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EP4590311A2 - Efficacité du saut d'exon médié par un oligonucléotide antisens dans le traitement de la dmd - Google Patents

Efficacité du saut d'exon médié par un oligonucléotide antisens dans le traitement de la dmd

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Publication number
EP4590311A2
EP4590311A2 EP23789830.9A EP23789830A EP4590311A2 EP 4590311 A2 EP4590311 A2 EP 4590311A2 EP 23789830 A EP23789830 A EP 23789830A EP 4590311 A2 EP4590311 A2 EP 4590311A2
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EP
European Patent Office
Prior art keywords
seq
exon
antisense oligonucleotide
oligomer
conjugate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23789830.9A
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German (de)
English (en)
Inventor
Chia-Ling Wu
Frederick Joseph Schnell
Remko GOOSSENS
Nisha VERWEY
Annemieke Aartsma-Rus
Yavuz ARIYUREK
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Sarepta Therapeutics Inc
Original Assignee
Sarepta Therapeutics Inc
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Filing date
Publication date
Application filed by Sarepta Therapeutics Inc filed Critical Sarepta Therapeutics Inc
Publication of EP4590311A2 publication Critical patent/EP4590311A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • Antisense technology provides a means for modulating the expression of one or more specific gene products, including alternative splice products, and is uniquely useful in a number of therapeutic, diagnostic, and research applications.
  • the principle behind antisense technology is that an antisense compound, e.g., an oligonucleotide, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing, or translation through any one of a number of antisense mechanisms.
  • the sequence specificity of antisense compounds makes them attractive as tools for target validation and gene functionalization, as well as therapeutics, to selectively modulate the expression of genes involved in disease.
  • modified antisense oligonucleotides wherein the modified antisense oligonucleotide is 18-40 subunits in length, comprising a targeting sequence complementary to a target region of a Duchenne muscular dystrophy (DMD) gene.
  • DMD Duchenne muscular dystrophy
  • the modified antisense oligonucleotide can comprise a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo- DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2’0-Me-phosphorotioate oligomer, or any combination of the foregoing.
  • PMO phosphoramidate or phosphorodiamidate morpholino oligomer
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • a phosphorothioate oligomer a tricyclo- DNA oligomer
  • a tricyclo-phosphorothioate oligomer a 2’0-Me-phosphorotioate oligomer, or any combination
  • Each subunit of the antisense oligonucleotide is taken together in order from the 5’ end of the antisense oligonucleotide to the 3’ end of the antisense oligonucleotide to form the targeting sequence, and the target region is within an exon of human dystrophin pre-mRNA, wherein the exon is flanked at the upstream 5' splice site of the exon by a slow intron, wherein the slow intron is an intron that is retained in the dystrophin pre-mRNA for a longer period of time compared to an average retention time for an intron in the dystrophin pre-m RNA that is downstream of the slow intron.
  • the antisense oligonucleotides are useful for the treatment of a neuromuscular disease.
  • the neuromuscular disease is Duchenne muscular dystrophy.
  • the exon is selected from Exon 10, Exon 14, Exon 17, Exon 18, Exon 21 , Exon 22, Exon 42, Exon 50, Exon 53, and Exon 70.
  • the antisense oligonucleotide is an oligonucleotide conjugate of Formula I: or a pharmaceutically acceptable salt thereof, wherein A', E', R 1 , R 2 , and z are as defined herein.
  • the antisense oligonucleotide of Formula I is an oligonucleotide conjugate selected from: (la); and
  • each R 1 is N(CH 3 )2.
  • each R 2 is independently selected from a naturally or non-naturally occurring nucleobase, and the sequence formed by the combination of each R 2 from 5’ to 3’ is a targeting sequence.
  • J is a cell-penetrating peptide.
  • composition comprising an antisense oligonucleotide provided herein and a pharmaceutically acceptable carrier.
  • Also provided herein is a method of treating a disease associated with dysregulation of peripheral myelin protein 22 comprising administering to a subject in need thereof an antisense oligomer provided herein.
  • the use of any of the antisense oligonucleotide provided herein for treating a neuromuscular disease is Duchenne muscular dystrophy.
  • FIG. 1A depicts a graphical representation of the splicing order of introns in the DMD gene (adapted from Gazzoli et. al. 2016).
  • Introns denoted by a line between exons are spliced slow, while exons shown directly adjacent are separated by ‘fast introns’.
  • Different exon classes based on flanking introns are shown in orange, green, yellow and pink as indicated on the right. Different shades of blue are a visual aid with no relevance to the hypothesis. Reading frame continuity of the exon is indicated by shape.
  • FIG. 1 D depicts data presented in FIG. 1 B reanalyzed and grouped to separate exons based on their downstream intron class as indicated. 3’Slow exons and 3’Fast exons show no difference in skipping efficiency.
  • FIG. 2A depicts the relative expression of various genes measured by RT-qPCR after nucleofection of an exon 51 targeting PMO with Lonza buffer systems and Amaxa pulse programs as indicated.
  • the DMD exon 50-52F_52R primer set is to measure the presence of the DMD gene when exon 51 is skipped, while the exon 49-50 primer set shows the presence of both skipped and unskipped DMD.
  • MYOG and MYH3 are measured as an indication for myogenic proliferation. Data is normalized to housekeeping genes GUSB and GAPDH.
  • FIG. 2B depicts a comparison of the same set of RT-PCR samples measuring exon 51 skipping percentages as the ratio of skipped over total (skip+full-length) PCR product, derived from a set of HC myotubes after nucleofection with an exon 51 targeting PMO. Adjacent bars show the same PCR product measured on either system as indicated
  • FIG. 3 depicts separated skipping data presented in FIG. 1 B-1D. Error bars indicate SD of two independent samples.
  • FIG. 4A depicts a graphical representation of the splicing order of introns in the DMD gene (adapted from Gazzoli et. al. 2016).
  • Introns denoted by a line between exons are spliced slow, while exons shown directly adjacent are separated by “fast introns.”
  • Different exon classes targeted for AON mediated exon skipping based on flanking introns are shown in green and yellow as indicated on the right. Different shades of blue are a visual aid with no relevance to the hypothesis. Reading frame continuity of the exon is indicated by the shape.
  • FIG. 4B depicts exon skipping efficiency of the DMD transcript with AONs for various exons from the 5’Slow-3’Fast flanking intron class as indicated in FIG. 4A.
  • Individual points indicate the average of two independently nucleofected samples.
  • the X-axis represents the possible window of targeting a 25-mer AON within the exonic sequence of the indicated exon, scaled from 0 to 100 for each exon to normalize exon size. Lines matching the symbol colors represent the results of a linear regression analysis of the skipping efficiency as a function of the targeting position. Individual slopes of each regression analysis are indicated below the plot.
  • FIG. 4D depicts a reanalysis and summarizing plot of the DMD exon skipping efficiencies of all AONs used in FIGs. 1 B, 4B and 4C, as a function of their position within the exon as indicated on the X-axis. Circles indicate data from FIG. 1 B, triangles represent data from FIGs. 4B-4C. Linear regression analysis (red line) shows a negative slope, indicating that in general, an AON targeting closer to the 5’-end of the exon will be more efficient at skipping the target exon than a more distally targeted AON.
  • FIG. 5A depicts separated skipping data presented in FIG. 4B. Error bars indicate SD of two independent samples.
  • FIG. 5C depicts data presented in FIGs. 4B and 4C reanalyzed and grouped to separate exons based on their upstream and downstream intron class as indicated.
  • FIG. 6 depicts separated skipping data presented in FIG. 4. Error bars indicate SD of two independent samples.
  • FIG. 7A depicts exon skipping efficiency of exon 51 of the DMD gene in HC myotubes after treatment with exon 51 targeting AONs. Skipping of exon 51 will lead to an out of frame transcript.
  • FIG. 7B depicts exon skipping efficiency of exon 53 of the DMD gene in HC myotubes after treatment with exon 53 targeting AONs. Skipping of exon 53 will lead to an out of frame transcript.
  • FIG. 7C depicts exon skipping efficiency of exon 51 of the DMD gene in DMD patient (AExon 48-50) myotubes after treatment with exon 51 targeting AONs. Skipping of exon 51 will lead to an in frame transcript.
  • FIG. 7D depicts exon skipping efficiency of exon 53 of the DMD gene in DMD patient (AExon 45-52) myotubes after treatment with exon 53 targeting AONs. Skipping of exon 53 will lead to an in frame transcript.
  • FIGs. 8A-8D depict RT-qPCR expression of DMD levels in samples presented in FIGs. 7A-7D. Measurements for the DMD transcript using a primer set upstream (Ex38-39) and downstream (Ex55-56) of the skipped exons are shown. MYH3 expression is used as an indication of myogenic proliferation in the sample. Data is normalized to housekeeping genes GUSB and GAPDH.
  • FIG. 9A depicts exon skipping efficiency of HC and DMD cells nucleofected with no PMOs (WT) or a mix of 12 exon 65 targeting PMOs, and treated with DMSO or CHX as indicated.
  • FIG. 9B depicts RT-qPCR expression of DMD levels in samples presented in FIG. 9A. Measurements for the DMD transcript spanning exon 38-39 and exon 55-56 are shown. MYH3 expression is used as an indication of myogenic proliferation in the sample. Data is normalized to housekeeping genes GUSB and GAPDH.
  • FIG. 9C depicts exon skipping efficiency of exon 51 of the DMD gene in HC myotubes after treatment with exon 51 targeting AONs and CHX as indicated.
  • FIG. 9D depicts exon skipping efficiency of exon 53 of the DMD gene in HC myotubes after treatment with exon 53 targeting AONs and CHX as indicated.
  • FIG. 9E depicts exon skipping efficiency of exon 51 of the DMD gene in DMD patient (AExon 48-50) myotubes after treatment with exon 51 targeting AONs and CHX as indicated
  • FIG. 9F depicts exon skipping efficiency of exon 53 of the DMD gene in DMD patient (AExon 45-52) myotubes after treatment with exon 53 targeting AONs and CHX as indicated.
  • modified antisense oligonucleotides wherein the modified antisense oligonucleotide is 18-40 subunits in length, comprising a targeting sequence complementary to a target region of a Duchenne muscular dystrophy (DMD) gene, wherein the modified antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2’0-Me- phosphorotioate oligomer, or any combination of the foregoing; each subunit is taken together in order from the 5’ end of the antisense oligonucleotide to the 3’ end of the antisense oligomer, or any
  • the antisense oligonucleotide is covalently linked to a cell-penetrating peptide.
  • the antisense oligonucleotides are useful for the treatment for various diseases in a subject in need thereof, including, but not limited to, Duchenne muscular dystrophy.
  • Morpholino-based oligomers are detailed, for example, in U.S. Patent Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,185,444; 5,521 ,063; 5,506,337; and PCT Publication Nos. WO/2009/064471 , WO/2012/043730, WO 2008/036127; and Summerton et al. 1997, Antisense and Nucleic Acid Drug Development, 7, 187-195; all of which are hereby incorporated by reference in their entirety.
  • heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized.
  • the heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group.
  • heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, e.g., 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, e.g., 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, e.g., 3- and 5-pyrazolyl), isothiazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, 1 ,3,4-triazolyl, tetrazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,3-oxadiazolyl, 1 ,3,4-thiadiazolyl and 1 ,3,4-oxadiazolyl.
  • protecting group or “chemical protecting group” refers to chemical moieties that block some or all reactive moieties of a compound and prevent such moieties from participating in chemical reactions until the protective group is removed, for example, those moieties listed and described in T.W. Greene, P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). It may be advantageous, where different protecting groups are employed, that each (different) protective group be removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions allow differential removal of such protecting groups. For example, protective groups can be removed by acid, base, and hydrogenolysis.
  • Groups such as trityl, monomethoxytrityl, dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile and may be used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile.
  • Carboxylic acid moieties may be blocked with base labile groups such as, without limitation, methyl, or ethyl, and hydroxy reactive moieties may be blocked with base labile groups such as acetyl in the presence of amines blocked with acid labile groups such as tert-butyl carbamate or with carbamates that are both acid and base stable but hydrolytically removable.
  • base labile groups such as, without limitation, methyl, or ethyl
  • hydroxy reactive moieties may be blocked with base labile groups such as acetyl in the presence of amines blocked with acid labile groups such as tert-butyl carbamate or with carbamates that are both acid and base stable but hydrolytically removable.
  • Carboxylic acid and hydroxyl reactive moieties may also be blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups may be blocked with base labile groups such as Fmoc.
  • a particularly useful amine protecting group for the synthesis of compounds of Formula I is the trifluoroacetamide.
  • Carboxylic acid reactive moieties may be blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while coexisting amino groups may be blocked with fluoride labile silyl carbamates.
  • Allyl blocking groups are useful in the presence of acid- and base-protecting groups since the former are stable and can be subsequently removed by metal or pi-acid catalysts.
  • an allyl-blocked carboxylic acid can be deprotected with a palladium(O)- catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups.
  • Yet another form of protecting group is a resin to which a compound or intermediate may be attached. As long as the residue is attached to the resin, that functional group is blocked and cannot react. Once released from the resin, the functional group is available to react.
  • base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine and hypoxanthine having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5- iodouracil, 2, 6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products).
  • base pairing moieties include, but are not limited to, expanded- size nucleobases in which one or more benzene rings has been added. Nucleic base replacements described in the Glen Research catalog (www.glenresearch.com); Krueger AT et al. (2007) Acc. Chem. Res. 40:141-150; Kool ET (2002) Acc. Chem. Res. 35:936-943; Benner SA et al. (2005) Nat. Rev. Genet. 6:553-543; Romesberg FE et al. (2003) Curr. Opin. Chem. Biol. 7:723-733; Hirao, I (2006) Curr. Opin. Chem. Biol. 10:622-627, the contents of which are incorporated herein by reference, are contemplated as useful for the synthesis of the oligomers described herein. Examples of expanded-size nucleobases are shown below:
  • oligonucleotide or “oligomer” refer to a compound comprising a plurality of linked nucleosides, nucleotides, or a combination of both nucleosides and nucleotides.
  • an oligonucleotide is a morpholino oligonucleotide.
  • antisense oligomer As used herein, the terms “antisense oligomer,” “antisense compound,” and
  • antisense oligonucleotides are used interchangeably and refer to a sequence of subunits, each having a base carried on a backbone subunit composed of ribose or other pentose sugar or morpholino group, and where the backbone groups are linked by intersubunit linkages that allow the bases in the compound to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence.
  • the oligomer may have exact sequence complementarity to the target sequence or nearly exact complementarity.
  • antisense oligonucleotides are designed to block or inhibit translation of the mRNA containing the target sequence, and may be said to be “directed to” a sequence with which it hybridizes. Also contemplated herein as types of “antisense oligomer,” “antisense compound,” or
  • antisense oligonucleotide are phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), phosphorothioate-modified oligomers, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), 2’-fluoro-modified oligomers, 2’-O,4’-C-ethylene-bridged nucleic acids (ENAs), tricyclo-DNAs, tricylo-DNA phosphorothioate-modified oligomers, 2’-O-[2-(N- methylcarbamoyl) ethyl] modified oligomers, 2’-O-methyl phosphorothioate modified oligomers, 2’-O-methoxyethyl (2’-O-MOE) modified oligomers, and 2’-O-Methyl oligonucleotides, or combinations thereof, as well as other antisense agents known in the art.
  • morpholino oligonucleotide or “PMO” refers to a modified oligonucleotide having morpholino subunits linked together by phosphoramidate or phosphorodiamidate linkages, joining the morpholino nitrogen of one subunit to the 5'- exocyclic carbon of an adjacent subunit.
  • Each morpholino subunit comprises a nucleobase- pairing moiety effective to bind, by nucleobase-specific hydrogen bonding, to a nucleobase in a target.
  • An antisense oligonucleotide “specifically hybridizes” to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions, with a Tm greater than 37°C, greater than 45°C, preferably at least 50°C, and typically 60°C-80°C or higher.
  • the “Tm” of an oligomer is the temperature at which 50% hybridizes to a complementary polynucleotide. Tm is determined under standard conditions in physiological saline, as described, for example, in Miyada et al. (1987) Methods Enzymol. 154:94-107. Such hybridization may occur with “near” or “substantial” complementarity of the antisense oligonucleotide to the target sequence, as well as with exact complementarity.
  • complementarity refers to oligonucleotides (i.e., a sequence of nucleotides) related by base-pairing rules.
  • sequence “T-G-A (5'-3')” is complementary to the sequence “T-C-A (5'-3').”
  • Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to base pairing rules. Or, there may be “complete,” “total,” or “perfect” (100%) complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • variations in sequence near the termini of an oligomer are generally preferable to variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5'-terminus, 3'-terminus, or both termini.
  • Naturally occurring nucleotide bases include adenine, guanine, cytosine, thymine, and uracil, which have the symbols A, G, C, T, and II, respectively. Nucleotide bases can also encompass analogs of naturally occurring nucleotide bases. Base pairing typically occurs between purine A and pyrimidine T or II, and between purine G and pyrimidine C.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Oligonucleotides containing a modified or substituted base include oligonucleotides in which one or more purine or pyrimidine bases most commonly found in nucleic acids are replaced with less common or non-natural bases. In some embodiments, the nucleobase is covalently linked at the N9 atom of the purine base, or at the N1 atom of the pyrimidine base, to the morpholine ring of a nucleotide or nucleoside.
  • Purine bases comprise a pyrimidine ring fused to an imidazole ring, as described by the general formula:
  • Adenine and guanine are the two purine nucleobases most commonly found in nucleic acids. These may be substituted with other naturally-occurring purines, including but not limited to N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.
  • Pyrimidine bases comprise a six-membered pyrimidine ring as described by the general formula:
  • Cytosine, uracil, and thymine are the pyrimidine bases most commonly found in nucleic acids. These may be substituted with other naturally-occurring pyrimidines, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain thymine bases in place of uracil.
  • modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g.
  • 5-halouracil 5-propynyluracil, 5- propynylcytosine, 5-aminomethyluracil, 5- hydroxym ethyl uracil, 5-aminomethylcytosine, 5- hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6- diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6- diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2- cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2- aminopurine (Pr-AP), pseudouracil or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like
  • Pseudouracil is a naturally occurring isomerized version of uracil, with a C-glycoside rather than the regular N-glycoside as in uridine.
  • nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the disclosure. These include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • nucleobases may include 5-methylcytosine substitutions, which have been shown to increase nucleic acid duplex stability by 0.6-1.2°C.
  • modified or substituted nucleobases are useful for facilitating purification of antisense oligonucleotides.
  • antisense oligonucleotides may contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases.
  • a string of three or more consecutive guanine bases can result in aggregation of the oligonucleotides, complicating purification.
  • one or more of the consecutive guanines can be substituted with hypoxanthine. The substitution of hypoxanthine for one or more guanines in a string of three or more consecutive guanine bases can reduce aggregation of the antisense oligonucleotide, thereby facilitating purification.
  • the oligonucleotides provided herein are synthesized and do not include antisense compositions of biological origin.
  • the molecules of the disclosure may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution, or absorption, or a combination thereof.
  • nucleic acid analog refers to a non-naturally occurring nucleic acid molecule.
  • a nucleic acid is a polymer of nucleotide subunits linked together into a linear structure. Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached to a phosphate group. Successive phosphate groups are linked together through phosphodiester bonds to form the polymer.
  • the two common forms of naturally occurring nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • a nucleic acid analog can include one or more non-naturally occurring nucleobases, sugars, and/or internucleotide linkages, for example, a phosphorodiamidate morpholino oligomer (PMO).
  • PMO phosphorodiamidate morpholino oligomer
  • a “morpholino oligomer” or “PMO” refers to a polymeric molecule having a backbone that supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose sugar backbone moiety, and more specifically a ribose backbone linked by phosphodiester bonds which is typical of nucleotides and nucleosides, but instead contains a ring nitrogen with coupling through the ring nitrogen.
  • An exemplary “morpholino” oligomer comprises morpholino subunit structures linked together by phosphoramidate or phosphorodiamidate linkages, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, each subunit comprising a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • Morpholino oligomers are detailed, for example, in U.S. Pat. Nos.
  • a preferred morpholino oligomer is a phosphorodiamidate-linked morpholino oligomer, referred to herein as a PMO.
  • PMO phosphorodiamidate-linked morpholino oligomer
  • Such oligomers are composed of morpholino subunit structures such as shown below: where X is NH 2 , NHR, or NR 2 (where R is lower alkyl, preferably methyl), Yi is O, and Z is O, and Pj and Pj are purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • structures having an alternate phosphorodiamidate linkage where X is lower alkoxy, such as methoxy or ethoxy, Yi is NH or NR, where R is lower alkyl, and Z is O.
  • Representative PMOs include PMOs wherein the intersubunit linkages are linkage (A1). See Table 1. Table 1 : Representative Intersubunit Linkages
  • a “phosphoramidate” group comprises phosphorus having three attached oxygen atoms and one attached nitrogen atom
  • a “phosphorodiamidate” group comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms.
  • a representative phosphorodiamidate example is below:
  • each Pj is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting-group, wherein the nucleobase independently at each occurrence comprises a C3-6 heterocyclic ring selected from pyridine, pyrimidine, triazinane, purine, and deaza-purine; and n is an integer of 6-38.
  • one nitrogen is always pendant to the backbone chain.
  • the second nitrogen, in a phosphorodiamidate linkage, is typically the ring nitrogen in a morpholino ring structure.
  • PMOs are water-soluble, uncharged or substantially uncharged antisense molecules that inhibit gene expression by preventing binding or progression of splicing or translational machinery components. PMOs have also been shown to inhibit or block viral replication (Stein, Skilling et al. 2001; McCaffrey, Meuse et al. 2003). They are highly resistant to enzymatic digestion (Hudziak, Barofsky et al. 1996). PMOs have demonstrated high antisense specificity and efficacy in vitro in cell-free and cell culture models (Stein, Foster et al. 1997; Summerton and Weller 1997), and in vivo in zebrafish, frog and sea urchin embryos (Heasman, Kofron et al.
  • Antisense PMO oligomers have been shown to be taken up into cells and to be more consistently effective in vivo, with fewer nonspecific effects, than other widely used antisense oligonucleotides (see e.g. P. Iversen, “Phosphoramidite Morpholino Oligomers,” in Antisense Drug Technology, S.T. Crooke, ed., Marcel Dekker, Inc., New York, 2001). Conjugation of PMOs to arginine-rich peptides has been shown to increase their cellular uptake (see e.g., U.S. Patent No. 7,468,418, incorporated herein by reference in its entirety).
  • Charged,” “uncharged,” “cationic,” and “anionic” as used herein refer to the predominant state of a chemical moiety at near-neutral pH, e.g., about 6 to 8.
  • the term may refer to the predominant state of the chemical moiety at physiological pH, that is, about 7.4.
  • a “cationic PMO” or “PMO+” refers to a phosphorodiamidate morpholino oligomer comprising any number of (l-piperazino)phosphinylideneoxy, (1-(4-(o-guanidino-alkanoyl))- piperazino)phosphinylideneoxy linkages (A2 and A3; see Table 1) that have been described previously (see e.g., PCT publication WO 2008/036127 which is incorporated herein by reference in its entirety).
  • the “backbone” of an oligonucleotide analog refers to the structure supporting the base-pairing moieties; e.g., for a morpholino oligomer, as described herein, the “backbone” includes morpholino ring structures connected by intersubunit linkages (e.g., phosphorus-containing linkages).
  • a “substantially uncharged backbone” refers to the backbone of an oligonucleotide analogue wherein less than 50% of the intersubunit linkages are charged at near-neutral pH.
  • a substantially uncharged backbone may comprise less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or even 0% intersubunit linkages which are charged at near neutral pH.
  • the substantially uncharged backbone comprises at most one charged (at physiological pH) intersubunit linkage for every four uncharged (at physiological pH) linkages, at most one for every eight or at most one for every sixteen uncharged linkages.
  • the nucleic acid analogs described herein are fully uncharged.
  • targeting base sequence or simply “targeting sequence” is the sequence in the nucleic acid analog that is complementary (meaning, in addition, substantially complementary) to a target sequence, e.g., a target sequence in the RNA genome of human Duchenne muscular dystrophy (DMD) gene.
  • a target sequence e.g., a target sequence in the RNA genome of human Duchenne muscular dystrophy (DMD) gene.
  • the entire sequence, or only a portion, of the analog compound may be complementary to the target sequence.
  • the targeting sequence is formed of contiguous bases in the analog, but may alternatively be formed of non-contiguous sequences that when placed together, e.g., from opposite ends of the analog, constitute sequence that spans the target sequence.
  • a “target sequence” refers to a nucleotide sequence within the genome of human Duchenne muscular dystrophy (DMD) gene to which the antisense compound will bind under conditions suitable for such binding, e.g., physiological conditions.
  • Examples of potential target sequences include sequences which comprise all or at least a portion of a 5' terminal region, a transcription regulatory sequence (TRS), a translation initiation region, or an AUG region.
  • TRS transcription regulatory sequence
  • a target sequence can typically encompass about 10 to about 30, about 20 to about 30, or about 20 to about 25 contiguous nucleotides of viral genome sequence.
  • a “slow intron” is an intron that is retained in the dystrophin pre- mRNA for a longer period of time compared to an average retention time for an intron in the dystrophin pre-mRNA that is downstream of the slow intron.
  • the “fast intron” the fast intron is an intron that is retained in the dystrophin pre-mRNA for a shorter period of time compared to an average retention time for an intron in the dystrophin pre-mRNA that is upstream of the intron.
  • the identification of slow and fast introns may be determined by the relative frequency that a particular intron appears in a sample (e.g., a pre-mRNA sample isolated from a plurality of cells expressing the gene of interest, e.g., DMD). Determination of intron frequency may be determined by any method known in the art, including, but not limited to, high-throughput sequencing. An intron may be classified as a slow intron if the intron’s normalized frequency is high. An intron may be classified as a fast intron is the intron’s normalized frequency is low.
  • a “cell-penetrating peptide” (CPP) or “carrier peptide” is a relatively short peptide capable of promoting uptake of PMOs by cells, thereby delivering the PMOs to the interior (cytoplasm) of the cells.
  • the CPP or carrier peptide typically is about 12 to about 40 amino acids long.
  • the length of the carrier peptide is not particularly limited and varies in different embodiments.
  • the carrier peptide comprises from 4 to 40 amino acid subunits.
  • the carrier peptide comprises from 6 to 30, from 6 to 20, from 8 to 25 or from 10 to 20 amino acid subunits.
  • the carrier peptide when conjugated to an antisense oligonucleotide having a substantially uncharged backbone, is effective to enhance the activity of the antisense oligonucleotide, relative to the antisense oligonucleotide in unconjugated form, as evidenced by:
  • conjugation of the peptide provides this activity in a cell-free translation assay, as described herein.
  • activity is enhanced by a factor of at least two, a factor of at least five or a factor of at least ten.
  • the carrier peptide is effective to enhance the transport of the nucleic acid analog into a cell, relative to the analog in unconjugated form.
  • transport is enhanced by a factor of at least two, a factor of at least two, a factor of at least five or a factor of at least ten.
  • a “peptide-conjugated phosphorodiamidate-linked morpholino oligomer,” “conjugate” or “PPMO” refers to a PMO covalently linked to a peptide, such as a cell-penetrating peptide (CPP) or carrier peptide.
  • CPP cell-penetrating peptide
  • the cell-penetrating peptide promotes uptake of the PMO by cells, thereby delivering the PMO to the interior (cytoplasm) of the cells.
  • a CPP can be generally effective or it can be specifically or selectively effective for PMO delivery to a particular type or particular types of cells.
  • PMOs and CPPs are typically linked at their ends, e.g., the C-terminal end of the CPP can be linked to the 5' end of the PMO, or the 3' end of the PMO can be linked to the N- terminal end of the CPP.
  • PPMOs can include uncharged PMOs, charged (e.g., cationic) PMOs, and mixtures thereof.
  • the carrier peptide may be linked to the nucleic acid analog either directly or via an optional linker, e.g., one or more additional amino acids, e.g., cysteine (C), glycine (G), or proline (P), or additional amino acid analogs, e.g., 6-aminohexanoic acid (X), beta-alanine (B), or XB.
  • an optional linker e.g., one or more additional amino acids, e.g., cysteine (C), glycine (G), or proline (P), or additional amino acid analogs, e.g., 6-aminohexanoic acid (X), beta-alanine (B), or XB.
  • amino acid subunit is generally an a-amino acid residue (-CO-CHR-NH-); but may also be a - or other amino acid residue (e.g., -CO-CH 2 CHR-NH-), where R is an amino acid side chain.
  • naturally occurring amino acid refers to an amino acid present in proteins found in nature; examples include Alanine (A), Cysteine (C), Aspartic acid (D), Glutamic acid (E), Phenyalanine (F), Glycine (G), Histidine (H), Isoleucine (I), Lysine (K), Leucine (L). Methionine (M), Asparagine (N), Proline (P), Glutamine (Q), Arginine (R), Serine (S), Threonine (T), Valine (V), Tryptophan (W), and Tyrosine (Y).
  • non-natural amino acids refers to those amino acids not present in proteins found in nature; examples include beta-alanine (p-Ala) and 6-aminohexanoic acid (Ahx).
  • each morpholino oligomer is conjugated to a carrier peptide at the 5' or 3’ end.
  • W represents O; each X is independently selected from OH and -NR 3 R 4 , wherein each R 3 and R 4 is independently at each occurrence -Ci-e alkyl; Y represents O; each Pi is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting- group, wherein the nucleobase independently at each occurrence comprises a C3-6 heterocyclic ring selected from pyridine, pyrimidine, triazinane, purine, and deaza-purine; and x is an integer of 6-38.
  • agent is “actively taken up by mammalian cells” when the agent can enter the cell by a mechanism other than passive diffusion across the cell membrane.
  • the agent may be transported, for example, by “active transport,” referring to transport of agents across a mammalian cell membrane by e.g. an ATP-dependent transport mechanism, or by “facilitated transport,” referring to transport of antisense agents across the cell membrane by a transport mechanism that requires binding of the agent to a transport protein, which then facilitates passage of the bound agent across the membrane.
  • a “subject” is a mammal, which can include a mouse, rat, hamster, guinea pig, rabbit, goat, sheep, cat, dog, pig, cow, horse, monkey, non-human primate, or human. In certain embodiments, a subject is a human.
  • treating refers to inhibiting or ameliorating a disease, condition or disorder in a subject who is experiencing or displaying the pathology or symptoms of the disease, condition or disorder.
  • inhibiting a disease, condition, or disorder refers to arresting further development of the pathology and/or symptoms of said disease, condition or disorder.
  • ameliorating a disease, condition or disorder refers to reversing the pathology and/or symptoms, such as decreasing the severity of the disease.
  • prevent comprises the prevention of at least one symptom associated with or caused by the disease, condition or disorder being prevented.
  • subject refers to an animal, preferably a mammal, and in particular a human or a non-human animal including livestock animals and domestic animals including, but not limited to, cattle, horses, sheep, swine, goats, rabbits, cats, dogs, and other mammals in need of treatment.
  • the subject is a human.
  • Administering of the antisense oligonucleotide to the subject includes both selfadministration and administration to the subject by another.
  • the subject may be in need of, or desire, treatment for an existing disease or medical condition, or may be in need of or desire prophylactic treatment to prevent or reduce the risk of occurrence of the disease or medical condition.
  • a subject “in need” of treatment of an existing condition or of prophylactic treatment encompasses both a determination of need by a medical professional as well as the desire of a patient for such treatment.
  • targeting an exon of which the upstream intron is retained in the transcript longer leads to generally higher efficiency of exon skipping than when the exon is preceded by a rapidly spliced (“fast”) intron.
  • targeting an exon closer to the 5’ end leads to more efficient exon skipping than targeting the 3’ of the same exon.
  • the optimal exon targeting region is the 5’, which can guide future design for AON based exon skipping therapies.
  • the modified antisense oligonucleotide may be 20-40 subunits, 18-35 subunits, 20-35 subunits, 18-30 subunits, 20-30 subunits, 18-26 subunits, 20-26 subunits, 22-26 subunits, 23-26 subunits, or 24-25 subunits including all integers in between these ranges.
  • the modified antisense oligonucleotide is 24-25 subunits.
  • the modified antisense oligonucleotide may have a melting temperature of about 60-80°C, about 62-80°C, about 64-80°C, about 60-78°C, about 62- 78°C, about 64-78°C, about 60-76°C, about 62-76°C, about 64-76°C, about 65-75°C, about 64-74°C, or about 65-74°C including all integers in between these ranges.
  • the modified antisense oligonucleotide has a melting temperature of about 64- 75°C.
  • the exon is selected from Exon 10, Exon 14, Exon 17, Exon 18,
  • the exon is Exon 10.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 14.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 17.
  • the targeting sequence comprises or consist of a sequence selected from:
  • SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC .
  • the exon is Exon 18.
  • the targeting sequence comprises or consist of a sequence selected from:
  • SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC
  • SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC.
  • the exon is Exon 21.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 22.
  • the targeting sequence comprises or consist of a sequence selected from:
  • SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT.
  • the exon is Exon 42.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 53.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 70.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the downstream 3’ splice site of the exon is flanked with a slow intron or a fast intron, wherein the fast intron is an intron that is retained in the dystrophin pre-mRNA for a shorter period of time compared to an average retention time for an intron in the dystrophin pre-mRNA that is upstream of the intron.
  • the conjugate comprising a modified antisense oligonucleotide and a cell-penetrating peptide may be 20-40 subunits, 18-35 subunits, 20-35 subunits, 18-30 subunits, 20-30 subunits, 18-26 subunits, 20-26 subunits, 22-26 subunits, 23-26 subunits, or 24-25 subunits including all integers in between these ranges. In a particular embodiment, the conjugate is 24-25 subunits.
  • the conjugate comprising a modified antisense oligonucleotide and a cell-penetrating peptide comprises a Guanine/Cytosine (G/C) content of 30-70%. In a further embodiment, the conjugate comprises a G/C content of 40-60%.
  • the conjugate comprising a modified antisense oligonucleotide and a cell-penetrating peptide may have a melting temperature of about 60-80°C, about 62- 80°C, about 64-80°C, about 60-78°C, about 62-78°C, about 64-78°C, about 60-76°C, about 62-76°C, about 64-76°C, about 65-75°C, about 64-74°C, or about 65-74°C including all integers in between these ranges.
  • the conjugate oligonucleotide has a melting temperature of about 64-75°C.
  • the exon is selected from Exon 10, Exon 14, Exon 17, Exon 18, Exon 21, Exon 22, Exon 42, Exon 50, Exon 53, and Exon 70.
  • the exon is Exon 10.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 14.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 17.
  • the targeting sequence comprises or consist of a sequence selected from: SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT
  • SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC .
  • the exon is Exon 18.
  • the targeting sequence comprises or consist of a sequence selected from:
  • SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC
  • SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC.
  • the exon is Exon 21.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the targeting sequence comprises or consist of a sequence selected from:
  • SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT.
  • the exon is Exon 42.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 50.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the targeting sequence comprises or consist of a sequence selected from:
  • the exon is Exon 70.
  • the targeting sequence comprises or consist of a sequence selected from:
  • the cell-penetrating peptide is selected from rTAT, TAT, R9F2, R5F2R4, R 4 , Rs, Re, R7, Re, Rg, (RXR) 4 , (RXR) 5 , (RXRRBR) 2 , (RAR) 4 F 2 , (RGR) 4 F 2 .
  • a phosphorodiamidate morpholino oligomer or a pharmaceutically acceptable salt thereof wherein:
  • A' is selected from ,
  • R 5 is -C(O)(O-alkyl) x -OH, wherein x is 3-10 and each alkyl group is, independently at each occurrence, C 2 .6-alkyl, or R 5 is selected from H, -C(O)Ci-6-alkyl, trityl, monomethoxytrityl, -(Ci-6-alkyl)-R 6 , - (Ci-6-heteroalkyl)-R 6 , aryl-R 6 , heteroaryl- R 6 , -C(O)O-(Ci-6-alkyl)-R 6 , -C(O)O-aryl-R 6 , -C(O)O- heteroaryl-R 6 , and
  • R 6 is selected from , each of which is covalently linked to a solid support; each R 1 is independently selected from OH and -N(R 3 )(R 4 ), wherein each R 3 and R 4 are, independently at each occurrence, H or -Ci-e-alkyl; each R 2 is independently, at each occurrence, selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein the nucleobase, independently at each occurrence, comprises a Cs-e-heterocyclic ring selected from pyridine, pyrimidine, purine, and deaza-purine; wherein each R 2 taken together form a targeting sequence; z is 8-40;
  • E' is selected from H, -Ci-e-alkyl, -C(O)Ci-e-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, wherein
  • Q is -C(O)(CH 2 ) 6 C(O)- or -C(O)(CH 2 )2S 2 (CH 2 )2C(O)-;
  • L is selected from glycine, proline, W, W-W, or R 9 , wherein L is covalently linked by an amide bond to the N-terminus or C-terminus of J;
  • W is -C(O)-(CH 2 ) m -NH-, wherein m is 2 to 12;
  • R 9 is selected from the group consisting of: n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5;
  • R 10 is selected from a bond, glycine, proline, W, or W-W;
  • R 11 is selected from the group consisting of glycine, proline, W, W-W, and
  • R 16 is selected from a bond, glycine, proline, W, or W-W; wherein R 16 is covalently linked by an amide bond to the N-terminus or C-terminus of J; J is a cell-penetrating peptide; and
  • G is selected from H, -C(O)Ci-6-alkyl, benzoyl, and stearoyl, wherein G is covalently linked to J; and wherein the targeting sequence comprises or consist of a sequence selected from:
  • SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC
  • one of the following definitions occurs in the oligomer of
  • E' is selected from H, -Ci-e-alkyl, -C(O)Ci-6-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, tri methoxytrityl, and
  • A' is selected from -N(Ci-6-alkyl)CH2C(O)NH2,
  • E' is selected from H, -C(O)CH3, benzoyl, stearoyl, trityl,
  • A' is selected from -N(Ci-6-alkyl)CH2C(O)NH2,
  • A' is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • 5 E' is selected from H, -C(O)CH3, trityl, 4-methoxytrityl, benzoyl, and stearoyl.
  • the compound of Formula I is selected from: wherein E' is selected from H, Ci-e-alkyl, -C(O)CH3, benzoyl, and stearoyl.
  • the phosphorodiamidate morpholino oligomer is of the Formula
  • the phosphorodiamidate morpholino oligomer is of the Formula
  • the cell-penetrating peptide is selected from rTAT, Tat, R9F2, R5F2R4, R4, Rs, Re, R7, Rs, Rg,
  • each R 1 is N(CHs)2.
  • L is glycine. In another embodiment, L is proline. In yet another embodiment, L is -C(O)-(CH 2 )s-NH-. In still another embodiment, L is -C(O)-(CH2)2-NH-. In an embodiment, L is -C(O)
  • bond; and R 11 is selected from: glycine and
  • bond; and R 11 is selected from: glycine and
  • bond; and R 11 is selected from: glycine and
  • J is selected from rTAT, TAT, R9F2, R5F2R4, R4, Rs, Re, R7, Rs, Rg,
  • G is selected from H, C(O)CHs, benzoyl, and stearoyl.
  • G is H or -C(O)CH3.
  • G is H.
  • G is -C(O)CH3.
  • an antisense oligomer compound and a pharmaceutically acceptable carrier.
  • the antisense oligonucleotides of the disclosure can employ a variety of antisense oligonucleotide chemistries.
  • oligomer chemistries include, without limitation, morpholino oligomers, phosphorothioate modified oligomers, 2'-O-methyl modified oligomers, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate oligomers, 2'-O-MOE modified oligomers, 2'-fluoro-modified oligomer, 2'-O,4'-C-ethylene- bridged nucleic acids (ENAs), tricyclo-DNAs, tricyclo-DNA phosphorothioate subunits, 2'-O- [2-(N- methylcarbamoyl)ethyl] modified oligomers, including combinations of any of the foregoing.
  • Phosphorothioate and 2'-O-Me-modified chemistries can be combined to generate a 2'-O-Me-phosphorothioate backbone. See, e.g., PCT Publication Nos. WO/2013/112053 and WO/2009/008725, which are hereby incorporated by reference in their entireties.
  • the nucleobases of the modified antisense oligonucleotides are linked to morpholino ring structures, wherein the morpholino ring structures are joined by phosphorous-containing intersubunit linkages joining a morpholino nitrogen of one ring structure to a 5' exocyclic carbon of an adjacent ring structure.
  • the nucleobases of the antisense oligonucleotide are linked to a peptide nucleic acid (PNA), wherein the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked.
  • PNA peptide nucleic acid
  • at least one of the nucleobases of the antisense oligonucleotide is linked to a locked nucleic acid (LNA), wherein the locked nucleic acid structure is a nucleotide analog that is chemically modified where the ribose moiety has an extra bridge connecting the 2' oxygen and the 4' carbon.
  • LNA locked nucleic acid
  • At least one of the nucleobases of the antisense oligonucleotide is linked to a bridged nucleic acid (BNA), wherein the sugar conformation is restricted or locked by introduction of an additional bridged structure to the furanose skeleton.
  • BNA bridged nucleic acid
  • at least one of the nucleobases of the antisense oligonucleotide is linked to a 2'-O,4'-C-ethylene- bridged nucleic acid (ENA).
  • the modified antisense oligonucleotide may contain unlocked nucleic acid (UNA) subunits.
  • UNAs and UNA oligomers are an analogue of RNA in which the C2'-C3' bond of the subunit has been cleaved.
  • the modified antisense oligonucleotide contains one or more phosphorothioates (or S-oligos), in which one of the nonbridging oxygens is replaced by a sulfur.
  • the modified antisense oligonucleotide contains one or more 2' O- Methyl, 2' O-MOE, MCE, and 2'-F in which the 2'-OH of the ribose is substituted with a methyl, methoxy ethyl, 2-(N-methylcarbamoyl)ethyl, or fluoro group, respectively.
  • the modified antisense oligonucleotide is a tricyclo-DNA (tc- DNA) which is a constrained DNA analog in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle g.
  • tc- DNA tricyclo-DNA
  • At least one of the nucleobases of the antisense oligonucleotide is linked to a bridged nucleic acid (BNA), wherein the sugar conformation is restricted or locked by introduction of an additional bridged structure to the furanose skeleton.
  • BNA bridged nucleic acid
  • at least one of the nucleobases of the antisense oligonucleotide is linked to a 2'-O,4'-C-ethylene- bridged nucleic acid (ENA).
  • each nucleobase which is linked to a BNA or ENA comprises a 5-methyl group.
  • PNAs Peptide Nucleic Acids
  • PNAs Peptide nucleic acids
  • the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached.
  • PNAs containing natural pyrimidine and purine bases hybridize to complementary oligomers obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition.
  • the backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well- suited for antisense applications (see structure below).
  • the backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.
  • PNAs are capable of sequence-specific binding in a helix form to DNA or RNA.
  • Characteristics of PNAs include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by singlebase mismatch, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triplex formation with homopurine DNA.
  • PANAGENETM has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2- sulfonyl group) and proprietary oligomerization process.
  • Bts benzothiazole-2- sulfonyl group
  • the PNA oligomerization using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping.
  • PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos.: 6,969,766; 7,211 ,668; 7,022,851 ; 7,125,994; 7,145,006; and 7,179,896. See also U.S. Pat.
  • LNAs Locked Nucleic Acids
  • Antisense oligonucleotides may also contain "locked nucleic acid” subunits (LNAs).
  • LNAs are a member of a class of modifications called bridged nucleic acid (BNA).
  • BNA is characterized by a covalent linkage that locks the conformation of the ribose ring in a C30- endo (northern) sugar pucker.
  • the bridge is composed of a methylene between the 2'-0 and the 4'-C positions. LNA enhances backbone preorganization and base stacking to increase hybridization and thermal stability.
  • LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54:3607; Jesper Wengel, Accounts of Chem. Research (1999) 32:301; Obika, et al, Tetrahedron Letters (1997) 38:8735; Obika, et al, Tetrahedron Letters (1998) 39:5401 ; and Obika, et al, Bioorganic Medicinal Chemistry (2008) 16:9230, which are hereby incorporated by reference in their entirety.
  • a non-limiting example of an LNA is depicted below.
  • Antisense oligonucleotides of the disclosure may incorporate one or more LNAs; in some cases, the antisense oligonucleotide may be entirely composed of LNAs.
  • Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligomers are described, for example, in U.S. Pat.: Nos. 7,572,582; 7,569,575; 7,084,125; 7,060,809; 7,053,207; 7,034,133; 6,794,499; and 6,670,461; each of which is incorporated by reference in its entirety.
  • Typical intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed.
  • inventions include an LNA containing antisense oligonucleotide where each LNA subunit is separated by a DNA subunit.
  • Certain antisense oligonucleotide are composed of alternating LNA and DNA subunits where the intersubunit linker is phosphorothioate.
  • ENAs 2'-O,4'-C-ethylene-bridged nucleic acids
  • Antisense oligonucleotide of the disclosure may incorporate one or more ENA subunits.
  • Antisense oligonucleotide may also contain unlocked nucleic acid (UNA) subunits.
  • UNAs and UNA oligomers are analogues of RNA in which the C2'-C3' bond of the subunit has been cleaved. Whereas LNA is conformationally restricted (relative to DNA and RNA), UNA is very flexible. UNAs are disclosed, for example, in WO 2016/070166. A non-limiting example of a UNA is depicted below.
  • Typical intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed.
  • Phosphorothioates are a variant of normal DNA in which one of the nonbridging oxygens is replaced by a sulfur.
  • a non-limiting example of a phosphorothioate is depicted below.
  • the sulfurization of the internucleotide bond reduces the action of endo-and exonucleases including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases SI and PI, RNases, serum nucleases and snake venom phosphodiesterase.
  • Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1, 2-benzodithiol-3-one 1 , 1 -dioxide (BDTD) (see, e.g., Iyer et al, J. Org. Chem. 55, 4693-4699, 1990, which is hereby incorporated by reference in its entirety).
  • TETD tetraethylthiuram disulfide
  • BDTD 2-benzodithiol-3-one 1 , 1 -dioxide
  • the latter methods avoid the problem of elemental sulfur's insolubility in most organic solvents and the toxicity of carbon disulfide.
  • the TETD and BDTD methods also yield higher purity phosphorothi
  • Tricyclo-DNAs are a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle g.
  • Homobasic adenine- and thymine-containing tc-DNAs form extraordinarily stable A-T base pairs with complementary RNAs.
  • Tricyclo-DNAs and their synthesis are described in International Patent Application Publication No. WO 2010/115993, which is hereby incorporated by reference in its entirety.
  • Antisense oligomers of the disclosure may incorporate one or more tricycle-DNA subunits; in some cases, the antisense oligomers may be entirely composed of tricycle-DNA subunits.
  • Tricyclo-phosphorothioate subunits are tricyclo-DNA subunits with phosphorothioate intersubunit linkages. Tricyclo-phosphorothioate subunits and their synthesis are described in International Patent Application Publication No. WO 2013/053928, which is hereby incorporated by reference in its entirety. Antisense oligomers of the disclosure may incorporate one or more tricycle-DNA subunits; in some cases, the antisense oligomers may be entirely composed of tricycle-DNA subunits. A non-limiting example of a tricycle- DNA/tricycle- phosphorothioate subunit is depicted below. tricyclo-DNA
  • 2'-O-Me oligomer molecules carry a methyl group at the 2'-OH residue of the ribose molecule.
  • 2'-O-Me-RNAs show the same (or similar) behavior as DNA, but are protected against nuclease degradation.
  • 2'-O-Me-RNAs can also be combined with phosphorothioate oligomers (PTOs) for further stabilization.
  • PTOs phosphorothioate oligomers
  • 2'-O-Me oligomers phosphodiester or phosphorothioate
  • a non-limiting example of a 2'-O-Me oligomer is depicted below.
  • 2'-O-Methoxyethyl Oligomers (2'-O-MOE) carry a methoxy ethyl group at the 2'-OH residue of the ribose molecule and are discussed in Martin et al., Helv. Chim. Acta, 78, 486- 504, 1995, which is hereby incorporated by reference in its entirety.
  • a non-limiting example of a 2'-O-MOE subunit is depicted below.
  • 2'-Fluoro (2'-F) oligomers have a fluoro radical in at the 2' position in place of the 2'- OH.
  • a non-limiting example of a 2'-F oligomer is depicted below. 2'-fluoro oligomers are further described in WO 2004/043977, which is hereby incorporated by reference in its entirety.
  • 2'-O-Methyl, 2'-O-MOE, and 2'-F oligomers may also comprise one or more phosphorothioate (PS) linkages as depicted below.
  • PS phosphorothioate
  • 2'-O-Methyl, 2'-0-M0E, and 2'-F oligomers may comprise PS intersubunit linkages throughout the oligomer, for example, as in the 2'-O-methyl PS oligomer drisapersen depicted below.
  • 2'-0-Methyl, 2'-0-M0E, and/or 2'-F oligomers may comprise PS linkages at the ends of the oligomer, as depicted below.
  • R is CH2CH2OCH3 (methoxyethyl or MOE).
  • X, Y, and Z denote the number of nucleotides contained within each of the designated 5'-wing, central gap, and 3'-wing regions, respectively.
  • Antisense oligomers of the disclosure can incorporate one or more 2'-O-Methyl, 2'-O- MOE, and 2'-F subunits and can utilize any of the intersubunit linkages described here.
  • an antisense oligomer of the disclosure can be composed of entirely 2'-O- Methyl, 2'-O-MOE, or 2'-F subunits.
  • One embodiment of an antisense oligomers of the disclosure is composed entirely of 2'-O-methyl subunits.
  • MCEs are another example of 2'-0 modified ribonucleosides useful in the antisense oligomers of the disclosure.
  • the 2'-OH is derivatized to a 2-(N-methylcarbamoyl)ethyl moiety to increase nuclease resistance.
  • a non-limiting example of an MCE oligomer is depicted below.
  • Antisense oligonucleotides of the disclosure may incorporate one or more MCE subunits.
  • Stereo-specific oligomers are those in which the stereo chemistry of each phosphorous-containing linkage is fixed by the method of synthesis such that a substantially stereo-pure oligomer is produced.
  • a non-limiting example of a stereo-specific oligomer is depicted below.
  • each phosphorous of the oligomer has the same stereo configuration.
  • Additional examples include the oligomers described herein.
  • LNAs, ENAs, Tricyclo-DNAs, MCEs, 2'-O-Methyl, 2 -O-MOE, 2'-F, and morpholino-based oligomers can be prepared with stereo-specific phosphorous-containing internucleoside linkages such as, for example, phosphorothioate, phosphodiester, phosphoramidate, phosphorodiamidate, or other phosphorous-containing internucleoside linkages.
  • Stereo specific oligomers, methods of preparation, chiral controlled synthesis, chiral design, and chiral auxiliaries for use in the preparation of such oligomers are detailed, for example, in WO2017192664, WO2017192679, WO2017062862, WO2017015575, WO2017015555, WO2015107425, W02015108048, W02015108046, W02015108047, WO2012039448, W02010064146, WO2011034072, W02014010250, W02014012081, WO20130127858, and WO2011005761, each of which is hereby incorporated by reference in its entirety.
  • Stereo-specific oligomers can have phosphorous-containing internucleoside linkages in an R or SP configuration. Chiral phosphorous-containing linkages in which the stereo configuration of the linkages is controlled is referred to as "stereopure,” while chiral phosphorous-containing linkages in which the stereo configuration of the linkages is uncontrolled is referred to as "stereorandom.”
  • the oligomers of the disclosure comprise a plurality of stereopure and stereorandom linkages, such that the resulting oligomer has stereopure subunits at pre-specified positions of the oligomer. An example of the location of the stereopure subunits is provided in international patent application publication number WO 2017/062862 A2 in Figures 7A and 7B.
  • all the chiral phosphorous-containing linkages in an oligomer are stereorandom.
  • all the chiral phosphorous-containing linkages in an oligomer are stereopure.
  • an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), all n of the chiral phosphorous-containing linkages in the oligomer are stereorandom. In an embodiment of an oligomer with n chiral phosphorous- containing linkages (where n is an integer of 1 or greater), all n of the chiral phosphorous- containing linkages in the oligomer are stereopure. In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 10% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure.
  • an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 20% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure. In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 30% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure.
  • an oligomer with n chiral phosphorous- containing linkages (where n is an integer of 1 or greater), at least 40% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure. In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 50% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure.
  • an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 60% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure. In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 70% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure.
  • an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 80% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure. In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), at least 90% (to the nearest integer) of the n phosphorous-containing linkages in the oligomer are stereopure.
  • the oligomer contains at least 2 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 3 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 4 contiguous stereopure phosphorous- containing linkages of the same stereo orientation (/.e. either SP or RP . In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 5 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 6 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 7 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 8 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 9 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 10 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 11 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 12 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 13 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP .
  • the oligomer contains at least 14 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 15 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 16 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 17 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 18 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP). In an embodiment of an oligomer with n chiral phosphorous-containing linkages (where n is an integer of 1 or greater), the oligomer contains at least 19 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 20 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP).
  • the oligomer contains at least 2 contiguous stereopure phosphorous-containing linkages of the same stereo orientation (/.e. either SP or RP) and at least 2 contiguous stereopure phosphorous-containing linkages of the other stereo orientation.
  • the oligomer can contain at least 2 contiguous stereopure phosphorous-containing linkages of the SP orientation and at least 2 contiguous stereopure phosphorous-containing linkages of the RP orientation.
  • the oligomer contains at least 2 contiguous stereopure phosphorous-containing linkages of the same stereo orientation in an alternating pattern.
  • the oligomer can contain the following in order: 2 or more RP, 2 or more SP, and 2 or more RP, etc.
  • Exemplary embodiments of the disclosure relate to phosphorodiamidate morpholino oligomers of the following general structure: and as described in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997). Morpholinos as described herein are intended to cover all stereoisomers and tautomers of the foregoing general structure. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S.
  • a morpholino is conjugated at the 5' or 3' end of the oligomer with a "tail" moiety to increase its stability and/or solubility.
  • exemplary tails include:
  • the disclosure provides antisense oligomers according to Formula I, or a pharmaceutically acceptable salt thereof.
  • the oligonucleotide can be 100% complementary to the nucleic acid target sequence, or it may include mismatches, e.g., to accommodate variants, as long as a heteroduplex formed between the oligonucleotide and nucleic acid target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo. Mismatches, if present, are less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • an antisense oligonucleotide is not necessarily 100% complementary to the nucleic acid target sequence, it is effective to stably and specifically bind to the target sequence, such that a biological activity of the nucleic acid target, e.g., expression of encoded protein(s), is modulated.
  • the stability of the duplex formed between an oligonucleotide and the target sequence is a function of the binding T m and the susceptibility of the duplex to cellular enzymatic cleavage.
  • the T m of an antisense compound with respect to complementary- sequence RNA may be measured by conventional methods, such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp.107-108 or as described in Miyada CG. and Wallace RB (1987) Oligonucleotide hybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107.
  • each antisense oligonucleotide has a binding T m , with respect to a complementary-sequence RNA, of greater than body temperature or in other embodiments greater than 50°C. In other embodiments T m 's are in the range 60-80°C or greater.
  • T m of an oligonucleotide compound, with respect to a complementary-based RNA hybrid can be increased by increasing the ratio of C:G paired bases in the duplex, and/or by increasing the length (in base pairs) of the heteroduplex.
  • the targeting sequence bases may be normal DNA bases or analogues thereof, e.g., uracil and inosine that are capable of Watson-Crick base pairing to target-sequence RNA bases.
  • An antisense oligonucleotide can be designed to block or inhibit or modulate translation of mRNA or to inhibit or modulate pre-mRNA splice processing, or induce degradation of targeted mRNAs, and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes.
  • the target sequence includes a region including a 3’ or 5’ splice site of a pre-processed mRNA, a branch point, or other sequence involved in the regulation of splicing.
  • the target sequence may be within an exon or within an intron or spanning an intron/exon junction.
  • An antisense oligonucleotide having a sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the antisense agent has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise modulate splicing and/or alters the three-dimensional structure of the targeted RNA.
  • an oligonucleotide reagent having a sufficient sequence complementary to a target RNA sequence to modulate splicing of the target RNA means that the oligonucleotide reagent has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise modulate splicing and/or alters the three- dimensional structure of the targeted RNA.
  • the degree of complementarity between the target sequence and antisense targeting sequence is sufficient to form a stable duplex.
  • the region of complementarity of the antisense oligonucleotides with the target RNA sequence may be as short as 8-11 bases, but can be 12-15 bases or more, e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers in between these ranges.
  • An antisense oligonucleotide of about 14-15 bases is generally long enough to have a unique complementary sequence. In certain embodiments, a minimum length of complementary bases may be required to achieve the requisite binding Tm, as discussed herein.
  • oligonucleotides as long as 40 bases may be suitable, where at least a minimum number of bases, e.g., 10-12 bases, are complementary to the target sequence.
  • facilitated or active uptake in cells is optimized at oligonucleotide lengths of less than about 30 bases.
  • an optimum balance of binding stability and uptake generally occurs at lengths of 18-25 bases.
  • antisense oligonucleotides e.g., PMOs, PMO-X, PNAs, LNAs, 2’-OMe
  • PMOs, PMO-X, PNAs, LNAs, 2’-OMe oligonucleotides that consist of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases, in which at least about 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous or noncontiguous bases are complementary to the desired target sequences.
  • antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to accommodate variants, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo.
  • certain oligonucleotides may have substantial complementarity, meaning, about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
  • Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein.
  • Mismatches are typically less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • an antisense oligonucleotide is not necessarily 100% complementary to the target sequence, it is effective to stably and specifically bind to the target sequence, such that splicing of the target pre-RNA is modulated.
  • the stability of the duplex formed between an oligonucleotide and a target sequence is a function of the binding Tm and the susceptibility of the duplex to cellular enzymatic cleavage.
  • the Tm of an oligonucleotide with respect to complementary-sequence RNA may be measured by conventional methods, such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C. G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107.
  • antisense oligonucleotides may have a binding Tm, with respect to a complementary-sequence RNA, of greater than body temperature and preferably greater than about 45°C or 50°C. Tm’s in the range 60-80°C or greater are also included.
  • Tm the Tm of an oligonucleotide, with respect to a complementary-based RNA hybrid, can be increased by increasing the ratio of C:G paired bases in the duplex, and/or by increasing the length (in base pairs) of the heteroduplex.
  • the disclosure provides an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, capable of binding a selected target to induce exon skipping in the human dystrophin gene, wherein the antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises a sequence of bases that is complementary to an exon target region of the dystrophin pre-m RNA designated as an annealing site, wherein each nucleobase R 2 , as recited in Formula I and described throughout the specification, from 1 to t and 5’ to 3’ can be selected from: ,
  • An antisense oligomer can be designed to block or inhibit or modulate translation of mRNA or to inhibit or modulate pre-mRNA splice processing, or induce degradation of targeted mRNAs, and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes.
  • the target sequence includes a region including a 3’ or 5’ splice site of a pre-processed mRNA, a branch point, or other sequence involved in the regulation of splicing.
  • the target sequence may be within an exon or within an intron or spanning an intron/exon junction.
  • An antisense oligomer having a sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the antisense agent has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise modulate splicing and/or alters the three- dimensional structure of the targeted RNA.
  • an oligomer reagent having a sufficient sequence complementary to a target RNA sequence to modulate splicing of the target RNA means that the oligomer reagent has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise modulate splicing and/or alters the three-dimensional structure of the targeted RNA.
  • the target region is within an exon of human dystrophin pre- mRNA wherein the exon is flanked at the upstream 5' splice site of the exon by a slow intron, wherein the slow intron is an intron that is retained in the dystrophin pre-mRNA for a longer period of time compared to an average retention time for an intron in the dystrophin pre- mRNA that is downstream of the slow intron.
  • Table 2 shows exemplary targeting sequences (in a 5’-to-3’ orientation) complementary to pre-mRNA sequences of the Duchenne muscular dystrophy gene .
  • Table 2 Antisense oligonucleotide sequences for targeting a region of a Duchenne muscular dystrophy (DMD) gene
  • the subject oligomer is conjugated to a peptide transporter moiety, for example a cell-penetrating peptide transport moiety (also referred to as a cell- penetrating peptide), which is effective to enhance transport of the oligomer into cells.
  • a cell-penetrating peptide transport moiety also referred to as a cell- penetrating peptide
  • the cell-penetrating peptide moiety is an arginine-rich peptide.
  • the peptide moiety is attached to either the 5' or 3' terminus of the oligomer. When such peptide is conjugated to either terminus, the opposite terminus is then available for further conjugation to a modified terminal group as described herein.
  • the cell-penetrating peptide moiety comprises 6 to 16 subunits selected from X’ subunits, Y’ subunits, and Z’ subunits, where
  • each Y’ subunit independently represents a neutral amino acid -C(O)-(CHR) n -NH-, where n is 2 to 7 and each R is independently H or methyl;
  • each Z’ subunit independently represents an a-amino acid having a neutral aralkyl side chain; wherein the peptide comprises a sequence represented by one of (X’Y’X’) P , (X’Y’)m, and (X’Z’Z’)p, where p is 2 to 5 and m is 2 to 8.
  • each Y’ is -CO-(CH2)n-CHR-NH-, where n is 2 to 7 and R is H.
  • Y’ is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx (or simply X); when n is 2 and R is H, Y’ is a - alanine subunit (referred to herein as B).
  • peptides of this type include those comprising arginine dimers alternating with single Y’ subunits, where Y’ is Ahx.
  • Examples include peptides having the formula (RY’R) P or the formula (RRY’) P , where Y’ is Ahx.
  • Y’ is a 6-aminohexanoic acid subunit
  • R is arginine
  • p is 4.
  • each Z’ is phenylalanine, and m is 3 or 4.
  • the conjugated peptide is linked to a terminus of the oligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunit and B is a p-alanine subunit.
  • the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg (R)).
  • the Y’ subunits are either contiguous, in that no X’ subunits intervene between Y’ subunits, or interspersed singly between X’ subunits.
  • the linking subunit may be between Y’ subunits.
  • the Y’ subunits are at a terminus of the cell-penetrating peptide moiety; in other embodiments, they are flanked by X’ subunits.
  • each Y’ is -CO-(CH2)n-CHR-NH-, where n is 2 to 7 and R is H.
  • Y’ is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx.
  • each X’ comprises a guanidyl side chain moiety, as in an arginine subunit.
  • Exemplary peptides of this type include those comprising arginine dimers alternating with single Y’ subunits, where Y’ is preferably Ahx. Examples include peptides having the formula (RY’R) 4 or the formula (RRY’) 4 , where Y’ is preferably Ahx.
  • the nucleic acid analog is linked to a terminal Y’ subunit, preferably at the C-terminus.
  • the linker is of the structure AhxB, where Ahx is a 6-aminohexanoic acid subunit and B is a p-alanine subunit.
  • the cell-penetrating peptide moieties as described above have been shown to greatly enhance cell entry of attached oligomers, relative to uptake of the oligomer in the absence of the attached transport moiety, and relative to uptake by an attached transport moiety lacking the hydrophobic subunits Y’.
  • Such enhanced uptake may be evidenced by at least a two-fold increase, or in other embodiments a four-fold increase, in the uptake of the compound into mammalian cells relative to uptake of the agent by an attached transport moiety lacking the hydrophobic subunits Y’.
  • uptake is enhanced at least twenty-fold or at least forty-fold, relative to the unconjugated compound.
  • a further benefit of the cell-penetrating peptide moiety is its expected ability to stabilize a duplex between an antisense oligomer and its target nucleic acid sequence. While not wishing to be bound by theory, this ability to stabilize a duplex may result from the electrostatic interaction between the positively charged transport moiety and the negatively charged nucleic acid.
  • the number of charged subunits in the transporter is less than 14, or in other embodiments between 8 and 11, since too high a number of charged subunits may lead to a reduction in sequence specificity.
  • CPP cell-penetrating peptides
  • Sequences assigned to SEQ ID NOs do not include the linkage portion (e.g., proline, beta-alanine, and glycine).
  • X and B refer to 6-aminohexanoic acid and beta-alanine, respectively.
  • an aspect of the present disclosure is a pharmaceutical composition comprising an oligonucleotide as disclosed herein and a pharmaceutically acceptable carrier.
  • oligonucleotide delivery include, but are not limited to, various systemic routes, including oral and parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as inhalation, transdermal and topical delivery.
  • oral and parenteral routes e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as inhalation, transdermal and topical delivery.
  • the appropriate route may be determined by one of skill in the art, as appropriate to the condition of the subject under treatment.
  • the antisense oligonucleotide can be administered in any convenient vehicle which is physiologically and/or pharmaceutically acceptable.
  • a composition can include any of a variety of standard pharmaceutically acceptable carriers employed by those of ordinary skill in the art. Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water (e.g., sterile water for injection), aqueous ethanol, emulsions such as oil/water emulsions or triglyceride emulsions, tablets and capsules.
  • PBS phosphate buffered saline
  • water e.g., sterile water for injection
  • aqueous ethanol emulsions
  • emulsions such as oil/water emulsions or triglyceride emulsions
  • tablets and capsules emulsions
  • suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.
  • the instant compounds e.g., a oli
  • the instant compounds may be used in the form of acid or base addition salts.
  • Acid addition salts of the free amino compounds may be prepared by methods well known in the art, and may be formed from organic and inorganic acids. Suitable organic acids include maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids.
  • Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids.
  • Base addition salts included those salts that form with the carboxylate anion and include salts formed with organic and inorganic cations such as those chosen from the alkali and alkaline earth metals (for example, lithium, sodium, potassium, magnesium, barium and calcium), as well as the ammonium ion and substituted derivatives thereof (for example, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, and the like).
  • the term “pharmaceutically acceptable salt” of Formula I is intended to encompass any and all acceptable salt forms.
  • prodrugs are also included within the context of this invention.
  • Prodrugs are any covalently bonded carriers that release a compound of Formula I in vivo when such prodrug is administered to a patient.
  • Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound.
  • Prodrugs include, for example, compounds of this invention wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amine or sulfhydryl groups.
  • prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol and amine functional groups of the compounds of Formula I.
  • esters may be employed, such as methyl esters, ethyl esters, and the like.
  • Morpholino subunits the modified intersubunit linkages, and oligomers comprising the same can be prepared as described, for example, in U.S. Patent Nos. 5,185,444, and 7,943,762, which are incorporated by reference in their entireties.
  • the morpholino subunits can be prepared according to the following general Reaction Scheme 1. Reaction Scheme 1. Preparation of Morpholino Subunit
  • the morpholino subunits may be prepared from the corresponding ribonucleoside (1) as shown.
  • the morpholino subunit (2) may be optionally protected by reaction with a suitable protecting group precursor, for example trityl chloride.
  • the 3’ protecting group is generally removed during solid-state oligomer synthesis as described in more detail below.
  • the base pairing moiety may be suitably protected for sold phase oligomer synthesis.
  • Suitable protecting groups include benzoyl for adenine and cytosine, phenylacetyl for guanine, and pivaloyloxymethyl for hypoxanthine (I).
  • the pivaloyloxymethyl group can be introduced onto the N1 position of the hypoxanthine heterocyclic base.
  • an unprotected hypoxanthine subunit may be employed, yields in activation reactions are far superior when the base is protected.
  • Other suitable protecting groups include those disclosed in U.S. Patent No. 8,076,476, which is hereby incorporated by reference in its entirety.
  • Compounds of structure 4 can be prepared using any number of methods known to those of skill in the art. For example, such compounds may be prepared by reaction of the corresponding amine and phosphorous oxychloride. In this regard, the amine starting material can be prepared using any method known in the art, for example those methods described in the Examples and in U.S. Patent No. 7,943,762.
  • a compound of structure 5 can be modified at the 5’ end to contain a linker to a solid support.
  • compound 5 may be linked to a solid support by a linker.
  • the protecting group e.g., trityl
  • the free amine is reacted with an activated phosphorous moiety of a second compound of structure 5. This sequence is repeated until the desired length of oligo is obtained.
  • the protecting group in the terminal 3’ end may either be removed or left on if a 3’-modification is desired.
  • modified morpholino subunits and morpholino oligomers are described in more detail in the Examples.
  • the morpholino oligomers containing any number of modified linkages may be prepared using methods described herein, methods known in the art and/or described by reference herein. Also described in the examples are global modifications of morpholino oligomers prepared as previously described (see e.g., PCT publication WO 2008/036127).
  • PMO with a 3’ trityl modification are synthesized essentially as described in PCT publication number WO 2009/064471 with the exception that the detritylation step is omitted.
  • a method of treating a neuromuscular disease comprises administering to a patient in need thereof a therapeutically effective amount of an oligonucleotide conjugate disclosed herein, or a pharmaceutical composition thereof.
  • the neuromuscular disease is Duchenne muscular dystrophy.
  • the method is an in vitro method. In certain other embodiments, the method is an in vivo method.
  • the host cell is a mammalian cell. In certain embodiments, the host cell is a non-human primate cell. In certain embodiments, the host cell is a human cell.
  • the host cell is a naturally occurring cell. In certain other embodiments, the host cell is an engineered cell.
  • the conjugate is administered to a mammalian subject, e.g., a human or a laboratory or domestic animal, in a suitable pharmaceutical carrier.
  • the conjugate is administered to a mammalian subject, e.g., a human or laboratory or domestic animal, together with an additional agent.
  • the conjugate and the additional agent can be administered simultaneously or sequentially, via the same or different routes and/or sites of administration.
  • the conjugate and the additional agent can be co-formulated and administered together.
  • the conjugate and the additional agent can be provided together in a kit.
  • the oligonucleotide is a phosphorodiamidate morpholino oligomer, contained in a pharmaceutically acceptable carrier, and is delivered intramuscularly.
  • the oligonucleotide is a peptide-conjugated phosphorodiamidate morpholino oligomer, contained in a pharmaceutically acceptable carrier, and is delivered intramuscularly.
  • the oligonucleotide is a phosphorodiamidate morpholino oligomer, contained in a pharmaceutically acceptable carrier, and is delivered intravenously (i.v.).
  • the oligonucleotide is a peptide-conjugated phosphorodiamidate morpholino oligomer, contained in a pharmaceutically acceptable carrier, and is delivered intravenously.
  • Additional routes of administration e.g., oral, subcutaneous, intraperitoneal, and pulmonary, are also contemplated by the instant disclosure.
  • the subject is a livestock animal, e.g., a pig, cow, or goat, etc.
  • the treatment is either prophylactic or therapeutic.
  • a livestock animal e.g., a pig, cow, or goat, etc.
  • the treatment is either prophylactic or therapeutic.
  • a method of feeding livestock with a food substance an improvement in which the food substance is supplemented with an effective amount of a conjugate composition as described above.
  • the conjugate is administered in an amount and manner effective to result in a peak blood concentration of at least 200 nM conjugate. In one embodiment, the conjugate is administered in an amount and manner effective to result in a peak plasma concentration of at least 200 nM conjugate. In one embodiment, the conjugate is administered in an amount and manner effective to result in a peak serum concentration of at least 200 nM conjugate.
  • the conjugate is administered in an amount and manner effective to result in a peak blood concentration of at least 400 nM conjugate. In one embodiment, the conjugate is administered in an amount and manner effective to result in a peak plasma concentration of at least 400 nM conjugate. In one embodiment, the conjugate is administered in an amount and manner effective to result in a peak serum concentration of at least 400 nM conjugate.
  • one or more doses of conjugate are administered, generally at regular intervals, for a period of about one to two weeks.
  • Preferred doses for oral administration are from about 0.01-15 mg conjugate per kg body weight. In some cases, doses of greater than 15 mg conjugate/kg may be necessary. For i.v. administration, preferred doses are from about 0.005 mg to 15 mg conjugate per kg body weight.
  • the conjugate may be administered at regular intervals for a short time period, e.g., daily for two weeks or less. However, in some cases the conjugate is administered intermittently over a longer period of time. Administration may be followed by, or accompanied by, administration of an antibiotic or other therapeutic treatment.
  • the treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests, and physiological examination of the subject under treatment.
  • An effective in vivo treatment regimen using the conjugates may vary according to the duration, dose, frequency and route of administration, as well as the condition of the subject under treatment (i.e., prophylactic administration versus administration in response to localized or systemic infection). Accordingly, such in vivo therapy will often require monitoring by tests under treatment, and corresponding adjustments in the dose or treatment regimen, in order to achieve an optimal therapeutic outcome.
  • the conjugate is actively taken up by mammalian cells.
  • the conjugate can be conjugated to a transport moiety (e.g., transport peptide) as described herein to facilitate such uptake.
  • the disease is a neuromuscular disease.
  • the neuromuscular disease is Duchenne muscular dystrophy.
  • a method of identifying a targeting sequence complementary to a target region of a Duchenne muscular dystrophy (DMD) gene wherein a target region is within an exon of human dystrophin pre-m RNA wherein the exon is flanked at the upstream 5' splice site of the exon by a slow intron, wherein the slow intron is an intron that is retained in the dystrophin pre-mRNA for a longer period of time compared to an average retention time for an intron in the dystrophin pre-mRNA that is downstream of the slow intron.
  • DMD Duchenne muscular dystrophy
  • the targeting sequences provided herein are capable of higher exon skipping efficiency than when the downstream 3’ splice site of the exon is flanked with a slow intron or a fast intron, wherein the fast intron is an intron that is retained in the dystrophin pre-mRNA for a shorter period of time compared to an average retention time for an intron in the dystrophin pre-mRNA that is upstream of the intron.
  • a Lonza Amaxa 4D nucleofector with accompanying X unit was utilized, as previously described. 14
  • cells were trypsinized and resuspended in nucleofection P1 buffer at a concentration of 1*10 6 cells per 20 pl of buffer. Then, 20 pl of P1 cell suspension was transferred to each well of the 16 well nucleocuvette, after which 1 pl of 1 mM PMO was added to the well, for a final concentration of 50 pM.
  • Cells were electroporated in the X unit using program CM-137 and allowed to recover for 10 min at room temperature (RT). After recovery, cells were carefully resuspended in 200 pl growth medium and transferred to 6-well plates containing equilibrated growth medium. Cells were grown for at least 48h before confluent cultures were allowed to differentiate to myotubes for 72h as described.
  • RNA isolation myotubes in each well were lysed in 500 pl Tri-sure lysis reagent, after which 200 pl chloroform was added and phases were separated by centrifugation at 16.200 relative centrifugal force (RCF) for 15 min at 4°C. The aqueous phase was transferred to 500 l of 2-propanol and RNA was precipitated by centrifugation for 15 min at 4°C at 16.200 RCF. The pellet was washed twice with 70% ethanol, air-dried and resuspended in 25 pl RNAse-free milli-Q (MQ). RNA concentration and purity was determined using a ND-1000 Nanodrop (thermo-scientific) and provided as A260/A230 and A260/A280 ratios. For cDNA synthesis, 1 pg of total RNA was used using bioscript Tetro (bioline BIO-65050), according to the manufacturer’s instructions in a 20 pl reaction. Samples were diluted to 100 pl final volume with MQ.
  • RT-PCR analysis was used to determine DMD exon skipping efficiency. Ten percent of generated cDNA was used per reaction using specific sets of intron spanning primers (see Table 4A), and Dreamtaq polymerase (Thermo scientific EP0713). Reactions of 25 pl total consisted of 2.5 pl 10x reaction buffer (green), 0.2 pl DreamTaq polymerase (1 Unit), 1 pl 10 pM forward primer, 1 pl 10 pM reverse primer, 1 pl DNTP mix (10 pM each nucleotide) and 9.3 pl MQ.
  • Amplification was performed in T-100 thermal cyclers (Bio-Rad) using the following parameters: 1: 95°C 2 min,; 2: 95°C 30 sec.; 3: 60°C 30 sec.; 4: 72°C 45 sec.; 5: Go to step 2, 34 additional cycles; 6: 72°C 5 min.
  • a fraction of each sample was analyzed using standard agarose TRIS-Borate-EDTA (TBE) gel-electrophoresis. DNA quantity in the PCR samples was assessed using Qubit dsDNA broad range reagent, measured in a Spectramax ID3 instrument. Samples were diluted to 0.2 ng/pl and analyzed using an Agilent Femto Pulse with NGS separation gel.
  • Peaks were called using the accompanying Prosize Data analysis software version 4.0.2.7. Skipping efficiency was calculated by determining the ratio of concentration (in nmole/L) of the skipped product over the total concentration (in nmole/L) of the sum of the full-length and skipped product. Statistical analysis was performed in Graphpad Prism Version 8.
  • the RT-qPCR reaction consisted of 4 pl SensiMix 2x SYBR master mix (bioline QT605-05), 2 pl cDNA, 1 pl forward primer (10 pM) and 1 pl reverse primer (10 pM) (see Table 4B). Each sample/primer combination was measured in a technical triplicate. Samples were manually pipetted in 384-well plates (Framestar 480/384, 4ti-0381 4titude) and run in a CFX-384 Real-time PCR system (Bio-Rad).
  • RNAstructure 6.2 was used for determination of the free energy of potential secondary structures (intramolecular and homodimer formation).
  • Table 5A Antisense oligonucleotide sequences for targeting a region of a Duchenne muscular dystrophy (DMD) gene
  • the condition with the highest detectable skipping of exon 51 was selected using RT-qPCR (FIG. 2A), and this condition was used (Buffer set P1, pulse program CM- 137) for the remainder of the project. Electroporation of myoblasts still allowed for differentiation to DMD expressing myotubes, as determined by RT-qPCR for myogenic markers MYOG and MYH3 (FIG. 2A), The foremost used method for semiquantification of exon skipping PCR products was the Agilent Bioanalyzer 2100 using a DNA-1000 chip. To facilitate the high amount of generated samples, the Bioanalyzer 2100 data was compared with data generated by the Agilent Femto pulse system, which allowed higher automated throughput of samples. Running the same RT-PCR sample on both systems showed no discernable differences in estimated exon skipping efficiency (FIG. 2B). Example 4 - Exons with slow 5’-introns appear to be more skippable
  • the set of PMOs targeting the 12 selected exons outlined above was electroporated in HC myoblasts and cells were allowed to differentiate to myotubes.
  • RT-PCR and analysis of exon skipping efficiency FIG. 3
  • the 5’Slow-‘3Slow and 5’Slow-3’Fast showed higher average skipping efficiencies than the 5’Fast-3’Slow and 5’Fast-3’Fast exon classes.
  • Example 5 An AON targeting the 5’ region within the exon is more efficient
  • skipping efficiency of each PMO was analyzed as before, using suitable RT-PCR primer sets for each targeted exon.
  • the relative binding position of each of the AONs was calculated as the most proximal or distal exonic position possible for a 25-mer AON on an arbitrary 1-100 scale, and skipping efficiency was plotted on this coordinate for the corresponding PMO.
  • Example 6 Exon skipping can lead to transcript loss in control cells

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Abstract

L'invention concerne des oligonucléotides, des peptides de pénétration cellulaire et des conjugués peptide-oligonucléotide. L'invention concerne également des méthodes de traitement d'une maladie musculaire, d'une infection virale, ou d'une infection bactérienne chez un sujet ayant besoin d'un tel traitement, comprenant l'administration au sujet d'oligonucléotides, de peptides et de conjugués peptide-oligonucléotide décrits dans la description.
EP23789830.9A 2022-09-21 2023-09-20 Efficacité du saut d'exon médié par un oligonucléotide antisens dans le traitement de la dmd Pending EP4590311A2 (fr)

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