (V) are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide.

7 . A modified universal 3′ end RNA sequence for stabilization of an RNA oligonucleotide, the RNA sequence comprising at least two modified intersubunit linkages of Formula Ia:

wherein:

B is a base moiety;

W is O or O(CH 2 )n 1 , wherein n 1 is an integer between 1 and 10;

X is selected from the group consisting of OH, OR 1 , R 1 , F, Cl, Br, I, SH, SR 1 , NH 2 , NHR 1 , NR 1 2 and COOR 1 ;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z 1 is O or O(CH 2 ) n 2 , wherein n 2 is an integer between 1 and 10;

Z 2 is O or O(CH 2 ) n 3 , wherein n 3 is an integer between 1 and 10;

R 1 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof,

wherein:

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O, and Y is O − ;

Z 1 is O, W is O(CH 2 ) n 1 , n 1 is 1, and Y is O − ;

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O, and Y is S − ;

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O(CH 2 ) n 1 , and Y is O − ; or

Z 1 is O(CH 2 ) n 2 , n 2 is 1, W is O(CH 2 ) n 1 , and Y is S − .

8 . The modified universal 3′ end RNA sequence of claim 7 , wherein the RNA sequence is three to ten consecutive nucleotides in length.

9 . The modified universal 3′ end RNA sequence of claim 7 , wherein:

the RNA sequence comprises at least two consecutive modified intersubunit linkages of Formula Ia;

the RNA sequence comprises four consecutive modified intersubunit linkages of Formula Ia; or

the RNA sequence comprises five consecutive modified intersubunit linkages of Formula Ia.

10 . The modified universal 3′ end RNA sequence of claim 7 , wherein the RNA sequence comprises consecutive nucleotides selected from the group consisting of UUUU, AAAA, CCCC, UUUUU, AAAAA, and CCCCC.

11 . The modified universal 3′ end RNA sequence of claim 7 , wherein the base moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

12 . A modified universal 3′ end RNA sequence for stabilization of an RNA oligonucleotide, the RNA sequence comprising at least two modified intersubunit linkages of Formula X:

wherein:

W is O or O(CH 2 )n 1 , wherein n 1 is an integer between 1 and 10;

Y is selected from the group consisting of O − , OH, OR 2 , NH − , NH 2 , NR 2 2 , BH 3 , S − , R 2 , and SH;

Z is O or O(CH 2 ) n 2 , wherein n 2 is an integer between 1 and 10; and

R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, or mixtures thereof, wherein:

Z is O(CH 2 ) n 2 , n 2 is 1, W is O, and Y is O − ;

Z is O, W is O(CH 2 ) n 1 , n 1 is 1, and Y is O − ;

Z is O(CH 2 ) n 2 , n 2 is 1, W is O, and Y is S − ;

Z is O(CH 2 ) n 2 , n 2 is 1, W is O(CH 2 ) n , and Y is O − ; or

Z is O(CH 2 ) n 2 , n 2 is 1, W is O(CH 2 ) n , and Y is S − .

13 . The modified RNA oligonucleotide of claim 1 , wherein between two and five of the at least two modified intersubunit linkages are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide.

14 . The modified RNA oligonucleotide of claim 1 , wherein three of the at least two modified intersubunit linkages are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide.

15 . The modified RNA oligonucleotide of claim 1 , wherein four of the at least two modified intersubunit linkages are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide.

16 . The modified RNA oligonucleotide of claim 1 , wherein five of the at least two modified intersubunit linkages are present at one or both of the 5′ end and the 3′ end of the modified RNA oligonucleotide.

17 . The modified RNA oligonucleotide of claim 3 , wherein the at least two intersubunit linkages are consecutive.

18 . The modified RNA oligonucleotide of claim 5 , wherein the siRNA comprises an antisense strand and a sense strand, and wherein one or both of the antisense A strand and the sense strand comprise the at least two modified intersubunit linkages.

19 . The modified universal 3′ end RNA sequence of claim 7 , wherein the RNA sequence consists of four consecutive nucleotides in length.

20 . The modified universal 3′ end RNA sequence of claim 7 , wherein the modified universal sequence consists of five consecutive nucleotides in length.

21 . The universal 3′ end RNA sequence of claim 12 , wherein each of the at least two intersubunit linkages of Formula X bridges two ribonucleosides.

Assignments (4)

Continuity (3)

References Cited (400)