MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FRIEDREICH'S ATAXIA
The present application relates to oligonucleotides (e.g., antisense oligonucleotides such as gapmers) designed to target FXN RNAs and targeting complexes for delivering the oligonucleotides to cells (e.g., muscle cells) and uses thereof, particularly uses relating to treatment of disease. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload increases expression or activity of a FXN allele comprising a disease-associated-repeat.
1 . A complex comprising a muscle-targeting agent covalently linked to an oligonucleotide configured for increasing FXN expression, wherein the oligonucleotide comprises a region of complementarity to a repeat region of an FXN RNA, wherein the repeat region comprises a target sequence as set forth in any one of SEQ ID NOs: 162-164, and wherein the region of complementarity is at least 12 nucleotides in length.
2 . The complex of claim 1 , wherein the muscle-targeting agent is an anti-transferrin receptor 1 (TfR1) antibody.
3 . The complex of claim 1 , wherein the oligonucleotide comprises at least 16 consecutive nucleotides of any one of SEQ ID NOs: 165-176, wherein each of the Us are optionally and independently Ts, optionally wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 165-176, and wherein each of the Us are optionally and independently Ts.
4 . The complex of claim 1 , wherein the oligonucleotide comprises a 5′-X-Y-Z-3′ configuration, wherein
X comprises 3-5 linked nucleosides, wherein at least one of the nucleosides in X is a 2′-modified nucleoside;
Y comprises 6-14 linked 2′-deoxyribonucleosides, wherein each cytidine in Y is optionally and independently a 5-methyl-cytidine; and
Z comprises 3-5 linked nucleosides, wherein at least one of the nucleosides in Z is a 2′-modified nucleoside.
5 . The complex of claim 4 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 165-167, wherein X comprises 5 linked nucleosides and each nucleoside in X is a 2′-MOE modified nucleoside; Y comprises 10 linked 2′-deoxyribonucleosides, wherein each cytidine in Y is optionally and independently a 5-methyl-cytidine; and Z comprises 5 linked nucleosides and each nucleoside in Z is a 2′-MOE modified nucleoside.
6 . The complex of claim 4 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 165-167, wherein X comprises 5 linked nucleosides, wherein each nucleoside in X is a LNA nucleoside; Y comprises 10 linked 2′-deoxyribonucleosides, wherein each cytidine in Y is optionally and independently a 5-methyl-cytidine; and Z comprises 5 linked nucleosides, wherein each nucleoside in Z is a LNA nucleoside.
7 . The complex of claim 4 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 171-173, wherein X comprises 3 linked nucleosides, wherein each nucleoside in X is a LNA nucleoside; Y comprises 14 linked 2′-deoxyribonucleosides, wherein each cytidine in Y is optionally and independently a 5-methyl-cytidine; and Z comprises 3 linked nucleosides, wherein each nucleoside in Z is a LNA nucleoside.
8 . The complex of claim 1 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 168-170, and wherein each nucleoside of the oligonucleotide is a 2′-MOE modified nucleoside.
9 . The complex of claim 1 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 168-170, and wherein each T in the oligonucleotide is a LNA nucleoside, and each C in the oligonucleotide is a 5-methyl-deoxycytidine.
10 . The complex of claim 1 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 174-176, and wherein each C in the oligonucleotide is a LNA nucleoside and each T is a deoxythymidine.
11 . The complex of claim 1 , wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages, optionally wherein the each internucleoside linkage in the oligonucleotide is a phosphorothioate internucleoside linkage.
12 . The complex of claim 1 , wherein the oligonucleotide is selected from:
(SEQ ID NO: 165)
oC*oU*oU*oC*oU*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*
dT*oC*oU*oU*oC*oU
(SEQ ID NO: 166)
oU*oU*oC*oU*oU*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*
xdC*oU*oU*oC*oU*oU
(SEQ ID NO: 167)
oU*oC*oU*oU*oC*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*
dT*oU*oC*oU*oU*oC
(SEQ ID NO: 168)
oC*oU*oU*C*oU*oU*C*oU*oU*C*oU*oU*oC*oU*oU*oC*
oU*oU*oC*oU
(SEQ ID NO: 169)
oU*oU*oC*oU*oU*C*U*oU*C*oU*oU*C*oU*oU*oC*oU*o
U*oC*oU*oU
(SEQ ID NO: 170)
oU*oC*oU*oU*C*oU*U*C*oU*oU*C*oU*oU*oC*oU*oU*o
C*oU*oU*oC
(SEQ ID NO: 171)
+C*+U*+U*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT
*dT*xdC*dT*+U*+C*+U
(SEQ ID NO: 172)
+U*+U*+C*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*
xdC*dT*dT*+C*+U*+U
(SEQ ID NO: 173)
+U*+C*+U*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC
*dT*dT*xdC*+U*+U*+C
(SEQ ID NO: 165)
+C*+U*+U*+C*+U*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*
dT*+C*+U*+U*+C*+U
(SEQ ID NO: 166)
+U*+U*+C*+U*+U*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*
xdC*+U*+U*+C*+U*+U
(SEQ ID NO: 167)
+U*+C*+U*+U*+C*T*dT*xdC*dT*dT*xdC*dT*dT*xdC*d
T*+U*+C*+U*+U*+C
(SEQ ID NO: 168)
xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+
U*+U*xdC*+U*+U*xdC*+U
(SEQ ID NO: 169)
+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U
*xdC*+U*+U*xdC*+U*+U
(SEQ ID NO: 170)
+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xd
C*+U*+U*xdC*+U*+U*xdC
(SEQ ID NO: 174)
+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*
+C*dT*dT*+C*dT
(SEQ ID NO: 175)
dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*
dT*dT*+C*dT*dT
(SEQ ID NO: 176)
dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*
dT*+C*dT*dT*+C
wherein “xdC” indicates a 5-methyl-deoxycytidine; “dN” indicates a 2′-deoxyribonucleoside; “+N” indicates a LNA nucleoside; “oN” indicates a 2′-MOE modified ribonucleoside; “oC” indicates a 5-methyl-2′-MOE-cytidine; “+C” indicates a 5-methyl-2′-4′-bicyclic-cytidine (2′-4′ methylene bridge); “oU” indicates a 5-methyl-2′-MOE-uridine; “+U” indicates a 5-methyl-2′-4′-bicyclic-uridine (2′-4′ methylene bridge); “*” indicates a phosphorothioate internucleoside linkage.
13 .- 15 . (canceled)
16 . The complex of claim 1 , wherein the muscle targeting agent and the oligonucleotide are covalently linked via a linker, optionally wherein the linker comprises a valine-citrulline sequence.
17 . The complex of claim 1 , wherein the FXN RNA contains disease-associated expanded GAA repeats.
18 . A method of increasing FXN expression in a muscle cell, the method comprising contacting the muscle cell with an effective amount of the complex of claim 1 for promoting internalization of the oligonucleotide to the muscle cell.
19 . A method of treating Friedreich's Ataxia (FA), the method comprising administering to a subject in need thereof an effective amount of the complex of claim 1 , wherein the subject has a mutant FXN allele comprising disease-associated GAA repeats.
20 . The method of claim 18 , wherein the complex results in an increase of FXN protein level.
21 . An oligonucleotide selected from:
(SEQ ID NO: 165)
oC*oU*oU*oC*oU*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*
dT*oC*oU*oU*oC*oU
(SEQ ID NO: 166)
oU*oU*oC*oU*oU*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*
xdC*oU*oU*oC*oU*oU
(SEQ ID NO: 167)
oU*oC*oU*oU*oC*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*
dT*oU*oC*oU*oU*oC
(SEQ ID NO: 168)
oC*oU*oU*C*oU*oU*C*oU*U*oC*oU*oU*oC*oU*oU*oC*
oU*oU*oC*oU
(SEQ ID NO: 169)
oU*oU*C*U*oU*C*oU*oU*C*oU*oU*C*oU*oU*C*oU*oU*
oC*oU*oU
(SEQ ID NO: 170)
oU*oC*oU*oU*C*U*oU*C*oU*oU*C*oU*oU*oC*oU*oU*o
C*oU*oU*oC
(SEQ ID NO: 171)
+C*+U*+U*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT
*dT*xdC*dT*+U*+C*+U
(SEQ ID NO: 172)
+U*+U*+C*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*
xdC*dT*dT*+C*+U*+U
(SEQ ID NO: 173)
+U*+C*+U*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC
*dT*dT*xdC*+U*+U*+C
(SEQ ID NO: 165)
+C*+U*+U*+C*+U*dT*xdC*dT*dT*xdC*dT*dT*xdC*dT*
dT*+C*+U*+U*+C*+U
(SEQ ID NO: 166)
+U*+U*+C*+U*+U*xdC*dT*dT*xdC*dT*dT*xdC*dT*dT*
xdC*+U*+U*+C*+U*+U
(SEQ ID NO: 167)
+U*+C*+U*+U*+C*dT*dT*xdC*dT*dT*xdC*dT*dT*xdC*
dT*+U*+C*+U*+U*+C
(SEQ ID NO: 168)
xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+
U*+U*xdC*+U*+U*xdC*+U
(SEQ ID NO: 169)
+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U
*xdC*+U*+U*xdC*+U*+U
(SEQ ID NO: 170)
+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xdC*+U*+U*xd
C*+U*+U*xdC*+U*+U*xdC
(SEQ ID NO: 174)
+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*
+C*dT*dT*+C*dT
(SEQ ID NO: 175)
dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*
dT*dT*+C*dT*dT
(SEQ ID NO: 176)
dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*dT*+C*dT*
dT*+C*dT*dT*+C
wherein “xdC” indicates a 5-methyl-deoxycytidine; “dN” indicates a 2′-deoxyribonucleoside; “+N” indicates a LNA nucleoside; “oN” indicates a 2′-MOE modified ribonucleoside; “oC” indicates a 5-methyl-2′-MOE-cytidine; “+C” indicates a 5-methyl-2′-4′-bicyclic-cytidine (2′-4′ methylene bridge); “oU” indicates a 5-methyl-2′-MOE-uridine; “+U” indicates a 5-methyl-2′-4′-bicyclic-uridine (2′-4′ methylene bridge); “*” indicates a phosphorothioate internucleoside linkage.
22 . A composition comprising the oligonucleotide of claim 21 in sodium salt form.
23 . The method of claim 19 , wherein the complex results in an increase of FXN protein level.