EXON SKIPPING OLIGOMER CONJUGATES FOR MUSCULAR DYSTROPHY
Antisense oligomers complementary to a selected target site in the human dystrophin gene to induce exon 50 skipping are described. In various aspects, antisense oligomers are described according to Formula (I): or a pharmaceutically acceptable salt thereof, wherein T, Nu, n, and R 100 are defined herein.
1 . An antisense oligomer according to Formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together form a targeting sequence;
T is a moiety selected from:
and the distal —OH or —NH 2 of the T moiety is optionally linked to a cell-penetrating peptide;
R 100 is hydrogen or a cell-penetrating peptide;
each Nu from 1 to n and 5′ to 3′ corresponds to the nucleobases in one of the following:
Annealing
Targeting Sequence
SEQ ID
Site
[5′ to 3′]
NO:
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+04 - 18)
AGG C
NO: 1
H50D
GAT CCA GTA TAC TTA CAG
SEQ ID
(+07 - 16)
GCT CC
NO: 3
H50D
GGA TCC AGT ATA CTT ACA
SEQ ID
(+07 - 17)
GGC TCC
NO: 4
H50A
ACT TCC TCT TTA ACA GAA
SEQ ID
(-19 + 07)
AAG CAT AC
NO: 5
H50D
ATC CAG TAT ACT TAC AGG
SEQ ID
(+07 - 15)
CTC C
NO: 6
H50A
GAG CTC AGA TCT TCT AAC
SEQ ID
(-02 + 23)
TTC CTC T
NO: 7
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+06 - 18)
AGG CTC
NO: 8
H50D
ATG GGA TCC AGT ATA CTT
SEQ ID
(+07 - 20)
ACA GGC TCC
NO: 9
wherein A is
C is
G is
and T is
2 . The antisense oligomer of claim 1 , wherein each Nu from 1 to n and 5′ to 3′ corresponds to SEQ ID NO: 3.
3 . The antisense oligomer of claim 2 , wherein the antisense oligomer contains one cell penetrating peptide.
4 . The antisense oligomer of claim 3 , wherein T is a moiety selected from:
5 . The antisense oligomer of claim 4 , wherein T is a moiety selected from:
and
R 100 is a cell-penetrating peptide.
6 . The antisense oligomer of claim 5 , wherein:
T is;
and
R 100 is a cell-penetrating peptide.
7 . The antisense oligomer of claim 6 , wherein the cell-penetrating peptide is an arginine-rich peptide.
8 . The antisense oligomer of claim 7 , wherein the arginine-rich peptide is selected from the group consisting of —(RXR) 4 —R a (SEQ ID NO: 15), R—(FFR) 3 —R a (SEQ ID NO: 16), —B—X—(RXR) 4 —R a (SEQ ID NO: 17), —B—X—R—(FFR) 3 —R a (SEQ ID NO: 18), -GLY-R—(FFR) 3 —R a (SEQ ID NO: 19), -GLY-R 5 —R a (SEQ ID NO: 20), —R 5 —R a (SEQ ID NO: 21), -GLY-R 6 —R a (SEQ ID NO: 11) and —R 6 —R a (SEQ ID NO: 10), wherein R a is selected from H, acyl, benzoyl, and stearoyl, and wherein R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine and GLY (or G) is glycine.
9 . The antisense oligomer of claim 8 , wherein the antisense oligomer is in free base form.
10 . The antisense oligomer of claim 8 , wherein the antisense oligomer is a pharmaceutically acceptable salt thereof.
11 . An antisense oligomer according to Formula (III):
or a pharmaceutically acceptable salt thereof, where each Nu from 1 to n and 5′ to 3′ corresponds to the nucleobases in one of the following:
Annealing
Targeting Sequence
SEQ ID
Site
[5′ to 3′]
NO:
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+04 - 18)
AGG C
NO: 1
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+07 - 18)
AGG CTC C
NO: 2
H50D
GAT CCA GTA TAC TTA CAG
SEQ ID
(+07 - 16)
GCT CC
NO: 3
H50D
GGA TCC AGT ATA CTT ACA
SEQ ID
(+07 - 17)
GGC TCC
NO: 4
H50A
ACT TCC TCT TTA ACA GAA
SEQ ID
(-19 + 07)
AAG CAT AC
NO: 5
H50D
ATC CAG TAT ACT TAC AGG
SEQ ID
(+07 - 15)
CTC C
NO: 6
H50A
GAG CTC AGA TCT TCT AAC
SEQ ID
(-02 + 23)
TTC CTC T
NO: 7
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+06 - 18)
AGG CTC
NO: 8
H50D
ATG GGA TCC AGT ATA CTT
SEQ ID
(+07 - 20)
ACA GGC TCC
NO: 9
wherein A is
C is
G is
and T is
and the distal —OH of formula (III) is optionally linked to a cell penetrating peptide.
12 . The antisense oligomer of claim 11 , wherein each Nu from 1 to n and 5′ to 3′ of Formula (III) corresponds to SEQ ID NO: 3.
13 . The antisense oligomer of claim 12 , wherein the antisense oligomer is in free base form.
14 . The antisense oligomer of claim 12 , wherein the antisense oligomer is a pharmaceutically acceptable salt thereof.
15 . An antisense oligomer according to Formula (IV):
where each Nu from 1 to n and 5′ to 3′ corresponds to the nucleobases in one of the following:
Annealing
Targeting Sequence
SEQ ID
Site
[5′ to 3′]
NO:
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+04 - 18)
AGG C
NO: 1
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+07 - 18)
AGG CTC C
NO: 2
H50D
GAT CCA GTA TAC TTA CAG
SEQ ID
(+07 - 16)
GCT CC
NO: 3
H50D
GGA TCC AGT ATA CTT ACA
SEQ ID
(+07 - 17)
GGC TCC
NO: 4
H50A
ACT TCC TCT TTA ACA GAA
SEQ ID
(-19 + 07)
AAG CAT AC
NO: 5
H50D
ATC CAG TAT ACT TAC AGG
SEQ ID
(+07 - 15)
CTC C
NO: 6
H50A
GAG CTC AGA TCT TCT AAC
SEQ ID
(-02 + 23)
TTC CTC T
NO: 7
H50D
GGG ATC CAG TAT ACT TAC
SEQ ID
(+06 - 18)
AGG CTC
NO: 8
H50D
ATG GGA TCC AGT ATA CTT
SEQ ID
(+07 - 20)
ACA GGC TCC
NO: 9
wherein A is
C is
G is
and T is
and the distal —OH of formula (IV) is optionally linked to a cell penetrating peptide.
16 . The antisense oligomer of claim 15 , wherein each Nu from 1 to n and 5′ to 3′ of Formula (IV) corresponds to SEQ ID NO: 3.
17 . The antisense oligomer of claim 15 , wherein the antisense oligomer is according to the structure of Formula (IVa)
18 . A pharmaceutical composition comprising an antisense oligomer of claim 17 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
19 . A method for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of the antisense oligomer of claim 17 .
20 . The method of claim 19 , wherein the subject has a mutation of the dystrophin gene that is amenable to exon 50 skipping.
21 . A method of restoring an mRNA reading frame to induce dystrophin production in a subject, the method comprising administering to the subject a therapeutically-effective amount of the antisense oligomer of claim 17 .
22 . The method of claim 21 , wherein the subject has a mutation of the dystrophin gene that is amenable to exon 50 skipping.