Antisense oligonucleotides having one or more abasic units

Abstract

Provided herein are oligonucleotides, peptide-oligonucleotide conjugates, and targeting sequences complementary to a target region within intron 1 of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene having at least one purine- and pyrimidine-free abasic subunit. Also provided herein are methods of treating a muscular disorder, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject the oligonucleotides, peptides, and peptide-oligonucleotide conjugates described herein.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 408,277, filed September 20, 2022, and U.S. Provisional Patent Application No. 63 / 261,860, filed September 30, 2021, the entire contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Antisense technology provides a means for regulating the expression of one or more specific gene products, including alternative splicing products, and is uniquely useful in many therapeutic, diagnostic, and research applications. The principle behind antisense technology is that antisense compounds, such as oligonucleotides that hybridize to target nucleic acids, regulate gene expression activities such as transcription, splicing, or translation through any one of many antisense mechanisms. The sequence specificity of antisense compounds makes them attractive as tools for target validation and gene function, as well as therapeutic agents that selectively regulate the expression of genes involved in disease.

[0003] Glycogen storage disease type II (GSD-II) (also known as Pompe disease, glycogen storage disease II, acid maltase deficiency (AMD)) is an inherited autosomal recessive lysosomal storage disorder caused by a deficiency of an enzyme called acid alpha-glucosidase (GAA). The role of GAA in the body is to break down glycogen. Reduced levels or absence of GAA activity leads to the accumulation of glycogen in affected tissues, including the heart, skeletal muscles (including those involved in breathing), liver, and nervous system. This glycogen accumulation is thought to cause progressive muscle weakness and respiratory failure in individuals with GSD-II. GSD-II can occur in infants, young children, or adults, and the prognosis varies depending on the time of onset and severity of symptoms. Clinically, GSD-II can manifest in a wide and continuous range of severity, ranging from severe (infantile) to mild late-onset adult forms. Patients ultimately die from respiratory failure. There is a good correlation between disease severity and residual acid alpha-glucosidase activity, with activity being 10-20% of normal in the late-onset form of the disease and less than 2% in the early-onset form. GSD-II is estimated to affect approximately 5,000-10,000 people worldwide.

[0004] The most common mutation associated with the adult-onset form of the disease is IVS1-13T>G. This mutation, found in more than two-thirds of adult-onset GSD-II patients, may confer a selective advantage in heterozygous individuals or may be a very old mutation. The wide racial differences in adult-onset GSD-II individuals carrying this mutation argue against a common proband.

[0005] The GAA gene consists of 20 exons spanning approximately 20 kb. The 3.4 kb mRNA encodes a protein with a molecular weight of approximately 105 kD. The IVS1-13T>G mutation results in the complete or partial loss of exon 2 (577 bases) containing the initiation AUG codon.

[0006] Treatment of GSD-II has involved drug strategies, dietary therapy, and bone marrow transplantation, but without great success. In recent years, enzyme replacement therapy (ERT) has brought new hope to GSD-II patients. For example, Myozyme®, a recombinant GAA protein drug, was approved for use in patients with GSD-II disease in 2006 in both the United States and Europe. Myozyme® relies on mannose-6-phosphate (M6P) on the surface of the GAA protein for delivery to lysosomes. The U.S. Food and Drug Administration has also approved Nexviazyme® (avaruglucosidase alfa-ngpt) for the treatment of patients with late-onset Pompe disease. Nexviazyme is an enzyme replacement therapy (ERT) designed to specifically target the M6P receptor.

[0007] Antisense technology, which is primarily used to downregulate RNA, has recently been adapted to modify the splicing process. Processing the primary gene transcript (pre-mRNA) of many genes involves the removal of introns and the precise splicing of exons where donor splice sites are joined to acceptor splice sites. Splicing is a precise process that involves the coordinate recognition of donor and acceptor splice sites, as well as branch points (upstream of the pre-acceptor site) with a balance of positive exon splice enhancers (mostly located within exons) and negative splice motifs (splice silencers are mostly located within introns).

[0008] Although significant advances have been made in the field of antisense technology, there remains a need for peptide-oligonucleotide-conjugates with improved antisense or antigene capabilities for improved treatment of GSD-II. Summary of the Invention [Means for solving the problem]

[0009] 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, Glycogen Storage Disease II, Acid Maltase Deficiency (AMD), Acid Alpha-Glucosidase Deficiency, and Lysosomal Alpha-Glucosidase Deficiency), and more specifically to inducing inclusion of exon 2 and thereby restoring levels of enzymatically active acid alpha-glucosidase (GAA) protein encoded by the GAA gene.

[0010] Thus, there is provided herein an antisense oligomer, or a pharma- ceutically acceptable salt thereof, the antisense oligomer being 18-40 subunits in length and comprising a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; each subunit of the antisense oligomer comprises a nucleobase or is an abasic subunit; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0011] The antisense oligomers are useful for treating a variety of disorders in subjects in need of such treatment, including, but not limited to, disorders such as Pompe disease.

[0012] The antisense oligomer may be a phosphorodiamidate morpholino oligomer. The antisense oligomer may further comprise a cell membrane penetrating peptide. The peptide may be any of those provided herein or known in the art.

[0013] In one embodiment, the targeting region comprises a sequence selected from the group consisting of SEQ ID NO:2 (GAA-IVS1(-189-167)) and SEQ ID NO:3 (GAA-IVS1(-80-24)). In another embodiment, the targeting region is selected from GAA-IVS1(-189-167), GAA-IVS1(-72,-48), GAA-IVS1(-71,-47), GAA-IVS1(-70,-46), GAA-IVS1(-69-45), GAA-IVS1(-65,-41), GAA-IVS1(-66,-42). In a further embodiment, the targeting region is GAA-IVS1(-189-167). In another embodiment, the targeting region is GAA-IVS1(-72,-48). In yet another embodiment, the targeting region is GAA-IVS1(-71,-47). In yet another embodiment, the targeting region is GAA-IVS1(-70,-46). In one embodiment, the targeting region is GAA-IVS1(-69-45). In another embodiment, the targeting region is GAA-IVS1(-65,-41). In yet another embodiment, the targeting region is GAA-IVS1(-66,-42).

[0014] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 7-1] [Table 7-2] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0015] In one embodiment, B is H.

[0016] In further embodiments, the targeting region comprises or consists of any one of the following sequences: [Table 8-1] [Table 8-2]

[0017] In one embodiment, B is H.

[0018] In some embodiments, the nucleobases of the antisense oligomer are linked to morpholino ring structures which are joined by phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one ring structure to the 5' exocyclic carbon of an adjacent ring structure.

[0019] In some embodiments, the nucleobases of the antisense oligomer are linked to peptide nucleic acids (PNAs), in which the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked.

[0020] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a locked nucleic acid (LNA), a locked nucleic acid structure that is a chemically modified nucleotide analog in which a ribose moiety has an extra bridge connecting the 2' oxygen and the 4' carbon.

[0021] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a bridged nucleic acid (BNA), where the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone. In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a 2'-O,4'-C-ethylene bridged nucleic acid (ENA).

[0022] In some embodiments, modified antisense oligomers may contain unlocked nucleic acid (UNA) subunits. UNA and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit has been truncated.

[0023] In some embodiments, modified antisense oligomers contain one or more phosphorothioates (or S-oligos), in which one of the non-bridging oxygens is replaced by sulfur. In some embodiments, modified antisense oligomers contain one or more 2'O-methyl, 2'O-MOE, MCE, and 2'-F, in which the 2'-OH of the ribose is replaced with a methyl, methoxyethyl, 2-(N-methylcarbamoyl)ethyl, or fluoro group, respectively.

[0024] In some embodiments, the modified antisense oligomer is tricyclo-DNA (tc-DNA), a constrained DNA analog in which each nucleotide is modified by the introduction of a cyclopropane ring to limit the conformational flexibility of the backbone and optimize the backbone geometry for torsion angle γ.

[0025] In one aspect, the antisense oligomer is a modified antisense oligonucleotide, The modified antisense oligonucleotide is 18 to 40 subunits in length and contains a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; The antisense oligonucleotide comprises a morpholino oligomer, each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0026] In one embodiment, the present disclosure provides an antisense oligomer according to formula I: [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, A' is -N(H)CH 2 C(O)NH 2 , -N(C 1-6 -alkyl)CH 2 C(O)NH 2 , [ka] is selected from the group consisting of R 5 is -C(O)(O-alkyl) x -OH, where x is 3 to 10, and each alkyl group, in each occurrence, is independently selected from the group consisting of C 2-6 - alkyl or or R 5 is H, -C(O)C 1-6 -Alkyl, trityl, monomethoxytrityl, -(C 1-6 -alkyl)-R 6 , -(C 1-6 -heteroalkyl)-R 6 , aryl-R 6 , heteroaryl-R 6 , -C(O)O-(C 1-6 -alkyl)-R 6 , -C(O)O-aryl-R 6 , -C(O)O-heteroaryl-R 6 , and [ka] is selected from R 6 are OH, SH, and NH 2 or R 6 is O, S, or NH, each of which is covalently attached to a solid support; Each R 1 are independently OH and -N(R 3 )(R 4 ) wherein each R 3 and R 4 is independently in each occurrence H or -C 1-6 -alkyl, Each R 2 is independently selected at each occurrence from H (abasic), a nucleobase, and a nucleobase functionalized with a chemical protecting group, and the nucleobase is independently selected at each occurrence from pyridine, pyrimidine, purine, and deaza-purine. 3-6 Contains a heterocycle, t is 8 to 40; E' is H, -C 1-6 -Alkyl, -C(O)C 1-6 -Alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, [ka] is selected from During the ceremony, Q is -C(O)(CH 2 ) 6 -C(O)- or -C(O)(CH 2 ) 2 S 2 (CH 2 ) 2 C(O)-, R 7 is -(CH 2 ) 2 O C (O) N (R 8 ) 2 where R 8 is -(CH 2 ) 6 NHC(=NH)NH 2 and L is glycine, proline, W, WW, or R 9 and L is covalently linked by an amide bond to the N-terminus or C-terminus of J; W is -C(O)-(CH 2 ) m -NH-, where m is 2 to 12; R 9 teeth, [ka] is selected from the group consisting of n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5; R 10 is selected from a bond, glycine, proline, W, or WW; R 11 is glycine, proline, W, WW, and [ka] is selected from the group consisting of R 16 is selected from a bond, glycine, proline, W, or WW; R 16 is covalently attached by an amide bond to the N-terminus or C-terminus of J, where J is a cell membrane penetrating peptide; G is H, -C(O)C 1-6 -alkyl, benzoyl, and stearoyl, and G is covalently linked to J.

[0027] In some embodiments, the antisense oligomer of the present disclosure is according to formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 9]

[0028] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0029] In one embodiment, B is H.

[0030] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 10-1] [Table 10-2]

[0031] In one embodiment, B is H.

[0032] In one embodiment, the antisense oligomer is a conjugate comprising a modified antisense oligonucleotide and a cell membrane penetrating peptide; The modified antisense oligonucleotide is 18 to 40 subunits in length and contains a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; The antisense oligonucleotide comprises a morpholino oligomer, The antisense oligonucleotide is covalently linked to a cell membrane-permeable peptide, each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0033] Thus, in one embodiment of formula I or a salt thereof, A' is [ka] or E' is [ka] It is.

[0034] In some embodiments, the antisense oligomer of the present disclosure conforms to formula (IIIa): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 11-1] [Table 11-2] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0035] In one embodiment, B is H.

[0036] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 12-1] [Table 12-2]

[0037] In one embodiment, B is H. In some embodiments, the antisense oligomer of the present disclosure conforms to formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 13-1] [Table 13-2]

[0038] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0039] In one embodiment, B is H.

[0040] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 14-1] [Table 14-2]

[0041] In one embodiment, B is H.

[0042] The antisense oligomer may promote retention of exon 2 in the GAA mRNA upon binding of the targeting sequence to the target region. The antisense oligomer retains the potency of GAA enzyme activity compared to a second antisense oligonucleotide that is fully complementary to the target region within SEQ ID NO: 1. In another aspect, provided herein is a pharmaceutical composition comprising an antisense oligomer provided herein and a pharma- ceutically acceptable carrier.

[0043] Also provided herein are methods of treating a disease comprising administering to a subject a therapeutically effective amount of an antisense oligomer provided herein.

[0044] In some embodiments, the antisense oligomers described herein may be used to treat Pompe disease. [Brief description of the drawings]

[0045] [Figure 1] 1 shows a bar graph depicting the GAA enzyme activity (enzyme assay) observed for various PMO compounds during screening. The Y-axis represents the fold increase in GAA enzyme activity compared to untreated controls. Individual compounds were administered at 10 μM. [Diagram 2] FIG. 2A shows a bar graph depicting the GAA enzyme activity (enzyme assay) observed for various PMO compounds during screening. The Y-axis represents enzyme activity (mmol / mg hr) and fold increase in GAA enzyme activity compared to untreated control (UT). FIG. 2B shows a bar graph depicting the GAA enzyme activity (enzyme assay) observed for various PMO compounds during screening. The Y-axis represents fold increase in GAA enzyme activity compared to untreated control. Individual compounds were administered at 20 μM. [Diagram 3] 1 shows a bar graph depicting antisense microwalk data in the −65 region of intron 1 of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene. Individual compounds were dosed at 20 μM. [Figure 4] FIG. 4A shows a bar graph depicting the GAA enzyme activity (enzyme assay) observed for various PPMO compounds during screening. The X-axis represents the fold increase in GAA enzyme activity compared to the non-targeted control. Individual compounds were administered at 20 μM. FIG. 4B shows a bar graph depicting the GAA mRNA transcript levels (qPCR assay) observed for various PPMO compounds during screening. The X-axis represents the fold increase in GAA mRNA transcript measured at two locations within GAA mRNA compared to the non-targeted control and the untreated control. Individual compounds were administered at 30 μM. [Diagram 5] 1 shows a bar graph depicting antisense microwalk data in the −169 region of intron 1 of the pre-mRNA of the GAA gene. Individual compounds were administered at 10 μM. [Figure 6] 1 shows a graph showing the dose-dependent increase in GAA enzyme activity in patient fibroblasts following gymnotic treatment with PPMO numbers 33, 34, 5, and 7. [Figure 7]1 shows a bar graph depicting GAA mRNA transcript levels (qPCR assay) observed for PPMO compounds during screening. The Y-axis represents the fold increase in GAA mRNA transcript compared to non-targeted and untreated controls. Individual compounds were dosed at 1, 2.5, 5, 10, 20, and 30 μM. [Figure 8] A graph showing the dose-dependent increase in GAA expression measured across the exon 1-2 junction in patient iPSC-derived myotubes following gymnotic treatment with selected PPMO numbers 34, 5, and 7 is shown. [Figure 9] 1 shows digital gel images and graphs showing increased amounts of GAA protein normalized to total protein in patient iPSC-derived myotubes following treatment with PPMO numbers 5, 7, and 34. [Figure 10] 1 shows digital gel images and a graph showing increased amounts of GAA protein normalized to total protein in patient iPSC-derived myotubes following treatment with PPMO nos. 12 and 15. [Figure 11] 1 shows a graph depicting increased amounts of GAA enzyme activity in patient iPSC-derived myotubes following treatment with PPMO nos. 7, 5, and 12. [Figure 12] FIG. 1 shows a graph illustrating the aggregation ability of selected PPMO compounds. The results are plotted as size / intensity distribution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Provided herein is an antisense oligomer, or a pharma- ceutically acceptable salt thereof, the antisense oligomer being 18-40 subunits in length and comprising a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene, wherein at least one subunit is an abasic subunit. The antisense oligomer is useful for treating a variety of diseases in subjects in need of such treatment, including, but not limited to, Pompe disease.

[0047] Certain embodiments relate to methods for enhancing levels of exon 2-containing GAA-encoding mRNA relative to exon-2 deleted GAA mRNA in a cell, the method comprising contacting the cell with an antisense oligomer of sufficient length and complementarity to specifically hybridize to a region within the GAA gene, thereby enhancing levels of exon 2-containing GAA mRNA relative to exon-2 deleted GAA mRNA in the cell. In some embodiments, the cell is in a subject and the method comprises administering the antisense oligomer to the subject.

[0048] In one embodiment, provided herein is an antisense oligomer comprising a cell membrane-permeable peptide, the antisense oligomer comprising a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene, and at least one subunit is an abasic subunit.

[0049] Also provided herein are methods for treating Pompe disease.

[0050] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this disclosure, preferred methods and materials are described. For purposes of this disclosure, the following terms are defined below.

[0051] The term "about" will be understood by those of skill in the art and will vary to some extent in the context in which it is used. As used herein, when referring to measurable values, such as amounts, durations, and the like, the term "about" is meant to encompass variations of ±10%, including ±5%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0052] The term "alkyl" refers, in certain embodiments, to a saturated, straight-chain or branched-chain hydrocarbon moiety containing 1 to 6 or 1 to 8 carbon atoms. 1-6 Examples of -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, and n-hexyl moieties; 1-8 Examples of -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

[0053] The number of carbon atoms in an alkyl substituent is defined as "C x-y " where x is the minimum and y is the maximum number of carbon atoms in the substituent. x Chain means an alkyl chain containing x carbon atoms.

[0054] The term "heteroalkyl," by itself or in combination with another term, unless otherwise stated, refers to a stable straight or branched chain alkyl group containing the specified number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom may be located at any position of the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH 2 -CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -OH, -CH 2 -CH 2 -NH-CH 3 , -CH 2 -S-CH 2 -CH 3 , and -CH 2 -CH 2 -S(=O)-CH 3。Up to two heteroatoms, e.g., -CH 2 -NH-OCH 3 , or -CH 2 -CH 2 -SS-CH 3 It can be continuous like this.

[0055] The term "aryl", when used alone or in combination with other terms, unless otherwise specified, refers to a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), which may be attached in a pendant fashion, such as biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. In various embodiments, examples of aryl groups include phenyl (e.g., C 6 -aryl) and biphenyl (e.g., C 12 In some embodiments, the aryl group has 6 to 16 carbon atoms. In some embodiments, the aryl group has 6 to 12 carbon atoms (e.g., C 6-12 In some embodiments, the aryl group has 6 carbon atoms (e.g., C 6 -aryl).

[0056] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. Heteroaryl substituents include, for example, C 1-9 Heteroaryl may be defined by the number of carbon atoms, not including the number of heteroatoms, to indicate the number of carbon atoms contained in the heteroaryl group. For example, C 1-9-Heteroaryl will contain 1 to 4 additional heteroatoms. Polycyclic heteroaryls can contain one or more rings that are partially saturated. Non-limiting examples of heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, for example, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, for example, 2-pyrrolyl), imidazolyl, thiazolyl, pyrazolyl (including, for example, 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0057] Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (e.g., 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (e.g., including 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (e.g., including 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofur ...isoquinolyl), and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (e.g., 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (e.g., 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (including, for example, 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbonyl, acridinyl, pyrrolidinyl, and quinolizidinyl.

[0058] The term "protecting group" or "chemical protecting group" refers to a chemical moiety that blocks some or all reactive moieties of a compound, preventing such moieties from participating in a chemical reaction until the protecting group is removed, for example, such moieties are listed and described in TW Greene, PG M Huts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). When different protecting groups are used, it may be advantageous for each (different) protecting group to be removable by different means. Protecting groups that are cleaved under completely different reaction conditions allow for differential removal of such protecting groups. For example, protecting groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, monomethoxytrityl, dimethoxytrityl, acetal, and tert-butyldimethylsilyl are acid labile and can be used to protect carboxy and hydroxy reactive moieties in the presence of hydrogenolysis-removable Cbz groups, and amino groups protected with base-labile Fmoc groups. Carboxylic acid moieties may be blocked with base labile groups such as, but not limited to, methyl or ethyl, and hydroxy reactive moieties may be blocked with base labile moieties such as acetyl in the presence of acid labile groups such as tert-butyl carbamate, or amines blocked with both acid and base stable but hydrolytically removable carbamates.

[0059] Carboxylic acid and hydroxyl reactive moieties may also be blocked with hydrolytically removable protecting groups such as benzyl groups, while amine groups may be blocked with base labile groups such as Fmoc. A particularly useful amine protecting group for the synthesis of compounds of formula I and formula IV is trifluoroacetamide. Carboxylic acid reactive moieties may also be blocked with oxidatively removable protecting groups such as 2,4-dimethoxybenzyl, while existing amino groups may be blocked with fluoride labile silyl carbamates.

[0060] Allyl blocking groups are useful in the presence of acid- and base-protecting groups because the former are stable and can be subsequently removed by metal or pi-acid catalysts. For example, allyl-blocked carboxylic acids can be deprotected with a palladium(0)-catalyzed reaction in the presence of acid-labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate can be attached. As long as the residue is attached to the resin, that functional group is blocked and cannot react. Once released from the resin, the functional group becomes available to react.

[0061] The terms "nucleobase", "base pairing moiety", "nucleobase-pairing moiety", or "base" refer to the heterocyclic portion of a nucleoside, nucleotide, and / or morpholino subunit. The nucleobase can be naturally occurring (e.g., uracil, thymine, adenine, cytosine, and guanine) or a modification or analog of these naturally occurring nucleobases, e.g., one or more nitrogen atoms of the nucleobase can be replaced by carbon, independently at each occurrence. Exemplary analogs include hypoxanthine (the base component of the nucleotide inosine), 2,6-diaminopurine; 5-methylcytosine; C5-propynyl-modified pyrimidines, 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp), and the like.

[0062] Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine and pyrimidine analogues such as acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine and pseudouracil, and 8-substituted purines, xanthine, or hypoxanthine, the latter two being natural decomposition products, and other modified nucleobases such as hypoxanthine. Modified nucleobases disclosed in Chiu and Rana (2003) RNA 9:1034-1048, Limbach et al. (1994) Nucleic Acids Res. 22:2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, the contents of which are incorporated herein by reference, are also contemplated.

[0063] Further examples of base pairing moieties include, but are not limited to, the expanded nucleobases with one or more benzene rings added.The nucleobase substitutions described in Glen Research catalog (www.glenresearch.com), Krueger AT et al. (2007) Acc.Chem.Res.40:141-150, Kool ET (2002) Acc.Chem.Res.35:936-943, Benner SA et al. (2005) Nat.Rev.Genet.6:553-543, Romesberg FE et al. (2003) Curr.Opin.Chem.Biol.7:723-733, Hirao, I (2006) Curr.Opin.Chem.Biol.10:622-627 (the contents of which are incorporated herein by reference) are contemplated to be useful in the synthesis of oligomers described herein. Examples of extended size nucleobases are shown below: [ka]

[0064] The term "oligonucleotide" or "oligomer" refers to a compound that includes a plurality of linked nucleosides, nucleotides, or a combination of both nucleosides and nucleotides. In certain embodiments provided herein, the oligonucleotide is a morpholino oligonucleotide.

[0065] As used herein, the terms "antisense oligomer" or "antisense compound" are used interchangeably and refer to an array of subunits, each having bases carried on a backbone subunit composed of a ribose or other pentose sugar or morpholino group, the backbone groups being linked by intersubunit linkages that allow the bases in the compound to hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or near exact complementarity to the target sequence. Such an antisense oligomer is designed to block or inhibit translation of the mRNA containing the target sequence and may be said to be "directed" to the sequence to which it hybridizes.

[0066] Also contemplated herein as types of "antisense oligomers" or "antisense compounds" are phosphorothioate modified oligomers, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), 2'-fluoro modified oligomers, 2'-O,4'-C-ethylene bridged nucleic acids (ENAs), tricyclo-DNA, tricyclo-DNA phosphorothioate modified oligomers, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers, 2'-O-methyl phosphorothioate modified oligomers, 2'-O-methoxyethyl (2'-O-MOE) modified oligomers, and 2'-O-methyl oligonucleotides, or combinations thereof, as well as other antisense agents known in the art.

[0067] An antisense oligomer "specifically hybridizes" to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions with a Tm of greater than 37°C, greater than 45°C, preferably at least 50°C, and typically 60°C to 80°C or greater. The "Tm" of an oligomer is the temperature at which it is 50% hybridized to a complementary polynucleotide. Tm is determined under standard conditions in saline, for example, as described in Miyada et al. (1987) Methods Enzymol. 154:94-107. Such hybridization can occur by "near" or "substantial" complementarity of the antisense oligomer to the target sequence, as well as complete complementarity.

[0068] The terms "complementary" and "complementarity" refer to oligonucleotides (i.e., nucleotide sequences) related by base-pairing rules. For example, the sequence "TGA(5'-3')" is complementary to the sequence "TCA(5'-3')". Complementarity may be "partial", in which only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there may be "complete", "total", or "perfect" (100%) complementarity between nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desired, some embodiments may include one or more mismatches to the target RNA, preferably 6, 5, 4, 3, 2, or 1 mismatch. Such hybridization can occur with "near" or "substantial" complementarity of the antisense oligomer to the target sequence, as well as complete complementarity. In some embodiments, the oligomer can hybridize to the target sequence with about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementarity. Variations at any position within the oligomer are included. In certain embodiments, sequence variations near the ends of the oligomer are generally preferred over variations in the interior, and when present, are typically about 6, 5, 4, 3, 2, or 1 nucleotide at the 5' end, 3' end, or both ends.

[0069] The terms "TEG", "EG3", or "triethylene glycol tail" refer to a triethylene glycol moiety conjugated to an oligomer, e.g., at its 3' or 5' end. For example, in some embodiments, "TEG" refers to, e.g., A' of a conjugate of Formula I or Formula IV is of the following formula: [ka]

[0070] Naturally occurring nucleotide bases include adenine, guanine, cytosine, thymine, and uracil, which have the symbols A, G, C, T, and U, respectively. Nucleotide bases can also include analogs of the naturally occurring nucleotide bases. Base pairing typically occurs between purine A and pyrimidines T or U, and between purine G and pyrimidine C.

[0071] Oligonucleotides may also contain nucleobase (simply referred to in the art as "base") modifications or substitutions. Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more of the most common purine or pyrimidine bases found in nucleic acids are replaced with less common or unnatural bases. In some embodiments, the nucleobase is covalently linked to the morpholine ring of the nucleoside at the N9 atom of a purine base or the N1 atom of a pyrimidine base.

[0072] Purine bases contain a pyrimidine ring fused to an imidazole ring, as described by the general formula: [ka]

[0073] Adenine and guanine are the two most commonly found purine nucleobases in nucleic acids. These can be substituted with other naturally occurring purines, including but not limited to, N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0074] The pyrimidine base contains a six-membered pyrimidine ring, as described by the general formula: [ka]

[0075] Cytosine, uracil, and thymine are the most commonly found pyrimidine bases in nucleic acids. They may be substituted with other naturally occurring pyrimidines, including, but not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain a thymine base instead of uracil.

[0076] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamps and derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaaden ... These include aza-7-deaza-2,6-diaminopurine, super-G, super-A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil, or derivatives thereof; and abasic bases such as 2,6-difluorotoluene or abasic sites, such as degenerate or universal bases (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is replaced by nitrogen (azaribose)). Pseudouracil is a naturally occurring isomerized version of uracil, with a C-glycoside rather than the usual N-glycoside in uridine.

[0077] Certain modified or substituted nucleobases are particularly useful for improving the binding affinity of the antisense oligonucleotides of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcitrine. In various embodiments, the nucleobases may include 5-methylcytosine substituents, which have been shown to improve nucleic acid duplex stability by 0.6-1.2°C.

[0078] In some embodiments, modified or substituted nucleobases are useful for facilitating the purification of antisense oligonucleotides.For example, in certain embodiments, antisense oligonucleotides can contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In certain antisense oligonucleotides, a string of three or more consecutive guanine bases can cause oligonucleotide aggregation, complicating purification. In such antisense oligonucleotides, one or more consecutive guanines can be replaced with hypoxanthine. Substitution of one or more guanines in a string of three or more consecutive guanine bases with hypoxanthine can reduce the aggregation of antisense oligonucleotides, thereby facilitating purification.

[0079] The term "abasic subunit" refers to a subunit in an antisense oligomer that does not contain a purine or pyrimidine. In one embodiment, an "abasic subunit" is hydrogen. Abasic subunits incorporated herein retain the antisense backbone but do not contain a purine or pyrimidine base. Non-limiting examples of antisense oligomers that contain abasic subunits are provided below. [ka]

[0080] The oligonucleotides provided herein are synthetic and do not include antisense compositions of biological origin. The molecules of the present disclosure may be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as, for example, liposomes, receptor targeting molecules, oral, rectal, topical, or other formulations, to aid in uptake, distribution, or absorption, or combinations thereof.

[0081] As used herein, "nucleic acid analog" refers to a non-naturally occurring nucleic acid molecule. Nucleic acids are polymers of nucleotide subunits linked together in a linear structure. Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is attached to a phosphate group. Successive phosphate groups are linked together through phosphodiester bonds to form a polymer. Two common forms of naturally occurring nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). One end of the chain bears a free phosphate group attached to the 5'-carbon atom of the sugar moiety, which is referred to as the 5'-end of the molecule. The other end has a free hydroxyl (-OH) group at the 3'-carbon of the sugar moiety, which is referred to as the 3'-end of the molecule. Nucleic acid analogs can contain one or more non-naturally occurring nucleobases, sugars, and / or internucleotide linkages, such as phosphorodiamidate morpholino oligomers (PMOs). As disclosed herein, in certain embodiments, a "nucleic acid analog" is a PMO, and in certain embodiments, a "nucleic acid analog" is a positively charged cationic PMO.

[0082] "Morpholino oligomer" or "PMO" refers to a polymeric molecule having a backbone supporting bases capable of hydrogen bonding to a typical polynucleotide, where the polymer lacks a pentose sugar backbone moiety and, more specifically, a ribose backbone linked by phosphodiester bonds that are typical of nucleotides and nucleosides, but instead contains ring nitrogens that are linked through the ring nitrogens. An exemplary "morpholino" oligomer comprises morpholino subunit structures linked together by phosphoramidate or phosphorodiamidate linkages, with the morpholino nitrogen of one subunit attached to the 5' exocyclic carbon of an adjacent subunit, and each subunit contains a purine or pyrimidine base pairing moiety effective to bind to a base in a polynucleotide by base-specific hydrogen bonding. Morpholino oligomers (including antisense oligomers) are described in detail in, e.g., U.S. Patent Nos. 5,034,506, 5,142,047, 5,166,315, 5,185,444, 5,217,866, 5,506,337, 5,521,063, 5,698,685, 8,076,476, and 8,299,206, and PCT Publication No. 2009 / 064471, all of which are incorporated by reference herein in their entireties.

[0083] Preferred morpholino oligomers are phosphorodiamidate-linked morpholino oligomers, referred to herein as PMOs. Such oligomers are composed of morpholino subunit structures such as those shown below: [ka] In the formula, X is NH 2 , NHR, or NR 2 (R is lower alkyl, preferably methyl) and Y 1 is O, Z is O, and P i and P jis a purine or pyrimidine base pairing moiety effective to bind to bases in a polynucleotide by base-specific hydrogen bonding. Also preferred are structures having alternating phosphorodiamidate linkages, where X is a lower alkoxy, such as methoxy or ethoxy, and Y is a 1 is NH or NR, R is lower alkyl, and Z is O.

[0084] Representative PMOs include those in which the intersubunit linkage is linkage(A1). See Table 1. [Table 1]

[0085] A "phosphoramidate" group contains a phosphorus with three attached oxygen atoms and one attached nitrogen atom, while a "phosphorodiamidate" group contains a phosphorus with two attached oxygen atoms and two attached nitrogen atoms. Representative examples of phosphorodiamidates are shown below: [ka] Each P i is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, and the nucleobase is independently selected at each occurrence from pyridine, pyrimidine, triazinane, purine, and deazapurine. 3-6 Heterocyclic rings, where n is an integer from 6 to 38. The ring nitrogen of the subunit at the 3' end of the PMO can be capped with a capping group such as acetyl, or can be decapped with free hydrogen.

[0086] In the uncharged or modified intersubunit linkages of the oligomers described herein, one nitrogen is always pendant to the backbone. The second nitrogen in the phosphorodiamidate linkage is typically a ring nitrogen in a morpholino ring structure.

[0087] PMOs are water-soluble, uncharged, or substantially uncharged antisense molecules that inhibit gene expression by preventing the binding or progression of splicing or translation machinery components. PMOs have also been shown to inhibit or block viral replication (Stein, Skilling et al. 2001; McCaffrey, Meuse et al. 2003). They are highly resistant to enzymatic digestion (Hudziak, Barofsky et al. 1996). PMOs have demonstrated high antisense specificity and efficacy in cell-free and cell culture models in vitro (Stein, Foster et al. 1997; Summerton and Weller 1997), and in zebrafish, frog, and sea urchin embryos in vivo (Heasman, Kofron et al. 2000; Nasevicius and Ekker 2000), as well as in adult animal models such as rats, mice, rabbits, dogs, and pigs (see, e.g., Arora and Iversen 2000; Qin, Taylor et al. 2000; Iversen 2001; Kipshidze, Keane et al. 2001; Devi 2002; Devi, Oldenkamp et al. 2002; Kipshidze, Kim et al. 2002; Ricker, Mata et al. 2002).

[0088] Antisense PMO oligomers have been shown to be taken up into cells with fewer non-specific effects and to be more consistently effective in vivo than other widely used antisense oligonucleotides (see, for example, P. Iversen, "Phosphoramidite Morpholino Oligomers," in Antisense Drug Technology, S.T. Crooke, ed., Marcel Dekker, Inc., New York, 2001). Conjugation of PMOs to arginine-rich peptides has been shown to increase their cellular uptake (see, for example, U.S. Patent No. 7,468,418, which is incorporated herein by reference in its entirety).

[0089] As used herein, "charged," "uncharged," "cationic," and "anionic" refer to the prevailing state of a chemical moiety at about neutral pH, e.g., about 6 to 8. For example, the terms can refer to the prevailing state of a chemical moiety at physiological pH, i.e., about 7.4.

[0090] "Cationic PMO" or "PMO+" refers to a phosphorodiamidate morpholino oligomer containing any number of (1-piperazino)phosphinylideneoxy, (1-(4-(ω-guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages (A2 and A3, see Table 1) previously described (see, e.g., PCT Publication No. 2008 / 036127, which is incorporated by reference in its entirety).

[0091] The "backbone" of an oligonucleotide analog (e.g., an uncharged oligonucleotide analog) refers to the structure that supports the base-pairing moieties, e.g., for the morpholino oligomers described herein, the "backbone" includes morpholino ring structures linked by intersubunit linkages (e.g., phosphorus-containing linkages). A "substantially uncharged backbone" refers to the backbone of an oligonucleotide analog in which less than 50% of the intersubunit linkages are charged at about neutral pH. For example, a substantially uncharged backbone may include less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or even less than 0% of the intersubunit linkages that are charged at about neutral pH. In some embodiments, a substantially uncharged backbone includes up to one charged (at physiological pH) intersubunit linkage for every four uncharged (at physiological pH), up to one for every eight uncharged linkages, or up to one for every sixteen uncharged linkages. In some embodiments, the nucleic acid analogs described herein are completely uncharged.

[0092] The term "targeting base sequence", or simply the term "targeting sequence", is a sequence in a nucleic acid analog that is complementary (meaning, in addition, substantially complementary) to a target sequence, for example, a target sequence in the human RNA genome. The entire sequence of the analog compound, or only a portion thereof, may be complementary to the target sequence. For example, in an analog having 20 bases, only 12-14 may be the targeting sequence. Typically, the targeting sequence is formed of consecutive bases in the analog, but may alternatively be formed of non-consecutive sequences that, for example, when placed together from both ends of the analog, constitute a sequence that spans the target sequence.

[0093] The term "peptide" refers to a compound that includes multiple linked amino acids. The peptides provided herein can be considered cell membrane penetrating peptides.

[0094] As used herein, a "cell membrane penetrating peptide" (CPP) or "carrier peptide" is a relatively short peptide that can facilitate uptake of a PMO by a cell, thereby delivering the PMO to the interior (cytoplasm) of the cell. A CPP or carrier peptide is typically about 12 to about 40 amino acids in length. The length of the carrier peptide is not particularly limited and varies in different embodiments. In some embodiments, the carrier peptide comprises 4 to 40 amino acid subunits. In other embodiments, the carrier peptide comprises 6 to 30, 6 to 20, 8 to 25, or 10 to 20 amino acid subunits. In various embodiments, CPP embodiments of the present disclosure may include an arginine-rich peptide, as further described below.

[0095] As used herein, "peptide-conjugated phosphorodiamidate-linked morpholino oligomer" or "PPMO" refers to a PMO covalently attached to a peptide, such as a cell membrane-penetrating peptide (CPP) or carrier peptide. The cell membrane-penetrating peptide facilitates uptake of the PMO by the cell, thereby delivering the PMO to the interior (cytoplasm) of the cell. Depending on its amino acid sequence, the CPP can be generally effective or can be specifically or selectively effective in delivering the PMO to a particular cell type. The PMO and CPP are typically linked at their termini, e.g., the C-terminus of the CPP can be linked to the 5'-terminus of the PMO, or the 3'-terminus of the PMO can be linked to the N-terminus of the CPP. The PPMO can include uncharged PMOs, charged (e.g., cationic) PMOs, and mixtures thereof. In one embodiment, the linking moiety of the conjugates described herein can be cleaved to release the PPMO.

[0096] The carrier peptide may be linked to the nucleic acid analog directly or via an optional linker, e.g., one or more additional naturally occurring amino acids such as cysteine ​​(C), glycine (G), or proline (P), or additional amino acid analogs such as 6-aminohexanoic acid (X), beta-alanine (B), or XB. Other linking moieties known in the art may also be used.

[0097] An "amino acid subunit" is generally an α-amino acid residue (-CO-CHR-NH-), but may also be a β- or other amino acid residue (e.g., -CO-CH 2 CHR-NH-), where R is an amino acid side chain.

[0098] The term "naturally occurring amino acid" refers to an amino acid present in proteins found in nature, examples of which include alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y). The term "unnatural amino acid" refers to an amino acid that does not occur in proteins found in nature, examples of which include beta-alanine (β-Ala) and 6-aminohexanoic acid (Ahx).

[0099] An agent is "actively taken up by a mammalian cell" if the agent can enter the cell by a mechanism other than passive diffusion across the cell membrane. An agent can be transported, for example, by "active transport," which refers to the transport of an agent across a mammalian cell membrane by an ATP-dependent transport mechanism, or by "facilitated transport," which refers to the transport of an antisense agent across the cell membrane by a transport mechanism that requires binding of the agent to a transport protein, which in turn facilitates passage of the bound agent across the membrane.

[0100] As used herein, an "effective amount" refers to any amount of a substance that is sufficient to achieve a desired biological result. A "therapeutically effective amount" refers to any amount of a substance that is sufficient to achieve a desired therapeutic result.

[0101] As used herein, a "subject" is a mammal, which may include a mouse, rat, hamster, guinea pig, rabbit, goat, sheep, cat, dog, pig, cow, horse, monkey, non-human primate, or human. In certain embodiments, the subject is a human.

[0102] "Treatment" of an individual (e.g., a mammal, such as a human) or cell is any type of intervention used to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed prophylactically or following the initiation of a pathological event or contact with a pathogen.

[0103] II. Peptide-oligonucleotides In some embodiments, provided herein are antisense oligomers comprising modified antisense oligonucleotides, The modified antisense oligonucleotide is 18 to 40 subunits in length and contains a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; The antisense oligonucleotide comprises a morpholino oligomer, each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0104] In one embodiment, the abasic subunit is internal to the targeting sequence.

[0105] In one embodiment, the modified antisense oligonucleotide is 20-40 subunits in length. In another embodiment, the modified antisense oligonucleotide is 19-29 subunits in length. In another embodiment, the modified antisense oligonucleotide is 18-40, 19-30, 19-29, 20-40, 20-30, 20-25, 21-40, 21-30, 21-25, 22-40, 22-30, 22-25, 23-40, 23-30, or 23-25 ​​subunits in length. In yet another embodiment, the modified antisense oligonucleotide is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 subunits in length.

[0106] In one embodiment, the modified antisense oligonucleotide is an antisense oligomer of Formula I: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, A' is -N(H)CH 2 C(O)NH 2 , -N(C 1-6 -alkyl)CH 2 C(O)NH 2 , [ka] is selected from the group consisting of R 5 is -C(O)(O-alkyl) x -OH, where x is 3 to 10, and each alkyl group, in each occurrence, is independently selected from the group consisting of C 2-6 - alkyl or or R 5 is H, -C(O)C 1-6 -Alkyl, trityl, monomethoxytrityl, -(C 1-6 -alkyl)-R 6 , -(C 1-6 -heteroalkyl)-R 6 , aryl-R6 , heteroaryl-R 6 , -C(O)O-(C 1-6 -alkyl)-R 6 , -C(O)O-aryl-R 6 , -C(O)O-heteroaryl-R 6 , and [ka] is selected from R 6 are OH, SH, and NH 2 or R 6 is O, S, or NH, each of which is covalently attached to a solid support; Each R 1 are independently OH and -N(R 3 )(R 4 ) wherein each R 3 and R 4 is independently in each occurrence H or -C 1-6 -alkyl, Each R 2 is independently selected at each occurrence from H (abasic), a nucleobase, and a nucleobase functionalized with a chemical protecting group, and the nucleobase is independently selected at each occurrence from pyridine, pyrimidine, purine, and deaza-purine. 3-6 Contains a heterocycle, t is 8 to 40; E' is H, -C 1-6 -Alkyl, -C(O)C 1-6 -Alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, [ka] is selected from During the ceremony, Q is -C(O)(CH 2 ) 6 -C(O)- or -C(O)(CH 2 ) 2 S 2 (CH 2 ) 2C(O)-, R 7 is -(CH 2 ) 2 O C (O) N (R 8 ) 2 where R 8 is -(CH 2 ) 6 NHC(=NH)NH 2 and L is glycine, proline, W, WW, or R 9 and L is covalently linked by an amide bond to the N-terminus or C-terminus of J; W is -C(O)-(CH 2 ) m -NH-, where m is 2 to 12; R 9 teeth, [ka] is selected from the group consisting of n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5; R 10 is selected from a bond, glycine, proline, W, or WW; R 11 is glycine, proline, W, WW, and [ka] is selected from the group consisting of R 16 is selected from a bond, glycine, proline, W, or WW; R 16 is covalently attached by an amide bond to the N-terminus or C-terminus of J, where J is a cell membrane penetrating peptide; G is H, -C(O)C 1-6 -alkyl, benzoyl, and stearoyl, and G is covalently linked to J.

[0107] In one aspect, disclosed herein is an antisense oligomer, the antisense oligomer being a conjugate comprising a modified antisense oligonucleotide and a cell membrane penetrating peptide, The modified antisense oligonucleotide is 18 to 40 subunits in length and contains a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; The antisense oligonucleotide comprises a morpholino oligomer, The antisense oligonucleotide is covalently linked to a cell membrane-permeable peptide, each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0108] In one embodiment, the modified antisense oligonucleotide is 20 to 40 subunits in length. In another embodiment, the modified antisense oligonucleotide is 19 to 29 subunits in length.

[0109] In one embodiment, the target region comprises a sequence selected from the group consisting of SEQ ID NO:2 (GAA-IVS1(-189-167)) and SEQ ID NO:3 (GAA-IVS1(-80-24)). In a further embodiment, the target region comprises the sequence set forth in SEQ ID NO:2. In another embodiment, the target region comprises the sequence set forth in SEQ ID NO:3.

[0110] In one embodiment, the target region is selected from GAA-IVS1(-189-167), GAA-IVS1(-80-56), GAA-IVS1(-76-52), GAA-IVS1(-74-55), GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), GAA-IVS1(-65-41), and GAA-IVS1(-49-24). In a further embodiment, the target region is GAA-IVS1(-189-167). In another embodiment, the target region is GAA-IVS1(-72,-48). In yet another embodiment, the targeting region is GAA-IVS1(-71,-47). In yet another embodiment, the targeting region is GAA-IVS1(-70,-46). In one embodiment, the targeting region is GAA-IVS1(-69-45). In another embodiment, the targeting region is GAA-IVS1(-65,-41). In yet another embodiment, the targeting region is GAA-IVS1(-66,-42).

[0111] In one embodiment, the targeting sequence comprises the sequence CCA GAA GGA AXX XCG AGA AAA GC (SEQ ID NO: 4), where each X is independently selected from guanine (G) or non-basic (B), and at least one X is B. In another embodiment, the targeting sequence comprises i) SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC); ii) Sequence number 6 (CCA GAA GGA AGB GCG AGA AAA GC), iii) SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC), iv) SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC), v) SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC), and vi) SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

[0112] In one embodiment, B is H.

[0113] In one embodiment, the targeting sequence comprises SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC). In another embodiment, the targeting sequence comprises SEQ ID NO: 6 (CCA GAA GGA AGB GCG AGA AAA GC). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC). In one embodiment, the targeting sequence comprises SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC). In another embodiment, the targeting sequence comprises SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

[0114] In one embodiment, the targeting sequence consists of the sequence CCA GAA GGA AXX XCG AGA AAA GC (SEQ ID NO: 4). In another embodiment, the targeting sequence consists of SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 6 (CCA GAA GGA AGB GCG AGA AAA GC). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC). In one embodiment, the targeting sequence consists of SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC). In another embodiment, the targeting sequence consists of SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

[0115] In one embodiment, the target region is selected from the group consisting of GAA-IVS1(-80-56), GAA-IVS1(-76-52), GAA-IVS1(-74-55), GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), GAA-IVS1(-65-41), and GAA-IVS1(-49-24). In another embodiment, the target region is selected from the group consisting of GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), and GAA-IVS1(-65-41).

[0116] In one embodiment, the targeting region is: i) Sequence number 11 (CTC ACX XXX CTC TCA AAG CAG CTC T), ii) Sequence number 12 (ACT CAC XXX XCT CTC AAA GCA GCT C), iii) Sequence number 13 (CAC TCA CXX XXC TCT CAA AGC AGC T), iv) SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C); v) Sequence number 15 (GCG GCA CTC ACX XXX CTC TCA AAG C), vi) comprising a sequence selected from the group consisting of SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G); Each X is independently selected from guanine (G) or abasic (B), and at least one X is B. In one embodiment, the targeting sequence is i) SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C); ii) SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C); iii) SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C); iv) SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C); v) SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C); vi) SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C); vii) SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C), and viii) SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0117] In one embodiment, B is H.

[0118] In one embodiment, the targeting sequence comprises SEQ ID NO: 11 (CTC ACX XXX CTC TCA AAG CAG CTC T). In another embodiment, the targeting sequence comprises SEQ ID NO: 12 (ACT CAC XXX XCT CTC AAA GCA GCT C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C). In one embodiment, the targeting sequence comprises SEQ ID NO: 15 (GCG GCA CTC ACX XXX CTC TCA AAG C). In another embodiment, the targeting sequence comprises SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C). In one embodiment, the targeting sequence comprises SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C). In another embodiment, the targeting sequence comprises SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C). In one embodiment, the targeting sequence comprises SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C). In another embodiment, the targeting sequence comprises SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0119] In one embodiment, the targeting sequence consists of SEQ ID NO: 11 (CTC ACX XXX CTC TCA AAG CAG CTC T). In another embodiment, the targeting sequence consists of SEQ ID NO: 12 (ACT CAC XXX XCT CTC AAA GCA GCT C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C). In one embodiment, the targeting sequence consists of SEQ ID NO: 15 (GCG GCA CTC ACX XXX CTC TCA AAG C). In another embodiment, the targeting sequence consists of SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C). In one embodiment, the targeting sequence consists of SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C). In another embodiment, the targeting sequence consists of SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C). In one embodiment, the targeting sequence consists of SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C). In another embodiment, the targeting sequence consists of SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0120] In one embodiment, the targeting sequence is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent complementary to the target region, excluding the abasic subunit. In another embodiment, the targeting sequence is at least 84%, at least 88%, or at least 92% complementary to the target region, excluding the abasic subunit. In yet another embodiment, the targeting sequence is at least 90% complementary to the target region, excluding the abasic subunit. In yet another embodiment, the targeting sequence is at least 95% complementary to the target region, excluding the abasic subunit. In yet another embodiment, the targeting sequence is 100% complementary to the target region, excluding the abasic subunit.

[0121] In one embodiment, each abasic subunit is at least 8 subunits from the 5' or 3' end of the targeting sequence.

[0122] The antisense oligonucleotide may contain from 1 to 5 abasic subunits, hi one embodiment, the antisense oligonucleotide contains 1, 2, 3, or 4 abasic subunits.

[0123] In another embodiment, the antisense oligomer is an antisense-oligomer conjugate having formula IV: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, A' is -N(H)CH 2 C(O)NH 2 , -N(C 1-6 -alkyl)CH 2 C(O)NH 2 , [ka] is selected from the group consisting of R5 is -C(O)(O-alkyl) x -OH, where x is 3 to 10, and each alkyl group, in each occurrence, is independently selected from the group consisting of C 2-6 - alkyl or or R 5 is H, -C(O)C 1-6 -Alkyl, trityl, monomethoxytrityl, -(C 1-6 -alkyl)-R 6 , -(C 1-6 -heteroalkyl)-R 6 , aryl-R 6 , heteroaryl-R 6 , -C(O)O-(C 1-6 -alkyl)-R 6 , -C(O)O-aryl-R 6 , -C(O)O-heteroaryl-R 6 , and [ka] is selected from R 6 are OH, SH, and NH 2 or R 6 is O, S, or NH, each of which is covalently attached to a solid support; Each R 1 are independently OH and -N(R 3 )(R 4 ) wherein each R 3 and R 4 is independently in each occurrence H or -C 1-6 -alkyl, Each R 2 is independently selected at each occurrence from H (abasic), a nucleobase, and a nucleobase functionalized with a chemical protecting group, and the nucleobase is independently selected at each occurrence from pyridine, pyrimidine, purine, and deaza-purine. 3-6 Contains a heterocycle, t is 8 to 40; E' is H, -C 1-6 -Alkyl, -C(O)C 1-6-Alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, [ka] is selected from During the ceremony, Q is -C(O)(CH 2 ) 6 -C(O)- or -C(O)(CH 2 ) 2 S 2 (CH 2 ) 2 C(O)-, R 7 is -(CH 2 ) 2 O C (O) N (R 8 ) 2 where R 8 is -(CH 2 ) 6 NHC(=NH)NH 2 and L is glycine, proline, W, WW, or R 9 and L is covalently linked by an amide bond to the N-terminus or C-terminus of J; W is -C(O)-(CH 2 ) m -NH-, where m is 2 to 12; R 9 teeth, [ka] is selected from the group consisting of n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5; R 10 is selected from a bond, glycine, proline, W, or WW; R 11 is glycine, proline, W, WW, and [ka] is selected from the group consisting of R 16 is selected from a bond, glycine, proline, W, or WW; R 16 is covalently attached by an amide bond to the N-terminus or C-terminus of J, where J is a cell membrane penetrating peptide; G is H, -C(O)C 1-6 -alkyl, benzoyl, and stearoyl, and G is covalently bonded to J; however, A' is [ka] or E' is [ka] This is subject to the condition that:

[0124] In one embodiment, E' is H, -C 1-6 -Alkyl, -C(O)C 1-6 -alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and [ka] is selected from.

[0125] In one embodiment, A' is N(C 1-6 -alkyl)CH 2 C(O)NH 2 , [ka] is selected from.

[0126] In one embodiment, E' is H, -C(O)CH 3 , benzoyl, stearoyl, trityl, 4-methoxytrityl, and [ka] is selected from.

[0127] In one embodiment, A' is N(C 1-6 -alkyl)CH 2 C(O)NH 2 , [ka] is selected from E' is [ka] It is.

[0128] In one embodiment, A' is [ka] and E' is H, -C(O)CH 3 , trityl, 4-methoxytrityl, benzoyl, and stearoyl. In one embodiment, the conjugate of formula IV is [ka] [ka] is a conjugate selected from In the formula, E' is H, C 1-6 -Alkyl, -C(O)CH 3 , benzoyl, and stearoyl.

[0129] In one embodiment, the conjugate is of formula (IVa): In one embodiment, the conjugate is of formula (IVb):

[0130] In one embodiment, each R 1 is -N(CH 3 ) 2 It is.

[0131] In one embodiment, each nucleobase, at each occurrence, is independently selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine. In one embodiment, L is glycine. In one embodiment, L is proline. In one embodiment, L is -C(O)-(CH 2 ) 5 In one embodiment, L is -C(O)-(CH 2 ) 2 In one embodiment, L is -C(O)-(CH 2 ) 2 -NH-C(O)-(CH 2 ) 5 -NH-.

[0132] In one embodiment, L is [ka] where R 10 is a bond, and R 11 Glycine and [ka] is selected from.

[0133] In one embodiment, L is [ka] where R 10 is a bond, and R 11 Glycine and [ka] is selected from.

[0134] In one embodiment, L is [ka] where R 10 is a bond, and R 11 Glycine and [ka] is selected from.

[0135] In one embodiment, J is rTAT, TAT, R 9 F 2 , R 5 F 2 R 4 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , (RXR) 4 , (RXR) 5 , (RXRRBR) 2 , (RAR) 4 F 2 , (RGR) 4 F 2 is selected from.

[0136] In one embodiment, G is H, C(O)CH 3 , benzoyl, and stearoyl. In one embodiment, G is H or -C(O)CH 3 In one embodiment, G is H. In one embodiment, G is -C(O)CH 3 It is.

[0137] In one embodiment, the targeting sequence is complementary to a target region within intron 1 (SEQ ID NO: 1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene, and at least one subunit is an abasic subunit. In another embodiment, the target region comprises a sequence selected from the group consisting of SEQ ID NO: 2 (GAA-IVS1(-189-167)) and SEQ ID NO: 3 (GAA-IVS1(-80-24)).

[0138] In one embodiment, the targeting sequence comprises the sequence: i) Sequence number 4 (CCA GAA GGA AXX XCG AGA AAA GC), ii) Sequence number 11 (CTC ACX XXX CTC TCA AAG CAG CTC T), iii) Sequence number 12 (ACT CAC XXX XCT CTC AAA GCA GCT C), iv) SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T), v) SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C); vi) Sequence number 15 (GCG GCA CTC ACX XXX CTC TCA AAG C), vii) SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G),

[0139] Each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

[0140] In one embodiment, B is H.

[0141] In a further embodiment, the targeting region is: i) SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC); ii) Sequence number 6 (CCA GAA GGA AGB GCG AGA AAA GC), iii) SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC), iv) SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC), v) SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC), vi) Sequence number 10 (CCA GAA GGA ABG BCG AGA AAA GC), vii) SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C); viii) SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C); ix) SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C); x) SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C); xi) SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C); xii) SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C); xiii) SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C), and xiv) SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0142] In one embodiment, B is H.

[0143] In one embodiment, the conjugate is a pharma- ceutically acceptable salt thereof and at least one pharma- ceutically acceptable carrier.

[0144] In one embodiment, provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate or pharmaceutical composition.

[0145] In one embodiment, the disease is Pompe disease. In one embodiment, the subject is a human. In a further embodiment, the human is a child. In another embodiment, the human is an adult.

[0146] III. Characteristics of Oligomeric Chemicals Also provided herein is an antisense oligomer, wherein the antisense oligomer is a modified antisense oligomer.Examples of modified antisense oligomers include, but are not limited to, morpholino oligomers, phosphorothioate modified oligomers, 2'-O-methyl modified oligomers, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate oligomers, 2'-O-MOE modified oligomers, 2'-fluoro modified oligomers, 2'-O,4'-C-ethylene bridged nucleic acid (ENA), tricyclo-DNA, tricyclo-DNA phosphorothioate subunits, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers, including any combination of the above.Phosphorothioate and 2'-O-Me modified chemistries can be combined to produce 2'-O-Me-phosphorothioate backbones. See, e.g., PCT Publication Nos. 2013 / 112053 and 2009 / 008725, which are incorporated by reference in their entireties.

[0147] In some embodiments, the nucleobases of the modified antisense oligomer are linked to morpholino ring structures which are joined by phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one ring structure to the 5' exocyclic carbon of an adjacent ring structure.

[0148] In some embodiments, the nucleobases of the antisense oligomer are linked to a peptide nucleic acid (PNA), in which the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudopeptide polymer to which the nucleobases are linked. In some aspects, at least one of the nucleobases of the antisense oligomer is linked to a locked nucleic acid (LNA), in which the locked nucleic acid structure is a chemically modified nucleotide analog in which the ribose moiety has an extra bridge connecting the 2' oxygen and the 4' carbon.

[0149] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a bridged nucleic acid (BNA), where the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone. In some aspects, at least one of the nucleobases of the antisense oligomer is linked to a 2'-O,4'-C-ethylene bridged nucleic acid (ENA).

[0150] In some embodiments, modified antisense oligomers may contain unlocked nucleic acid (UNA) subunits. UNAs and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit has been truncated.

[0151] In some embodiments, modified antisense oligomers contain one or more phosphorothioates (or S-oligos) in which one of the non-bridging oxygens is replaced by sulfur. In some aspects, modified antisense oligomers contain one or more 2'O-methyl, 2'O-MOE, MCE, and 2'-F in which the 2'-OH of the ribose is replaced with a methyl, methoxyethyl, 2-(N-methylcarbamoyl)ethyl, or fluoro group, respectively.

[0152] In some embodiments, the modified antisense oligomer is tricyclo-DNA (tc-DNA), a constrained DNA analog in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry for torsion angle g.

[0153] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a bridged nucleic acid (BNA), and the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone. In some aspects, at least one of the nucleobases of the antisense oligomer is linked to a 2'-O,4'-C-ethylene bridged nucleic acid (ENA). In such aspects, each nucleobase linked to a BNA or ENA comprises a 5-methyl group. Exemplary embodiments of oligomer chemistries of the present disclosure are further described below.

[0154] 1. Peptide Nucleic Acid (PNA) Peptide nucleic acids (PNAs) are analogs of DNA whose backbone is structurally isomorphous with a deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs, which contain natural pyrimidine and purine bases, hybridize to complementary oligomers according to Watson-Crick base pairing rules, mimicking DNA in terms of base pair recognition. The backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases. Non-limiting examples of PNAs are shown below. [ka]

[0155] Despite the radical structural change relative to the natural structure, PNAs are capable of sequence-specific binding to DNA or RNA in a helical form. PNA features include high binding affinity to complementary DNA or RNA, destabilizing effects caused by single base mismatches, resistance to nucleases and proteases, salt-independent hybridization to DNA or RNA, and triplex formation with homopurine DNA. PANAGENE™ has developed its proprietary Bts PNA monomer (Bts, benzothiazole-2-sulfonyl group) and proprietary oligomerization process. PNA oligomerization using Bts PNA monomer consists of repeated cycles of deprotection, coupling, and capping. PNAs can be produced synthetically using any technique known in the art. See, for example, U.S. Patent Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. See also, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262 for the preparation of PNAs. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the above is incorporated by reference in its entirety.

[0156] 2. Locked Nucleic Acid (LNA) Antisense oligomers may also contain "locked nucleic acid" subunits (LNA). "LNA" is a member of a class of modifications called bridged nucleic acids (BNA). BNAs are characterized by a covalent bond that locks the conformation of the ribose ring to a C30-endo (Northern) sugar pucker. In the case of LNA, the bridge consists of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking to enhance hybridization and thermal stability.

[0157] The structures of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54:3607; Jesper Wengel, Accounts of Chem. Research (1999) 32:301; Obika, et al., Tetrahedron Letters (1997) 38:8735; Obika, et al., Tetrahedron Letters (1998) 39:5401; and Obika, et al., Bioorganic Medicinal Chemistry (2008) 16:9230, which are incorporated herein by reference in their entireties. Non-limiting examples of LNAs are provided below. [ka]

[0158] The antisense oligomer of the present disclosure may incorporate one or more LNAs. In some cases, the antisense oligomer may be entirely composed of LNA. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligomers are described, for example, in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Exemplary intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers may be used. Further embodiments include LNA-containing antisense oligomers, in which each LNA subunit is separated by a DNA subunit. Certain antisense oligomers are composed of alternating LNA and DNA subunits, where the intersubunit linker is phosphorothioate.

[0159] 3. Ethylene-bridged nucleic acid (ENA) 2'O,4'C-Ethylene-bridged Nucleic Acids (ENAs) are another member of the BNA class. Non-limiting examples are provided below. [ka]

[0160] ENA oligomers and their preparation are described in Obika et al., Tetrahedron Lett (1997) 38(50):8735, which is incorporated herein by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more ENA subunits.

[0161] 4. Unlocked Nucleic Acid (UNA) Antisense oligomers can also contain unlocked nucleic acid (UNA) subunits. UNA and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit is broken. LNA is conformationally restricted (relative to DNA and RNA), whereas UNA is highly flexible. UNA is disclosed, for example, in WO2016 / 070166. Non-limiting examples of UNA are shown below. [ka] Exemplary intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers can be used.

[0162] 5. Phosphorothioates "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens is replaced with a sulfur. Non-limiting examples of phosphorothioates are shown below. [ka]

[0163] Sulfurization of internucleotide bonds reduces the action of endonucleases and exonucleases, including 5' to 3' and 3' to 5' DNA POL1 exonuclease, nucleases S1 and P1, RNases, serum nucleases, and snake venom phosphodiesterases. Phosphorothioates are made by two major routes: the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonates, or sulfurization of phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990, which is incorporated herein by reference in its entirety). The latter method avoids the problems of the insolubility of elemental sulfur in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also result in phosphorothioates of higher purity.

[0164] 6. Tricyclo-DNA and tricyclo-phosphorothioate subunits Tricyclo-DNA (tc-DNA) is a class of constrained DNA analogues in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry of the torsion angle γ. Homobasic adenine and thymine-containing tc-DNA forms highly stable AT base pairs with complementary RNA. Tricyclo-DNA and its synthesis are described in International Patent Application Publication No. 2010 / 115993, which is incorporated herein by reference in its entirety. The antisense oligomers of the present disclosure may incorporate one or more tricyclo-DNA subunits. In some cases, the antisense oligomers may be entirely composed of tricyclo-DNA subunits.

[0165] Tricyclo-phosphorothioate subunits are tricyclo-DNA subunits with phosphorothioate intersubunit linkages. Tricyclo-phosphorothioate subunits and their synthesis are described in International Patent Application Publication No. 2013 / 053928, which is incorporated herein by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more tricyclo-DNA subunits. In some cases, antisense oligomers may be entirely composed of tricyclo-DNA subunits. Non-limiting examples of tricyclo-DNA / tricyclo-phosphorothioate subunits are shown below. [ka]

[0166] 7. 2'-O-Methyl, 2'-O-MOE, and 2'-F Oligomers A "2'-O-Me oligomer" molecule has a methyl group at the 2'-OH residue of the ribose molecule. 2'-O-Me-RNA behaves the same (or similar) as DNA, but is protected from nuclease degradation. 2'-O-Me-RNA can also be combined with phosphorothioate oligomers (PTO) for further stabilization. 2'O-Me oligomers (phosphodiester or phosphorothioate) can be synthesized according to conventional techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004, which is incorporated herein by reference in its entirety). Non-limiting examples of 2'O-Me oligomers are shown below. [ka] 2'O-Me

[0167] 2'-O-Methoxyethyl oligomers (2'-O-MOE) have a methoxyethyl group at the 2'-OH residue of the ribose molecule and are discussed in Martin et al., Helv. Chim. Acta, 78, 486-504, 1995, which is incorporated herein by reference in its entirety. Non-limiting examples of 2'-O-MOE subunits are shown below. [ka]

[0168] 2'-Fluoro (2'-F) oligomers have a fluorescent radical at the 2' position instead of 2'-OH. Non-limiting examples of 2'-F oligomers are shown below. [ka]

[0169] 2'-Fluoro oligomers are further described in WO2004 / 043977, which is incorporated herein by reference in its entirety.

[0170] The 2'-O-Methyl, 2'-O-MOE, and 2'-F oligomers may also contain one or more phosphorothioate (PS) linkages, as shown below. [ka]

[0171] Additionally, 2'-O-methyl, 2'-O-MOE, and 2'-F oligomers may contain PS intersubunit linkages throughout the oligomer, such as, for example, the 2'-O-methyl PS oligomer drisapersen shown below. [ka]

[0172] Alternatively, the 2'-O-methyl, 2'-O-MOE, and / or 2'-F oligomers may contain a PS linkage at the terminus of the oligomer, as shown below. [ka] During the ceremony, R is CH 2 CH 2 OCH 3 (methoxyethyl or MOE), X, Y, and Z represent the number of nucleotides contained within the designated 5'-wing, central gap, and 3'-wing regions, respectively.

[0173] The antisense oligomers of the present disclosure may incorporate one or more 2'-O-methyl, 2'-O-MOE, and 2'-F subunits and may utilize any of the intersubunit linkages described herein. In some cases, the antisense oligomers of the present disclosure may be composed entirely of 2'-O-methyl, 2'-O-MOE, or 2'-F subunits. One embodiment of the antisense oligomers of the present disclosure is composed entirely of 2'-O-methyl subunits.

[0174] 8. 2'-O-[2-(N-methylcarbamoyl)ethyl] oligomer (MCE) MCE is another example of a 2'-O modified ribonucleoside useful in the antisense oligomers of the present disclosure, where the 2'-OH is derivatized to a 2-(N-methylcarbamoyl)ethyl moiety to enhance nuclease resistance. A non-limiting example of an MCE oligomer is shown below. [ka]

[0175] MCE and its synthesis are described in Yamada et al., J. Org. Chem. (2011) 76(9):3042-53, which is incorporated by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more MCE subunits.

[0176] 9. Stereospecific Oligomers Stereospecific oligomers are those in which the stereochemistry of each phosphorus-containing linkage is fixed by synthetic methods such that substantially stereopure oligomers are produced. Non-limiting examples of stereospecific oligomers are shown below. [ka]

[0177] In the above examples, each phosphorus of the oligomer has the same stereochemistry.Additional examples include the oligomers described herein.For example, LNA, ENA, tricyclo-DNA, MCE, 2'-O-methyl, 2'-O-MOE, 2'-F, and morpholino-based oligomers can be prepared using stereospecific phosphorus-containing internucleoside linkages, such as phosphorothioate, phosphodiester, phosphoramidate, phosphorodiamidate, or other phosphorus-containing internucleoside linkages. Stereospecific oligomers, methods of preparation, chiral controlled synthesis, chiral designs, and chiral auxiliaries for use in the preparation of such oligomers are described, for example, in WO2017 / 192664, WO2017 / 192679, WO2017 / 062862, WO2017 / 015575, WO2017 / 015555, WO2015 / 107425, WO2015 / 108048, WO No. 2015 / 108046, WO2015 / 108047, WO2012 / 039448, WO2010 / 064146, WO2011 / 034072, WO2014 / 010250, WO2014 / 012081, WO2013 / 0127858, and WO2011 / 005761, each of which is incorporated herein by reference in its entirety.

[0178] The stereospecific oligomer is R P Placement or S PThe oligomer may have phosphorus-containing internucleoside linkages in any configuration. Chiral phosphorus-containing linkages in which the configuration of the linkage is controlled are referred to as "stereo-pure" and chiral phosphorus-containing linkages in which the configuration of the linkage is not controlled are referred to as "stereoirregular". In certain embodiments, the oligomers of the present disclosure include a plurality of stereo-pure and stereoirregular linkages, such that the resulting oligomer has stereo-pure subunits at pre-specified positions in the oligomer. Examples of the positions of stereo-pure subunits are provided in WO 2017 / 062862(A2) in Figures 7A and 7B. In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereoirregular. In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereo-pure.

[0179] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), all n chiral phosphorus-containing linkages in the oligomer are stereoirregular. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), all n chiral phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 10% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 20% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 30% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 40% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 50% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 60% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 70% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 80% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 90% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure.

[0180] In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomers have the same stereo-orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least two consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least three consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least four consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least five consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least six consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 7 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 8 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R PIn one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 9 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 10 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 11 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 12 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 13 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 14 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 15 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 16 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., SP or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 17 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 18 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 19 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P The compound contains at least 20 consecutive stereopure phosphorus-containing linkages of the formula (I) or (II).

[0181] In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomers have the same stereo-orientation (i.e., S P or R P (either S P At least two consecutive stereopure phosphorus-containing linkages of the orientation R P and at least two consecutive stereopure phosphorus-containing linkages in the oriented direction.

[0182] In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least two consecutive stereopure phosphorus-containing linkages of the same stereoorientation in an alternating pattern. For example, the oligomer may contain, in order, the following: two or more R P , 2 or more S P , and two or more R P etc.

[0183] 10. Morpholino Oligomers Exemplary embodiments of the present disclosure include compounds having the following general structure: [ka] and as shown in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997). The morpholino described herein is intended to include all stereoisomers and tautomers of the above general structure. The synthesis, structure and binding properties of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476 and 8,299,206, all of which are incorporated herein by reference.

[0184] In certain embodiments, the morpholinos are conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] Includes.

[0185] In various aspects, the disclosure provides an antisense oligomer according to formula (IV), or a pharma- ceutically acceptable salt thereof.

[0186] In one embodiment, the targeting sequence is complementary to a target region within intron 1 (SEQ ID NO: 1) of the pre-mRNA of the human acid alpha glucosidase (GAA) gene, and at least one subunit is an abasic subunit. In another embodiment, the target region comprises a sequence selected from the group consisting of SEQ ID NO: 2 (GAA-IVS1(-189-167)) and SEQ ID NO: 3 (GAA-IVS1(-80-24)). In a further embodiment, the target region comprises the sequence set forth in SEQ ID NO: 2. In another embodiment, the target region comprises the sequence set forth in SEQ ID NO: 3.

[0187] In one embodiment, the target region is selected from GAA-IVS1(-189-167), GAA-IVS1(-80-56), GAA-IVS1(-76-52), GAA-IVS1(-74-55), GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), GAA-IVS1(-65-41), and GAA-IVS1(-49-24). In a further embodiment, the target region is GAA-IVS1(-189-167). In another embodiment, the target region is GAA-IVS1(-72,-48). In yet another embodiment, the targeting region is GAA-IVS1(-71,-47). In yet another embodiment, the targeting region is GAA-IVS1(-70,-46). In one embodiment, the targeting region is GAA-IVS1(-69-45). In another embodiment, the targeting region is GAA-IVS1(-65,-41). In yet another embodiment, the targeting region is GAA-IVS1(-66,-42).

[0188] In one embodiment, the targeting sequence comprises the sequence CCA GAA GGA AXX XCG AGA AAA GC (SEQ ID NO: 4), where each X is independently selected from guanine (G) or non-basic (B), and at least one X is B. In another embodiment, the targeting sequence comprises i) SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC); ii) Sequence number 6 (CCA GAA GGA AGB GCG AGA AAA GC), iii) SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC), iv) SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC), v) SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC), and vi) SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

[0189] In one embodiment, B is H.

[0190] In one embodiment, the targeting sequence comprises SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC). In another embodiment, the targeting sequence comprises SEQ ID NO: 6 (CCA GAA GGA AGB GCG AGA AAA GC). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC). In one embodiment, the targeting sequence comprises SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC). In another embodiment, the targeting sequence comprises SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

[0191] In one embodiment, the targeting sequence consists of the sequence CCA GAA GGA AXX XCG AGA AAA GC (SEQ ID NO: 4). In another embodiment, the targeting sequence consists of SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 6 (CCA GAA GGA AGB GCG AGA AAA GC). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC). In one embodiment, the targeting sequence consists of SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC). In another embodiment, the targeting sequence consists of SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

[0192] In one embodiment, the target region is selected from the group consisting of GAA-IVS1(-80-56), GAA-IVS1(-76-52), GAA-IVS1(-74-55), GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), GAA-IVS1(-65-41), and GAA-IVS1(-49-24). In another embodiment, the target region is selected from the group consisting of GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), and GAA-IVS1(-65-41).

[0193] In one embodiment, the targeting region is: i) Sequence number 11 (CTC ACX XXX CTC TCA AAG CAG CTC T), ii) Sequence number 12 (ACT CAC XXX XCT CTC AAA GCA GCT C), iii) Sequence number 13 (CAC TCA CXX XXC TCT CAA AGC AGC T), iv) SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C); v) Sequence number 15 (GCG GCA CTC ACX XXX CTC TCA AAG C), vi) comprising a sequence selected from the group consisting of SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G); Each X is independently selected from guanine (G) or abasic (B), and at least one X is B. In one embodiment, the targeting sequence is i) SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C); ii) SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C); iii) SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C); iv) SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C); v) SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C); vi) SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C); vii) SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C), and viii) SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0194] In one embodiment, B is H.

[0195] In one embodiment, the targeting sequence comprises SEQ ID NO: 11 (CTC ACX XXX CTC TCA AAG CAG CTC T). In another embodiment, the targeting sequence comprises SEQ ID NO: 12 (ACT CAC XXX XCT CTC AAA GCA GCT C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C). In one embodiment, the targeting sequence comprises SEQ ID NO: 15 (GCG GCA CTC ACX XXX CTC TCA AAG C). In another embodiment, the targeting sequence comprises SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C). In one embodiment, the targeting sequence comprises SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C). In another embodiment, the targeting sequence comprises SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence comprises SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C). In one embodiment, the targeting sequence comprises SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C). In another embodiment, the targeting sequence comprises SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0196] In one embodiment, the targeting sequence consists of SEQ ID NO: 11 (CTC ACX XXX CTC TCA AAG CAG CTC T). In another embodiment, the targeting sequence consists of SEQ ID NO: 12 (ACT CAC XXX XCT CTC AAA GCA GCT C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C). In one embodiment, the targeting sequence consists of SEQ ID NO: 15 (GCG GCA CTC ACX XXX CTC TCA AAG C). In another embodiment, the targeting sequence consists of SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C). In one embodiment, the targeting sequence consists of SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C). In another embodiment, the targeting sequence consists of SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C). In yet another embodiment, the targeting sequence consists of SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C). In one embodiment, the targeting sequence consists of SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C). In another embodiment, the targeting sequence consists of SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

[0197] In some embodiments, the antisense oligomer of the present disclosure is according to formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 15]

[0198] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0199] In one embodiment, B is H.

[0200] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 16-1] [Table 16-2]

[0201] In one embodiment, B is H.

[0202] In some embodiments, the antisense oligomer of formula (II) is in free base form. In some embodiments, the antisense oligomer of formula (II) is in its pharma- ceutically acceptable salt form. In some embodiments, the antisense oligomer of formula (II) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is 1 HCl, 2 HCl, 3 HCl, 4 HCl, 5 HCl, or 6 HCl salt. In certain embodiments, the HCl salt is 6 HCl salt.

[0203] In some embodiments, the antisense oligomer of the present disclosure conforms to formula (IIIa): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 17] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0204] In one embodiment, B is H.

[0205] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 18-1] [Table 18-2]

[0206] In one embodiment, B is H.

[0207] In some embodiments, the antisense oligomer of formula (IIIa) is in free base form. In some embodiments, the antisense oligomer of formula (IIIa) is its pharma- ceutically acceptable salt. In some embodiments, the antisense oligomer of formula (IIIa) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 5-HCl salt. In certain embodiments, the HCl salt is a 6-HCl salt.

[0208] In some embodiments, the antisense oligomer of the present disclosure conforms to formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 19] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0209] In one embodiment, B is H.

[0210] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 20]

[0211] In one embodiment, B is H.

[0212] In some embodiments, the antisense oligomer of formula (III) is in free base form. In some embodiments, the antisense oligomer of formula (III) is its pharma- ceutically acceptable salt. In some embodiments, the antisense oligomer of formula (III) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 5-HCl salt. In certain embodiments, the HCl salt is a 6-HCl salt.

[0213] In some embodiments, the antisense oligomer of the present disclosure is according to formula (V): [ka] wherein each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 21] Each X is independently selected from guanine (G) or abasic (B), where at least one X is B. When X is abasic (B), a hydrogen is present in place of nucleobase A, C, T, or G.

[0214] In one embodiment, B is H.

[0215] In some embodiments of formula (V), one instance of X is abasic and each of the other instances of X is G. In certain embodiments, two instances of X are abasic and one instance is G. In some embodiments of formula (V), the first instance of X from 5' to 3' is abasic and the other two instances of X are G. In some embodiments of formula (V), the second instance of X from 5' to 3' is abasic and the first and third instances of X are G. In certain embodiments of formula (V), the third instance of X from 5' to 3' is abasic and the first and second instances of X are G.

[0216] In some embodiments of formula (V), two instances of X are abasic and the other instance of X is G. In some embodiments of formula (V), the first and second instances of X from 5' to 3' are abasic and the third instance of X is G. In some embodiments of formula (V), the first and third instances of X from 5' to 3' are abasic and the second instance of X is G. In some embodiments of formula (V), the second and third instances of X from 5' to 3' are abasic and the first instance of X is G.

[0217] In one embodiment, the targeting region comprises or consists of any one of the following sequences: [Table 22-1] [Table 22-2]

[0218] In one embodiment, B is H.

[0219] For example, in some embodiments, including some embodiments of Formula (V), the antisense oligomer is according to Formula (Va): [ka] wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

[0220] In one embodiment, B is H.

[0221] In some embodiments of Formula (Va), one instance of X is abasic and each of the other instances of X is G. In certain embodiments of Formula (Va), two instances of X are abasic and one instance is G. In some embodiments of Formula (Va), the first instance of X from 5' to 3' is abasic and the other two instances of X are G. In some embodiments of Formula (Va), the second instance of X from 5' to 3' is abasic and the first and third instances of X are G. In certain embodiments of Formula (Va), the third instance of X from 5' to 3' is abasic and the first and second instances of X are G.

[0222] In some embodiments of Formula (Va), two instances of X are abasic and the other instance of X is G. In some embodiments of Formula (Va), the first and second instances of X from 5' to 3' are abasic and the third instance of X is G. In some embodiments of Formula (Va), the first and third instances of X from 5' to 3' are abasic and the second instance of X is G. In some embodiments of Formula (Va), the second and third instances of X from 5' to 3' are abasic and the first instance of X is G.

[0223] For example, in some embodiments, including some embodiments of Formula (V), the antisense oligomer is according to Formula (Vb): [ka] wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

[0224] In one embodiment, B is H.

[0225] In some embodiments of formula (Vb), one instance of X is abasic and each of the other instances of X is G. In certain embodiments, two instances of X are abasic and one instance is G. In some embodiments of formula (Vb), the first instance of X from 5' to 3' is abasic and the other two instances of X are G. In some embodiments of formula (Vb), the second instance of X from 5' to 3' is abasic and the first and third instances of X are G. In certain embodiments of formula (Vb), the third instance of X from 5' to 3' is abasic and the first and second instances of X are G.

[0226] In some embodiments of formula (Vb), two instances of X are abasic and the other instance of X is G. In some embodiments of formula (Vb), the first and second instances of X from 5' to 3' are abasic and the third instance of X is G. In some embodiments of formula (Vb), the first and third instances of X from 5' to 3' are abasic and the second instance of X is G. In some embodiments of formula (Vb), the second and third instances of X from 5' to 3' are abasic and the first instance of X is G.

[0227] For example, in some embodiments, including some embodiments of Formula (V), the antisense oligomer conforms to Formula (VII). [ka]

[0228] For example, in some embodiments, including some embodiments of Formula (V), the antisense oligomer conforms to Formula (VIII). [ka]

[0229] For example, in some embodiments, including some embodiments of Formula (V), the antisense oligomer is according to Formula (Vc): [ka] wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

[0230] In one embodiment, B is H.

[0231] In some embodiments of Formula (Vc), one instance of X is abasic and each of the other instances of X is G. In certain embodiments of Formula (Vc), two instances of X are abasic and one instance is G. In some embodiments of Formula (Vc), the first instance of X from 5' to 3' is abasic and the other two instances of X are G. In some embodiments of Formula (Vc), the second instance of X from 5' to 3' is abasic and the first and third instances of X are G. In certain embodiments of Formula (Vc), the third instance of X from 5' to 3' is abasic and the first and second instances of X are G.

[0232] In some embodiments of Formula (Vc), two instances of X are abasic and the other instance of X is G. In some embodiments of Formula (Vc), the first and second instances of X from 5' to 3' are abasic and the third instance of X is G. In some embodiments of Formula (Vc), the first and third instances of X from 5' to 3' are abasic and the second instance of X is G. In some embodiments of Formula (Vc), the second and third instances of X from 5' to 3' are abasic and the first instance of X is G.

[0233] For example, in some embodiments, including some embodiments of Formula (V), the antisense oligomer is according to Formula (Vd): [ka] wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

[0234] In one embodiment, B is H.

[0235] In some embodiments of Formula (Vd), one instance of X is abasic and each of the other instances of X is G. In certain embodiments of Formula (Vd), two instances of X are abasic and one instance is G. In some embodiments of Formula (Vd), the first instance of X from 5' to 3' is abasic and the other two instances of X are G. In some embodiments of Formula (Vd), the second instance of X from 5' to 3' is abasic and the first and third instances of X are G. In certain embodiments of Formula (Vd), the third instance of X from 5' to 3' is abasic and the first and second instances of X are G.

[0236] In some embodiments of Formula (Vd), two instances of X are abasic and the other instance of X is G. In some embodiments of Formula (Vd), the first and second instances of X from 5' to 3' are abasic and the third instance of X is G. In some embodiments of Formula (Vd), the first and third instances of X from 5' to 3' are abasic and the second instance of X is G. In some embodiments of Formula (Vd), the second and third instances of X from 5' to 3' are abasic and the first instance of X is G.

[0237] IV. Target Sequences and Target Regions In some embodiments for antisense applications, the oligomers may be 100% complementary to the nucleic acid target sequence (except for at least one abasic subunit) or may contain mismatches, for example to accommodate variants, so long as the heteroduplex formed between the oligomer and the nucleic acid target sequence is stable enough to withstand the action of cellular nucleases and other degradation modes that may occur in vivo. If present, mismatches are less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches tolerated depends on the length of the oligomer, the proportion of G:C base pairs in the duplex, and the location of the mismatch in the duplex according to well-understood principles of duplex stability. Such antisense oligomers are not necessarily 100% complementary to the nucleic acid target sequence, but are effective to stably and specifically bind to the target sequence such that the biological activity of the nucleic acid target, for example, expression of the encoded protein, is modulated.

[0238] The stability of the duplex formed between the oligomer and the target sequence is determined by the binding T m The T of an antisense compound with respect to a complementary sequence RNA is a function of the susceptibility of the duplex to enzymatic cleavage in cells. m can be measured by conventional methods such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or Miyada CG. and Wallace RB (1987) Oligonucleotide hybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107.

[0239] In some embodiments, each antisense oligomer has a binding T with respect to the complementary sequence RNA that is greater than body temperature, or in other embodiments, greater than 50° C. m In another embodiment, T m The T of the oligomeric compound with respect to the complementary-based RNA hybrid is in the range of 60-80° C. or more according to well-known principles. mcan be increased by increasing the ratio of C:G paired bases in the duplex and / or by increasing the length (in base pairs) of the heteroduplex. At the same time, it may be advantageous to limit the size of the oligomer in order to optimize cellular uptake. For this reason, high T m (50℃ or higher) m These are generally preferred over compounds that require more than 20 bases for their value. In some applications, longer oligomers, e.g., greater than 20 bases, may have certain advantages.

[0240] The targeted sequence bases can be normal DNA bases or their analogs, such as uracil and inosine, which are capable of Watson-Crick base pairing to the targeted sequence RNA bases.

[0241] An antisense oligomer may be designed to block, inhibit or modulate translation of an mRNA, or to inhibit or modulate pre-mRNA splice processing, or to induce degradation of a targeted mRNA, and may be said to be "directed" or "targeted" to the target sequence to which it hybridizes. In certain embodiments, the target sequence includes a region containing a 3' or 5' splice site, branch point, or other sequence involved in the control of splicing of a pre-processed mRNA. The target sequence may be within an exon, or within an intron, or may span an intron / exon junction.

[0242] An antisense oligomer having sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the antisense agent has sufficient sequence to induce masking of a binding site of a natural protein that would otherwise modulate splicing and / or alter the three-dimensional structure of the targeted RNA. Similarly, an oligomeric reagent having sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the oligomeric reagent has sufficient sequence to induce masking of a binding site of a natural protein that would otherwise modulate splicing and / or alter the three-dimensional structure of the targeted RNA.

[0243] In certain embodiments, the antisense oligomer has sufficient length and complementarity to a sequence in intron 1 of the human GAA pre-mRNA. The sequence of intron 1 (SEQ ID NO:1) of the human GAA gene is shown below in Table 2 (the highlighted T / G near the 3' end of SEQ ID NO:1 is the IVS1-13T>G mutation described above, where the nucleotide at this position is either T or G). [Table 2-1] [Table 2-2] [Table 2-3]

[0244] In certain embodiments, the degree of complementarity between the target sequence and the antisense targeting sequence is sufficient to form a stable duplex. The region of complementarity of the antisense oligomer, excluding the abasic units, including the target RNA sequence, can be as short as 8-11 bases, but can be 12-15 bases or more, such as 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers between these ranges. An antisense oligomer of about 14-15 bases is generally long enough to have a unique complementary sequence. In certain embodiments, a minimum length of complementary bases may be required to achieve the required binding Tm, as discussed herein.

[0245] In certain embodiments, oligomers as long as 40 bases may be suitable, with at least a minimum number of bases, e.g., 10-12 bases, being complementary to the target sequence. In some embodiments, facilitated or active uptake in cells is optimized at oligomer lengths of less than about 30 bases. For the PMO oligomers described further herein, the optimal balance of binding stability and uptake generally occurs at lengths of 18-25 bases. Antisense oligomers (e.g., PMO, PMO-X, PNA, LNA, 2'-OMe) of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases are suitable. , at least about 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous or non-contiguous bases that are included in the present disclosure and are complementary to a desired target sequence.

[0246] In certain embodiments, antisense oligomers may be 100% complementary to the target sequence (except for at least one abasic nucleotide) or may contain mismatches, for example to accommodate variants, so long as the heteroduplex formed between the oligomer and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation methods that may occur in vivo. Thus, certain oligomers may have substantial complementarity, i.e., about or at least about 70% sequence complementarity, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity, between the oligomer (except for at least one abasic nucleotide) and the target sequence. Oligomer backbones that are less susceptible to nuclease cleavage are contemplated herein. When mismatches exist, they are typically less destabilizing toward the end regions of the hybrid duplex than toward the center. The number of mismatches tolerated depends on the length of the oligomer, the ratio of G:C base pairs in the duplex, and the position of the mismatch in the duplex according to well-understood principles of duplex stability. Such antisense oligomers are not necessarily 100% complementary to the target sequence, but are effective in binding stably and specifically to the target sequence so that splicing of the target pre-RNA is regulated. The stability of the duplex formed between the oligomer and the target sequence is a function of the binding Tm, which indicates the susceptibility of the duplex to cellular enzymatic cleavage. The Tm of an oligomer with respect to a complementary sequence RNA can be measured by conventional methods such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or those described in Miyada CG and Wallace RB, 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107.In certain embodiments, the antisense oligomer may have a binding Tm with respect to the complementary sequence RNA that is higher than body temperature, preferably about 45°C or more than 50°C. Tms in the range of 60-80°C are also included. In accordance with well-known principles, the Tm of the oligomer with respect to the complementary-based RNA hybrid may be increased by increasing the ratio of C:G paired bases in the duplex and / or by increasing the length (in base pairs) of the heteroduplex. At the same time, it may be advantageous to limit the size of the oligomer in order to optimize cellular uptake. For this reason, compounds that exhibit high Tm (45-50°C or higher) at lengths of 25 bases or less are generally preferred over compounds that require more than 25 bases for high Tm values.

[0247] In certain embodiments, the antisense targeting sequence is designed to hybridize to one or more regions of the target sequence listed in Table 2. The selected antisense targeting sequence can be made shorter, for example, about 12 or more bases, for example, about 40 bases, and can contain a small number of mismatches, as long as the sequence is sufficiently complementary to effect splice regulation upon hybridization to the target sequence, and optionally forms a heteroduplex with RNA having a Tm of 45°C or higher.

[0248] V. Cell-penetrating peptides (CPPs) For example, arginine-rich cell membrane-penetrating peptides (CPPs) discussed herein within the scope of substituent J may be effective in enhancing cellular penetration of antisense oligomers and in inducing exon skipping in different muscle groups in animal models.

[0249] Exemplary arginine-rich peptides are shown in Table 3 below. [Table 3]

[0250] In another embodiment, exemplary cell membrane penetrating peptides within the scope of substituent J are provided in Table 4. The point of attachment to substituent L is as indicated in the table.

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

[0251] L is as recited in formula (I) and as described throughout the specification, β=3-alanine, α=6-aminohexanoic acid, tg=unmodified amino terminus or an amino terminus capped with an acetyl, benzoyl, or stearoyl group (i.e., acetylamide, benzoylamide, or stearoylamide), and Y is NH-(CHR)-C(O)-, where n is 2-7 and each R is independently at each occurrence hydrogen or methyl. For simplicity, not all sequences are depicted with a terminal td group, but each of the above sequences can include an unmodified amino terminus or an amino terminus capped with an acetyl, benzoyl, or stearoyl group.

[0252] VI. Pharmaceutical Compositions The present disclosure also provides for the formulation and delivery of the disclosed antisense oligomers. Accordingly, one aspect of the present disclosure is a pharmaceutical composition comprising an antisense oligomer disclosed herein and a pharma- ceutical acceptable carrier.

[0253] Effective delivery of antisense oligomers to target nucleic acid is an important aspect of treatment. Antisense oligomer delivery routes include, but are not limited to, oral and parenteral routes, such as intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as various systemic routes, including inhalation, transdermal, and topical delivery. Appropriate routes can be determined by those skilled in the art as necessary for the condition of the subject being treated. For example, the appropriate route for delivery of antisense oligomers in the treatment of viral infections of the skin is topical delivery, while delivery of antisense oligomers for the treatment of viral respiratory infections can be intravenous or by inhalation. Antisense oligomers can also be delivered directly to any particular site of viral infection.

[0254] Antisense oligomers can be administered in any convenient vehicle that is physiologically and / or pharmaceutical acceptable. Such compositions can include any of a variety of standard pharmaceutical acceptable carriers used by those skilled in the art. Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water (e.g., sterile water for injection), aqueous ethanol, emulsions such as oil / water emulsions or triglyceride emulsions, tablets, and capsules. The choice of suitable physiologically acceptable carriers depends on the method of administration selected.

[0255] The compounds (e.g., antisense oligomers) can generally be utilized as free acids or free bases. Alternatively, the compounds can be used in the form of acid addition salts or base addition salts. Acid addition salts of free amino compounds can be prepared by methods well known in the art and can be formed from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, and benzenesulfonic acid. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid. Base addition salts include salts formed with carboxylate anions, and include salts formed with organic and inorganic cations such as those selected from alkali metals and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium, and calcium), as well as ammonium ion and its substituted derivatives (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, etc.).Thus, the term "pharmaceutically acceptable salt" of structure (I) is intended to encompass any and all acceptable salt forms.

[0256] In addition, prodrugs are also included within the context of the present invention. Prodrugs are any covalently bonded carriers that release a compound of structure (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in such a way that the modification is cleaved, either by routine manipulation or in vivo, to obtain the parent compound. Prodrugs include, for example, compounds of the present invention in which a hydroxy, amine, or sulfhydryl group is bonded to any group that cleaves to form a hydroxy, amine, or sulfhydryl group when administered to a patient. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate, and benzoate derivatives of alcohol and amine functional groups of compounds of structure (I). Furthermore, in the case of carboxylic acids (-COOH), esters such as methyl esters, ethyl esters, etc. may be used.

[0257] VII. Preparation method Preparation of Oligomers with Basic Nitrogenous Internucleotide Linkers Morpholino subunits, modified intersubunit linkages, and oligomers containing them can be prepared, for example, as described in U.S. Patent Nos. 5,185,444 and 7,943,762, which are incorporated by reference in their entireties. Morpholino subunits can be prepared according to the following General Reaction Scheme 1. [ka]

[0258] Referring to reaction scheme 1, where B represents the base-pairing moiety and PG represents the protecting group, the morpholino subunit can be prepared from the corresponding ribonucleoside (1) as shown. The morpholino subunit (2) can be optionally protected by reaction with a suitable protecting group precursor, for example, trityl chloride. The 3' protecting group is generally removed during solid-phase oligomer synthesis, as described in more detail below. The base-pairing moiety can be suitably protected for solid-phase oligomer synthesis. Suitable protecting groups include benzoyl for adenine and cytosine, phenylacetyl for guanine, and pivaloyloxymethyl for hypoxanthine (I). The pivaloyloxymethyl group can be introduced at the N1 position of the hypoxanthine heterocyclic base. It was used without isolation in the preparation. Although unprotected hypoxanthine subunits can be used, the yield in the activation reaction is much better when the base is protected. Other suitable protecting groups include those disclosed in US Pat. No. 8,076,476, which is incorporated herein by reference in its entirety.

[0259] Reaction of 3 with an activated phosphorus compound 4 results in a morpholino subunit having the desired linking moiety 5. Compounds of structure 4 can be prepared using any number of methods known to those of skill in the art. For example, such compounds can be prepared by reaction of the corresponding amine and phosphorus oxychloride. In this regard, the amine starting material can be prepared using any method known in the art, such as those described in the Examples and in U.S. Pat. No. 7,943,762.

[0260] Compounds of structure 5 can be used in solid-phase automated oligomer synthesis to prepare oligomers containing intersubunit linkages. Such methods are well known in the art. Briefly, compounds of structure 5 can be modified at the 5' end to contain a linker to a solid support. For example, compound 5 can be linked to a solid support by a linker. Once supported, the protecting group (e.g., trityl) is removed and the free amine is reacted with the activated phosphorus moiety of a second compound of structure 5. This sequence is repeated until an oligo of the desired length is obtained. The protecting group at the terminal 5' end can either be removed or left in place if a 5' modification is desired.

[0261] The preparation of modified morpholino subunits and morpholino oligomers is described in more detail in the Examples. Morpholino oligomers containing any number of modified linkages can be prepared using methods described herein, known in the art, and / or described herein by reference. Also described in the Examples are overall modifications of morpholino oligomers prepared as described above (see, for example, PCT Publication No. 2008 / 036127).

[0262] The synthesis of PMOs, PMO+, PPMOs, and PMO-X containing the additional linkage modifications described herein can be carried out using methods known in the art and are described in pending U.S. Patent Nos. 8,299,206 and 8,076,476, and PCT Publication Nos. 2009 / 064471, 2011 / 150408, and 2012 / 150960, which are incorporated by reference in their entireties.

[0263] PMOs bearing 3' trityl modifications are essentially synthesized as described in PCT Publication No. 2009 / 064471, except that the detritylation step is omitted.

[0264] VIII. Treatment method Provided herein are methods for increasing expression of exon 2-containing GAA mRNA and / or protein for therapeutic purposes (e.g., treating a subject with GSD-II) using antisense oligomers of the present disclosure. The methods include administering to a patient in need thereof a therapeutically effective amount of an antisense oligomer disclosed herein or a pharmaceutical composition thereof. In one embodiment, the disease is Pompe disease. In some embodiments, the antisense oligomer comprises a nucleotide sequence of sufficient length and complementarity to specifically hybridize to a region within the pre-mRNA of the acid alpha-glucosidase (GAA) gene, such that binding of the antisense oligomer to the region increases the level of exon 2-containing GAA mRNA in cells and / or tissues of the subject. Exemplary antisense targeting sequences are provided in Tables 6A-6C herein.

[0265] Also included is an antisense oligomer for use in preparing a medicament for the treatment of glycogen storage disease type II (GSD-II, Pompe disease), comprising a nucleotide sequence of sufficient length and complementarity to specifically hybridize to a region within the pre-mRNA of the acid alpha-glucosidase (GAA) gene, such that binding of the antisense oligomer to that region increases levels of exon 2-containing GAA mRNA.

[0266] In some embodiments of the method of treating GSD-II or the medicament for the treatment of GSD-II, the antisense oligomeric compound comprises: an antisense oligomer having a length of 18 to 40 subunits, the antisense oligomer comprising a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; each subunit of the antisense oligomer comprises a nucleobase or is an abasic subunit; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0267] In some embodiments of the method of treating GSD-II or the medicament for the treatment of GSD-II, the antisense oligomeric compound comprises: an antisense oligomer having a length of 18 to 40 subunits, the antisense oligomer comprising a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; The antisense oligonucleotide comprises a morpholino oligomer, each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0268] In some embodiments of the method of treating GSD-II or the medicament for the treatment of GSD-II, the antisense oligomeric compound comprises: a modified antisense oligonucleotide having a length of 18 to 40 subunits, the modified antisense oligonucleotide comprising a targeting sequence complementary to a target region within intron 1 (SEQ ID NO:1) of the pre-mRNA of the human acid alpha glucosidase (GAA) gene; The antisense oligonucleotide comprises a morpholino oligomer, The antisense oligonucleotide is covalently linked to a cell membrane-permeable peptide, each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence; at least one subunit is an abasic subunit; Excluding the abasic subunits, the targeting sequence is at least 80% complementary to the target region.

[0269] As noted above, "GSD-II" refers to glycogen storage disease type II (GSD-II or Pompe disease), a human autosomal recessive disorder often characterized by underexpression of the GAA protein in affected individuals. Subjects with infantile GSD-II and subjects with later-onset forms of the disease are included.

[0270] In certain embodiments, the subject has reduced expression and / or activity of GAA protein in one or more tissues (e.g., compared to healthy subjects or earlier time points), including heart, skeletal muscle, liver, and nervous system tissue. In some embodiments, the subject has increased glycogen accumulation in one or more tissues (e.g., compared to healthy subjects or earlier time points), including heart, skeletal muscle, liver, and nervous system tissue. In certain embodiments, the subject has at least one IVS1-13T>G mutation (also referred to as c.336-13T>G), optionally in combination with other mutations that result in reduced expression of functional GAA protein. A summary of the molecular genetic tests used in GSD-II is provided below in Table 5. [Table 5]

[0271] Certain embodiments relate to methods of increasing expression of exon 2 containing GAA mRNA or protein in a cell, tissue, and / or subject, as described herein. In some cases, the exon-2 containing GAA mRNA or protein is increased by about or at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control, e.g., a control cell / subject, a control composition not including an antisense oligomer, the absence of treatment, and / or an earlier time point. Also included are methods of maintaining expression of the containing GAA mRNA or protein compared to the levels of a healthy control.

[0272] Some embodiments relate to methods of increasing expression of functional / active GAA protein in cells, tissues, and / or subjects as described herein. In certain instances, the level of functional / active GAA protein is increased by about or at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control, e.g., a control cell / subject, a control composition not including an antisense oligomer, the absence of treatment, and / or an earlier time point. Also included are methods of maintaining expression of functional / active GAA protein compared to the level of a healthy control.

[0273] Certain embodiments relate to methods of reducing glycogen accumulation in one or more cells, tissues, and / or subjects as described herein. In certain cases, glycogen accumulation is reduced by about or at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control, e.g., a control cell / subject, a control composition not including an antisense oligomer, the absence of treatment, and / or an earlier time point. Also included are methods of maintaining normal or otherwise healthy glycogen levels (e.g., subclinical levels or levels associated with reduced symptoms of GSD-II) in cells, tissues, and / or subjects.

[0274] Also included are methods for reducing one or more symptoms of GSD-II in a subject in need thereof.Specific examples include symptoms of infantile GSD-II, such as cardiac hypertrophy, hypotonia, cardiomyopathy, left ventricular outflow obstruction, respiratory distress, motor delay / weakness, and feeding difficulties / disorders.Additional examples include symptoms of late-onset GSD-II, such as muscle weakness (e.g., skeletal muscle weakness, including progressive muscle weakness), cough disorders, recurrent chest infections, hypotonia, delayed motor milestones, difficulty swallowing or chewing, and reduced lung capacity or respiratory failure.

[0275] The antisense oligomers of the present disclosure can be administered to a subject to treat (prophylactically or therapeutically) GSD-II. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) can be considered. Differences in the metabolism of therapeutic drugs can lead to severe toxicity or treatment failure by altering the relationship between the dose and blood concentration of pharmacologically active drugs.

[0276] Thus, a physician or clinician may consider applying knowledge gained from relevant pharmacogenomic studies to decisions about whether to administer a therapeutic agent, as well as to adjusting the dosage and / or treatment regimen of treatment with the therapeutic agent.

[0277] Effective delivery of antisense oligomers to target nucleic acid is one aspect of treatment. Antisense oligomer delivery routes include, but are not limited to, oral and parenteral routes, such as intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as various systemic routes, including inhalation, transdermal, and local delivery. Appropriate routes can be determined by those skilled in the art as required for the condition of the subject being treated. Vascular or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are some non-limiting sites where RNA can be introduced. Direct CNS delivery can be used, for example, intraventricular or intrathecal administration can be used as an administration route.

[0278] In certain embodiments, antisense oligomers are administered to subjects by intramuscular injection (IM), i.e., they are administered or delivered intramuscularly.Non-limiting examples of intramuscular injection sites include the deltoid muscle of the arm, the vastus lateralis muscle of the leg, and the ventrogluteal muscle of the hip joint, and the dorsogluteal muscle of the buttocks.In certain embodiments, PMO, PMO-X, or PPMO is administered by IM.

[0279] In certain embodiments, subjects in need thereof as glycogen stores in central nervous system tissue. Examples include cases where central nervous system pathology contributes to respiratory impairment in GSD-II (see, e.g., DeRuisseau et al., PNAS USA. 106:9419-24, 2009). Thus, the antisense oligomers described herein can be delivered to the nervous system of a subject by any art-recognized method, for example, when the subject has GSD-II with CNS involvement. For example, peripheral blood injection of the antisense oligomers of the present disclosure can be used to deliver the reagent to peripheral neurons via diffusive and / or active means. Alternatively, the antisense oligomers can be modified to facilitate crossing the blood-brain barrier (BBB) ​​to achieve delivery of the reagent to neuronal cells of the central nervous system (CNS). Specific recent advances in antisense oligomer technology and delivery strategies have broadened the scope of antisense oligomer use for neuronal disorders (see, e.g., Forte, A., et al. 2005. Curr. Drug Targets 6:21-29; Jaeger, LB, and WABanks. 2005. Methods Mol. Med. 106:237-251; Vinogradov, SV, et al. 2004. Bioconjug. Chem. 5:50-60, which are incorporated herein by reference in their entirety). For example, the antisense oligomers of the present disclosure can be produced as peptide nucleic acid (PNA) compounds. Each of the PNA reagents has been identified to cross the BBB (Jaeger, LB, and WABanks. 2005. Methods Mol. Med. 106:237-251). For example, treatment of a subject with a vasoactive agent has also been described to promote transport across the BBB. (Id.) Conjugation of the antisense oligomers of the present disclosure to agents that are actively transported across the BBB can also be used as a delivery mechanism.Administering an antisense agent together with an imaging agent such as iohexol (e.g., separately, simultaneously, in the same formulation) can also facilitate delivery across the BBB, as described in PCT Publication No. 2013 / 086207, which is incorporated by reference in its entirety.

[0280] In certain embodiments, the antisense oligomers of the present disclosure may be delivered by transdermal methods (e.g., via incorporation of the antisense oligomer, for example, in an emulsion, optionally packaging such antisense oligomer in a liposome). Such transdermal and emulsion / liposome mediated delivery methods have been described in the art for delivery of antisense oligomers, for example, in U.S. Patent No. 6,965,025, the contents of which are incorporated herein by reference in their entirety.

[0281] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is an art-recognized process, for example, using the artificial implant designs described in U.S. Patent No. 6,969,400, the contents of which are incorporated herein by reference in their entirety.

[0282] Antisense oligomers can be introduced into cells using art-recognized techniques (e.g., transfection, electroporation, fusion, liposomes, colloidal polymer particles, and viral and non-viral vectors, as well as other means known in the art). The delivery method selected will depend at least on the oligomer chemistry, the cells to be treated, and the location of the cells, and will be apparent to those skilled in the art. For example, localization can be achieved by liposomes with specific markers on the surface to direct the liposomes, direct injection into tissues containing target cells, specific receptor-mediated uptake, etc.

[0283] As is known in the art, antisense oligomers may be delivered using methods involving, for example, liposome-mediated uptake, exosome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake, receptor-mediated endocytosis, as well as additional non-endocytotic delivery methods such as microinjection, permeabilization (e.g., streptolysin-O permeabilization, anionic peptide permeabilization), electroporation, and a variety of non-invasive non-endocytotic delivery methods known in the art (see Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49, incorporated by reference in its entirety).

[0284] Antisense oligomers can be administered in any convenient vehicle or carrier that is physiologically and / or pharma- ceutically acceptable. Such compositions can include any of a variety of standard pharma-ceutically acceptable carriers used by those skilled in the art. Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions such as oil / water emulsions or triglyceride emulsions, tablets, and capsules. The selection of a suitable physiologically acceptable carrier depends on the selected method of administration. Pharmaceutically acceptable carriers are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0285] The compounds of the present disclosure (e.g., antisense oligomers) can generally be utilized as free acid or free base. Alternatively, the compounds of the present disclosure can be used in the form of acid addition salts or base addition salts. The acid addition salts of the free amino compounds of the present disclosure can be prepared by methods well known in the art and can be formed from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, and benzenesulfonic acid.

[0286] Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid. Base addition salts include salts formed with carboxylate anions, including organic and inorganic cations such as those selected from alkali metals and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium, and calcium), and ammonium ion and its substituted derivatives (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, etc.). Thus, the term "pharmaceutically acceptable salt" is intended to encompass any and all acceptable salt forms.

[0287] In addition, prodrugs are also included within the context of the present disclosure. Prodrugs are any covalently bonded carriers that release a compound in vivo when such prodrugs are administered to a patient. Prodrugs are generally prepared by modifying functional groups in such a way that the modification is cleaved, either by routine manipulation or in vivo, to obtain the parent compound. Prodrugs include, for example, compounds of the present disclosure in which a hydroxyl, amine, or sulfhydryl group is bonded to any group that cleaves to form a hydroxyl, amine, or sulfhydryl group when administered to a patient. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate, and benzoate derivatives of alcohol and amine functional groups of the antisense oligomers of the present disclosure. Furthermore, in the case of carboxylic acids (-COOH), esters such as methyl esters, ethyl esters, etc. can be used.

[0288] In some cases, liposomes can be used to facilitate uptake of antisense oligomers into cells (see, e.g., Williams, SA, Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res. 23:119, 1994; Uhlmann et al., antisense oligomers: a new therapeutic principle, Chemical Reviews, Volume 90, No. 4, 25 pages 544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels can also be used as vehicles for antisense oligomer administration, e.g., as described in WO93 / 01286. Alternatively, oligomers can be administered in microspheres or microparticles. (See, e.g., Wu, GY and Wu, CH, J. Biol. Chem. 262:4429-4432, 30 1987). Alternatively, the use of gas-filled microbubbles complexed with antisense oligomers can enhance delivery to target tissues, as described in U.S. Patent No. 6,245,747. Sustained-release compositions can also be used. These can include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules.

[0289] In one embodiment, the antisense oligomer is administered in a suitable pharmaceutical carrier to a mammalian subject, e.g., a human or a domestic animal, that exhibits symptoms of a lysosomal storage disorder. In one aspect of the method, the subject is a human subject, e.g., a patient diagnosed with GSD-II (Pompe disease). In a preferred embodiment, the antisense oligomer is delivered orally in a pharmaceutically acceptable carrier. In another preferred embodiment, the oligomer is delivered intravenously (iv) in a pharmaceutically acceptable carrier.

[0290] In one embodiment, the antisense compound is administered in an amount and manner effective to produce a peak blood concentration of the antisense oligomer of at least 200-400 nM. Typically, one or more doses of the antisense oligomer are administered, generally at regular intervals, for a period of about 1-2 weeks. A preferred dose for oral administration is about 1-1000 mg of oligomer per 70 kg. In some cases, doses of more than 1000 mg / patient may be necessary. For iv administration, a preferred dose is about 0.5 mg-1000 mg of oligomer per 70 kg. The antisense oligomer may be administered at regular intervals for a short period of time, for example, daily, for up to 2 weeks. In some cases, however, the oligomer is administered intermittently for a longer period of time. Administration may be followed by or may be simultaneous with administration of an antibiotic or other therapeutic treatment. The treatment regimen may be adjusted (dosage, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests, and physiological tests of the subject being treated.

[0291] In certain embodiments, the method is an in vitro method. In certain other embodiments, the method is an in vivo method.

[0292] In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is a non-human primate cell. In certain embodiments, the host cell is a human cell.

[0293] In certain embodiments, the host cell is a naturally occurring cell. In certain other embodiments, the host cell is an engineered cell.

[0294] In certain embodiments, the antisense oligomer is administered in a suitable pharmaceutical carrier to a mammalian subject, such as a human or a laboratory or domestic animal.

[0295] In certain embodiments, the antisense oligomer is administered to a mammalian subject, such as a human or an experimental or domestic animal, together with an additional agent. The antisense oligomer and the additional agent may be administered simultaneously or sequentially via the same or different routes and / or sites of administration. In certain embodiments, the antisense oligomer and the additional agent may be co-formulated and administered together. In certain embodiments, the antisense oligomer and the additional agent may be provided together in a kit.

[0296] In one embodiment, the antisense oligomer contained in a pharma- ceutically acceptable carrier is delivered orally.

[0297] In one embodiment, the antisense oligomer contained in a pharma- ceutically acceptable carrier is delivered intravenously (iv).

[0298] Additional routes of administration, such as subcutaneous, intraperitoneal, and pulmonary, are also contemplated by the present disclosure.

[0299] In another application of the method, the subject is a livestock animal, such as a pig, cow, or goat, and the treatment is either prophylactic or therapeutic. Also contemplated is an improvement in a method of feeding livestock, in which the food is supplemented with an effective amount of the antisense oligomer composition described above.

[0300] In one embodiment, the antisense oligomer is administered in an amount and manner effective to provide a peak blood concentration of the antisense oligomer of at least 200 nM. In one embodiment, the antisense oligomer is administered in an amount and manner effective to provide a peak plasma concentration of the antisense oligomer of at least 200 nM. In one embodiment, the antisense oligomer is administered in an amount and manner effective to provide a peak serum concentration of the antisense oligomer of at least 200 nM.

[0301] In one embodiment, the antisense oligomer is administered in an amount and manner effective to provide a peak blood concentration of the antisense oligomer of at least 400 nM. In one embodiment, the antisense oligomer is administered in an amount and manner effective to provide a peak plasma concentration of the antisense oligomer of at least 400 nM. In one embodiment, the antisense oligomer is administered in an amount and manner effective to provide a peak serum concentration of the antisense oligomer of at least 400 nM.

[0302] Typically, one or more doses of antisense oligomer are administered, generally at regular intervals, for a period of about 1-2 weeks. A preferred dose for oral administration is about 0.01-15 mg of antisense oligomer per kg body weight. In some cases, doses of more than 15 mg of antisense oligomer / kg may be necessary. For iv administration, a preferred dose is about 0.005 mg-15 mg of antisense oligomer per kg body weight. The antisense oligomer may be administered at regular intervals for a short period of time, for example, daily for up to two weeks. In some cases, however, the antisense oligomer is administered intermittently for a longer period of time. Administration may be followed or accompanied by administration of antibiotics or other therapeutic treatments. The treatment regimen may be adjusted (dosage, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests, and physiological tests of the subject being treated.

[0303] Effective in vivo therapeutic regimens using antisense oligomers may vary depending on the duration, dose, frequency, and route of administration, as well as the condition of the subject being treated (i.e., prophylactic administration versus administration in response to a local or systemic infection). Thus, such in vivo therapies often require laboratory monitoring during treatment and corresponding adjustments of the dose or therapeutic regimen to achieve optimal therapeutic outcomes.

[0304] In some embodiments, the antisense oligomer is actively taken up by mammalian cells, hi further embodiments, the antisense oligomer may be conjugated to a transport moiety (e.g., a transport peptide) described herein to facilitate such uptake.

[0305] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0306] Examples are described below for illustrative purposes to describe certain embodiments of the present disclosure.However, the scope of the claims is in no way limited by the examples described herein.Various changes and modifications to the disclosed embodiments are obvious to those skilled in the art, and such changes and modifications can be made without departing from the spirit of the present disclosure and the scope of the appended claims, including but not limited to those related to the chemical structures, substituents, derivatives, preparations or methods of the present disclosure.The definitions of the variables of the structures in the schemes herein are equivalent to those of the corresponding positions of the formulas presented herein.

[0307] Example 1 - Design of antisense targeting sequences Antisense oligomer targeting sequences were designed for therapeutic splice switching applications associated with the IVS1-13T>G mutation in the human GAA gene, where splice switching oligomers are predicted to suppress intronic and exonic splice silencer elements (ISS and ESS elements, respectively), thereby promoting exon 2 retention in the mature GAA mRNA. Restoration of normal or near-normal GAA expression would then allow a functional enzyme to be synthesized, thereby providing clinical benefit to GSD-II patients.

[0308] Exemplary oligomers containing the targeting sequences set forth in Tables 6A-6C were prepared as PPMOs (oligomers conjugated to a CPP, such as an arginine-rich CPP). As described below, these antisense oligomers were introduced into GSD-II patient-derived fibroblasts and patient iPSC-derived myotubes using a gymnotic uptake protocol, as also described in Example 2 below.

[0309] Example 2 - Materials and Methods GSD-II cells. Patient-derived fibroblasts from individuals with GSD-II (Coriell cell lines GM00443 and GM11661) were cultured according to standard protocols in Eagle's DMEM with 10%-15% FBS. Cells were passaged at least twice prior to experimentation and are approximately 80% confluent at the time of transfection. GM00443 and GM11661 patient-derived fibroblasts were reprogrammed into iPSC lines, which were then differentiated into myoblasts, expanded, and banked. Patient iPSC-derived myoblasts were cultured and passaged twice in myoblast growth medium prior to use. Myoblasts were allowed to differentiate into myotubes for 2 days prior to treatment.

[0310] GM00443 fibroblasts are from a 30-year-old male; adult type; onset in 30s; normal size and amount of mRNA for GAA, GAA protein detected by antibody but only 9-26% of normal acid alpha-1,4 glucosidase activity; passage 3 in CCR; donor subject is heterozygous with one allele carrying a T>G transversion at position -13 of the acceptor site in intron 1 of the GAA gene, resulting in an alternatively spliced ​​transcript with a deletion of the first coding exon [exon 2 (IVS1-13T>G)].

[0311] GM11661 fibroblasts were derived from a 38-year-old male with abnormal liver function tests; occasional leg muscle stiffness during physical activity, morning headaches; intolerance to fatty foods; abdominal cysts; deficient fibroblast and WBC acid alpha-1,4 glucosidase activity; the donor subject was a compound heterozygote, with allele 1 carrying a T>G transversion at position -13 of the acceptor site in intron 1 of the GAA gene (IVS1-13T>G), the resulting alternatively spliced ​​transcript having an in-frame deletion of exon 2 containing the start codon, and allele 2 carrying an in-frame deletion of exon 18.

[0312] Treatment protocol. Patient-derived fibroblasts were passaged twice before use. Cells were treated at approximately 80% confluency by replacing the medium containing various concentrations of PPMO. Patient iPSC-derived myoblasts were passaged / expanded at least twice before use. Myoblasts were cultured for 1 day and differentiated into myotubes for 2 days, then treated with differentiation medium containing various concentrations of PPMO.

[0313] GAA qPCR. For quantitative PCR experiments, a multiplex TaqMan qPCR assay was used that simultaneously amplifies GAA mRNA at the junctions of exons 1-2 and 3-4 in addition to the reference gene. 100-500 ng of total RNA from treated patient iPSC-derived myotubes was reverse transcribed using the SuperScript VILO cDNA synthesis kit (Thermo Fisher). cDNA was diluted 3-10-fold before amplification using TaqMan Multiplex Master Mix (Thermo Fisher) using a Quantstuio 7 Pro thermocycler (Thermo Fisher). Each qPCR reaction contained a FAM probe to detect the GAA exon 1-2 junction, a VIC probe to detect the GAA exon 3-4 junction, and a JUN probe to detect the reference gene. Relative standard curves for each assay and probe set up in the multiplex were generated and used to calculate the starting amount of each species in the treated samples normalized to the reference gene.

[0314] GAA Enzyme Assay and Protein Simple Wes. Patient-derived fibroblasts were cultured to approximately 80% confluency and then treated with PPMO compounds via gymnotic uptake. Treatment continued for 6 days, at which point GAA activity was measured using the Abcam GAA Activity Assay Kit (ab252887).

[0315] Western blots to GAA protein were performed using the ProteinSimple® Jes™ system. GAA was detected using recombinant anti-GAA antibody (EPR4716(2)) (Abcam ab137068) and the ProteinSimple® anti-rabbit detection module (DM-001) and 12-230 kDa separation module (SM-W004). GAA protein concentrations were normalized to total protein using the ProteinSimple® Protein Normalization Kit (AM-PN01).

[0316] Example 3 - Preparation of antisense PPMO (R 1 is --N(CH 3 ) 2 is) An antisense PPMO was designed to target human GAA pre-mRNA (e.g., intron 1 of human GAA pre-mRNA), synthesized as described herein, and used to treat GSD-II patient-derived fibroblasts and GSD-II patient iPSC-derived myotubes. [Table 6A-1] [Table 6A-2] [Table 6B] [Table 6C] [Table 6D]

[0317] Example 4 - GAA activity and protein in fibroblasts from GSD-II patients The above antisense PPMOs were delivered to GM00443 or GM11661 fibroblasts and post-patient iPSC-derived myotubes by gymnotic uptake. 2 The cells were incubated at 4-6 days, the cells were lysed, and GAA activity or GAA protein expression in the lysates was measured by immunoassay as described above. In general, protein expression of the GAA enzyme in cells treated with the antisense oligonucleotides of the present disclosure was higher than the GAA expression levels in untreated cells. These results indicate that the oligonucleotides of the present disclosure increase the expression and / or activity of the GAA enzyme in cells from patients with late-onset Pompe disease. The targeting sequence of the variant oligonucleotide is complementary to a target region within intron 1 (SEQ ID NO: 1) of the pre-mRNA of the human alpha-glucosidase (GAA) gene, the target region comprising abasic subunits that are free of purines and pyrimidines. Surprisingly, the use of these abasic subunits facilitated the synthesis of low-yield oligonucleotides while retaining the potency of the parent sequence.

[0318] Example 5 - Small scale synthesis of activated morpholino abasic subunits General preparation: Compound 1 (1.10 equiv.) was suspended in dichloromethane (7.00 mL / g). To this suspension, tetramethylguanidine (0.50 equiv.) and diisopropylethylamine (1.80 equiv.) were added and the mixture was warmed to 30° C. and held for 60 min to dissolve the material, then cooled to room temperature. Separately, trityl chloride (1.00 equiv.) was dissolved in dichloromethane (2.42 mL / g) and this solution was added slowly to the first solution, keeping the temperature below 30° C. Upon reaction completion (1-2 h), the reaction mixture was washed with citrate buffer (pH 4) and water. The organic phase was separated and assayed for compound 2 content (yield: 93%).

[0319] To a solution of compound 2 (1.00 equiv.), 2,6-lutidine (1.15 equiv.) and N-methylimidazole (0.38 equiv.) were added and the solution was concentrated to a volume of 6.00 mL / g by atmospheric distillation. Dichloromethane (5.00 mL / g) was added to the solution, which was again concentrated to the same volume by atmospheric distillation. This process was repeated until the water content was undetectable by Karl Fischer titration, and the solution was cooled to 0-5 °C. Dimethylaminophosphoryl dichloride (1.05 equiv.) was added in a thin stream and the reaction mixture was allowed to warm to room temperature overnight.

[0320] After confirmation of reaction completion, the mixture was passed through a molecular sieve column and the resulting solution was purified by silica gel chromatography using a step gradient of ethyl acetate in heptane. The product-containing fractions were pooled and the pool was evaporated to dryness to produce the final product solid (yield: 73% for the final step and 68% for the two-step process). [ka]

[0321] Because the activated morpholino subunits are unstable in the aqueous HPLC mobile phase, quench derivatization with 1-(4-nitrophenyl)piperazine (NPP) was used to convert the subunits to stable diamidate derivatives for analysis using HPLC. NPP absorbs strongly at 391 nm, so in addition to providing stability, analysis at 391 nm can be used to see impurities that are likely to react with the growing chain of the oligomer.

[0322] Since the product for analysis is the NPP-derivatized activated morpholino base subunit, a standard of this material was synthesized and characterized by HPLC, mass spectrometry, and NMR. This allows for identification of the activated subunit by comparison with the HPLC chromatogram of the synthesized standard with mass spectrometry and NMR confirmed structure. The product peak in the HPLC analysis is slightly split due to partial resolution of the two diastereomers. [ka]

[0323] Example 6 - GAA activity in fibroblasts from GSD-II patients Fibroblast culture. Human fibroblast cell lines were cultured in modified Eagle's medium (MEM, Thermo Fisher) containing 15% fetal bovine serum (FBS) and 2 mM L-glutamine at 37 °C for 2 h at 2 °C under 5% CO. 2 The cells were maintained in a 37°C incubator at 4°C. The fibroblast cell lines currently used were obtained from the Coriell Institute and include the following lines: GM08402 (healthy control), GM08400 (healthy control), GM00443 (late Pompe onset), GM11661 (late Pompe onset), GM20089 (Pompe infantile onset), and GM20123 (healthy Pompe carrier). One day prior to treatment, cells were plated at 30,000 cells / well in 24-well cell culture plates and incubated overnight. The cells were then washed with PBS and treatments of PPMO supplemented with medium were added to the wells. The cells were allowed to incubate for 6 days with treatment and without medium change. For GAA activity assay lysis, the cells were washed once with PBS and then lysed in ice-cold GAA activity assay buffer (Abcam). Figure 6 shows the dose-dependent increase in GAA expression in patient fibroblasts after gymnotic treatment with selected PPMO compounds.

[0324] Example 7 - GAA activity and protein in myotubes from GSD-II patients Patient iPSC-derived myotubes. Patient fibroblasts were reprogrammed into iPSCs using a feeder-free and footprint-free method. Pluripotency was verified by immunostaining with the markers Oct3 / 4, NANOG, and TRA-1-60. iPSCs retained normal karyotype and alkaline phosphatase activity. iPSC lines were differentiated into myoblasts, frozen, and recovered. Myogenic lineage was confirmed by immunofluorescence for the myoblast markers desmin and MyoD, as well as expression of key markers measured by qPCR. Terminal differentiation of myoblasts was performed over 3-6 days of culture and confirmed by expression of the myogenic markers MHC and MyoG measured by immunofluorescence.

[0325] Non-integrative reprogramming of fibroblasts to iPSCs. Fibroblasts were maintained in DMEM 10% FCS. After overnight incubation, the culture medium was replaced with fresh one and cells were transfected with 2 μg of episomal plasmid from Epi5™ iPSC reprogramming kit (Themofisher) by using FuGENE6 transfection reagent (Promega). The next day, the culture medium was replaced with mTeSR-plus medium (StemCell Technologies). During the reprogramming process, transfected cells were cultured in mTeSR plus and the medium was changed every other day for up to 2 weeks after transfection. Colonies were transferred to new culture dishes covered with Geltrex matrix by using a pipette tip. One hour before the procedure, 10 μM of Y-27632 was added to the culture medium. iPSCs were further expanded and maintained in mTeSR plus medium as described in Alonso-Barroso et al., Stem Cell Res. 23, 173-177; 2017.

[0326] SKM Differentiation. Myogenic progenitor cells were differentiated from hiPSCs according to a previously described protocol [Chal, J et. Al. Nat. Biotech. 2015, 33, 962-969]. Briefly, myogenic progenitor cells were generated through a multi-step small molecule differentiation protocol. Myogenic progenitor cells were expanded in 60 μg / mL collagen I-coated 6-well plates, passaged, and cryopreserved. For myoblast differentiation, frozen myogenic progenitor cells were thawed in myoblast growth medium (iXCells, Cat. No. MD-0102A). Growth medium was refreshed every 2 days for 8 days and then cryopreserved. For myotube differentiation, myoblasts were harvested, seeded at a density of 32,000 / cm2, and cultured using myoblast growth medium to reach 100% confluency. For skeletal muscle cell differentiation, confluent myoblast cultures were switched to myoblast differentiation medium (iXCells, Cat. No. MD-0102B) with medium changes every 2 days. Elongated myotubes were evident after 72 hours in myoblast differentiation medium.

[0327] PPMO increases GAA expression in LOPD patient iPSC-derived myotubes. Patient iPSC-derived myoblasts were seeded in 96-well or 24-well collagen-coated plates (Corning) and grown for 48 h in iPSC-derived myoblast growth medium (iXCells Biotechnologies). The medium was replaced with myotube differentiation medium (iXCells Biotechnologies) and differentiation continued for 48 h. The medium was then replaced with fresh differentiation medium containing the indicated concentrations of PPMO. RNA was extracted from cell cultures after 72 h of gymnotic treatment using the Quick-RNA 96 Kit (Zymo) according to the manufacturer's protocol. 100–300 ng of RNA was reverse transcribed using the superscript VILO kit (Thermo Fisher) according to the manufacturer's protocol. Multiplex qPCR assays measuring GAA expression at the exon 1–2 locus on the FAM channel (Hs.PT.58.24962380, Integrated DNA Technologies, 900 nM primers, 250 nM probe), GAA at the exon 3–4 locus on the VIC channel (Hs01089834_m1, Thermo Fisher, 1.8 μM primers, 500 nM probe), and HPRT on the JUN channel (Hs99999909_m1_qsy, Thermo Fisher, 900 nM primers, 250 nM probe) were used with Multiplex Master Mix (Thermo Fisher) on a Quantstudio 7 Pro PCR thermocycler (Thermo Fisher). qPCR cycling conditions consisted of an initial denaturation step at 95° C. for 20 s, followed by 40 cycles of 95° C. for 3 s and 58° C. for 20 s with a ramp rate of 1.92° C. per s. Figures 7 and 8 show a dose-dependent increase in GAA expression in patient iPSC-derived myotubes following gymnotic treatment with selected PPMOs.

[0328] PPMO increases GAA protein in LOPD patient iPSC-derived myotubes. Patient iPSC-derived myoblasts were seeded in 24-well collagen-coated plates (Corning) and grown in iPSC-derived myoblast growth medium (iXCells Biotechnologies) for 24 h. The medium was replaced with myotube differentiation medium (iXCells Biotechnologies) and differentiation continued for 24 h. The medium was then replaced with fresh differentiation medium containing the indicated concentrations of PPMO. Cell lysates were prepared after 96 h of gymnotic treatment using RIPA lysis buffer (Thermo Fisher). Protein concentrations were measured using the Pierce BCA Assay Kit (Thermo Fisher). Cell lysates were prepared using an automated capillary western blot system, Sample Preparation Kit for the JESS system (Proteinsimple) (Proteinsimple). Cell lysates were diluted to the same protein concentration using 0.1x sample buffer (Proteinsimple) and mixed with 5x fluorescence master mix (Proteinsimple) as per protocol instructions. Samples were denatured at 95°C as per protocol instructions. JESS was performed using 1:400 diluted anti-GAA primary antibody (Abcam ab137068) diluted in milk-free antibody diluent, protein normalization substrate, horseradish peroxidase (HRP)-conjugated secondary antibody, chemiluminescent substrate, and wash buffer dispensed into the indicated wells of the assay plate. Samples were loaded in triplicate with biotinylated ladder markers at the indicated locations on the JESS plate, and the assay plate was placed in the JESS instrument. Protein signal intensity (peak area) was normalized to the peak area of ​​total protein contained in the capillary wells using a protein normalization kit and analysis in Compass Software (Proteinsimple). Quantitative analysis of GAA protein bands was performed using Compass Software (ProteinSimple). Figures 9 and 10 show the increase in GAA protein in patient iPSC-derived myotubes following treatment with selected PPMO compounds.

[0329] PPMO increases GAA protein in LOPD patient iPSC-derived myotubes. Patient iPSC-derived myoblasts were plated in 24-well collagen-coated plates (Thermo Fisher) at 80,000 cells / well in growth medium (EM, iXCells Biotechnologies). After 48 h of growth in EM, cells were washed in PBS and the medium was replaced with differentiation medium (DM, iXCells Biotechnologies). Cells were incubated in DM for 48 h, then treated with PPMO-supplemented DM and incubated for 4 days without medium replacement. For GAA activity assay lysis, cells were washed once with PBS and then lysed in ice-cold GAA activity assay buffer (Abcam). Figure 11 shows a dose-dependent increase in GAA enzyme activity in patient iPSC-derived myotubes after treatment with selected PPMO compounds.

[0330] Example 8 - Abasic Substitutions Reduce PPMO Aggregation Aggregation of PPMO samples in constant concentration solutions in PBS (Gibco) was measured by dynamic light scattering using a Zetasizer Nano (Malvern) using the manufacturer's standard protocol. Figure 12 shows that abasic substitution reduces PPMO aggregation. The ratio of free PPMO increases with abasic substitution as measured by dynamic light scattering (DLS).

[0331] In summary, the PPMO compounds provided herein consistently corrected GAA splicing in LOPD patient-derived myotubes and increased GAA protein and enzyme activity levels. Target engagement of human IVS1-GAA was confirmed in a mouse model of LOPD. Surprisingly, replacement of the abasic subunit is nearly as effective as the parent sequence in restoring the GAA enzyme to an active state (e.g., PPMO7 vs. PPMO33). Interestingly, DLS data indicates some alteration of aggregation or secondary structure formation in these sequences by including the abasic subunit.

Claims

1. A conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide, the modified antisense oligonucleotide is 18 to 40 subunits in length and comprises a targeting sequence complementary to a target region within intron 1 (SEQ ID NO: 1) of the pre-mRNA of the human acid alpha-glucosidase (GAA) gene; the antisense oligonucleotide comprises a morpholino oligomer; the antisense oligonucleotide is covalently bound to the cell membrane-permeable peptide; each subunit of the antisense oligonucleotide comprises a nucleobase or is an abasic subunit, and the subunits are joined together in order from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form the targeting sequence; at least one subunit is an abasic subunit; A conjugate wherein the targeting sequence, excluding the abasic subunit, is at least 80% complementary to the target region.

2. 2. The conjugate of claim 1, wherein the target region comprises a sequence selected from the group consisting of SEQ ID NO: 2 (GAA-IVS1(-189-167)) and SEQ ID NO: 3 (GAA-IVS1(-80-24)).

3. The conjugate of claim 2 , wherein the target region comprises the sequence set forth in SEQ ID NO:

2.

4. The conjugate of claim 2 , wherein the target region comprises the sequence set forth in SEQ ID NO:

3.

5. 3. The conjugate of claim 1 or 2, wherein the target region is selected from GAA-IVS1(-189-167), GAA-IVS1(-80-56), GAA-IVS1(-76-52), GAA-IVS1(-74-55), GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), GAA-IVS1(-65-41), and GAA-IVS1(-49-24).

6. 6. The conjugate of claim 1 or 5, wherein the target region is GAA-IVS1 (-189-167).

7. 10. The conjugate of claim 1 or 6, wherein the targeting sequence comprises the sequence CCA GAA GGA AXX XCG AGA AAA GC (SEQ ID NO: 4), wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

8. the targeting sequence is i) SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC); ii) Sequence number 6 (CCA GAA GGA AGB GCG AGA AAA GC), iii) SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC), iv) SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC), v) SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC), and vi) SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC).

9. 6. The conjugate of claim 1 or 5, wherein the target region is selected from the group consisting of GAA-IVS1(-80-56), GAA-IVS1(-76-52), GAA-IVS1(-74-55), GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), GAA-IVS1(-65-41), and GAA-IVS1(-49-24).

10. 10. The conjugate of claim 1 or 9, wherein the target region is selected from the group consisting of GAA-IVS1(-72-48), GAA-IVS1(-71-47), GAA-IVS1(-70-46), GAA-IVS1(-69-45), GAA-IVS1(-66-42), and GAA-IVS1(-65-41).

11. the targeting sequence is i) SEQ ID NO: 11 (CTC ACX XXX CTC TCA AAG CAG CTC T), ii) SEQ ID NO: 12 (ACT CAC XXX XCT CTC AAA GCA GCT C), iii) SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T), iv) SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C); v) SEQ ID NO: 15 (GCG GCA CTC ACX XXX CTC TCA AAG C), vi) SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G); 11. The conjugate of claim 1 or 10, wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

12. the targeting sequence is i) SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C); ii) SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C); iii) SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C); iv) SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C); v) SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C); vi) SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C); vii) SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C), and viii) SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC 12. The conjugate of claim 1 or 11, wherein the conjugate is selected from the group consisting of:

13. 13. The conjugate of any one of claims 1 to 12, wherein the targeting sequence is at least 84%, at least 88%, or at least 92% complementary to the target region, excluding the abasic subunit.

14. The conjugate of any one of claims 1 to 12, wherein the targeting sequence is at least 90% complementary to the target region, excluding the abasic subunit.

15. 6. The conjugate of any one of claims 1 to 5, wherein the targeting sequence, excluding the abasic subunit, is at least 95% complementary to the target region.

16. 6. The conjugate of any one of claims 1 to 5, wherein the targeting sequence is 100% complementary to the target region, except for the abasic subunit.

17. The conjugate of any one of claims 1 to 4, wherein each abasic subunit is at least 8 subunits from the 5' or 3' end of the targeting sequence.

18. The conjugate of any one of claims 1 to 4, wherein the antisense oligonucleotide comprises 1 to 5 abasic subunits.

19. 19. The conjugate of any one of claims 1 to 4 or 18, wherein the antisense oligonucleotide comprises one, two, three, or four abasic subunits.

20. The conjugate is a compound of formula IV: 【Chemistry 81】 or a pharmaceutically acceptable salt thereof; During the ceremony, A' is -N(H)CH ２ C(O)NH ２ , -N(C １－６ -alkyl)CH ２ C(O)NH ２ , 【Chemistry 82】 is selected from: R ５ is —C(O)(O-alkyl) ｘ -OH, where x is 3 to 10, and each alkyl group in each occurrence is independently selected from the group consisting of C ２－６ - alkyl or or R ５ is H, -C(O)C １－６ -Alkyl, trityl, monomethoxytrityl, -(C １－６ -alkyl)-R ６ , -(C １－６ -heteroalkyl)-R ６ , aryl-R ６ , heteroaryl-R ６ , -C(O)O-(C １－６ -alkyl)-R ６ , —C(O)O-aryl-R ６ , —C(O)O-heteroaryl-R ６ , and 【Chemistry 83】 is selected from R ６ are OH, SH, and NH ２ or R ６ is O, S, or NH, each of which is covalently attached to a solid support; Each R １ are independently OH and —N(R ３ ) (R ４ ) wherein each R ３ and R ４ is independently in each occurrence H or —C １－６ - alkyl, Each R ２ is independently selected at each occurrence from H (abasic), a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein said nucleobase is independently selected at each occurrence from pyridine, pyrimidine, purine, and deaza-purine. ３－６ containing a heterocycle, t is 8 to 40; E' is H, -C １－６ -alkyl, -C(O)C １－６ - alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 【Chemistry 84】 is selected from During the ceremony, Q is —C(O)(CH ２ ) ６ C(O)- or -C(O)(CH ２ ) ２ S ２ (CH ２ ) ２ C(O)—, R ７ is -(CH ２ ) ２ OC(O)N(R ８ ) ２ wherein R ８ is -(CH ２ ) ６ NHC (=NH)NH ２ and L is glycine, proline, W, WW, or R ９ and L is covalently attached by an amide bond to the N-terminus or C-terminus of J; W is —C(O)—(CH ２ ) ｍ -NH-, where m is 2 to 12; R ９ teeth, 【Chemistry 85】 is selected from the group consisting of n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5; R １０ is selected from a bond, glycine, proline, W, or W-W; R １１ is glycine, proline, W, W-W, and 【Chemistry 86】 is selected from the group consisting of R １６ is selected from a bond, glycine, proline, W, or WW; R １６ is covalently attached by an amide bond to the N-terminus or C-terminus of J, wherein J is a cell membrane penetrating peptide; G is H, -C(O)C １－６ - selected from alkyl, benzoyl, and stearoyl, wherein G is covalently bonded to J; however, A' is 【Hua 87】 or E' is 【Hua 88】 The conjugate of any one of claims 1 to 19, provided that:

21. E' is H, -C １－６ -alkyl, -C(O)C １－６ - alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and 【Chemistry 89】 21. The conjugate of claim 20, selected from:

22. A' is -N(C １－６ -alkyl)CH ２ C(O)NH ２ , 【Chemistry 90】 22. The conjugate of claim 20 or 21, selected from:

23. E' is H, -C(O)CH ３ , benzoyl, stearoyl, trityl, 4-methoxytrityl, and 【Chemistry 91】 The conjugate according to any one of claims 20 to 22, selected from:

24. A' is -N(C １－６ -alkyl)CH ２ C(O)NH ２ , 【Chemistry 92】 is selected from E' is 【Chemistry 93】 The conjugate according to any one of claims 20 to 23, wherein

25. A' is 【Chemistry 94】 and E' is H, -C(O)CH ３ 24. The conjugate of any one of claims 20 to 23, wherein the aryl group is selected from the group consisting of aryl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

26. The peptide-oligonucleotide conjugate of formula IV is 【Chemistry 95】 a peptide-oligonucleotide conjugate selected from In the formula, E′ is H, C １－６ -Alkyl, -C(O)CH ３ 21. The conjugate of claim 20, wherein the alkyl group is selected from the group consisting of benzoyl, benzoyl, and stearoyl.

27. The conjugate of any one of claims 20 to 26, wherein the conjugate is of formula (IVa):

28. The conjugate of any one of claims 20 to 26, wherein the conjugate is of formula (IVb):

29. Each R １ But -N(CH ３ ) ２ The conjugate according to any one of claims 20 to 26, wherein

30. 30. The conjugate of any one of claims 20 to 29, wherein each nucleobase, in each occurrence, is independently selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.

31. The targeting sequence has the sequence: i) SEQ ID NO: 4 (CCA GAA GGA AXX XCG AGA AAA GC), ii) SEQ ID NO: 11 (CTC ACX XXX CTC TCA AAG CAG CTC T), iii) SEQ ID NO: 12 (ACT CAC XXX XCT CTC AAA GCA GCT C), iv) SEQ ID NO: 13 (CAC TCA CXX XXC TCT CAA AGC AGC T), v) SEQ ID NO: 14 (GCA CTC ACX XXX CTC TCA AAG CAG C); vi) SEQ ID NO: 15 (GCG GCA CTC ACX XXX CTC TCA AAG C), vii) comprising SEQ ID NO: 16 (GGC GGC ACT CAC XXX XCT CTC AAA G); 31. The conjugate of any one of claims 20 to 30, wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

32. the targeting sequence is i) SEQ ID NO: 5 (CCA GAA GGA AGG BCG AGA AAA GC); ii) SEQ ID NO: 6 (CCA GAA GGA AGB GCG AGA AAA GC), iii) SEQ ID NO: 7 (CCA GAA GGA ABG GCG AGA AAA GC), iv) SEQ ID NO: 8 (CCA GAA GGA AGB BCG AGA AAA GC), v) SEQ ID NO: 9 (CCA GAA GGA ABB GCG AGA AAA GC), vi) SEQ ID NO: 10 (CCA GAA GGA ABG BCG AGA AAA GC), vii) SEQ ID NO: 17 (GCA CTC ACB GGG CTC TCA AAG CAG C); viii) SEQ ID NO: 18 (GCA CTC ACG BGG CTC TCA AAG CAG C); ix) SEQ ID NO: 19 (GCA CTC ACG GBG CTC TCA AAG CAG C); x) SEQ ID NO: 20 (GCA CTC ACG GGB CTC TCA AAG CAG C); xi) SEQ ID NO: 21 (GCA CTC ACB BGG CTC TCA AAG CAG C); xii) SEQ ID NO: 22 (GCA CTC ACG BBG CTC TCA AAG CAG C); xiii) SEQ ID NO: 23 (GCA CTC ACG GBB CTC TCA AAG CAG C), and xiv) The conjugate of any one of claims 20 to 31, comprising a sequence selected from the group consisting of SEQ ID NO: 24 (GGC GGC ACT CAC GBB GCT CTC AAA G).

33. The conjugate of any one of claims 20 to 32, wherein L is glycine.

34. The conjugate of any one of claims 20 to 32, wherein L is proline.

35. L is —C(O)—(CH ２ ) ５ The conjugate of any one of claims 20 to 32, which is -NH-.

36. L is —C(O)—(CH ２ ) ２ The conjugate of any one of claims 20 to 32, which is -NH-.

37. L is —C(O)—(CH ２ ) ２ -NH-C(O)-(CH ２ ) ５ The conjugate of any one of claims 20 to 32, which is -NH-.

38. L, 【Chemistry 96】 and R １０ is a bond, and R １１ However, glycine and 【Chemistry 97】 The conjugate of any one of claims 20 to 32, selected from:

39. L, 【Chemistry 98】 and R １０ is a bond, and R １１ However, glycine and 【Hua99】 The conjugate of any one of claims 20 to 32, selected from:

40. L, 【Chemistry 100】 and R １０ is a bond, and R １１ However, glycine and 【Chemistry 101】 The conjugate of any one of claims 20 to 32, selected from:

41. J is rTAT, TAT, R ９ F ２ , R ５ F ２ R ４ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , (RXR) ４ , (RXR) ５ , (RXRRBR) ２ , (RAR) ４ F ２ , (RGR) ４ F ２ The conjugate of any one of claims 20 to 38, selected from:

42. G is H, C(O)CH ３ 42. The conjugate of any one of claims 20 to 41, wherein the alkyl group is selected from the group consisting of benzoyl, benzoyl, and stearoyl.

43. G is H or —C(O)CH ３ The conjugate of any one of claims 20 to 42, wherein

44. The conjugate of any one of claims 20 to 43, wherein G is H.

45. G is —C(O)CH ３ The conjugate according to any one of claims 20 to 43, wherein

46. 46. ​​A pharmaceutical composition comprising the conjugate of any one of claims 1 to 45, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

47. A composition comprising a conjugate according to any one of claims 1 to 45 or a pharmaceutical composition according to claim 46 for treating a disease in a subject in need thereof.

48. 48. The composition of claim 47, wherein the disease is Pompe disease.

49. 48. The composition of claim 47, wherein the subject is a human.

50. 50. The composition of claim 49, wherein the human is a child.

51. 50. The composition of claim 49, wherein the human is an adult.

52. 1. An antisense oligomeric compound comprising: 【Chemistry 102】 is selected from wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

53. the antisense oligomeric compound 【Chemistry 103】 and 53. The antisense oligomeric compound of claim 52, wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B.

54. the antisense oligomeric compound 【Chemistry 104】 53. The antisense oligomeric compound of claim 52, wherein each X is independently selected from guanine (G) or abasic (B), and at least one X is B. In one embodiment, B is H.

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