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  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
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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
  • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
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  • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
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  • C12N2320/00—Applications; Uses
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  • C12N2320/33—Alteration of splicing
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  • C12N2320/00—Applications; Uses
  • C12N2320/50—Methods for regulating/modulating their activity
  • C12N2320/52—Methods for regulating/modulating their activity modulating the physical stability, e.g. GC-content

Definitions

• heteroaryl or “heteroaromatic” or “HetAr” refers to a 5- to 18- membered aromatic radical (e.g., Cs-Cnheteroaryl) that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system.
• a numerical range such as “5 to 18” refers to each integer in the given range - e.g., "5 to 18 ring atoms” means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms.
• Suitable “amino protecting groups” for the present invention include and are typically and preferably independently at each occurrence selected from methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 2,7-di-7-butyl-[9-( 10,10-dioxo- 10,10, 10, 10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1, 1- dimethyl-2,2-dibromoethyl carbamate (DB-i-BOC), 1,1 -dimethyl -2,2,2-trichloroethyl carbamate (TCBOC),
• nucleobase derivatives include methylated adenine, guanine, uracil and cytosine and nucleobase derivatives, preferably of (i), (ii), (iii) or (iv), wherein the respective amino groups, preferably the exocyclic amino groups, are protected by acyl protecting groups or dialkylformamidino, preferably dimethylformamidino (DMF), and further include nucleobase derivatives such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine and pyrimidine analogs such as pseudoisocytosine and pseudouracil.
• nucleobase derivatives such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine and pyrimidine analogs such as pseudoisocytosine and pseudo
• nucleosidic linkage group includes phosphorus linkage groups and non- phosphorus linkage groups.
• Non-phosphorus linkage groups do not contain a phosphorus atom and examples of non-phosphorus linkage groups include, and are typically and preferably selected from alkyl, aryl, preferably, phenyl, benzyl, or benzoyl, cycloalkyl, alkylenearyl, alkylenediaryl, alkoxy, alkoxyalkylene, alkylsulfonyl, alkyne, ether, each independently of each other optionally substituted with cyano, nitro, halogen; carboxyl, amide, amine, amino, imine, thiol, sulfide, sulfoxide, sulfone, sulfamate, sulfonate, sulfonamide, siloxane or mixtures thereof.
• said internucleosidic linkage group is selected from a phosphodiester linkage group, a phosphotriester linkage group, a phosphorothioate linkage group, or a phosphonate linkage group, wherein the phosphonate is preferably a H-phosphonate linkage group or methylphosphonate linkage group.
• Ti and T2 is an internucleosidic linkage group, and the other of Ti and T2 is ORi, OR2, a 5' terminal group, a 3' terminal group or a internucleosidic linkage group, wherein Ri is H or a hydroxyl protecting group, and R2 is a phosphorus moiety;
• the tc-DNA nucleosides of the oligomeric compounds of the inventive compositions comprise a compound of Formula (3) (also known as a C(6')- functionalized tc-DNA):
• the tc-DNA nucleosides of the oligomeric compounds of the inventive compositions comprise a compound of Formula (4), wherein Bx is selected from the group consisting of thymine, adenine, guanine, and cytosine.
• the tc-DNA nucleosides of the oligomeric compounds of the invention comprise a compound of Formula (4), wherein Bx is a modified base.
• Ti and T2 is an internucleosidic linkage group
• the other of Ti and T2 is ORi, OR2, a 5' terminal group, a 3' terminal group or a internucleosidic linkage group, wherein Ri is H or a hydroxyl protecting group, and R2 is a phosphorus moiety.
• the tc-DNA nucleosides of the oligomeric compounds of the inventive compositions comprise a compound of Formula (5), wherein Bx is selected from the group consisting of thymine, adenine, guanine, and cytosine.
• the tc-DNA nucleosides of the oligomeric compounds of the invention comprise a compound of Formula (5), wherein Bx is a modified base.
• said one or more nucleosides tc-DNA nucleosides of the oligomeric compounds of the inventive compositions comprise a compound of Formula (5') (also known as 2'-fluoro-tc-ANA):
• said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are independently of each other 2' -modified ribonucleic acid (2' -modified- RNA) nucleosides.
• the one or more nucleosides other than tc-DNA nucleosides of the oligomeric compounds of the invention comprise a compound of Formula (13), wherein Bx is selected from the group consisting of cytosine, adenine, guanine, and uracil.
• the 2 '-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of Formula (13), wherein Bx is a modified base.
• Preferred inorganic bases or amines leading to said salt formation with the OH or SH groups are well known in the art and are typically and preferably trimethylamine, diethylamine, methylamine or ammonium hydroxide.
• These phosphorus moieties included in the present invention are, if appropriate, also abbreviated as " ⁇ + ", wherein said HB + refers to the counter cation formed.
• said one or more lipid moiety is independently of each other selected from a fatty acid moiety, a fatty diacid moiety, an alkylphosphate moiety and an alkylphosphonate moiety.
• said one or more lipid moiety is independently of each other a moiety of formula (I)
• said one or more lipid moiety is independently of each other a moiety of formula C3-32alkynyl-C(0)-*, wherein said asterisk (*) represents the point of said covalent linkage to said oligomeric compound or to said spacer, wherein preferably said composition comprises exactly one lipid moiety.
• said one or more lipid moiety is independently of each other a moiety of formula HOOC-C3-32alkylene-C(0)-*, wherein said asterisk (*) represents the point of said covalent linkage to said oligomeric compound or to said spacer, wherein preferably said composition comprises exactly one lipid moiety.
• said one or more lipid moiety is independently of each other a moiety of formula C3-32alkenyl-C(0)-*, wherein said asterisk (*) represents the point of said covalent linkage to said oligomeric compound or to said spacer, wherein preferably said composition comprises exactly one lipid moiety, and wherein said C3-32alkenyl is a branched C3-32alkenyl having an uneven number of carbon atoms.
• said spacer comprises, preferably is, independently selected from, any one of the formula
• one or more -Qrh-moieties in said C2-i2alkylene are optionally replaced independently by -0-, -S-, -NH-, -C(O)-, -C(0)O, an aryl, a heteroaryl, a cycloalkyl, a heterocycloalkyl, - OP(OH)0-, OP(0)(SH)0-, ⁇ (0)( ⁇ )0-, NHP(0)(OH)0-, NHP(0)(SH)0-, or -(O-CH2- CH2)k- with k being an integer of 1 to 8, and wherein one or more -Qrh-moieties in said C2- nalkylene are independently of each other optionally substituted with one or more -COOH, - NH2, -OP(0)(OH)2 or -OH (and thus meaning that one or both, preferably one, of the hydrogen atoms in one or more of the-CH2-moieties in said d-nalkylene are independently of each other optionally
• said spacer comprises, preferably is, independently selected from, any one of the formula
• one or more -Q-h-moieties in said C2-i2alkylene are optionally replaced independently by -0-, -S-, -NH-, -C(O)-, -C(0)0-, phenyl, triazolyl, cyclopentyl, cyclohexyl, succinimidyl, - OP(OH)0-, OP(0)(SH)0-, ⁇ (0)( ⁇ )0-, NHP(0)(OH)0-, NHP(0)(SH)0-, or -(O-CH2- CH2)k- with k being an integer of 1 to 8, and wherein one or more -Q-h-moieties in said C2- nalkylene are independently of each other optionally substituted with one or more -COOH, - NH2, -OP(0)(OH)2 or -OH, and wherein said (#) represents the point of covalent linkage to said lipid moiety and said ( ⁇ ) represents the point of covalent linkage to said oligomeric compound.
• said one or more lipid moiety is independently of each other linked to said oligomeric compound at the 5' terminus of said oligomeric compound.
• said oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 50% or more of all nucleosides are tc-DNA nucleosides.
• said oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 80% or more of all nucleosides are tc-DNA nucleosides.
• PNAs peptide nucleic acids
• PMO phosphorodiamidate morpholino
• said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are independently of each other 2'-deoxy 2'-fluoro-arabino nucleosides.
• said oligomeric compound has a length of, up to 40 monomer subunits, preferably up to 30 monomer subunits, more preferably up to 30 monomer subunits, again more preferably up to 20 monomer subunits or up to 15 monomer subunits.
• said oligomer comprises from 5 to 40 monomeric subunits, preferably from 8 to 30 monomer subunits, more preferably from 8 to 25 monomer subunits, again more preferably from 8 to 20 monomer subunits.
• said oligomeric compound is an oligonucleotide, said wherein said oligomeric compound comprises one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, wherein preferably said oligomeric compound comprises from 5 to 40 monomer subunits.
• said oligomeric compound is an oligonucleotide, said wherein said oligomeric compound comprises one or more tricyclo- deoxyribonucleic acid (tc-DNA) nucleosides, wherein said oligomeric compound comprises from 5 to 40 monomer subunits, and wherein said monomer subunits are linked by internucleosidic linkage groups,
• said monomer subunits are independently of each other selected from naturally occurring nucleosides, modified nucleosides and mimetics of nucleosides, wherein preferably said naturally occurring nucleosides, said modified nucleosides and said mimetics of nucleosides are independently of each other selected from tricyclic nucleosides, ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2' -modified ribonucleic acid (2' -modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acids (PNAs) nucleosides, 2'-deoxy 2'-fluoro-arabino nucleosides, hexitol nucleic acids (HNAs) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, and wherein said
• no more than 3 of said plurality of intemucleosidic linkage groups are phosphorothioate linkage groups. In another preferred embodiment, no more than 2 of said plurality of intemucleosidic linkage groups are phosphorothioate linkage groups. In another preferred embodiment, no more than 1 of said plurality of intemucleosidic linkage groups are phosphorothioate linkage groups. In another preferred embodiment, none (zero/0) of said plurality of intemucleosidic linkage groups are phosphorothioate linkage groups.
• said plurality of intemucleosidic linkage groups are independently selected from a phosphorothioate linkage group and a phosphorodiester linkage group, and wherein no more than 30% of said plurality of intemucleosidic linkage groups are phosphorothioate linkage groups.
• At least 70% of said plurality of internucleosidic linkage groups are phosphorodiester linkage groups. In another preferred embodiment, at least 75% of said plurality of internucleosidic linkage groups are phosphorodiester linkage groups. In another preferred embodiment, at least 80% of said plurality of internucleosidic linkage groups are phosphorodiester linkage groups. In another preferred embodiment, at least 85% of said plurality of internucleosidic linkage groups are phosphorodiester linkage groups. In another preferred embodiment, at least 90% of said plurality of internucleosidic linkage groups are phosphorodiester linkage groups.
• said plurality of internucleosidic linkage groups are independently selected from a phosphorothioate linkage group and a phosphorodiester linkage group, and wherein no more than 6 of said plurality of internucleosidic linkage groups are phosphorothioate linkage groups.
• said plurality of internucleosidic linkage groups are independently selected from a phosphorothioate linkage group and a phosphorodiester linkage group, and wherein no more than 5 of said plurality of internucleosidic linkage groups are phosphorothioate linkage groups.
• said plurality of internucleosidic linkage groups are independently selected from a phosphorodiester linkage group.
• all of said plurality of internucleosidic linkage groups are phosphorodiester linkage groups.
• said oligomeric compound comprises from 5 to 40 monomer subunits, wherein said monomer subunits are nucleosides, and wherein at most 2 of said nucleosides is a nucleoside other than a tc-DNA nucleoside, and wherein all the other of said nucleosides are said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and wherein said nucleosides are linked by a plurality of internucleosidic linkage groups, and wherein said at most 2 nucleoside other than a tc-DNA nucleoside is selected from a ribonucleic acid (R A) nucleoside, a deoxyribonucleic acid (DNA) nucleoside, a 2 '-modified ribonucleic acid (2' -modified-RNA) nucleoside, a locked nucleic acid (LNA) nucleoside,
• R A ribonucleic
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:4. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 4, wherein said 2'-modified- RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO : 8. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 8, wherein said 2'-modified- RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 11. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 11, wherein said 2'-modified-RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 19.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:20. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:20, wherein said 2'-modified-RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:25. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 25, wherein said 2'-modified-RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:28. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:28, wherein said 2'-modified-RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:29. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 29, wherein said 2'-modified-RNA is 2'-OMe-RNA.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:34.
• said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO:35.
• inventive composition is selected from any one the compositions listed in Table 3.
• an * between two nucleosides indicates a phosphorothioate intemucleoside linkage group
• the absence of an * between two nucleosides indicates a phosphorodiester intemucleoside linkage group
• the nucleobase at all C positions is 5- methylcytosine
• the nucleobase at all c positions is cytosine
• the primed lowercase letters a', u', g', and t' indicate deoxyribonucleosides;
• the inventive composition comprises, preferably is, SY-0343.
• the inventive composition comprises, preferably is, SY-0427.
• the inventive composition comprises, preferably is, SY-0440.
• the inventive composition comprises, preferably is, SY-0443.
• the inventive composition comprises, preferably is, SY-0448.
• the inventive composition comprises, preferably is, SY-0450.
• the inventive composition comprises, preferably is, SY-0451.
• the inventive composition comprises, preferably is, SY-0495.
• the inventive composition comprises, preferably is, SY-0511.
• the inventive composition comprises, preferably is, SY-0525.
• compositions described herein can be used in a method for treating a disease.
• the disease is a disease that may be treated using an exon-skipping oligomeric compound.
• the disease is a disease that may be treated using an antisense-mediated exon-inclusion oligomeric compound.
• the compositions described herein cross the blood-brain barrier, thus are useful in treating diseases of the central nervous system, behavioral disorders, psychiatric disorders, and/or behavioral symptoms of diseases.
• the disease is a disease of the central nervous system (CNS).
• the disease is amyotrophic lateral sclerosis (ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), epilepsy, Creutzfeldt- Jakob, (CJ), Menkes Disease, or Huntington's Disease (HD).
• ALS amyotrophic lateral sclerosis
• AD Alzheimer's Disease
• PD Parkinson's Disease
• MS Multiple Sclerosis
• epilepsy Creutzfeldt- Jakob
• CJ Menkes Disease
• Huntington's Disease Huntington's Disease
• the disease is a disease affecting cerebellar function, including, but not limited to, ataxia.
• the disease is a disease affecting amygdala function, including, but not limited to, Urbach-Wiethe Disease.
• the disease is a disease affecting hippocampal function, including, but not limited to, memory loss.
• the disease to be treated is a psychiatric or behavioral disorder, including, but not limited to, mood disorders, dementia, anxiety, bipolar disorder, schizophrenia, sleep disorders, post-traumatic stress disorder (PTSD), attention-deficit hyperactivity disorder (ADHD), and depression disorders.
• a psychiatric or behavioral disorder including, but not limited to, mood disorders, dementia, anxiety, bipolar disorder, schizophrenia, sleep disorders, post-traumatic stress disorder (PTSD), attention-deficit hyperactivity disorder (ADHD), and depression disorders.
• negative symptoms of schizophrenia include deficit in motivation, deficit in spontaneity, inability to think abstractly, deficit in mood expression, deficit in cognition, deficit in the ability to experience pleasure, affective flattening, alogia, avolition, dysphoric mood, including anger, anxiety, and depression), disturbances in sleep pattern, poor impulse control, lack of judgment, abnormal psychomotor activity, such as pacing or rocking, and movement disorders, such as tardive dyskinesia.
• areas of cognition such as verbal memory, verbal fluency, memory consolidation, and executive functions, are improved by administration of one or more of the oligonucleotide compounds of the present invention.
• slow wave sleep is increased, thereby improving cognition, with administration of one or more of the oligonucleotide compounds of the present invention.
• the disease is selected from the group consisting of Duchenne muscular dystrophy (DMD), familial dysautonomia, spinal muscular atrophy (SMA), ataxia telangiectasia, congenital disorder of glycosylation, fronto-temporal dementia (FTD), Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, megalencephalic leukoencephalopathy with subcortical cysts type 1, Pelizaeus-Merzbacher disease, Pompe disease, and myotonic dystrophy type 1.
• DMD Duchenne muscular dystrophy
• SMA spinal muscular atrophy
• FTD fronto-temporal dementia
• Parkinsonism linked to chromosome 17 Niemann-Pick disease type C
• neurofibromatosis type 1 neurofibromatosis type 2
• tc-DNA oligonucleotides for the treatment of Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), spinocerebellar ataxia type 3 (SCA3), and other diseases are known in the art and are described, e.g., in U.S. Patent Nos.
• Table A provides a listing of certain neurodegenerative diseases and their targets for which the compositions of the present invention are useful.
• Efficacy of the compositions described herein in treating, preventing and/or managing the indicated diseases or disorders can be tested using various models known in the art, which provide guidance for treatment of human disease.
• Models for diseases that may be treated using an exon-skipping oligomeric compound are described, e.g., in Siva, et al., Nucleic Acid Therapeutics 2014, 24, 69-86.
• Models for diseases that may be treated using an antisense- mediated exon-inclusion oligomeric compound are described, e.g., in Hua and Krainer, Methods Mol. Biol. 2012, 867, 307-323.
• DMD dystrophin/utrophin double knockout mice
• humanized DMD mice mdx52 mice (carrying a deletion of exon 52 in murine DMD), and 4CV mice (carrying a nonsense mutation in exon53).
• Goyenvalle et al, Mol. Ther. 2010, 18, 198-05; Bremmer-Bout, et al., Mol. Ther. 2004, 10, 232-240; Aoki, et al., Mol. Ther. 2010, 18, 1995-2005; Mitrpant, et al., J. Gene. Med. 2009, 11, 46-56.
• SMA Spinal muscular atrophy
• SMA is a class of inherited diseases that arise from a defect in a survival motor neuron gene (SMN 1) mapped to chromosome 5ql 1.2-13.3. Overall, SMA is characterized by a loss of spinal cord and brainstem motor neurons, resulting in muscular atrophy from the loss of neural contact. The various SMAs have an incidence of about 1 in 6,000. Type I SMA, which is also known as Werdnig-Hoffman disease or severe infantile SMA, affects babies in their first year of life, and is generally fatal. Type II SMA, also known as intermediate SMA, affects children and causes muscle weakness such that the patients are never able to stand and walk, but may be able to sit, although weakness increases with age. Type III SMA patients are able to walk at some point in their development.
• SMA is caused by the loss of a functional SMN1 gene, and a mutation in exon 7 of the SMN2 paralog that causes substantial skipping of this exon and production of only low levels of functional protein, such that SMN2 protein cannot compensate for the loss of SMN1.
• Oligonucleotide-mediated exon-inclusion methods for the treatment of SMA are being explored, including methods of compensating for the deleterious mutation in SMN2 by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield a modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein.
• WO 2010/115993 Al the disclosure of which is incorporated herein by reference.
• numerous other diseases can also be potentially treated by the exon inclusion approach provided by the inventive compositions, including those diseases described herein.
• the present invention provides further for the inventive composition for use as a medicament in the prevention, treatment or diagnosis of a disease, wherein preferably said disease is a neuromuscular or musculoskeletal disease, and wherein further preferably said neuromuscular or said musculoskeletal disease is selected from Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorder of glycosylation, fronto-temporal dementia, Parkinsonism linked to chromosome 17, Niemann- Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, megalencephalic leukoencephalopathy with subcortical cysts type 1, Pelizaeus-Merzbacher disease, Pompe disease, myotonic dystrophy type 2 (DM2 or proximal myotonic myopathy), and myotonic dystrophy type 1 (DM1 or Steinert disease), and again further preferably said neuromuscular or said musculoskeletal disease is selected from Duchenne muscular dyst
• mdx displays the classical features of dystrophic muscle characterized by numerous necrotic fibers with subsequent infiltration of scavenger cells (Coulton et al., Neuropathol. Appl. Neurobiol, 1988, 14, 299-314).
• an efficient unknown compensatory mechanism counteracts the degeneration thus up- keeping the regeneration process to restore unceasing mechanical damages.
• the number of revertant fibers is low, normally present at around 1% of total fibers, although their number increases with the age of the mice (Lu et al., J. Cell. Biol, 2000, 148, 985-995).
• translation of a shortened-dystrophin is possible by skipping exon 23 in the course of the mRNA splicing.
• Oligonucleotide synthesis Oligonucleotides for in vivo experiments were synthesized on 260 ⁇ scale using Aekta Oligo Pilot 10 synthesizer and NittoPhase® UnyLinkerTM 200, universal solid support.
• inventive compositions comprising one or more lipid moieties and/or said spacer at the 3' end, the corresponding modified, and typically and preferably Fmoc-protected, phosphoroamidite was introduced in the first cycle of the synthesis. Modified synthetic cycle using typically 2.2 equivalent of phosphoramidites and 4 min coupling time was employed.
• the corresponding biotin phosphoroamidite was introduced in the first cycle of the synthesis, followed by the C4 spacer phosphoramidite in the second cycle of the synthesis and corresponding, and typically and preferably Fmoc-protected, spacer phosphoroamidite in the third cycle of the synthesis. Modified synthetic cycle using typically 9 equivalent of phosphoramidites and 3.5 min coupling time was employed. In case of inventive compositions comprising said one or more lipid moiety and/or spacer at the 5' end, the corresponding modified, phosphoroamidite was used in the last cycle. Solid support was treated with 20% diethylamine in MeCN in order to remove Fmoc and cyanoethyl protecting groups.
• oligonucleotides for in vitro experiments were purified by isocratic RP HPLC (Waters x Bridge prep C18, 5 ⁇ , 4.6x 150 mm) using about 30% iPrOH in ammonium bicarbonate buffer (pH 7) at 60 °C and flow rate of 1 mL/min. Fractions containing the product of sufficient purity were combined and subsequently desalted using SEC (GE Healthcare, HiPrep 26/10) with water as eluent.
• Linking of the lipid moieties to the oligomeric compounds Multiple approaches exist for said linking said one or more lipid moiety to the oligonucleotides either directly on solid support or in solution in accordance with the present invention. These approaches follow classical conjugation chemistry well known to the skilled person in the art and extensively described in the literature (Bioconjugate Techniques, Greg T. Hermanson, Pierce Biotechnology; Singh, Chem. Soc. Rev. , 2010, 39, 2054-2070; H. Rosemeyer, Chem. Biodiversity, 2005, 2, 977-1062).
• Typical and preferred linking procedures for linking said one or more lipid moiety of formula (I), A-B-*, wherein said asterisk (*) represents the point of said covalent linkage to said oligomeric compound or to said spacer in accordance with the present invention include but are not limited to
• compositions of the present invention and used in the experimental section are characterized in particular in Table 3 by further reference to their names and abbreviations, respectively, which are typically used throughout this specification.
• Analysis of oligonucleotides by RP-HPLC-DAD-MS Following parameters were used for analytical HPLC: C18 column with a particle size of 1.7 ⁇ was used. The column temperature was set to 75 °C. Mobile phase A was 400 mM hexafluoroisopropanol (HFIP) and 15 mM triethylamine + 10 % methanol. Mobile phase B was methanol. A gradient of 32 to 52 % mobile phase B was applied. The flow rate was set to 0.25 mL/min. The oligonucleotides were detected using a UV photometer at 260 nm and a time-of-flight mass spectrometer.
• Detection of multimers Polyacrylamide-gel electrophoresis (PAGE) experiments were performed to detect self-multimers. The following chemicals were used: Tris(hydroxymethyl)aminomethane (Tris), (TCI A0321); acetic acid (Merck 1.00063); acrylamide/Bis solution, 29: 1 (40 %, Serva 10680.01); tetramethylethylenediamine (TEMED, Sigma-Aldrich T9281); ammonium persulfate (Sigma-Aldrich 248614); and glycerol (Sigma- Aldrich G9012). Buffer solution A was prepared by dissolving 60 g of Tris in 200 mL of water.
• test solution was 1 mg/mL in 10 % glycerol, and ⁇ 0 ⁇ ⁇ was applied (equal to 10 ⁇ g of oligonucleotide).
• the pre-migration settings were 40 min/90 V with buffer solution B.
• the migration settings were 90 min/90 V or 15 min/90 V plus 45-60 min/120 V with buffer solution B. 5-7 ⁇ ⁇ of 6X DNA loading dye was also migrated. Detection was performed by placing the gel on a TLC plate and examining under UV light at 254 nm.
• the peptide mixture was desalted using ZipTip ⁇ -Cl S Pipette Tip (Millipore) and separated with an Easy nano-LC Proxeon system (Thermo Fisher Scientific) equipped with a reversed phase C18 column (Easy-Column Proxeon C18, L 15 cm, ID 75 ⁇ ). Eluates were monitored by a LTQ VelosOrbitrap mass spectrometer (Thermo Fisher Scientific) and tandem MS (MS/MS) data were processed with Proteome Discoverer 1.4 software (Thermo Fisher scientific) coupled to an in house Mascot search server (Matrix Science, 2.3.2 213 version) using SwissProt database as described previously (Rouillon, 2015).
• the relative abundance of each protein identified was estimated by label-free quantification using the Progenesis LC MS software (Nonlinear Dynamics, 4.0 version). Average Normalized Abundances (ANA) reflecting the relative quantities of proteins in Progenesis analysis, were used to compare quantities of protein bound to a vector.
• ANA Average Normalized Abundances
• mice Six-eight week old mdx mice were injected intravenously in the retro-orbital sinus, under general anesthesia using 1.5-2% isoflurane, once a week with different AONs (15mer or 13mer, PO or PS, conjugated or not) for a period of ranging from 4-12 weeks.
• An age-matched C57/BL10 (WT) group and mdx group receiving an equivalent volume of sterile saline were included as controls.
• One hour after the first injection blood samples were collected from all mice to measure complement C3 and cytokines/chemokines levels. Additional blood samples were collected one week after the 6th injection (mid-treatment) and one week after the end of the treatment.
• Serum and urine analysis Blood samples were collected from tail bleeds under general anesthesia. Analyses of serum creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, creatinine, urea and albumin levels were performed by the pathology laboratory at Mary Lyon Centre, Medical Research Council, Harwell, Oxfordshire, UK. Cytokines and chemokines levels in serum were analyzed by multiplex assays, using the Luminex® technology.
• CK creatine kinase
• ALT alanine aminotransferase
• AST aspartate aminotransferase
• ALP alkaline phosphatase
• bilirubin creatinine, urea and albumin levels were performed by the pathology laboratory at Mary Lyon Centre, Medical Research Council, Harwell, Oxfordshire, UK. Cytokines and chemokines levels in serum were analyzed by multiplex assay
• Urine was collected using metabolic cages over 24h, directly in refrigerated tubes (4°C). Upon collection, urines were centrifuged at 10,000 x g for 10 min and supernatant was aliquoted and frozen at -80°C for further analysis. Urine creatinine was measured using Creatinine assay kit (R&D Systems, Inc, Minneapolis, MN) following manufacturer's instructions. Total protein in urine samples was measured as previously described (Swayze et al., Nucleic Acids Res., 2007, 35, 687-700). Briefly, proteins were precipitated from urine samples by adding 40 cUrhO and 200 of prechilled acetone to 10 of urine.
• the cDNA synthesis was carried out at 45 °C for 45 min, directly followed by the primary PCR of 30 cycles of 95 °C (30 s), 55 °C (1 min) and 72 °C (2 min). Two of these reactions were then re-amplified in nested PCRs by 22 cycles of 95 °C (30 s), 55 °C (1 min) and 72 °C (2 min) using the internal primers:
• Ex 20Fi 5'-CCCAGTCTACCACCCTATCAGAGC-3' - SEQ ID NO:40
• Ex 26Ri 5'- CCTGCCTTTAAGGCTTCCTT-3 ' - SEQ ID NO:41.
• SY-0252 also named "tcDNA-PO SYN51" interchangeably herein, corresponds to p- AGATGGCATTTCT-OH of SEQ ID NO: 19, with all nucleotides being tc-DNAs and all internucleosidic linkage groups being phosphorodiester linkage groups, and p being a phosphate moiety at the 5' end.
• SYN51 is known as a sequence to be highly toxic in the mouse in the presence of PS bonds.

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