COMPOSITIONS COMPRISING EXON SKIPPING OLIGONUCLEOTIDE CONJUGATES FOR TREATING MUSCULAR DYSTROPHY
Composition comprising: (a) an antisense oligonucleotide conjugate comprising a cell penetrating peptide covalently attached to a nucleic acid analog, or a pharmaceutically acceptable salt thereof, wherein the cell penetrating peptide includes at least two positively charged amino acids; (b) one or more surfactants; (c) one or more sugars; and (d) one or more buffering agents are provided herein.
1 . A composition, comprising:
a. an antisense oligonucleotide conjugate comprising a cell penetrating peptide covalently attached to a nucleic acid analog, or a pharmaceutically acceptable salt thereof, wherein the cell penetrating peptide includes at least two positively charged amino acids, or a pharmaceutically acceptable salt thereof;
b. one or more surfactants;
c. one or more sugars; and
d. one or more buffering agents.
2 - 3 . (canceled)
4 . The composition of claim 1 , wherein the one or more surfactants are selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80, polysorbate 20), polyvinylpyrrolidone (PVP, povidone, poloxamer, poloxamer F188), and any mixtures thereof.
5 - 6 . (canceled)
7 . The composition of claim 1 , wherein the one or more sugars are selected from the group consisting of trehalose, mannitol, sucrose, and any mixtures thereof.
8 - 9 . (canceled)
10 . The composition of claim 1 , wherein the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.0 to about 7.0 when the composition is dissolved or dispersed in an aqueous solvent.
11 . (canceled)
12 . The composition of claim 10 , wherein the one or more buffering agents are present in a range of about 10 mM to about 50 mM when the composition is dissolved or dispersed in an aqueous solvent.
13 . The composition of claim 10 , wherein the one or more buffering agents are selected from the group consisting of histidine, citrate, maleate, arginine, and mixtures thereof.
14 . (canceled)
15 . The composition of claim 1 , wherein the composition has an osmolality between about 250 mOsm/kg and about 650 mOsm/kg.
16 - 17 . (canceled)
18 . The composition of claim 1 , wherein the composition is in lyophilized form.
19 . The composition of claim 1 , wherein the composition dissolves within 30 minutes after addition of an aqueous solvent.
20 - 22 . (canceled)
23 . The composition of claim 1 , wherein the composition comprises about 50 mg to about 500 mg of the antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof.
24 . The composition of claim 1 , wherein the antisense oligonucleotide conjugate is capable of binding a selected target to induce exon skipping in the human dystrophin gene.
25 . (canceled)
26 . The composition of claim 24 , wherein the antisense oligonucleotide conjugate comprises a cell penetrating peptide that is an arginine-rich peptide, wherein the arginine-rich peptide is selected from the group consisting of —(RXR) 4 —R a (SEQ ID NO: 54), R-(FFR) 3 -R a (SEQ ID NO: 55), —B—X—(RXR) 4 —R a (SEQ ID NO: 56), —B—X—R-(FFR) 3 -R a (SEQ ID NO: 57), -GLY-R-(FFR) 3 -R a (SEQ ID NO: 58), -GLY-R 5 —R a (SEQ ID NO: 59), —R 5 —R a (SEQ ID NO: 60), -GLY-R 6 —R a (SEQ ID NO: 52) and —R 6 —R a (SEQ ID NO: 53), 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.
27 - 28 . (canceled)
29 . The composition of claim 1 , wherein the antisense oligonucleotide conjugate is of 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:
R 100 is the cell penetrating peptide;
each Nu from I to n and 5′ to 3′ corresponds to the nucleobases in one of the following:
SEQ ID
Annealing Site
Base Sequence [5′ to 3′]
NO.
H51A(+66+95)
CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG
1
H51A(+74+97)
ACC TCC AAC ATC AAG GAA GAT GGC
2
H51A(+70+99)
GTA CCT CCA ACA TCA AGG AAG ATG GCA TTT
3
H51A(+72+99)
GTA CCT CCA ACA TCA AGG AAG ATG GCA T
4
H51A(+68+87)
TCA AGG AAG ATG GCA TTT CT
5
H51A(+68+87)
UCA AGG AmAGm AmUGm GmCA UUU CU
6
H53A(+36+60)
GTT GCC TCC GGT TCT GAA GGT GTT C
7
H53A(+36+60)
GTT G5mC5mC T5mC5mC GGT T5mC T GAA GGT GTT 5mC
8
H53A(+36+56)
CCT CCG GTT CTG AAG GTG TTC
9
H53A(+23+47)
CTG AAG GTG TTC TTG TAC TTC ATC C
10
H53A(+32+56)
CCT CCG GTT CTG AAG GTG TTC TTG T
11
H53A(+33+60)
GTT GCC TCC GGT TCT GAA GGT GTT CTT G
12
H53A(+30+59)
TTG CCT CCG GTT CTG AAG GTG TTC TTG TAC
13
H53A(+39+62)
CTG TTG CCT CCG GTT CTG AAG GTG
14
H53A(+36+69)
CAT TCA ACT GTT GCC TCC GGT TCT GAA GGT G
15
H53A(+45+62)
CTG TTG CCT CCG GTT CTG
16
H45A(−03+19)
CAA TGC CAT CCT GGA GTT CCT G
17
H45A(−09+25)
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA T
18
H45A(−03+25)
GCT GCC CAA TGC CAT CCT GGA GTT CCT G
19
H45A(−06+25)
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA A
20
H45A(−12+19)
CAA TGC CAT CCT GGA GTT CCT GTA AGA TAC C
21
H45A(−09+19)
CAA TGC CAT CCT GGA GTT CCT GTA AGA T
22
H45A(−12+16)
TGC CAT CCT GGA GTT CCT GTA AGA TAC C
23
H45A(−14+25)
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA
24
TAC CAA
H45A(−08+19)
CAA TGC CAT CCT GGA GTT CCT GTA AGA
25
HM45A(−07+25)
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AG
26
H45A(−12+22)
GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA TAC C
27
H45A(−09+22)
GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA T
28
H45A(−09+30)
TTG CCG CTG CCC AAT GCC ATC CTG GAG TTC CTG TAA
29
GAT
H45A(−06+22)
GCC CAA TGC CAT CCT GGA GTT CCT GTA A
30
H45A(−06+28)
GCC GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA A
31
H45A(−03+22)
GCC CAA TGC CAT CCT GGA GTT CCT G
32
H45A(−03+28)
GCC GCT GCC CAA TGC CAT CCT GGA GTT CCT G
33
H45A(+9+26)
m5C-G-m5C-T-G-C-m5C-m5C-A-A-T-G-m5C-m5C-A-U-m5C-
34
m5C
H44A(−10+15)
GAT CTG TCA AAT CGC CTG CAG GTA A
35
H44A(−07+15)
GAT CTG TCA AAT CGC CTG CAG G
36
H44M(−07+17)
CAG ATC TGT CAA ATC GCC TGC AGG
37
H44A(−08+15)
GAT CTG TCA AAT CGC CTG CAG GT
38
H44A(−06+15)
GAT CTG TCA AAT CGC CTG CAG
39
H44A(−08+17)
CAG ATC TGT CAA ATC GCC TGC AGG T
40
H44A(−06+17)
CAG ATC TGT CAA ATC GCC TGC AG
41
H50D(+04−18)
GGG ATC CAG TAT ACT TAC AGG C
42
H50D(+07−18)
GGG ATC CAG TAT ACT TAC AGG CTC C
43
H50D(+07−16)
GAT CCA GTA TAC TTA CAG GCT CC
44
H50D(+07−17)
GGA TCC AGT ATA CTT ACA GGC TCC
45
H50A(−19+07)
ACT TCC TCT TTA ACA GAA AAG CAT AC
46
H50D(+07−15)
ATC CAG TAT ACT TAC AGG CTC C
47
H50A(−02+23)
GAG CTC AGA TCT TCT AAC TTC CTC T
48
H50D(+06−18)
GGG ATC CAG TAT ACT TAC AGG CTC
49
H50D(+07−20)
ATG GGA TCC AGT ATA CTT ACA GGC TCC
50
H52A(−01+24)
CTG TTC CAA ATC CTG CAT TGT TGC C
51
wherein A is
C is
G is
T is
U is
Gm is methylated guanine, Am is methylated adenine, and m5C is
30 - 35 . (canceled)
36 . The composition of claim 1 , wherein the antisense oligonucleotide conjugate is of Formula (II):
or a pharmaceutically acceptable salt thereof, each Nu from 1 to (n+1) and 5° to 3′ corresponds to the nucleobases in one of the following:
SEQ
ID
Base Sequence [5′ to 3′]
NO.
CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG
1
ACC TCC AAC ATC AAG GAA GAT GGC
2
GTA CCT CCA ACA TCA AGG AAG ATG GCA TTT
3
GTA CCT CCA ACA TCA AGG AAG ATG GCA T
4
TCA AGG AAG ATG GCA TTT CT
5
UCA AGG AmAGm AmUGm GmCA UUU CU
6
GTT GCC TCC GGT TCT GAA GGT GTT C
7
GTT G5mC5mC T5mC5mC GGT T5mC T GAA GGT GTT
8
5mC
CCT CCG GTT CTG AAG GTG TTC
9
CTG AAG GTG TTC TTG TAC TTC ATC C
10
CCT CCG GTT CTG AAG GTG TTC TTG T
11
GTT GCC TCC GGT TCT GAA GGT GTT CTT G
12
TTG CCT CCG GTT CTG AAG GTG TTC TTG TAC
13
CTG TTG CCT CCG GTT CTG AAG GTG
14
CAT TCA ACT GTT GCC TCC GGT TCT GAA GGT G
15
CTG TTG CCT CCG GTT CTG
16
CAA TGC CAT CCT GGA GTT CCT G
17
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA
18
T
GCT GCC CAA TGC CAT CCT GGA GTT CCT G
19
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA A
20
CAA TGC CAT CCT GGA GTT CCT GTA AGA TAC C
21
CAA TGC CAT CCT GGA GTT CCT GTA AGA T
22
TGC CAT CCT GGA GTT CCT GTA AGA TAC C
23
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA
24
TAC CAA
CAA TGC CAT CCT GGA GTT CCT GTA AGA
25
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AG
26
GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA TAC
27
C
GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA T
28
TTG CCG CTG CCC AAT GCC ATC CTG GAG TTC CTG
29
TAA GAT
GCC CAA TGC CAT CCT GGA GTT CCT GTA A
30
GCC GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA
31
A
GCC CAA TGC CAT CCT GGA GTT CCT G
32
GCC GCT GCC CAA TGC CAT CCT GGA GTT CCT G
33
m5C-G-m5C-T-G-C-m5C-m5C-A-A-T-G-m5C-m5C-A-U-
34
m5C-m5C
GAT CTG TCA AAT CGC CTG CAG GTA A
35
GAT CTG TCA AAT CGC CTG CAG G
36
CAG ATC TGT CAA ATC GCC TGC AGG
37
GAT CTG TCA AAT CGC CTG CAG GT
38
GAT CTG TCA AAT CGC CTG CAG
39
CAG ATC TGT CAA ATC GCC TGC AGG T
40
CAG ATC TGT CAA ATC GCC TGC AG
41
GGG ATC CAG TAT ACT TAC AGG C
42
GGG ATC CAG TAT ACT TAC AGG CTC C
43
GAT CCA GTA TAC TTA CAG GCT CC
44
GGA TCC AGT ATA CTT ACA GGC TCC
45
ACT TCC TCT TTA ACA GAA AAG CAT AC
46
ATC CAG TAT ACT TAC AGG CTC C
47
GAG CTC AGA TCT TCT AAC TTC CTC T
48
GGG ATC CAG TAT ACT TAC AGG CTC
49
ATG GGA TCC AGT ATA CTT ACA GGC TCC
50
CTG TTC CAA ATC CTG CAT TGT TGC C
51
wherein A is
C is
G is
T is
U is
Gm is methylated guanine, Am is methylated adenine, and m5C is
37 - 41 . (canceled)
42 . The composition of claim 1 , wherein the antisense oligonucleotide conjugate is of Formula (III):
or a pharmaceutically acceptable salt thereof, each Nu from 1 to (n+1) and 5′ to 3′ corresponds to the nucleobases in one of the following:
SEQ
ID
Base Sequence [5′ to 3′]
NO.
CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG
1
ACC TCC AAC ATC AAG GAA GAT GGC
2
GTA CCT CCA ACA TCA AGG AAG ATG GCA TTT
3
GTA CCT CCA ACA TCA AGG AAG ATG GCA T
4
TCA AGG AAG ATG GCA TTT CT
5
UCA AGG AmAGm AmUGm GmCA UUU CU
6
GTT GCC TCC GGT TCT GAA GGT GTT C
7
GTT G5mC5mC T5mC5mC GGT T5mC T GAA GGT GTT
8
5mC
CCT CCG GTT CTG AAG GTG TTC
9
CTG AAG GTG TTC TTG TAC TTC ATC C
10
CCT CCG GTT CTG AAG GTG TTC TTG T
11
GTT GCC TCC GGT TCT GAA GGT GTT CTT G
12
TTG CCT CCG GTT CTG AAG GTG TTC TTG TAC
13
CTG TTG CCT CCG GTT CTG AAG GTG
14
CAT TCA ACT GTT GCC TCC GGT TCT GAA GGT G
15
CTG TTG CCT CCG GTT CTG
16
CAA TGC CAT CCT GGA GTT CCT G
17
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA
18
T
GCT GCC CAA TGC CAT CCT GGA GTT CCT G
19
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA A
20
CAA TGC CAT CCT GGA GTT CCT GTA AGA TAC C
21
CAA TGC CAT CCT GGA GTT CCT GTA AGA T
22
TGC CAT CCT GGA GTT CCT GTA AGA TAC C
23
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA
24
TAC CAA
CAA TGC CAT CCT GGA GTT CCT GTA AGA
25
GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA AG
26
GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA TAC
27
C
GCC CAA TGC CAT CCT GGA GTT CCT GTA AGA T
28
TTG CCG CTG CCC AAT GCC ATC CTG GAG TTC CTG
29
TAA GAT
GCC CAA TGC CAT CCT GGA GTT CCT GTA A
30
GCC GCT GCC CAA TGC CAT CCT GGA GTT CCT GTA A
31
GCC CAA TGC CAT CCT GGA GTT CCT G
32
GCC GCT GCC CAA TGC CAT CCT GGA GTT CCT G
33
m5C-G-m5C-T-G-C-m5C-m5C-A-A-T-G-m5C-m5C-A-U-
34
m5C-m5C
GAT CTG TCA AAT CGC CTG CAG GTA A
35
GAT CTG TCA AAT CGC CTG CAG G
36
CAG ATC TGT CAA ATC GCC TGC AGG
37
GAT CTG TCA AAT CGC CTG CAG GT
38
GAT CTG TCA AAT CGC CTG CAG
39
CAG ATC TGT CAA ATC GCC TGC AGG T
40
CAG ATC TGT CAA ATC GCC TGC AG
41
GGG ATC CAG TAT ACT TAC AGG C
42
GGG ATC CAG TAT ACT TAC AGG CTC C
43
GAT CCA GTA TAC TTA CAG GCT CC
44
GGA TCC AGT ATA CTT ACA GGC TCC
45
ACT TCC TCT TTA ACA GAA AAG CAT AC
46
ATC CAG TAT ACT TAC AGG CTC C
47
GAG CTC AGA TCT TCT AAC TTC CTC T
48
GGG ATC CAG TAT ACT TAC AGG CTC
49
ATG GGA TCC AGT ATA CTT ACA GGC TCC
50
CTG TTC CAA ATC CTG CAT TGT TGC C
51
wherein A is
C is
G is
T is
U is
Gm is methylated guanine, Am is methylated adenine, and m5C is
43 - 47 . (canceled)
48 . The composition of claim 1 , wherein the antisense oligonucleotide conjugate is PPMO #1, PPMO #2, PPMO #3, or PPMO #4 or a pharmaceutically acceptable salt thereof.
49 . (canceled)
50 . The composition of claim 1 , wherein the antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.
51 - 53 . (canceled)
54 . The composition of claim 50 , wherein the antisense oligonucleotide conjugate is the 6HCl salt form of PPMO #1, PPMO #2, PPMO #3, or PPMO #4.
55 - 58 . (canceled)
59 . A method of treating a human patient having Duchenne muscular dystrophy, comprising dissolving or dispersing the composition of claim 1 in an aqueous solvent.
60 . The method of claim 59 , wherein the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of the antisense oligonucleotide conjugate of about 20 mg/mL to about 200 mg/mL.
61 - 63 . (canceled)