Antisense-induced exon2 inclusion in acid alpha-glucosidase

Abstract

The present disclosure relates to antisense oligomers and related compositions and methods for inducing exon inclusion as a treatment for glycogen storage disease type II (GSD-II) (also known as Pompe disease, glycogenosis II, acid maltase deficiency (AMD), acid alpha-glucosidase deficiency, and lysosomal alpha-glucosidase deficiency), and more specifically relates to inducing inclusion of exon 2 and thereby restoring levels of enzymatically active acid alpha-glucosidase (GAA) protein encoded by the GAA gene.

1 . An antisense oligomer compound of 10 to 40 nucleotides or nucleotide analogs, comprising:
 a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2′O-Me-phosphorothioate oligomer, or any combination of the foregoing; and   
       a targeting sequence complementary to a region within intron 2 (SEQ ID. NO: 3) of a pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T). 
     
     
         2 . (canceled) 
     
     
         3 . A compound of formula (I): 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         each Nu is a nucleobase which taken together forms a targeting sequence; 
         x is an integer from 8 to 38; 
         each Y is independently selected from O or —NR a , wherein R a  is selected from the group consisting of hydrogen, -T 1 -NR c R d R e , and a cell penetrating peptide, wherein:
 R c  is selected from the group consisting of hydrogen, C 1 -C 6  alkyl, aralkyl, and —C(═NH)NH 2 , 
 R d  is selected from the group consisting of hydrogen, aralkyl, and C 1 -C 6  alkyl, or 
 R c  and R d  taken together with the nitrogen atom to which they are attached form a 5-7 membered ring when R c  and R d  are each independently C 1 -C 6  alkyl or aralkyl, where the ring is optionally substituted with a substituent selected from the group consisting of C 1 -C 6  alkyl, phenyl, halogen, and aralkyl, and 
 R e  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
 
         each L is independently selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
       
       and a cell penetrating peptide, wherein w is an integer selected from 3-20, and S is an integer selected from 1 to 8;
 n is an integer from 0 to 3; 
 each R 1  is independently selected from the group consisting of —N(CH 3 ) 2 , —NR 5 R 6 , —OR 7 , 
 a moiety of formula (II): 
 
       
         
           
           
               
               
           
         
         
           wherein: 
           R 8  is selected from the group consisting of hydrogen, methyl, —C(═NH)NH 2 , —Z-T 2 -NHC(═NH)NH 2 , and a cell penetrating peptide, where Z is carbonyl or a direct bond, 
           R 9  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
           each R 10  is independently selected from hydrogen or methyl; and 
         
         a moiety of formula (III): 
       
       
         
           
           
               
               
           
         
         
           wherein: 
           q is an integer from 0 to 2, 
           R 11  is selected from the group consisting of hydrogen, C 1 -C 6  alkyl, aralkyl, and —C(═NH)NH 2 , 
           R 12  is selected from the group consisting of hydrogen, aralkyl, and C 1 -C 6  alkyl, or 
           R 11  and R 12  taken together with the nitrogen atom to which they are attached form a 5-7 membered ring where the ring is optionally substituted with a substituent selected from the group consisting of C 1 -C 6  alkyl, phenyl, halogen, and aralkyl, and 
           R 13  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
         
         R 2  is selected from the group consisting of hydrogen, OH, a nucleotide, a cell-penetrating peptide, a moiety of formula: 
       
       
         
           
           
               
               
           
         
       
       trityl, —C(═O)OR f , and acyl, wherein R f  is C 1 -C 30  alkyl optionally substituted by one or more oxygen or hydroxyl moieties, or R 2  is absent;
 R 3  is selected from the group consisting of hydrogen, a C 1 -C 6  alkyl, a nucleotide, a cell penetrating peptide, —C(═NH)NH 2 , trityl, —C(═O)OR g , acyl, and a moiety of formula: 
 
       
         
           
           
               
               
           
         
         wherein
 R g  is C 1 -C 30  alkyl optionally substituted by one or more oxygen or hydroxyl moieties; 
 
         R 4  is selected from the group consisting of an electron pair, hydrogen, a C 1 -C 6  alkyl, and acyl 
         R 5  is independently selected from hydrogen or methyl; 
         R 6  and R 7  is independently selected from hydrogen or -T 3 -NR c R d R e ; and 
         each of T 1 , T 2 , and T 3  is independently an optional linker of up to 18 atoms in length comprising alkyl, alkoxy, or alkylamino groups, or combinations thereof, 
         wherein the targeting sequence is complementary to a region within intron 2 (SEQ ID. NO: 3) of a pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T). 
       
     
     
         4 . (canceled) 
     
     
         5 . The compound of  claim 3 , wherein each R 1  is —N(CH 3 ) 2 . 
     
     
         6 . (canceled) 
     
     
         7 . The compound of  claim 3 , wherein at least one R 1  is selected from the group consisting of. 
       
         
           
           
               
               
           
         
       
     
     
         8 . The compound of  claim 3 , wherein 50-90% of the R 1  groups are —N(CH 3 ) 2 . 
     
     
         9 . The compound of  claim 3 , wherein 66% of the R 1  groups are —N(CH 3 ) 2 . 
     
     
         10 . The compound of  claim 3 , wherein:
 n is 2;   R 2  and L taken together are of the formula:   
       
         
           
           
               
               
           
         
       
       and
 Y is O at each occurrence. 
 
     
     
         11 . A compound of formula (IV): 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 each Nu is a nucleobase which taken together form a targeting sequence; 
 x is an integer from 15 to 25; 
 each Y is O; 
 each R 1  is independently selected from the group consisting of: 
 
       
       
         
           
           
               
               
           
         
         wherein at least one R 1  is —N(CH 3 ) 2 , and 
         wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T). 
       
     
     
         12 . A pharmaceutical composition, comprising a compound of  claim 1 , comprising an antisense oligomer compound of 10 to 40 nucleotides or nucleotide analogs and a pharmaceutically acceptable carrier, the compound comprising:
 a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2′O-Me-phosphorothioate oligomer, or any combination of the foregoing; and   a targeting sequence complementary to a region within intron 2 (SEQ ID. NO: 3) of a pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T).   
     
     
         13 . (canceled) 
     
     
         14 . A pharmaceutical composition, comprising a compound of  claim 3 , comprising a compound of formula (I): 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         each Nu is a nucleobase which taken together forms a targeting sequence; 
         x is an integer from 8 to 38; 
         each Y is independently selected from O or —NR a , wherein R a  is selected from the group consisting of hydrogen, -T 1 -NR c R d R e , and a cell penetrating peptide, wherein:
 R c  is selected from the group consisting of hydrogen, C 1 -C 6  alkyl, aralkyl, and —C(═NH)NH 2 , 
 R d  is selected from the group consisting of hydrogen, aralkyl, and C 1 -C 6  alkyl, or 
 R c  and R d  taken together with the nitrogen atom to which they are attached form a 5-7 membered ring when R c  and R d  are each independently C 1 -C 6  alkyl or aralkyl, where the ring is optionally substituted with a substituent selected from the group consisting of C 1 -C 6  alkyl, phenyl, halogen, and aralkyl, and 
 R e  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
 
         each L is independently selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
       
       and a cell penetrating peptide, wherein w is an integer selected from 3-20, and S is an integer selected from 1 to 8;
 n is an integer from 0 to 3; 
 each R 1  is independently selected from the group consisting of —N(CH 3 ) 2 , —NR 5 R 6 , —OR 7 , 
 a moiety of formula (II): 
 
       
         
           
           
               
               
           
         
         
           wherein: 
           R 8  is selected from the group consisting of hydrogen, methyl, —C(═NH)NH 2 , —Z-T 2 -NHC(═NH)NH 2 , and a cell penetrating peptide, where Z is carbonyl or a direct bond, R 9  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
           each R 10  is independently selected from hydrogen or methyl; and 
         
         a moiety of formula (III): 
       
       
         
           
           
               
               
           
         
         
           wherein: 
           q is an integer from 0 to 2, 
           R 11  is selected from the group consisting of hydrogen, C 1 -C 6  alkyl, aralkyl, and —C(═NH)NH 2 , 
           R 12  is selected from the group consisting of hydrogen, aralkyl, and C 1 -C 6  alkyl, or 
           R 11  and R 12  taken together with the nitrogen atom to which they are attached form a 5-7 membered ring where the ring is optionally substituted with a substituent selected from the group consisting of C 1 -C 6  alkyl, phenyl, halogen, and aralkyl, and 
           R 13  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
         
         R 2  is selected from the group consisting of hydrogen, OH, a nucleotide, a cell-penetrating peptide, a moiety of formula: 
       
       
         
           
           
               
               
           
         
       
       trityl, —C(═O)OR f , and acyl, wherein R f  is C 1 -C 30  alkyl optionally substituted by one or more oxygen or hydroxyl moieties, or R 2  is absent;
 R 3  is selected from the group consisting of hydrogen, a C 1 -C 6  alkyl, a nucleotide, a cell penetrating peptide, —C(═NH)NH 2 , trityl, acyl, and a moiety of formula: 
 
       
         
           
           
               
               
           
         
         
           R g  is C 1 -C 30  alkyl optionally substituted by one or more oxygen or hydroxyl moieties; 
         
         R 4  is selected from the group consisting of an electron pair, hydrogen, a C 1 -C 6  alkyl, and acyl; 
         R 5  is independently selected from hydrogen or methyl; 
         R 6  and R 7  is independently selected from hydrogen or -T 3 -NR c R d R e ; and 
         each of T 1 , T 2 , and T 3  is independently an optional linker of up to 18 atoms in length comprising alkyl, alkoxy, or alkylamino groups, or combinations thereof; 
         wherein the targeting sequence is complementary to a region within intron 2 (SEQ ID. NO: 3) of a pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T), and a pharmaceutically acceptable carrier. 
       
     
     
         15 - 17 . (canceled) 
     
     
         18 . A method of treating glycogen storage disease type II in a subject in need thereof, comprising a compound of  claim 1 , comprising administering to the subject an effective amount of an antisense oligomer compound of 10 to 40 nucleotides or nucleotide analogs comprising:
 a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2′O-Me-phosphorotioate oligomer, or any combination of the foregoing; and   a targeting sequence complementary to a region within intron 2 (SEQ ID. NO: 3) of a pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T).   
     
     
         19 . (canceled) 
     
     
         20 . A method of treating glycogen storage disease type II in a subject in need thereof, comprising a compound of  claim 3 , comprising administering to the subject an effective amount of an antisense oligomer compound of formula (I): 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         each Nu is a nucleobase which taken together forms a targeting sequence; 
         x is an integer from 8 to 38; 
         each Y is independently selected from O or —NR a , wherein R a  is selected from the group consisting of hydrogen, -T 1 -NR c R d R e , and a cell penetrating peptide, wherein:
 R c  is selected from the group consisting of hydrogen, C 1 -C 6  alkyl, aralkyl, and —C(═NH)NH 2 , 
 R d  is selected from the group consisting of hydrogen, aralkyl, and C 1 -C 6  alkyl, or 
 R c  and R d  taken together with the nitrogen atom to which they are attached form a 5-7 membered ring when R c  and R d  are each independently C 1 -C 6  alkyl or aralkyl, where the ring is optionally substituted with a substituent selected from the group consisting of C 1 -C 6  alkyl, phenyl, halogen, and aralkyl, and 
 R e  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
 
         each L is independently selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
       
       and a cell penetrating peptide, wherein w is an integer selected from 3-20, and S is an integer selected from 1 to 8;
 n is an integer from 0 to 3; 
 each R 1  is independently selected from the group consisting of —N(CH 3 ) 2 , —NR 5 R 6 , —OR 7 , 
 a moiety of formula (II): 
 
       
         
           
           
               
               
           
         
         
           wherein: 
           R 8  is selected from the group consisting of hydrogen, methyl, —C(═NH)NH 2 , —Z-T 2 -NHC(═NH)NH 2 , and a cell penetrating peptide, where Z is carbonyl or a direct bond, 
           R 9  is selected from the group consisting of an electron pair, hydrogen, a C 1 -C 6  alkyl, and aralkyl; 
           each R 10  is independently selected from hydrogen or methyl; and 
         
         a moiety of formula (III): 
       
       
         
           
           
               
               
           
         
         
           wherein: 
           q is an integer from 0 to 2, 
           R 11  is selected from the group consisting of hydrogen, C 1 -C 6  alkyl, aralkyl, and —C(═NH)NH 2 , 
           R 12  is selected from the group consisting of hydrogen, aralkyl, and C 1 -C 6  alkyl, or 
           R 11  and R 12  taken together with the nitrogen atom to which they are attached form a 5-7 membered ring where the ring is optionally substituted with a substituent selected from the group consisting of C 1 -C 6  alkyl, phenyl, halogen, and aralkyl, and 
           R 13  is selected from the group consisting of an electron pair, hydrogen, C 1 -C 6  alkyl, and aralkyl; 
         
         R 2  is selected from the group consisting of hydrogen, OH, a nucleotide, a cell-penetrating peptide, a moiety of formula: 
       
       
         
           
           
               
               
           
         
       
       trityl, —C(═O)OR f , and acyl, wherein R 1  is C 1 -C 30  alkyl optionally substituted by one or more oxygen or hydroxyl moieties, or R 2  is absent;
 R 3  is selected from the group consisting of hydrogen, a C 1 —C alkyl, a nucleotide, a cell penetrating peptide, —C(═NH)NH 2 , trityl, acyl, and a moiety of formula: 
 
       
         
           
           
               
               
           
         
         
           R g  is C 1 -C 30  alkyl optionally substituted by one or more oxygen or hydroxyl moieties; 
         
         R 4  is selected from the group consisting of an electron pair, hydrogen, a C 1 -C 6  alkyl, and acyl; 
         R 5  is independently selected from hydrogen or methyl; 
         R 6  and R 7  is independently selected from hydrogen or -T 3 -NR c R d R e ; n each of T 1 , T 2 , and T 3  is independently an optional linker of up to 18 atoms in length comprising alkyl, alkoxy, or alkylamino groups, or combinations thereof,
 wherein the targeting sequence is complementary to a region within intron 2 (SEQ ID. NO: 3) of a pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein the targeting sequence is selected from SEQ ID NOS: 11, 13, 16, 82, 83, 89 and 91, wherein X is selected from uracil (U) or thymine (T). 
 
       
     
     
         21 - 26 . (canceled)