Modified antisense oligomers for exon inclusion in spinal muscular atrophy
The present disclosure relates to modified antisense oligomers and related compositions and methods for increasing the expression of functional SMN protein and methods for treating spinal muscular atrophy and relates to inducing inclusion of exon 7 in SMN2 mRNA.
1. A compound of formula (V):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together forms a targeting sequence;
Z is an integer from 6 to 38;
each Y is independently selected from O and —NR 4 , wherein each R 4 is independently selected from H, C 1 -C 6 alkyl, aralkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) n NR 5 C(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 5 C(═NH)NH 2 , and G, wherein R 5 is selected from H and C 1 -C 6 alkyl and n is an integer from 1 to 5;
T is selected from OH and a moiety of the formula:
wherein:
A is selected from OH, —N(R 7 ) 2 R 8 , and R 1 wherein:
each R 7 is independently selected from H and C 1 -C 6 alkyl, and
R 8 is selected from an electron pair and H, and
R 6 is selected from OH, —N(R 9 )CH 2 C(O)NH 2 , and a moiety of the formula:
wherein:
R 9 is selected from H and C 1 -C 6 alkyl; and
R 10 is selected from G, —C(O)—R 11 OH, acyl, trityl, 4-methoxytrityl, —C(═NH)NH 2 , —C(O)(CH 2 ) m NR 12 C(═NH)NH 2 , and —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 12 C(═NH)NH 2 , wherein:
m is an integer from 1 to 5,
R 11 is of the formula —(O-alkyl) y - wherein y is an integer from 3 to 10 and
each of the y alkyl groups is independently selected from C 2 -C 6 alkyl; and
R 12 is selected from H and C 1 -C 6 alkyl;
each instance of R 1 is independently selected from:
—N(R 13 ) 2 R 14 wherein each R 13 is independently selected from H and C 1 -C 6 alkyl, and R 14 is selected from an electron pair and H;
a moiety of formula (II):
wherein:
R 15 is selected from H, G, C 1 -C 6 alkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) q NR 18 C(═NH)NH 2 , and —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 18 C(═NH)NH 2 , wherein:
R 18 is selected from H and C 1 -C 6 alkyl; and
q is an integer from 1 to 5,
R 16 is selected from an electron pair and H; and
each R 17 is independently selected from H and methyl; and
a moiety of formula (III):
wherein:
R 19 is selected from H, C 1 -C 6 alkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) r NR 22 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 3 NHC(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 22 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 4 NH 2 , and G wherein:
R 22 is selected from H and C 1 -C 6 alkyl; and
r is an integer from 1 to 5,
R 20 is selected from H and C 1 -C 6 alkyl; and
R 21 is selected from an electron pair and H; and
R 2 is selected from G, H, acyl, trityl, 4-methoxytrityl, and C 1 -C 6 alkyl,
wherein G is a cell penetrating peptide (“CPP”) and linker moiety selected from —C(O)(CH 2 ) 5 NH—CPP, —C(O)(CH 2 ) 2 NH—CPP, —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH—CPP, and —C(O)CH 2 NH—CPP, or G is of the formula:
wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that up to one instance of G is present,
wherein the targeting sequence comprises a sequence selected from SEQ ID NOS: 35 to 37, and wherein at least one of the following conditions is present:
a) at least one R 1 is of formula (II) or of formula (III), or
b) R 2 is G or T is:
2. The compound of claim 1 , wherein T is of the formula:
and R 2 is H.
3. The compound of claim 1 , wherein each R 1 is independently selected from
and —N(CH 3 ) 2 , T is:
and R 2 is H, wherein at least one R 1 is
4. The compound of claim 1 , each R 1 is independently selected from
and —N(CH 3 ) 2 , T is:
and R 2 is H, wherein at least one R 1 is
5. The compound of 1 , wherein each R 1 is —N(CH 3 ) 2 , R 2 is selected from H and acyl, and T is of the formula:
6. The compound of claim 1 , wherein R 2 is G, T is of the formula:
and each R 1 is —N(CH 3 ) 2 .
7. The compound of claim 1 , wherein R 2 is selected from H or acyl, T is of the formula:
and each R 1 is —N(CH 3 ) 2 .
8. The compound of claim 6 , wherein G is —C(O)CH 2 NH—CPP, wherein CPP comprises SEQ ID NO:23 and R a , wherein R a is attached to SEQ ID NO:23 at the SEQ ID NO:23 N-terminus,
and wherein R a is selected from H, acetyl, benzoyl, and stearoyl.
9. The compound of claim 1 , wherein Z is an integer from 16 to 23.
10. A pharmaceutical composition, comprising a compound of formula (V):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together forms a targeting sequence;
Z is an integer from 6 to 38;
each Y is independently selected from O and —NR 4 , wherein each R 4 is independently selected from H, C 1 -C 6 alkyl,
aralkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) n NR 5 C(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 5 C(═NH)NH 2 , and G, wherein R 5 is selected from H and C 1 -C 6 alkyl and n is an integer from 1 to 5;
T is selected from OH and a moiety of the formula:
wherein:
A is selected from —OH, —N(R 7 ) 2 R 8 , and R 1 wherein:
each R 7 is independently selected from H and C 1 -C 6 alkyl, and
R 8 is selected from an electron pair and H, and
R 6 is selected from OH, —N(R 9 )CH 2 C(O)NH 2 , and a moiety of the formula:
wherein:
R 9 is selected from H and C 1 -C 6 alkyl; and
R 10 is selected from G, —C(O)—R 11 OH, acyl, trityl, 4-methoxytrityl, —C(═NH)NH 2 , —C(O)(CH 2 ) m NR 12 C(═NH)NH 2 , and —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 12 C(═NH)NH 2 , wherein:
m is an integer from 1 to 5,
R 11 is of the formula —(O-alkyl) y - wherein y is an integer from 3 to 10 and
each of the y alkyl groups is independently selected from C 2 -C 6 alkyl; and
R 12 is selected from H and C 1 -C 6 alkyl;
each instance of R 1 is independently selected from:
—N(R 13 ) 2 R 14 wherein each R 13 is independently selected from H and C 1 -C 6 alkyl, and R 14 is selected from an electron pair and H;
a moiety of formula (II):
wherein:
R 15 is selected from H, G, C 1 -C 6 alkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) q NR 18 C(═NH)NH 2 , and —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 18 C(═NH)NH 2 , wherein:
R 18 is selected from H and C 1 -C 6 alkyl; and
q is an integer from 1 to 5,
R 16 is selected from an electron pair and H; and
each R 17 is independently selected from H and methyl; and
a moiety of formula (III):
wherein:
R 19 is selected from H, C 1 -C 6
alkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) r NR 22 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 3 NHC(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 22 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 4 NH 2 , and G wherein:
R 22 is selected from H and C 1 -C 6 alkyl; and
r is an integer from 1 to 5,
R 20 is selected from H and C 1 -C 6 alkyl; and
R 21 is selected from an electron pair and H; and
R 2 is selected from G, H, acyl, trityl, 4-methoxytrityl, and C 1 -C 6 alkyl,
wherein G is a cell penetrating peptide (“CPP”) and linker moiety selected from —C(O)(CH 2 ) 5 NH—CPP, —C(O)(CH 2 ) 2 NH—CPP, —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH—CPP, and —C(O)CH 2 NH—CPP, or G is of the formula:
wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that up to one instance of G is present,
wherein the targeting sequence comprises a sequence selected from SEQ ID NOS: 35 to 37, and wherein at least one of the following conditions is present:
c) at least one R 1 is of formula (II) or of formula (III), or
d) R 2 is G or T is:
11. A method of treating spinal muscular atrophy in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (V):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together forms a targeting sequence;
Z is an integer from 6 to 38;
each Y is independently selected from 0 and —NR 4 , wherein each R 4 is independently selected from H, C1-C6 alkyl, aralkyl, —C(═NH)NH2, —C(O)(CH 2 ) n NR 5 C(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 5 C(═NH)NH 2 , and G, wherein R 5 is selected from H and C 1 -C 6 alkyl and n is an integer from 1 to 5;
T is selected from OH and a moiety of the formula:
wherein:
A is selected from —OH, —N(R 7 ) 2 R 8 , and R 1 wherein:
each R 7 is independently selected from H and C 1 -C 6 alkyl, and
R 8 is selected from an electron pair and H, and
R 6 is selected from OH, —N(R 9 )CH 2 C(O)NH 2 , and a moiety of the formula:
wherein:
R 9 is selected from H and C1-C6 alkyl; and
R 10 is selected from G, —C(O)—R 11 OH, acyl, trityl, 4-methoxytrityl, —C(═NH)NH 2 , —C(O)(CH 2 ) m NR 12 C(═NH)NH 2 , and —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 12 C(═NH)NH 2 , wherein:
m is an integer from 1 to 5,
R 11 is of the formula —(O-alkyl) y - wherein y is an integer from 3 to 10 and
each of the y alkyl groups is independently selected from C2-C6 alkyl; and
R 12 is selected from H and C1-C6 alkyl;
each instance of R 1 is independently selected from:
—N(R 13 ) 2 R 14 wherein each R 13 is independently selected from H and C 1 -C 6 alkyl, and R 14 is selected from an electron pair and H;
a moiety of formula (II):
wherein:
R 15 is selected from H, G, C 1 -C 6 alkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) q NR 18 C(═NH)NH 2 , and —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 18 C(═NH)NH 2 , wherein:
R 18 is selected from H and C 1 -C 6 alkyl; and
q is an integer from 1 to 5,
R 16 is selected from an electron pair and H; and
each R 17 is independently selected from H and methyl; and
a moiety of formula(III):
wherein:
R 19 is selected from H, C1-C6 alkyl, —C(═NH)NH 2 , —C(O)(CH 2 ) r NR 22 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 3 NHC(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 22 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 4 NH 2 , and G wherein:
R 22 is selected from H and C 1 -C 6 alkyl; and
r is an integer from 1 to 5,
R 20 is selected from H and C 1 -C 6 alkyl; and
R 21 is selected from an electron pair and H; and
R 2 is selected from G, H, acyl, trityl, 4-methoxytrityl, and C1-C6 alkyl,
wherein G is a cell penetrating peptide (“CPP”) and linker moiety selected from —C(O)(CH 2 ) 5 NH—CPP, —C(O)(CH 2 ) 2 NH—CPP, —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH—CPP, and —C(O)CH 2 NH—CPP, or G is of the formula:
wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that up to one instance of G is present,
wherein the targeting sequence comprises a sequence selected from SEQ ID NOS: 35 to 37, and wherein at least one of the following conditions is present:
a) at least one R 1 is of formula (II) or of formula (III), or
b) R 2 is G or T is: