US20170182189A1 - Inhibiting or downregulating glycogen synthase by creating premature stop codons using antisense oligonucleotides - Google Patents

Inhibiting or downregulating glycogen synthase by creating premature stop codons using antisense oligonucleotides Download PDF

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US20170182189A1
US20170182189A1 US15/313,387 US201515313387A US2017182189A1 US 20170182189 A1 US20170182189 A1 US 20170182189A1 US 201515313387 A US201515313387 A US 201515313387A US 2017182189 A1 US2017182189 A1 US 2017182189A1
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glycogen synthase
antisense oligonucleotide
mrna
glycogen
nucleic acid
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Carol A. Nelson
Bruce M. Wentworth
Ronald K. Scheule
Timothy E. Weeden
Nicholas P. CLAYTON
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Genzyme Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01011—Glycogen(starch) synthase (2.4.1.11)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
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    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present disclosure relates to antisense oligonucleotides (AONs) for modulating the expression of glycogen synthase.
  • AONs of the present disclosure may be useful in treating diseases associated with the modulation of the expression of the enzyme glycogen synthase, such as Pompe disease.
  • compositions comprising AONs, as well as methods of down regulating mRNA coding for glycogen synthase, methods for reducing glycogen synthase in skeletal and cardiac muscle, and methods for treating Pompe disease.
  • Pompe disease is an inherited disorder caused by the accumulation of glycogen in the body's cells. This buildup of glycogen in the body, especially in skeletal and cardiac muscle, impacts the ability of the body's organs and tissues to function normally.
  • Pompe disease There are three known types of Pompe disease, including classic infantile-onset, non-classic infantile-onset, and late-onset.
  • the classic form of infantile-onset begins within a few months of birth, and infants with this disorder experience myopathy, hypotonia, hepatomegaly, and heart defects, and death from heart failure typically results within the first year of life if not treated.
  • Non-classic infantile-onset usually develops within the first year of life, and children with this disorder experience delayed motor skill development, progressive muscle weakness, and may have an enlarged heart.
  • Serious breathing problems can occur and children with this form of Pompe disease do not live past early childhood.
  • the last type of Pompe disease, late-onset may not become apparent until much later in a person's life, including even into adulthood. Late-onset Pompe disease is usually milder than the infantile-onset forms and typically does not involve the heart. However, people with this form of the disease experience progressive muscle weakness, which can lead to breathing problems and may eventually lead to respiratory failure.
  • Pompe disease is caused by mutations in the GAA gene.
  • the GAA gene encodes for acid alpha-glucosidase, which is an enzyme that breaks down glycogen into the simple sugar glucose. Mutations in the GAA gene lead to the genetic deletion of acid alpha-glucosidase. As a result, glycogen builds up in the cells and leads to the symptoms associated with Pompe disease.
  • Pompe disease is currently treated by enzyme replacement therapy using recombinant GAA. However, this method of treatment is not always entirely effective, and as a result, additional therapies for Pompe disease are needed.
  • Inhibiting the biosynthesis of glycogen is another potential means to treat patients with Pompe disease.
  • So-called substrate reduction therapy is based on the inhibition of the main enzyme isoform responsible for building the glycogen polymer in skeletal muscle, glycogen synthase 1.
  • Three methods have been reported that accomplish glycogen synthase 1 inhibition in Pompe mice: administration of a small interfering RNA (Douillard-Guilloux et al 2008); genetic knock down of the GSY1 gene in mice then crossed to Pompe mice (Douillard-Guilloux et al 2010); and inhibition of mTORC1 by administration of rapamycin (Ashe et al 2010). All three methods suppressed the accumulation of glycogen in Pompe mice.
  • glycogen synthase It is accordingly a primary object of the present disclosure to modulate the expression of glycogen synthase, resulting in beneficial effects for mammals who suffer from symptoms related to the buildup of glycogen in the body's cells.
  • Glycogen synthase is an enzyme involved in converting glucose into glycogen. In humans there are two different forms called isozymes or isoforms, glycogen synthase 1 and glycogen synthase 2, which are encoded for by the genes GYS1 and GYS2, respectively. Glycogen synthase 1 is expressed in muscles and other tissues, and glycogen synthase 2 is expressed only in the liver. GYS1 encodes for a pre-mRNA that has 16 exons.
  • Exemplary human GYS1 sequences may be found at NCBI Reference Sequence: NM_002103.4 also shown in SEQ ID NO.: 1 (DNA/RNA) and SEQ ID NO.: 2 (CDS) or via the Human Gene Nomenclature Committee at HGNC: 4706 (see FIG. 1 ).
  • Exemplary mouse GYS1 sequences may be found at NCBI Reference Sequence: NM_030678.3 also shown in SEQ ID NO.: 3 (DNA/RNA) and SEQ ID NO.: 4 (CDS) or via the Human Gene Nomenclature Committee at MGI: 101805 (see FIG. 2 ).
  • the modulation of pre-mRNA or mRNA transcribed from GYS1 may result in the down-regulation of glycogen synthase protein and reduce glycogen synthase enzyme activity in skeletal and cardiac muscle, as well as treat and/or prevent the symptoms associated with glycogen buildup in the muscles.
  • the present disclosure relates to a method of down regulating mRNA coding for glycogen synthase comprising administering an effective amount of an antisense oligonucleotide to an animal, wherein the antisense oligonucleotide forms a sequence complementary to a nucleic acid sequence encoding for glycogen synthase, and wherein the hybridization of the antisense oligonucleotide to the nucleic acid sequence encoding for glycogen synthase induces exon skipping.
  • the present disclosure relates to a method of down regulating mRNA coding for glycogen synthase comprising administering an effective amount of an antisense oligonucleotide to an animal, wherein the antisense oligonucleotide forms a sequence complementary to a nucleic acid sequence encoding for glycogen synthase, and wherein the hybridization of the antisense oligonucleotide to the nucleic acid sequence encoding for glycogen synthase induces translational inhibition.
  • the present disclosure relates to a method of down regulating mRNA coding for glycogen synthase comprising administering an effective amount of an antisense oligonucleotide to an animal, wherein the antisense oligonucleotide forms a sequence complementary to a nucleic acid sequence encoding for glycogen synthase, and wherein the hybridization of the antisense oligonucleotide to the nucleic acid sequence encoding for glycogen synthase induces suppression of polyadenylation.
  • One embodiment of the invention is method of down regulating mRNA coding for glycogen synthase comprising administering an effective amount of an antisense oligonucleotide to an animal, wherein the antisense oligonucleotide comprises a sequence complementary to a nucleic acid sequence encoding for glycogen synthase, and wherein the hybridization of the antisense oligonucleotide to the nucleic acid sequence encoding for glycogen synthase induces exon skipping.
  • a further embodiment is of the invention is a method of down regulating mRNA coding for glycogen synthase comprising administering an effective amount of an antisense oligonucleotide to an animal, wherein the antisense oligonucleotide comprises a sequence complementary to a nucleic acid sequence encoding for glycogen synthase, and wherein the hybridization of the antisense oligonucleotide to the nucleic acid sequence encoding for glycogen synthase induces exon skipping, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligo (also known as “PMO” or “morpholino”) or wherein the antisense oligonucleotide is a PMO linked to a cell penetrating peptide (“CPP”).
  • the antisense oligonucleotide comprises a sequence complementary to a nucleic acid sequence encoding for glycogen synthase
  • a further embodiment is of the invention is a method of down regulating mRNA coding for glycogen synthase comprising administering an effective amount of an antisense oligonucleotide to an animal, wherein the antisense oligonucleotide comprises a sequence complementary to a nucleic acid sequence encoding for glycogen synthase, and wherein the hybridization of the antisense oligonucleotide to the nucleic acid sequence encoding for glycogen synthase induces exon skipping, wherein mRNA coding for glycogen synthase is reduced by up to 80%, up to 90% or up to 95%.
  • FIG. 1 is the exemplary human GYS1 sequences as found at NCBI Reference Sequence: NM_002103.4 also shown in SEQ ID NO.: 1 (DNA/RNA) and SEQ ID NO.: 2 (CDS).
  • FIG. 2 is the exemplary mouse GYS1 sequences as found at NCBI Reference Sequence: NM_030678.3 also shown in SEQ ID NO.: 3 (DNA/RNA) and SEQ ID NO.: 4 (CDS).
  • FIG. 3 Gys1 mRNA levels are reduced in skeletal and cardiac muscles of Pompe mice treated with repeated intravenous injections of GS-PPMO.
  • A Semi-quantitative PCR analysis was performed on pooled samples of RNA prepared from tissues of wild type and Pompe mice that received the indicated treatments to determine Gys1 transcript levels. Gys1 mRNA levels (normalized to ⁇ -actin mRNA levels) were measured in the (B) quadriceps, (C) diaphragm, and (D) heart tissues.
  • FIG. 4 Gys1 protein levels are reduced in skeletal and cardiac muscles of Pompe mice treated with repeated intravenous injections of GS-PPMO.
  • A Western blot analysis was carried out on pooled samples of protein lysates to assess GYS1 protein levels in tissues of wild type and Pompe mice that received the indicated treatments. GYS1 protein levels (normalized to gapdh protein levels), in the (B) quadriceps, (C) diaphragm, and (D) heart tissues were measured.
  • FIG. 8 Serum chemistries of Pompe mice treated with GS-PPMO compared to control animals.
  • FIG. 9 Histopathological analysis of kidney and liver of Pompe and wild type mice. Hematoxylin and eosin stained slides were prepared from mice treated with either vehicle of GS-PPMO as indicated.
  • Kidney sections of GS-PPMO treated Pompe mice show a normal architecture of proximal convoluted tubules and glomeruli.
  • (B) Livers of GS-PPMO-treated Pompe mice exhibit the presence of Kupffer cells in hepatocytes. Magnification 40 ⁇ .
  • Glycogen synthase is an enzyme involved in converting glucose into glycogen. In humans there are two different forms called isozymes or isoforms, glycogen synthase 1 and glycogen synthase 2, which are encoded for by the genes GYS1 and GYS2, respectively. Glycogen synthase 1 is located in muscles and other tissues, and glycogen synthase 2 is found only in the liver. GYS1 encodes for a pre-mRNA that has 16 exons.
  • Exemplary human GYS1 sequences may be found at NCBI Reference Sequence: NM_002103.4 also shown in SEQ ID NO.: 1 (DNA/RNA) and SEQ ID NO.: 2 (CDS) or via the Human Gene Nomenclature Committee at HGNC: 4706 (see FIG. 1 ).
  • Exemplary mouse GYS1 sequences may be found at NCBI Reference Sequence: NM_030678.3 also shown in SEQ ID NO.: 3 (DNA/RNA) and SEQ ID NO.: 4 (CDS) or via the Human Gene Nomenclature Committee at MGI: 101805 (see FIG. 2 ).
  • RNA target refers to an RNA transcript to which a morpholino binds in a sequence specific manner.
  • the RNA target is one or more GSY1 mRNA or pre-mRNA molecules.
  • Morpholino or “morpholino antisense oligonucleotide” refer to an oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide.
  • the morpholino binds to an RNA target which blocks translation of the RNA target into a protein. In other embodiments, the morpholino prevents aggregation of the RNA target with itself or with other cellular RNAs, proteins, or riboproteins, such as, but not limited to, RNAs, proteins, and riboproteins associated with the cellular mRNA splicing apparatus.
  • mammals can be a mammal, such as any common laboratory model organism, or any other mammal. Mammals include, but are not limited to, humans and non-human primates, farm animals, sport animals, pets, mice, rats, and other rodents.
  • treatment refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • prevention includes providing prophylaxis with respect to occurrence or recurrence of a disease or the symptoms associated with a disease in an individual.
  • An individual may be predisposed to, susceptible to, or at risk of developing a disease, but has not yet been diagnosed with the disease.
  • an “effective amount” or “therapeutically effective amount” refers to an amount of therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.
  • an antisense oligomer this effect is typically brought about by inhibiting translation or natural splice-processing of a selected target sequence.
  • Acid maltase or ⁇ -glucosidase is a lysosomal enzyme that catalyzes the breakdown of glycogen to glucose. Mutations in the GAA gene that lead to a reduction in the amount or activity of the enzyme are the molecular basis of Pompe disease (glycogen storage disease type II). This autosomal recessive metabolic myopathy results as a consequence of the progressive accumulation of undegraded glycogen, primarily in the lysosomes of cardiac and skeletal muscle. Patients with Pompe disease (incidence of approximately 1 in 40,000) present with a broad spectrum of disease severity that is inversely correlated with the amount of residual enzyme activity. Complete loss of enzyme activity results in an infantile presentation (so called “floppy babies”) with affected individuals rarely living beyond 2 years of age. Varying degrees of residual enzyme activity lead to a progressive myopathy in young adults as well as older individuals that is invariably fatal.
  • Pompe disease is managed by periodic infusions of a recombinant enzyme (rhGAA) preparation that gained regulatory approval in 2006.
  • rhGAA recombinant enzyme
  • Systemic infusion of rhGAA has been shown in clinical trials to improve cardiomyopathy and prolong survival in children and to improve walking ability as well as stabilize pulmonary function in adults.
  • rhGAA recombinant enzyme
  • These residual deficits may be due in part to inefficient delivery of the enzyme to some of the affected tissues or to the host immune response to the administered protein.
  • modified forms of rhGAA conjugated with mannose 6-phosphate-bearing oligosaccharides or engineered to express a portion of IGF-1 have been developed that show improved delivery to muscle and bioactivity in animal studies.
  • a small molecule chaperone that reportedly improves the stability of the enzyme and enhances glycogen clearance in Pompe mice is being tested clinically.
  • Gene therapy with recombinant AAV vectors encoding the enzyme deficient in Pompe disease is also being evaluated as a treatment modality.
  • the instant invention is directed to an alternative approach for reducing muscle glycogen levels in Pompe mice.
  • Skeletal muscle glycogen synthase activity is the result of transcription of the Gys1 gene.
  • liver synthase activity is generated mostly by expression of the Gys2 gene and its encoded enzyme produces glycogen as a ready store of glucose for body-wide metabolism.
  • PMO phosphorodiamidate morpholino-based antisense oligonucleotide
  • the PMO dose needed to restore dystrophin synthesis can be greatly reduced if it is conjugated to a cell penetrating peptide (PPMO).
  • PPMO cell penetrating peptide
  • Delivery of a therapeutically relevant dose of a phosphorodiamidate morpholino-based antisense oligonucleotide (PMO) for the purpose of skipping mutant exons was utilized to induce exon skipping of the Gys1 mRNA for the purpose of reducing its transcript levels, presumably and without being limited as to theory, via nonsense mediated decay, with concomitant reductions in the skeletal muscle enzyme.
  • Treating Pompe mice with a PPMO targeting a specific sequence in exon 6 of Gys1 mRNA reduces in a dose dependent manner the Gys1 transcript in skeletal muscle and heart but not the Gys2 transcript in liver.
  • the glycogen synthase protein level is reduced in skeletal muscle and heart and synthase activity is restored. Consequently, glycogen accumulation is completely abated in skeletal muscle; the impact is less in the heart.
  • ASO antisense oligonucleotide
  • the present disclosure relates to oligomeric antisense compounds, i.e., AONs, such as phosphorodiamidate morpholino (PMO) compounds, peptide nucleic acids (PNAs), 2′-O alkyl (e.g., methyl) antisense oligonucleotides, and tricyclo-DNA antisense nucleotides for use in modulating pre-mRNA and mRNA transcribed from GYS1.
  • AONs oligomeric antisense compounds, i.e., AONs, such as phosphorodiamidate morpholino (PMO) compounds, peptide nucleic acids (PNAs), 2′-O alkyl (e.g., methyl) antisense oligonucleotides, and tricyclo-DNA antisense nucleotides for use in modulating pre-mRNA and mRNA transcribed from GYS1.
  • PMO phosphorodiamidate morpholino
  • PNAs
  • the present disclosure relates to any AON that specifically hybridize with one or more of pre-mRNA or mRNA transcribed from GYS1 and induce a reduction in glycogen accumulation in a disease state.
  • the AONs contemplated for use in the instant invention include those attached to a cell-penetrating peptide (CPP) to enhance delivery.
  • the AON-CPP may comprise multiple AON, including PMO AON, attached to a single CPP.
  • the multiple AON-CPP conjugate further comprises a cathepsin cleavable linker.
  • the cathepsin cleavable linker can occur in between the AON and the CPP or it can occur in a sequence such as AON-cathepsin linker-AON-cathepsin linker-CPP.
  • the multiple PMO-CPP conjugate further comprises a cathepsin cleavable linker.
  • the cathepsin cleavable linker can occur in between the PMO and the CPP or it can occur in a sequence such as PMO-cathepsin linker-PMO-cathepsin linker-CPP.
  • an AON specifically hybridizes to a target polynucleotide, such as pre-mRNA or mRNA, when the AON hybridizes to the target under physiological conditions.
  • hybridization occurs via hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary purine and pyrimidine bases.
  • hydrogen bonding may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary purine and pyrimidine bases.
  • adenine (A) and thymine (T) are complementary nucleobases which pair through the formation of hydrogen bonds.
  • AONs such as PMO compounds
  • AONs are complementary to a target pre-mRNA or mRNA when hybridization occurs according to generally accepted base-pairing rules, e.g., adenine (A)-thymine (T), cytosine (C)-guanine (G), adenine (A)-uracil (U).
  • base-pairing rules e.g., adenine (A)-thymine (T), cytosine (C)-guanine (G), adenine (A)-uracil (U).
  • “complementary” as used herein refers to the capacity for precise pairing between two nucleobases.
  • a base (B) at a certain position of an AON is capable of hydrogen binding with a nucleotide at the same position of a pre-mRNA or mRNA molecule
  • the AON and the pre-mRNA or mRNA molecule are considered to be complementary to each other at that position.
  • the AON and pre-mRNA or mRNA target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by bases that can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the AON, such as a PMO, and the pre-mRNA or mRNA target.
  • Absolute complementarity i.e., a 100% complementary base pair match, is not necessary as long as the heteroduplex formed between the target pre-mRNA or mRNA and the AON has sufficient stability to bring about the desired effect such as a reduction in glycogen accumulation.
  • an AON such as a PMO
  • an AON is specifically hybridizable when binding of the AON to the target pre-mRNA or mRNA molecule interferes with the normal function of the target pre-mRNA or mRNA molecule, and/or it brings about the desired effect, and there is a sufficient degree of complementarity to avoid intolerable non-specific binding of the AON to a non-target sequence under conditions in which specific binding is desired, for example under physiological conditions for in vivo applications or under conditions in which assays are performed for in vitro applications.
  • hybridization between an AON and pre-mRNA or mRNA interferes with their normal functions, such as translation of protein from the mRNA and splicing of the pre-mRNA to yield one or more mRNA species.
  • the hybridization between the AON and pre-mRNA affects the splicing of the pre-mRNA to form stable RNA.
  • the hybridization affects the translation of glycogen synthase 1 from mRNA.
  • AONs include PMO compounds as well as PNA compounds, phosphoramidate compounds, methylene methylimino (“MMI”) compounds, 2-O-methyl compounds and 2-methoxy ethyl compounds, wherein the oligonucleobase of each subunit are set forth in FIG. 1 .
  • the oligonucleotide compounds are synthetic analogs of natural nucleic acids.
  • the oligonucleotide compounds instead of deoxyribose rings and phosphate-linkages, the oligonucleotide compounds comprise subunits comprised of the respective oligonucleotide subunits shown below:
  • B is a nucleotide base.
  • the primary nucleobases are cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), abbreviated as C, G, A, T, and U, respectively.
  • A, G, C, and T appear in DNA, these molecules are called DNA-bases;
  • A, G, C, and U are called RNA-bases.
  • Uracil replaces thymine in RNA.
  • Adenine and guanine belong to the double-ringed class of molecules called purines (abbreviated as R). Cytosine, thymine, and uracil are all pyrimidines (abbreviated as Y).
  • AON compositions can comprise morpholino oligonucleotide compositions.
  • Morpholinos are synthetic molecules having a structure that closely resembles a naturally occurring nucleic acid. These nucleic acids bind to complementary RNA sequences by standard nucleic acid base pairing. Structurally, morpholinos differ from DNA or RNA in that these molecules have nucleic acid bases bound to morpholine rings instead of deoxyribose or ribose rings. Additionally, the backbone structure of morpholinos consists of non-ionic or cationic linkage groups instead of phosphates.
  • morpholinos in organisms or cells uncharged molecules.
  • Morpholinos are most commonly used as single-stranded oligos, though heteroduplexes of a morpholino strand and a complementary DNA strand may be used in combination with cationic cytosolic delivery reagents.
  • morpholinos do not degrade their target RNA molecules. Instead, morpholinos act by “steric blocking,” i.e., binding to a target sequence within an RNA and sterically hindering molecules which might otherwise interact with the RNA. Bound to the 5′-untranslated region of messenger RNA (mRNA), morpholinos can interfere with progression of the ribosomal initiation complex from the 5′ cap to the start codon. This prevents translation of the coding region of the targeted transcript (called “knocking down” gene expression). Some morpholinos knock down expression so effectively that after degradation of preexisting proteins the targeted proteins become undetectable by Western blot.
  • mRNA messenger RNA
  • Morpholinos can also interfere with pre-mRNA processing steps, usually by preventing splice-directing snRNP complexes from binding to their targets at the borders of introns on a strand of pre-RNA. Preventing U1 (at the donor site) or U2/U5 (at the polypyrimidine moiety and acceptor site) from binding can result in modified splicing, commonly leading to the exclusion of exons from a mature mRNA transcript. Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) and is seen as a band shift after gel electrophoresis of RT-PCR products.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • Morpholinos have also been used to block intronic splice silencers and splice enhancers. U2 and U12 snRNP functions have been inhibited by morpholinos. Morpholinos targeted to “slippery” mRNA sequences within protein coding regions can induce translational frame shifts. Activities of morpholinos against this variety of targets suggest that morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.
  • compositions of the present invention are composed of morpholino subunits linked together by uncharged phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5′ exocyclic carbon of an adjacent subunit, wherein the base attached to the morpholino group is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. Preparation of such oligomers is described in detail in U.S. Pat. No.
  • the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate).
  • the 5′ oxygen may be substituted with amino or lower alkyl substituted amino.
  • the pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl.
  • the purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine.
  • the morpholino antisense oligonucleotides of the present invention can be complementary to the pre mRNA sequences in the transcript emanating from the GSY1 locus. In some embodiments, the morpholino antisense oligonucleotide is at least any of about 90%, 95%, or 100%, inclusive, including any percentages in between these values, identical to an mRNA target. In another embodiment, the morpholino antisense oligonucleotide binds to the GSY1 mRNA transcript in a sequence-specific manner. In some embodiments, the morpholino antisense oligonucleotide comprises a 5′ amine modification. In another embodiment, the morpholino antisense oligonucleotide can be a phosphorodiamidate cationic peptide-linked morpholino antisense oligonucleotide.
  • a cationic peptide as described herein can be 8 to 30 amino acid residues in length and consist of subsequences selected from the group consisting of RXR, RX, RB, and RBR; where R is arginine (which may include D-arginine), B is ⁇ -alanine, and each X is independently —NH—(CHR 1 ) n —C(O)—, where n is 4-6 and each R 1 is independently H or methyl, such that at most two R 1 's are methyl.
  • each R 1 is hydrogen.
  • the cationic peptide can be any of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length.
  • the variable n is 5, e.g. as in 6-aminohexanoic acid.
  • the cationic peptide comprises the amino acid sequence Ac(RXRRBR) 2 XB-, where Ac is an acetyl group.
  • the cationic peptide comprises the amino acid sequence Ac(RXR) 4 XB-, where Ac is an acetyl group.
  • the cationic peptide is linked directly to the morpholino antisense oligonucleotide.
  • the cationic peptide is linked to the morpholino antisense oligonucleotide via a spacer moiety linked to the 5′ end of the morpholino antisense oligonucleotide.
  • the spacer moiety may be incorporated into the peptide during cationic peptide synthesis.
  • a spacer contains a free amino group and a second functional group (e.g., a carboxyl group or an amino group) that enables binding to another molecular moiety, the spacer may be conjugated to a solid support used for peptide synthesis.
  • the cationic peptide may be synthesized directly onto the spacer's free amino group by standard solid phase techniques.
  • the AON-CPP compound may comprise multiple AON, including PMO AON, attached to a single cationic peptide (CPP).
  • the multiple AON-CPP conjugate further comprises a cathepsin cleavable linker.
  • the cathepsin cleavable linker can occur in between the AON and the CPP or it can occur in a sequence such as AON-cathepsin linker-AON-cathepsin linker-CPP.
  • the multiple PMO-CPP conjugate further comprises a cathepsin cleavable linker.
  • the cathepsin cleavable linker can occur in between the PMO and the CPP or it can occur in a sequence such as PMO-cathepsin linker-PMO-cathepsin linker-CPP.
  • the spacer moiety may be conjugated to the cationic peptide after peptide synthesis. Such conjugation may be achieved by methods well established in the art.
  • the linker contains at least one functional group suitable for attachment to the target functional group of the synthesized cationic peptide. For example, a spacer with a free amine group may be reacted with the cationic peptide's C-terminal carboxyl group.
  • the spacer moiety comprises:
  • the cationic peptide-linked morpholino antisense oligonucleotides have the following structure:
  • R 2 is a cationic peptide (such as any of the cationic peptides disclosed herein), R 3 is H, CH 3 or CH 2 CONH 2 , and R 4 is a morpholino antisense oligonucleotide comprising the sequence 5′-(AGC) n -3′ (SEQ ID NO.: 5), 5′-(GCA) n -3′ (SEQ ID NO.: 6), or 5′-(CAG) n -3′ (SEQ ID NO.: 7), wherein n is any of about 5-25.
  • the cationic peptide-linked morpholino antisense oligonucleotides can further comprise 1 to 2 additional morpholino nucleotides on the 5′ and/or 3′ end of the oligonucleotides.
  • the cationic peptide linked morpholino antisense oligonucleotide comprises
  • Ac is acetyl
  • R is arginine (which may include D-arginine)
  • B is ⁇ -alanine
  • each X is independently —NH—(CHR 1 ) n —C(O)—, where n is 4-6 and each R 1 is H
  • R 4 is a morpholino antisense oligonucleotide comprising a therapeutic sequence.
  • the cationic peptide linked morpholino antisense oligonucleotide comprises
  • Ac is acetyl
  • R is arginine (which may include D-arginine)
  • B is ⁇ -alanine
  • each X is independently —NH—(CHR 1 ) n —C(O)—, where n is 4-6 and each R 1 is H
  • R 4 is a morpholino antisense oligonucleotide comprising a therapeutic sequence.
  • compound may include a variable sequence—spacer—linker according to any of the sequences of Table 2 or Table 3; wherein R is L-arginine or arginine; X is 3-cis-aminocyclohexane or 1,3 cis-aminocyclohexane carboxylic acid; and Z is cis-2-aminocyclopentane-1-carbonyl or cis-(1 R,2S)-2-aminocyclopentane carboxylic acid. In some embodiments, X can be any combination of 0, 1, or more residues that are R, X, and Z.
  • X can also include other types of residues, such as proline, glycine, or alanine, or additional modified or nonstandard amino acids.
  • the variable sequence includes alpha, beta, gamma, or delta amino acids, or cycloalkane structures.
  • the linker includes the sequence FS (SEQ ID NO.: 8).
  • the linker includes the sequence FSQ (SEQ ID NO.: 9) or FSQK (SEQ ID NO.: 10), wherein F is phenyalanine, S is serine, K is lysine and Q is glutamine.
  • the linker includes the sequence FxyB (SEQ ID NO.: 11), where x is any amino acid, standard or nonstandard, y is glutamic acid (E), aspartic acid (D), and lysine (K), serine (S), or threonine (T), and B is ⁇ -alanine or ⁇ -glycine.
  • the cationic peptide linked to the morpholino antisense oligonucleotide is one of the peptides in Table 3.
  • the antisense oligonucleotides including cationic peptide-linked morpholino antisense oligonucleotides, disclosed herein can be formulated with a pharmaceutically acceptable excipient or carriers to be formulated into a pharmaceutical composition.
  • the antisense oligonucleotides can be administered in the form of pharmaceutical compositions.
  • These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions.
  • Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • compositions which contain, as the active ingredient, one or more of the antisense oligonucleotides, including cationic peptide-linked morpholino antisense oligonucleotides, associated with one or more pharmaceutically acceptable excipients or carriers.
  • the active ingredient is usually mixed with an excipient or carrier, diluted by an excipient or carrier or enclosed within such an excipient or carrier which can be in the form of a capsule, sachet, paper or other container.
  • the excipient or carrier serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the active compound In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
  • excipients or carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 mg to about 100 mg or more, such as any of about 5 mg to about 10 mg, about 5 mg to about 20 mg, about 5 mg to about 30 mg, about 5 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 60 mg, about 5 mg to about 70 mg, about 5 mg to about 80 mg, or about 5 mg to about 90 mg, inclusive, including any range in between these values, of the active ingredient.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for individuals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient or carrier.
  • the cationic peptide-linked morpholino antisense oligonucleotides are effective over a wide dosage range and are generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the cationic peptide-linked morpholino antisense oligonucleotides actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the principal active ingredient/cationic peptide-linked morpholino antisense oligonucleotide is mixed with a pharmaceutical excipient or carrier to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • a pharmaceutical excipient or carrier to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.
  • the compositions can be administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may also be administered, orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. According to the present disclosure, inhibition is the preferred form of modulation of gene expression.
  • modulation of the expression of GYS1 is selective over the modulation of GYS2 according to the present disclosure because the pre-mRNA and RNA transcribed from GYS1 is targeted rather than pre-mRNA and RNA transcribed from GYS2.
  • the ASO compound has from 15-25 subunits of a subunit selected from Formulas (I)-(VI). In another embodiment, the ASO compound has from 20-25 subunits of a subunit selected from Formulas (I)-(VI). In yet another ASO, the ASO compound has about 25 subunits of a subunit selected from Formulas (I)-(VI), such as from 24-26 subunits.
  • the ASO including a PMO
  • Gys1 mRNA levels were assessed in tibialis anterior muscles of C57Bl/6 mice injected with individual PMOs as described in the Materials and Methods.
  • the PMO sequence (in line 10) targeting exon 6 for skipping and referred to herein as GS-PMO produced the greatest impact on Gys1 mRNA levels as assessed by semi-quantitative PCR and was selected for subsequent studies.
  • AONs such as PMO compounds, according to the present disclosure that specifically hybridize to a target sequence at or near a splice site of pre-mRNA transcribed from GYS1 can lead to inclusion of an intron in the mRNA or to skipping both the intron and the exon near the splice site target. Either event can lead to the introduction of a premature stop codon, or a frame shift producing a nonsense mRNA.
  • the inclusion of an exon usually leads to the inclusion of a stop codon in the reading frame of that intron.
  • a frame shift caused by exon skipping also often leads to a premature stop codon in the frame-shifted exon.
  • Premature stop codons are recognized and degraded by the nonsense-mediated machinery leading to exo and endo-nucleolytic mRNA degradation.
  • the degradation of mRNA transcribed from GYS1 may lead to down regulating mRNA coding for glycogen synthase 1, a reduced amount of glycogen synthase 1, and ultimately alleviation of symptoms associated with the buildup of glycogen in the cells.
  • the present disclosure includes a method of down regulating mRNA coding for glycogen synthase 1 comprising administering to an animal an AON, such as a PMO, according to the present disclosure.
  • the present disclosure also includes a method for reducing glycogen synthase 1 in skeletal and cardiac muscle comprising administering to an animal an AON, such as a PMO, according to the present disclosure.
  • Phosphorodiamidate morpholino oligomers were designed to hybridize to Gys1 mRNA so as to invoke either exon skipping or translation inhibition as described by Morcos.
  • the sequences designed to skip exons in Gys1 mRNA are as follows:
  • PMO 1 (5′-TCAGGGTTGTGGACTCAATCATGCC-3′) (SEQ ID NO.: 54) targeted the intronic sequence proximal to the splice acceptor site of intron 7;
  • PMO 2 (5′-AAGGACCAGGGTAAGACTAGGGACT-3′) (SEQ ID NO.: 55) targeted the intronic sequence proximal to the splice acceptor site of intron 4;
  • PMO 3 (5′-GTCCTGGACAAGGATTGCTGACCAT-3′) (SEQ ID NO.: 56) targeted the exon-intron boundary of exon 8;
  • PMO 4 (5′-CTGCTTCCTTGTCTACATTGAACTG-3′) (SEQ ID NO.: 57) targeted the intron-exon boundary of exon 5;
  • PMO 5 (5′-ATACCCGGCCCAGGTACTTCCAATC-3′) (SEQ ID NO.: 58) targeted the exon-intron boundary of exon 14;
  • PMO 6 (5′-CTGGACAAGGATTGCTGACCATAGT-3′) (SEQ ID NO.: 59), similar to PMO 3 also targeted the exon-intron boundary of exon 8;
  • PMO 7 (5′-AATTCATCCTCCCAGTCTTCCAATC-3′) (SEQ ID NO.: 60) was designed to inhibit translation initiation by targeting a sequence 3′ to the initiation codon of Gys1;
  • PMO 8 (5′-TCCCACCGAGCAGGCCTTACTCTGA-3′) (SEQ ID NO.: 61) targeted the exon-intron boundary of exon 7;
  • PMO 9 (5′-GACCACAGCTCAGACCCTACCTGGT-3′) (SEQ ID NO.: 62) targeted the exon-intron boundary of exon 5;
  • PMO 10 (5′-TCACTGTCTGGCTCACATACCCATA-3′) (SEQ ID NO.: 63) targeted the exon-intron boundary of exon 6.
  • Peptide B (Ac(RXRRBR)2XB-OH) (SEQ ID NO.: 64) was activated in dimethylformamide containing O-(6-Chlorobenzotriazol-1-yl)-N,N,N,N′′-tetramethyluronium hexafluorophosphate (HCTU)/diisopropylethylamine (DIEA) at molar ratios of 1:2 per moles of peptide at room temperature (RT).
  • HCTU O-(6-Chlorobenzotriazol-1-yl)-N,N,N,N′′-tetramethyluronium hexafluorophosphate
  • DIEA diisopropylethylamine
  • the Morpholino, GS1-ES6, (5′-TCACTGTCTGGCTCACATACCCATA-3′) (SEQ ID NO.: 63) with a 5′ primary amine modification was dissolved in dimethylsulfoxide and added to activated peptide at a 1.2-1.5:1 molar ratio of peptide:ASO and allowed to react at RT for 2 h; when completed the reaction was quenched with water.
  • PPMO conjugates were separated from unbound PMO by isolation over carboxymethyl sepharose and eluted in 2M guanidine-HCl, 1M NaCl, pH 7.5, 20% acetonitrile.
  • the eluate was dialyzed against several buffer exchanges of 0.1mM NaHCO3 in a dialysis cassette with molecular weight cut-off of 3,000 Da.
  • the dialyzed PPMO was quantified by spectrophotometric absorbance in 0.1N HCl at 265 nm, frozen, and lyophilized.
  • Molecular weight of conjugated GS1-ES6 PPMO was confirmed by MALDI mass spectrometry.
  • TA muscles were injected with 12 uL of 0.4 U/ ⁇ L bovine hyaluronidase 2 hours before PMO injection and electroporation.
  • One TA was injected with 20 ⁇ g (1 ⁇ g/ ⁇ l) of various PMOs (Table 4) and the contralateral TA with 20 ⁇ I phosphate buffered saline (PBS).
  • the muscle was electroporated using the parameters of 100 V/cm, 10 pulses at 1 Hz, and 20 ms duration per pulse. Mice were euthanized two weeks after electroporation and TA muscle collected and snap frozen until analysis.
  • RT-PCR products (25 cycles) were separated on 2% agarose gels containing ethidium bromide and scanned on a bio-imaging system. Band intensity was quantified using Image J software. Levels of Gys1 and Gys2 mRNA was determined relative to beta actin.
  • tissue homogenate 50-100 ⁇ g of tissue homogenate was boiled in 2 ⁇ sample buffer containing dithiothreitol. The lysate was then applied to a 4-15% precast Tris/HCl-polyacrylamide gel. Proteins were transferred to nitrocellulose with a dry blot apparatus. The blots were blocked overnight with 3% milk and the appropriate antibody added at a final concentration of 0.02-0.08 ng/ml and incubated for 1 hr at room temp. The blot was then incubated with an HRP-conjugated secondary antibody for 1 hr at room temperature and treated with an ECL substrate detection kit as described by the manufacturer. Protein band intensity was quantified using Image J software. Levels of glycogen synthase 1 and 2 protein was determined relative to GAPDH.
  • Glycogen synthase activity in tissue lysates was measured using a gel filtration radioactivity assay as described previously [Niederwanger A et al., J of Chromatography B, 2005; 820:143-145].
  • a 60 ⁇ L reaction solution consisting of 10 ⁇ g of protein lysate (2 ng/ ⁇ L), 4% glycogen, 30 mM UDP-glucose, 4.5 mM glucose-6-phosphate, homogenization buffer (described above) and labeled uridine diphosphate glucose [Glucose- 14 C-U] was incubated in a 37° C.
  • Tissue glycogen levels were determined as previously described [Ziegler R J et al., Hum Gene Ther. June 2008; 19(6):609-21]. Fluorescence was detected and analyzed using a micro-plate reader, 530 nm excitation and 590 nm emission, with acquisition and analysis software. Rabbit liver glycogen was used to construct the standard curve. Glycogen levels were determined by subtracting the glucose levels in the undigested samples from those in the digested samples.
  • Kidney and liver were collected from mice following euthanasia, fixed for up to 72 h in 10% neutral buffered formalin and processed for paraffin embedding. Serial 5 ⁇ m-thick sections were generated and stained with hemotoxylin and eosin solution. A board certified veterinary pathologist, blinded-to the treatments, evaluated the slides for qualitative analysis.
  • PMO-based antisense oligonucleotide confers selective knockdown of Gys1 mRNA in murine muscle.
  • a collection of PMO antisense oligonucleotides was designed to selectively reduce the expression of the isoform of glycogen synthase found mainly in skeletal muscle and heart with the potential to induce exon skipping in the cognate Gys1 but not the Gys2 transcript. Exon skipping was designed to introduce a premature stop codon into the Gys1 transcript to effect the production of an unstable mRNA prone to nonsense-mediated decay. Incorporation of a nonsense codon would also be expected, after translation, to lead to a non-functional enzyme.
  • Candidate ASOs were first tested by direct injection into the TA muscle of mice followed by electroporation. One week later Gys1 mRNA levels were quantified. Twelve ASOs were tested and two resulted in what appeared to be a substantial reduction in Gys1 mRNA (Table 4). One PMO sequence in particular (number 10 in Table 4) targeted the skipping of exon 6 and was evaluated further.
  • the selected PMO was synthesized with a 5′ primary amine to facilitate conjugation of a well-characterized arginine-rich cell penetrating sequence that had been shown previously to facilitate muscle delivery.
  • This conjugated PMO (GS-PPMO) was injected through a tail vein into Pompe mice once every two weeks for a total of 12 weeks.
  • Age-matched Pompe mice administered saline vehicle or 20 mg/kg recombinant ⁇ -glucosidase (rhGAA) on the same schedule served as treatment controls.
  • Age-matched wild type (57Bl/6) mice served as untreated controls.
  • FIGS. 3A, 3B and 3C Analysis of tissue extracts from Pompe mice at the end of the study showed that treatment with either 15 or 30 mg/kg GS-PPMO significantly reduced Gys1 mRNA levels in the quadriceps and diaphragm muscle ( FIGS. 3A, 3B and 3C ). Gys1 mRNA levels were also dramatically reduced in the heart, but only after treatment with the higher dose ( FIG. 3D ). No significant changes were seen in the steady state levels of Gys2 mRNA in the liver, indicating that GS-PPMO-mediated knockdown was specific for the muscle isoform of glycogen synthase ( FIG. 3E ). As expected, treating animals with rhGAA had no impact on Gys1 mRNA levels in skeletal muscle or heart or the Gys2 mRNA levels in liver.
  • GS-PPMO glycogen synthase activity in the skeletal muscle and heart of Pompe mice.
  • FIG. 5A A reduction of enzyme activity in the heart was observed only in Pompe mice treated at the higher dose of 30 mg/kg GS-PPMO ( FIG. 5B ) whereas an apparent increase in activity was noted at the lower dose.
  • Treating Pompe mice with rhGAA had no effect on glycogen synthase activity in the skeletal muscle but lowered that in the heart to normal levels. This differential response to treatment with the recombinant enzyme is consistent with previous reports in Pompe mice.
  • Tissue extracts were subjected to quantitative glycogen analysis to determine whether the noted reductions in glycogen synthase protein levels and activity in GS-PPMO-treated Pompe mice also resulted in a concomitant lowering of lysosomal glycogen accumulation.
  • Treating Pompe mice with GS-PPMO led to a dose dependent decrease in glycogen accumulation in the quadriceps and heart ( FIGS. 6A and 6C ), and a reduction to the level found in normal control mice in the diaphragm at both doses tested ( FIG. 6B ).
  • the levels of glycogen were reduced to those found in wild type C57Bl/6 mice.
  • GS-PPMO was capable of provoking Gys1 mRNA decreases in quadriceps, diaphragm and heart in a dose dependent manner.
  • the bioactivity seen in the heart was only significant at the higher dose tested, a finding consistent with PPMOs tested for exon skipping of dystrophin (data not shown).
  • GS-PPMO activity at the mRNA level appeared to be sequence specific as there was no impact on the liver isoform, Gys2. This finding was expected given that GS-PPMO is complementary to intron sequence in Gys1.
  • the fact that GS-PPMO appears specific for the muscle enzyme suggests that its action will not interfere with systemic glucose mobilization in Pompe patients, which is governed by the predominant liver enzyme encoded by the Gys2 gene.
  • administration of rhGAA had no impact on the steady state level of Gys1 or Gys2 mRNA in any tissue tested.
  • the GS-PPMO mediated knock down of Gys1 mRNA greatly reduced the amount of Gys1 protein in quadriceps and diaphragm at both doses tested, and also in the heart at the higher dose.
  • Glycogen synthase activity was considerably elevated in the quadriceps and heart of Pompe mice; a finding consistent with the protein levels cited above and previous reports. Treating Pompe mice with GS-PPMO reduced activity in these tissues to very near the wild type levels found in C57Bl/6 mice at both doses tested. This was also true in the heart but only with the higher dose employed. It is remarkable to note that even with the elevated level of GS-activity in the untreated Pompe heart, the administration of GS-PPMO (15 mg/kg) increased GS-activity further by 200-fold. Glycogen synthase activity is regulated at the level of protein phosphorylation that is controlled by environmental conditions through the mTOR pathway.
  • GS-PPMO treatment resulted in a modest dose-dependent decline of glycogen build up in heart.

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