Antisense oligomers and methods of using the same for treating diseases associated with the acid alpha-glucosidase gene

1. A modified antisense oligonucleotide of 24 nucleobases comprising: a targeting sequence complementary to a target region within intron 1 (SEQ ID NO: 1) of a pre-mRNA of the human alpha glucosidase (GAA) gene, wherein the target region comprises at least one additional nucleobase compared to the targeting sequence, wherein the at least one additional nucleobase has no complementary nucleobase in the targeting sequence, and wherein the at least one additional nucleobase is internal to the target region,

wherein the modified antisense oligonucleotide is an antisense oligomer compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

each Nu is a nucleobase which taken together form a targeting sequence;

Z is 22;

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 , and R 1 wherein each R 7 is independently selected from H and C 1 -C 6 alkyl, 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 , wherein each R 13 is independently selected from H and C 1 -C 6 alkyl;

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, 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 ,

 R 22 C(═NH)NH 2 , H

 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, and

 R 20 is selected from H and C 1 -C 6 alkyl; or

 R 19 and R 20 together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring having from 5 to 7 ring atoms and optionally containing an additional heteroatom selected from oxygen, nitrogen, and sulfur; and

R 2 is selected from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, stearoyl, C 1 -C 6 alkyl, —C(═NH)NH 2 , —C(O)—R 23 , —C(O)(CH 2 ) s NR 24 C(═NH)NH 2 , —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NR 24 C(═NH)NH 2 , —C(O)CH(NH 2 )(CH 2 ) 3 NHC(═NH)NH 2 , and a moiety of the formula:

wherein,

R 23 is of the formula —(O-alkyl) v -OH wherein v is an integer from 3 to 10 and each of the v alkyl groups is independently selected from C 2 -C 6 alkyl; and

R 24 is selected from H and C 1 -C 6 alkyl;

s is an integer from 1 to 5;

L is selected from —C(O)(CH 2 ) 6 C(O)— and —C(O)(CH 2 ) 2 S 2 (CH 2 ) 2 C(O)—; and

each R 25 is of the formula —(CH 2 ) 2 OC(O)N(R 26 ) 2 wherein each R 26 is of the formula —(CH 2 ) 6 NHC(═NH)NH 2 ,

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, —C(O)CH 2 NH-CPP, and:

wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, and

wherein G may be present in one occurrence or is absent; and wherein the targeting sequence is selected from the group consisting of:

a) SEQ ID NO: 13 (GGC CAG AAG GAA GGC GAG AAA AGC) wherein Z is 22;

b) SEQ ID NO: 14 (GCC AGA AGG AAG GCG AGA AAA GCX) wherein Z is 22;

c) SEQ ID NO: 15 (CCA GAA GGA AGG CGA GAA AAG CXC) wherein Z is 22;

d) SEQ ID NO: 16 (CAG AAG GAA GGC GAG AAA AGC XCC) wherein Z is 22;

e) SEQ ID NO: 17 (AGA AGG AAG GCG AGA AAA GCX CCA) wherein Z is 22; and

f) SEQ ID NO: 18 (GAA GGA AGG CGA GAA AAG CXC CAG) wherein Z is 22

wherein X is selected from uracil (U) or thymine (T).

2. The modified antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide promotes retention of exon 2 in the GAA mRNA upon binding of the targeting sequence to the target region.

3. The modified antisense oligonucleotide of claim 1 , wherein the target region comprises from one to three additional nucleobases compared to the targeting sequence.

4. The modified antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide induces GAA enzyme activity at least two fold according to an enzyme activity test as compared to a second antisense oligonucleotide that is fully complementary to the target region within SEQ ID NO: 1.

5. The antisense oligomer compound of claim 1 , wherein: (a) each R 1 is —N(CH 3 ) 2 ; or (b) at least one R 1 is selected from:

6. The antisense oligomer compound of claim 1 , wherein T is selected from:

and

Y is O at each occurrence.

7. The modified antisense oligonucleotide of claim 1 , wherein each Y is O;

T is:

each R 1 is N(CH 3 ) 2 ;

R 2 is:

R a is H; and

each Nu is a nucleobase, which taken together form the targeting sequence (5′ to 3′) of:

SEQ ID NO: 13 (GGC CAG AAG GAA GGC GAG AAA AGC) wherein Z is 22.

8. The modified antisense oligonucleotide of claim 1 , wherein each Nu is a nucleobase, which taken together form the targeting sequence (5′ to 3′) of: SEQ ID NO: 13 (GGC CAG AAG GAA GGC GAG AAA AGC) wherein Z is 22.