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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/33—Alteration of splicing

Definitions

• the present invention relates generally to pre-mRNA splice modulating peptide-conjugated antisense therapeutics for treatment of diseases and in particular, peptide-conjugated antisense therapeutics for the treatment of Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA).
• DMD Duchenne Muscular Dystrophy
• SMA Spinal Muscular Atrophy
• Antisense therapeutics that modulate pre-messenger RNA splicing by either inducing exon splicing or inhibiting exon splicing function by binding to the pre-messenger RNA and have been approved for the treatment of various diseases including Duchenne’s Muscular Dystrophy and Spinal Muscular Atrophy.
• DMD Duchenne Muscular Dystrophy
• DMD Duchenne Muscular Dystrophy
• Dystrophin stabilizes the sarcolemma by bridging cytoskeletal actin to the extracellular matrix, via forming a membrane- associated glycoprotein complex [1 , 2]
• Dystrophin loss results in progressive body-wide muscle degeneration, loss of ambulation before the teens, and cardiorespiratory malfunction during the twenties that typically leads to death [3]
• DMD is present in 19.8 per 100,000 male births and is the most common inherited neuromuscular disorder worldwide [4],
• Exon skipping based therapeutics are based on the observation that, at least -90% of the time, in-frame mutations in DMD give rise to milder phenotypes, as found in Becker Muscular Dystrophy (BMD) [5, 6], By excluding out-of-frame exons from the DMD transcript using antisense oligonucleotides, exon skipping converts out-of-frame into in-frame mutations, producing truncated but partially functional dystrophin.
• Four exon skipping therapies have been approved by the U.S.
• exons 45-55 Deletion of exons 45-55 is also commonly associated with asymptomatic to mild phenotypes.
• 90% of patients with the exons 45-55 deletion have BMD; in other databases, including UMD-TREAT-NMD, and clinical studies, the deletion is associated with BMD or asymptomatic individuals in all examined cases [12-15],
• Exons 45-55 skipping cocktails that restored dystrophin synthesis in the muscles of dystrophic mice have been developed [16]
• a PMO cocktail that skipped human DMD exons 45-55 in immortalized patient myotubes and humanized DMD mice have been developed [13, 17]
• Average skipping efficacies of 27-61% and 15-22% were observed, respectively, and treatment produced up to 14% dystrophin of normal levels in vitro [13, 17]
• this cocktail used one PMO per exon (except exon 48, which required two) to skip exons 45-55.
• Efficacy is reduced and off-target effects increased as all PMOs have to be present in the same nuclei to induce skipping of exons 45-55.
• PMO based exon skipping therapies efficacy is impacted by their rapid clearance from the bloodstream, poor uptake into muscle, and inability to escape from endosomes [19, 20], Eteplirsen for instance, only restored 0.93% dystrophin of normal levels in patients after 180 weeks of treatment with a 30 or 50 mg/kg/week dose [7],
• SMA Spinal muscular atrophy
• SMA can result in progressive muscular weakness, respiratory distress, or even paralysis, and is one of the leading genetic causes of infant mortality [46]
• SMA is characterized by mutations in the survival of the telomeric motor neuron 1 (SMN1) gene leading to its homozygous deletion [47].
• SMN2 The complete loss of SMN protein is embryonically lethal [48,49], Humans possess a paralog of SMN1 called SMN2, enabling patients to be born [47,50], However, SMN2 cannot fully compensate for SMN1 loss because of a C-to-T transition in exon 7, leading to its exclusion.
• SMN2 full-length SMN2
• A7 SMN2 transcripts without exon 7
• ISS-N1 intra splicing silencer N1 that binds to a repressor protein hnRNP A1 (heterogeneous ribonucleoprotein A1) [52-54]
• nusinersen brand name Spinraza
• ona shogene abeparvovec brand name Zolgensma
• risdiplam brand name Evrysdi
• Nusinersen is a splice-switching antisense oligonucleotide (AO) with a 2’-O- methoxyethyl (MOE) phosphorothioate chemistry. It modulates SMN2 splicing by targeting ISS-N1 and promotes the inclusion of exon 7.
• nusinersen does not cross the blood-brain barrier (BBB), it requires repeated intrathecal administration that ensures a robust restoration of SMN protein mainly to the central nervous system (CNS) tissues [67], Despite providing a safe profile, nearly one-third of the treated patients suffer from scoliosis, persistent lumbar- fluid leakage, thrombocytopenia, and other coagulation defects [68,69],
• PMOs phosphorodiamidate morpholino oligomers
• LNAs locked nucleic acid
• c-Et constrained ethyl
• Pip6a a peptide from the family of PMO internalizing peptide (Pip) into adult mice
• the FL-SMN2 expression in the peripheral and CNS tissues increased the FL-SMN2 expression in the peripheral and CNS tissues [71]
• the localization and distribution of AOs in the CNS remain unknown.
• Pip peptides are known to be highly toxic [74]
• the present invention provides pre-mRNA splice modulating peptide-conjugated antisense therapeutics for treatment of diseases.
• a conjugate comprising an antisense oligonucleotide capable of modulating exon splicing of pre-mRNA attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
• conjugate comprising an antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
• the conjugate capable of inducing exon skipping in human dystrophin has an antisense oligonucleotide that binds to a target in exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54 and/or exon 55 of human dystrophin pre-mRNA.
• the conjugate capable of inducing exon skipping in human dystrophin has an antisense oligonucleotide that comprises or consists of any one of the following oligonucleotides:
• composition comprising a first conjugate comprising a first antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
• YArVRRrGPRGYArVRRrGPRr L-amino acids
• lowercase D-amino acids
• a second conjugate comprising a second antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to the cell penetrating peptide (CPP) .
• CPP cell penetrating peptide
• the composition further comprises at least one other conjugate comprising another antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to the cell penetrating peptide (CPP).
• CPP cell penetrating peptide
• the composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 46, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 48, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 50, a peptide-conjugated antisense oligonucleotide targeting exon 51 , a peptide-conjugated antisense oligonucleotide targeting exon 52, a peptide- conjugated antisense oligonucleotide targeting exon
• the composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 51 , a peptide-conjugated antisense oligonucleotide targeting exon 53 and a peptide-conjugated antisense oligonucleotide targeting exon 55.
• the composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47 and a peptide-conjugated antisense oligonucleotide targeting exon 53.
• a method of treating a subject having DMD comprising administering a therapeutically effective amount one or more peptide conjugates of the invention capable of inducing exon skipping in human dystrophin.
• conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene covalently attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
• the conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene binds to intronic splicing silencer N1 of SMN2 pre-mRNA.
• the conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene includes the antisense oligonucleotide comprises or consists of the sequence 5'-TCACTTTCATAATGCTGG-3' and wherein the thymines are optionally replaced with uracil, optionally wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer.
• a method of treating spinal muscular atrophy (SMA) in a subject comprising administering a therapeutically effective amount of the conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene of the invention.
• SMA spinal muscular atrophy
• a pharmaceutical composition comprising one or more peptide conjugates of the invention, and a pharmaceutically acceptable excipient.
• FIG. 1 shows testing minimized exons 45-55 skipping cocktails in immortalized patient myotubes.
• A The DMD exons targeted by the “all” cocktail and its derivatives are indicated by circles.
• B Culture scheme for PMO transfection in immortalized patient myotubes.
• C RT- PCR exons 45- 55 skipping efficiency results upon transfection of PMO cocktails in KM155, KM571 , 6594, and 6311 myotubes. Black (upper) arrows indicate native/unskipped bands, blue (lower) arrows indicate exons 45-55-skipped bands. GAPDH is shown below as a control. Quantification is shown at the bottom.
• FIG. 2 shows single-dose exon 51 skipping treatment with DG9-PMO.
• A Male, 3- month-old hDMDdel52;mdx mice were given a single retro-orbital injection (1 * r.o.) of saline, 50 mg/kg PMO, or equimolar 64 mg/kg DG9-PMO for exon 51 skipping. Tissues were collected 1 week later.
• B RT-PCR exon 51 skipping efficiency results post-treatment in various muscles, with quantification on the right. Gapdh is shown as a control.
• C Western blot for dystrophin (DYS1), with wild-type (WT) shown for reference.
• Protein extracts were loaded at 40 pg for saline-injected and treated muscles, and at indicated percentages of this for WT tibialis anterior samples. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signals are shown relative to the intensity of the 5% WT band.
• FIG. 3 shows repeated-dose exon 51 skipping treatment with DG9-PMO.
• A Male, 2- month-old hDMDdel52;mdx mice were given three once-weekly retro-orbital injections (3* r.o.) of saline or 30 mg/kg DG9-PMO for exon 51 skipping. Purple arrows indicate functional testing. Tissues were collected 2 weeks later.
• B RT-PCR exon 51 skipping efficiency results post-treatment in various muscles, with quantification on the right.
• Gapdh is shown as a control.
• E Representative immunofluorescence images for dystrophin (DYS1 , green) and nuclei (DAPI, blue) in various muscles and conditions. Scale bar: 100 pm.
• F Body weights of saline- and DG9-PMO-treated mice throughout the experiment.
• G Forelimb grip strength for saline- and DG9-PMO-treated mice, normalized to body weight. The % change from baseline is on the right.
• FIG. 4 shows local treatment with the minimized “block” DG9-PMO exons 45-55 skipping cocktail.
• A Male, 5-6-month-old hDMDdel52;mdx mice were injected with the DG9-conjugated “block” cocktail at 5 pg/DG9-PMO (25 pg total dose) in the right tibialis anterior (R), and saline in the left (L). Tissues were collected 1 week later.
• C Western blot for dystrophin (DYS1), with wild-type (WT) and non-treated (NT) tibialis anterior samples used for reference. Protein extracts were loaded at 40 pg for NT, saline- and DG9-PMO-treated muscles, and at indicated percentages of this for WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signals are shown relative to the intensity of the 1% WT band.
• FIG. 5 details preliminary testing of minimized exons 45-55 skipping cocktails.
• Minimized derivatives of the “all” exons 45-55 skipping PMO cocktail were generated and tested in immortalized myotubes.
• Strategy #1 for minimization involved two rounds of sequential removal of individual PMOs from the “all” cocktail.
• FIG. 6 shows histological data from single-dose exon 51 skipping treatment with DG9- PMO.
• Male, 3-month-old hDMDdel52;mdx mice were injected once retro-orbitally with saline, 50 mg/kg PMO, or equimolar 64 mg/kg DG9-PMO for exon 51 skipping.
• Tissues were collected 1 week later for assessment, sectioned, and stained using hematoxylin and eosin (HE).
• HE hematoxylin and eosin
• CNF Centrally nucleated fiber quantification from HE images. Error bars: S.E.M.
• C Minimal Feret’s diameter quantification from HE images for the tibialis anterior and (D) diaphragm. The frequency distribution is shown on the left, while quantification of individual fibers are shown on the right. Box edg es , 25th an d 75th percentiles; central line, median; whiskers, range.
• FIG. 7 shows functional and histological data from repeated-dose exon 51 skipping treatment with DG9-PMO.
• Male, 2-month-old hDMDdel52;mdx mice were injected thrice retro-orbitally with saline or 30 mg/kg DG9-PMO for exon 51 skipping, once a week for 3 weeks. Functional testing was done at baseline and at 2 weeks after the final injection.
• C Tissues were collected after post-treatment functional testing and stained using hematoxylin and eosin (HE). Representative HE images of the tibialis anterior and diaphragm from wild-type, saline-, and DG9-PMO-treated mice are shown. Scale bar: 100 pm
• D Centrally nucleated fiber (CNF) quantification from HE images. Error bars: SIM.
• FIG. 9 shows subcutaneous administration of DG9-PMO at postnatal day 0 extends survival and improves motor function in severe SMA mice.
• A Survival curves of heterozygous mice (Het), non-treated (NT), unconjugated-PMO (PMO), DG9-PMO and MOE injected at PDO at a dose of either 40 or 80 mg/kg.
• n 15 (Hets)
• n 22 (NT)
• n 14 (DG9-PMO)
• n 29 (MOE).
• mice at PD7 administered with either 40 or 80 mg/kg doses. Each dot (symbol) indicates a neonatal pup.
• C Hindlimb suspension assay (HLS). Mice were treated with 40 mg/kg AOs at PDO. Scored is based on the position of the hindlimbs when suspended from a falcon tube .
• D Righting reflex test. Mice were treated with 40 mg/kg AOs at PDO.
• mice The ability of mice to right themselves on their paws was measured every alternate day between PD2 to PD20 (left). The mean righting reflex time at PD6 and PD8 was also indicated (right: box whiskers plots). Box edges, 25 th and 75 th percentiles; central line, median; whiskerslange.
• E Forelimb grip strength measured in adult mice at PD30 and PD60 from 40 mg/kg treatment groups normalized to the body weight.
• B,D box whisker plots
• E One-way ANOVA followed by post hoc Tukey’s test was performed. *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.005.
• C and D Two-way ANOVA followed by Sidak’s multiple comparison in A and B. *p ⁇ 0.03; **p ⁇ 0.002; ***p ⁇ 0.0002.
• FIG. 10 shows subcutaneous administration of DG9-PMO at postnatal day 0 increases SMN expression.
• A Relative expression levels of full length SMN2 (FL-SMN2) compared to deleted SMN2 transcripts (A7 SMN2) measured by qPCR.
• B Representative images from western blotting and the quantification SMN protein levels, relative to - Tubulin. The heterozygous mice were used as a control with the relative SMN expression set to 1. 40 mg/kg AOs were injected on PD 0. Tissues were collected at PD7. One-way ANOVA followed by post hoc Tukey’s test was performed *NT, #PMO, @DG9-PMO, &MOE. *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.005. Error bars: SEM.
• FIG. 11 shows DG9-PMO treatment improves breathing function at postnatal day 7 in SMA mice.
• B Respiratory frequency fR- (0) Tidal volume (VT) relative to the mean of heterozygotes (100%) in normoxia.
• D Minute ventilation (VE) relative to the mean of heterozygotes (100%) in normoxia.
• E Coefficient of variation of frequency (CV).
• F Total apnea duration (seconds in one minute).
• hypoxia was conducted with kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s method.
• the difference between hypoxia and normoxia was conducted with a signed rank test, p ⁇ 0.05 is taken as statistically significant difference; p ⁇ 0.05, p ⁇ 0.01 , p ⁇ 0.001 compared between groups indicated (*NT, #PMO, @DG9-PMO, &MOE); $ ⁇ 0.05, $$ ⁇ 0.01 , $$$ ⁇ 0,001 compared with normoxia.
• FIG. 12 shows systemic administration of DG9-PMO improves the muscle pathology in SMA mice.
• A Representative images from H&E staining of the quadriceps muscle (top row), diaphragm (middle row) and the intercostal muscle (bottom row) at PD7 in the heterozygous, NT control and treated groups. Scale bar: 100 pm.
• FIG. 13 shows DG9-PMO treatment leads to improvement in the neuromuscular junctions (NMJs).
• NMJ neuromuscular junction
• FIG. 13 shows DG9-PMO treatment leads to improvement in the neuromuscular junctions (NMJs).
• NMJ neuromuscular junction staining in quadriceps and intercostal muscles collected at PD30. Scale bar: 100 pm.
• Postsynaptic endplates were stained using a-bungarotoxin (red, a-BTX) while neurofilament (2H3) and synaptic vesicles (SV2) were indicative of neurons (green).
• Denervated endplates can be identified as a-BTX endplates without overlapping synaptophysin-stained axons, while partially denervated endplates are identified as ⁇ 50% occupancy of the pre-synaptic nerve terminals in an endplate.
• White arrowheads full innervation.
• Yellow arrows partial innervation.
• Blue arrows denervation.
• White arrows collapsed NMJs.
• FIG. 14 shows DG9 increases uptake of PMO in target tissues following subcutaneous administration at PD0.
• B Representative immunohistochemistry images from PD7 heart, quadriceps muscle, brain, and the spinal cord following fluorescently tagged DG9-PMO subcutaneous administration at PD0.
• FIG. 15 shows DG9-PMO penetrates the blood-brain barrier and increases FL-SMN2 expression in a mild SMA model.
• FIG. 16 shows DG9-PMO treatment induces SMN expression in a dose-dependent manner in SMA mice.
• A Relative expression levels of full length SMN2 (FL-SMN2) compared to deleted SMN2 transcripts (A7 SMN2) measured by qPCR in NT and treated mice in the quadriceps muscle, liver, heart, brain, and spinal cord following 80 mg/kg AO treatment.
• B Representative images from western blotting and the quantification of SMN levels relative to p- Tubulin. The heterozygous mice were used as a control relative SMN expression set to 1 . The tissues from the 80 mg/kg treated mice were collected at PD7.
• FIG. 17 shows DG9-PMO maintains SMN levels in SMA mice at postnatal day 30. Representative images from western blotting and the quantification of SMN levels. The tissues were collected at PD30 from 40 mg/kg groups, relative to - Tubulin. The heterozygous mice were used as a control with the relative SMN expression set to 1. Statistics performed using one- way ANOVA followed by post hoc Tukey’s test. #PMO, @DG9-PMO, &MOE. *p ⁇ 0.05, **p ⁇ 0.01 ,***p ⁇ 0.005. Error bars: SEM.
• FIG. 18 shows muscle strength and motor function tests in DG9-PMO treated adult SMA mice.
• A Forelimb grip strength measured in adult males and females at PD30. 40 mg/kg of AOs were injected at PD0.
• B Forelimb grip strength normalized to the body weight. Measured at PD30 and PD60. 80 mg/kg of AOs were injected at PD0.
• C Rotarod test with an acceleration profile. Measured at PD 30-35. Each mouse was subjected to three trials spaced 20 minutes from one other. The maximum time on the beam was noted down. 40 mg/kg of AOs were injected at PD0.
• FIG. 19 shows DG9-PMO improves the muscle pathology in SMA mice at postnatal day 7.
• CSA cross-sectional area
• FIG. 20 shows muscle pathology of DG9-PMO treated mice at postnatal day 30.
• A Representative images from H & E staining of the quadriceps muscle (top row), diaphragm (middle row) and the intercostal muscle (bottom row) at PD30 in the heterozygous, NT control and treated groups. 40 mg/kg of AOs were injected at PDO. Scale bar: 100 pm.
• B Frequency distribution of the minimal Feret’s diameter (pm) and the quantification of individual myofibers from tissues collected at PD30 shown below.
• C Frequency distribution of cross-sectional area (CSA) (pm 2 ) and the quantification of individual myofibers shown below.
• FIG. 21 shows DG9-PMO treatment does not lead to an apparent immune response and toxicity.
• A Representative images from immunostaining of CD68 + macrophages (green) and DAPI (blue) in the quadriceps muscle at PD7.
• FIG. 22 shows a DG9 PMO induced heart-specific exon-skipping in a transgenic zebrafish model.
• a transgenic zebrafish line is produced using the Tol2 transposon system by flanking the dual fluorescent protein switch with Tol2 inverted terminal repeats (ITR). Ubiquitous mRNA is produced using the carp beta-actin promoter, non-coding exon, and mini intron 1 (B-act). mRNA will be produced from two exons. The first exon contains a complete blue fluorescent protein (BFP) open reading frame including a stop codon. The second exon includes a red fluorescence protein (RFP) open reading frame with its stop codon, a polyadenylation signal and a transcription terminator.
• BFP blue fluorescent protein
• RFP red fluorescence protein
• This dual cistronic mRNA will normally only produce functional BFP. However, in the presence of a PMO targeted against the splice acceptor of the BFP (derived from exon 2 of the carp beta actin gene), the BFP exon will be skipped, and a shorter mRNA will be made. RFP can be translated from this shorter mRNA. The switch from expression of BFP to RFP will correspond with PMO activity in the nucleus and indicates successful delivery into the cells.
• B. qPCR data shows detection of the RFP transcript collected from the DG9 PPMO-treated zebrafish group (15 months old, - 8 fish per group, injected with 1 dose of 25mg/Kg PPMO via an intravenous route).
• FIG. 23 shows improved efficacy following DG9-PMO treatment compared to R6-PMO in SMA mice.
• A Survival curves of heterozygous mice (Het), non-treated (NT), unconjugated-PMO (PMO), DG9-PMO, MOE and R6G-PMO injected at PDO at a dose of 40 mg/kg.
• mice A representative image of heterozygous mouse, mice treated with either 80 mg/kg or 40 mg/kg of DG9-PMO, mouse injected with unconjugated- PMO (40 mg/kg), and saline-treated NT mouse at PD7 (left-to-right).
• C Weight of mice at PD7 administered with either 40 or 80 mg/kg doses. Each dot (symbol) indicates a neonatal pup.
• D Hindlimb suspension assay (HLS). Mice were treated with 40 mg/kg AOs at PDO. Scored is based on the position of the hindlimbs when suspended from a falcon tube.
• E Righting reflex test. Mice were treated with 40 mg/kg AOs at PDO.
• mice The ability of mice to right themselves on their paws was measured every alternate day between PD2 to PD20 (left). The mean righting reflex time at PD6 and PD8 was also indicated (right: box whiskers plots). Box edges, 25 th and 75 th percentiles; central line, median; whiskers, range.
• F Relative expression levels of full length SMN2 (FL- SMN2) compared to deleted SMN2 transcripts (A7 SMN2) measured by qPCR following 40 mg/kg ASO or PBS treatments.
• C E (box whisker plots)
• F One-way ANOVA followed by post hoc Tukey’s test was performed. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005.
• the invention provides pre-mRNA splicing modulating therapeutics optionally suitable for systematic delivery.
• the therapeutics comprise antisense oligonucleotides conjugated to a cell penetrating peptide derived from the protein transduction domain (PTD) of the human Hph-1 transcription factor.
• PTD protein transduction domain
• the therapeutics are designed to either promote exon skipping or exon inclusion.
• the therapeutics includes peptide-conjugated antisense therapeutics for the treatment of Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA).
• DMD Duchenne Muscular Dystrophy
• SMA Spinal Muscular Atrophy
• DMD therapeutics promote exon skipping.
• such therapeutics comprise more than one conjugate and are configured to promote multi-exon skipping.
• the therapeutic comprises two, three, four, five, six, seven or more different conjugates wherein each conjugate comprises antisense oligonucleotides conjugated to a cell penetrating peptide derived from the protein transduction domain (PTD) of the human Hph-1 transcription factor.
• PTD protein transduction domain
• each different conjugate targets a different exon.
• different conjugates target the same exon.
• SMA therapeutics promote exon inclusion.
• Conjugates of the invention comprise an antisense oligonucleotide capable of modulating exon splicing of pre-mRNA attached to a cell penetrating peptide (CPP).
• the cell penetrating peptide of the invention also referred to as DG9, facilitates the delivery of the conjugated antisense oligonucleotide and has improved activity over the R6G cell penetrating peptide.
• conjugates of the invention are for intravenous delivery.
• the cell penetrating peptide of the invention is derived from the protein transduction domain of human HPH-1 transcription factor and comprises the amino acid sequence:
• one or two amino are deleted or substituted.
• amino acids are substituted with non-naturally occurring amino acids.
• one or more L-amino acids are replaced with D-amino acids.
• two, three, four, five or six L-amino acids are replaced with D-amino acids.
• two, three, four, five or six L-arginines are replaced with D-arginines
• the cell penetrating peptide has the amino acid sequence: YArVRRrGPRGYArVRRrGPRr; wherein uppercase represent L-amino acids and lowercase represent D-amino acids.
• the OPP is conjugated to the 5’ end of the oligonucleotide. In other embodiments, the OPP is conjugated at the 3’ of the oligonucleotide.
• antisense oligonucleotides refer to a sequence of subunits, each having a base carried on a backbone subunit , 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 by Watson-Crick base pairing, to form a nucleic acid:oligonucleotide heteroduplex within the target sequence.
• the oligonucleotides may have exact sequence complementarity to the target sequence or sufficient complementarity to selectively bind the target sequence.
• the antisense oligonucleotide are between about 20 to about 50 nucleotides in length, including at least 10, 12, 15, 17, or 20 consecutive nucleotides of complementary to the target sequence.
• the antisense oligonucleotide is 20 to 30 or 24 to 28 nucleotides in length.
• the antisense oligonucleotide is an antisense oligonucleotide analogue.
• oligonucleotide analogue and ‘nucleotide analogue’ refers to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art.
• oligonucleotide analogues include, but are not limited to, peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides, phosphoramidite oligonucleotides, tricyclo-DNA, and 2’methoxyethyl oligonucleogides.
• PNAs peptide nucleic acids
• morpholino oligonucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acy
• the antisense oligonucleotide comprises morpholino subunits.
• the antisense oligonucleotide is a morpholino antisense oligonucleotide.
• the antisense oligonucleotide comprises morpholino subunits linked together by phosphorus-containing linkages.
• the antisense oligonucleotide is a phosphoramidate or phosphorodiamidate morpholino antisense oligonucleotide.
• morpholino antisense oligonucleotide or ‘PMO’ (phosphoramidate or phosphorodiamidate morpholino oligonucleotide) refer to an antisense oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, for example one to three atoms long, for example two atoms long, and for example uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide.
• PMO phosphoramidate or phosphorodiamidate morpholino oligonucleotide
• the antisense oligonucleotide comprises phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit.
• the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate).
• the 5' oxygen may be substituted with amino or lower alkyl substituted amino.
• the pendant nitrogen attached to the phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl.
• Antisense oligonucleotide may be made through the well-known technique of solid phase synthesis.
• the conjugate further comprises a label.
• the label provides a direct or indirect detectable signal.
• Appropriate labels are known in the art and include fluorescent or radioactive labels.
• DMD Duchenne Muscular Dystrophy
• peptide-conjugated antisense therapeutics for the treatment of Duchenne Muscular Dystrophy. Treatment of DMD with peptide-conjugated antisense therapeutics restores partially functional dystrophin to the DMD patient.
• a ‘functional’ dystrophin protein refers to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy when compared to the defective form of dystrophin protein that is present in subjects with a muscular disorder such as DMD.
• a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the in vitro or in vivo biological activity of wild-type dystrophin.
• the activity of dystrophin in muscle cultures in vitro can be measured according to myotube size, myofibril organization, contractile activity, and spontaneous clustering of acetylcholine receptors.
• the peptide-conjugated antisense therapeutics of the invention are for use in the treatment of DMD by inducing exon skipping in the human dystrophin pre-mRNA to restore functional dystrophin protein expression.
• the therapeutic comprises one peptide-conjugated antisense oligonucleotide.
• the therapeutic comprises more than one peptide conjugated antisense oligonucleotide, wherein each peptide conjugated antisense oligonucleotide targets a different sequence in the dystrophin pre-mRNA.
• peptide-conjugated antisense therapeutics that may be therapeutically effective for exon skipping therapy skipping in the human dystrophin (DMD) gene, of one or more of exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54 and/or exon 55.
• DMD human dystrophin
• the peptide-conjugate antisense therapeutic comprises a plurality of peptide-conjugated antisense oligonucleotides and promotes skipping of exons 45-55.
• the peptide-conjugate antisense therapeutic comprises a peptide- conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 46, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 48, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 50, a peptide-conjugated antisense oligonucleotide targeting exon 51 , a peptide- conjugated antisense oligonucleotide targeting exon 52, a peptide-conjugated antisense oligonucleotide targeting exon 53, a peptide-
• the peptide-conjugate antisense therapeutic comprises a peptide- conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 51 , a peptide-conjugated antisense oligonucleotide targeting exon 53 and a peptide-conjugated antisense oligonucleotide targeting exon 55.
• the peptide-conjugate antisense therapeutic comprises a peptide- conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47 and a peptide-conjugated antisense oligonucleotide targeting exon 53.
• the antisense oligonucleotide of the peptide-conjugated antisense therapeutics binds to a target sequence in exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54 or exon 55 of the human dystrophin (DMD) gene.
• the target sequence is proximal to the exon acceptor splice site in the pre- mRNA.
• the target sequence is at the exon acceptor splice site, within 10 bases of the exon splice site, within 15 bases of the exon splice site, within 20 bases of the exon splice site, within 30 bases of the exon splice site, within 40 bases of the exon splice site, within 50 bases of the exon splice site, within 75 bases of the exon splice site or within 100 bases of the exon splice site
• the anti-sense oligonucleotide of the conjugate comprises or consists of at least 10, 12, 14, 16, 18 or 20 consecutive nucleotides of any one of the following sequences wherein, thymine bases in the sequences are optionally uracil:
• the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
• the conjugates have increased delivery to, uptake or retention by cardiac cells.
• SMA Spinal Muscular Atrophy
• exon inclusion peptide-conjugated antisense therapeutics for the treatment of Spinal Muscular Atrophy. Treatment of SMA with the peptide-conjugated antisense therapeutics results in the production of stable SMN2 to compensate for the loss of SMN1 .
• the peptide-conjugated antisense therapeutics comprises DG-9 conjugated to an antisense oligonucleotide that binds to intronic splicing silencer N1 of SMN2 pre-mRNA.
• the antisense oligonucleotide comprises or consists of the sequence 5 -TCACTTTCATAATGCTGG- 3' and wherein the thymines are optionally replaced with uracil.
• the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer.
• composition comprising the antisense oligonucleotide(s) of the invention or a conjugate thereof, further comprising one or more pharmaceutically acceptable excipients.
• the pharmaceutical composition is prepared in a manner known in the art, with pharmaceutically inert inorganic and/or organic excipients being used.
• pharmaceutically acceptable refers to molecules and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction when administered to a patient.
• the pharmaceutical composition may be formulated as a pill, tablet, coated tablet, hard gelatin capsule, soft gelatin capsule and/or suppository, solution and/or syrup, injection solution, microcapsule, implant and/or rod, and the like. In some embodiments, the pharmaceutical composition may be formulated as an injection solution.
• pharmaceutically acceptable excipients for preparing pills, tablets, coated tablets and hard gelatin capsules may be selected from any of: Lactose, corn starch and/or derivatives thereof, talc, stearic acid and/or its salts, etc.
• pharmaceutically acceptable excipients for preparing soft gelatin capsules and/or suppositories may be selected from fats, waxes, semisolid and liquid polyols, natural and/or hardened oils, etc.
• pharmaceutically acceptable excipients for preparing solutions and/or syrups may be selected from water, sucrose, invert sugar, glucose, polyols, etc.
• pharmaceutically acceptable excipients for preparing injection solutions may be selected from water, saline, alcohols, glycerol, polyols, vegetable oils, etc.
• pharmaceutically acceptable excipients for preparing microcapsules, implants and/or rods may be selected from mixed polymers such as glycolic acid and lactic acid or the like.
• the pharmaceutical composition may comprise a liposome formulation.
• the pharmaceutical composition may comprise two or more different antisense oligonucleotides or conjugates thereof.
• the antisense oligonucleotide and/or conjugate may be present in the pharmaceutical composition as a physiologically tolerated salt.
• physiologically tolerated salts retain the desired biological activity of the antisense oligonucleotide and/or conjugate thereof and do not impart undesired toxicological effects.
• suitable examples of pharmaceutically acceptable salts include (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesul
• a pharmaceutical composition may also comprise additives, such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizing agents, emulsifiers, preservatives, sweeteners, dyes, flavorings or aromatizing agents, thickeners, diluents or buffering substances, and, in addition, solvents and/or solubilizing agents and/or agents for achieving a slow release effect, and also salts for altering the osmotic pressure, coating agents and/or antioxidants.
• additives such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizing agents, emulsifiers, preservatives, sweeteners, dyes, flavorings or aromatizing agents, thickeners, diluents or buffering substances, and, in addition, solvents and/or solubilizing agents and/or agents for achieving a slow release effect, and also salts for altering the osmotic pressure, coating agents
• Suitable additives may include Tris-HCI, acetate, phosphate, Tween 80, Polysorbate 80, ascorbic acid, sodium metabisulfite, Thimersol, benzyl alcohol, lactose, mannitol, or the like.
• the peptide conjugated antisense oligonucleotide(s) and/or pharmaceutical composition is for topical, enteral or parenteral administration.
• the peptide conjugated antisense oligonucleotide(s) and/or pharmaceutical composition may be for administration orally, transdermally, intravenously, intrathecally, intramuscularly, subcutaneously, nasally, transmucosally or the like.
• the antisense oligonucleotide(s) and/or pharmaceutical composition is for intramuscular administration.
• the antisense oligonucleotide(s) and/or pharmaceutical composition is for intramuscular administration by injection.
• An ‘effective amount’ or ‘therapeutically effective amount’ refers to an amount of the antisense oligonucleotide, administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a desired physiological response or therapeutic effect in the subject.
• the antisense oligonucleotide(s) or conjugate thereof are administered daily, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 days, once every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 weeks, or once every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 months.
• the antisense oligonucleotide or conjugate thereof may be administered as two, three, four, five, six or more sub-doses separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
• Example 1 PEPTIDE-CON JUGATED PMOS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY
• Duchenne muscular dystrophy is primarily caused by out-of-frame deletions in the dystrophin gene.
• Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin.
• Skipping of exons 45-55 could treat 40-47% of all patients and is associated with improved clinical outcomes.
• the development of peptide-conjugated PMOs for exons 45-55 skipping is reported.
• Experiments with immortalized patient myotubes revealed that exons 45-55 could be skipped by targeting as few as 5 exons.
• the “base” cocktail showed significant exons 45-55 skipping (p ⁇ 0.05) compared to the mock, as well as its derivatives where exon 47 (p ⁇ 0.05) or 51 (p ⁇ 0.05) were not targeted (FIG. 5a).
• the “base” and “base -51” cocktail showed the highest skipping efficiencies in this batch (FIG. 1a).
• the “base -51”, “block”, and “3-PMO” cocktails induced significant exons 45-55 skipping in KM 155, KM571 , and 6594 myotubes compared to the mock (at least p ⁇ 0.05); the “base” cocktail only showed significant skipping in KM155 cells (p ⁇ 0.001).
• the “3-PMO” cocktail induced significantly higher skipping than the “all” cocktail. Intriguingly, none of the minimized cocktails showed any exons 45-55 skipping in 6311 myotubes.
• the dystrophin restoration capabilities of the “block” and “3-PMO” cocktails in KM571 myotubes were evaluated, since these cocktails induced the highest levels of exons 45-55 skipping in this line.
• DG9 As a conjugate for the minimized exons 45-55 skipping cocktail, its efficacy in vivo as applied to single-exon skipping was evaluated.
• DG9 was conjugated to Ex51_AcO, an exon 51 -skipping PMO from the “all” cocktail that was up to 7 times more effective than eteplirsen in restoring dystrophin production in vitro [13, 26).
• hDMDdel52;mdx mice at 3 months were given a single retro-orbital injection of either saline, 50 mg/kg unconjugated PMO, or 64 mg/kg DG9- PMO (equimolar to the PMO dose) and assessed a week later (FIG. 2a).
• DG9-PMO-treated mice had significantly higher levels of exon 51 skipping than the saline- or PMO-treated groups across various skeletal muscles (at least p ⁇ 0.05) and the heart (p ⁇ 0.001) (FIG. 2b).
• DG9-PMO induced 2.2 to 12.3-fold higher skipping in skeletal muscles and 14.4-fold higher skipping in the heart on average compared to unconjugated PMO.
• DG9-PM0 treatment significantly restored dystrophin production compared to the saline (p ⁇ 0.005) and PMO (p ⁇ 0.05) treatments in the gastrocnemius and quadriceps, reaching up to 3% of wild-type levels (FIG. 2c).
• DG9-PM0 restored an average 2.5% dystrophin of wild-type levels, significantly higher than that in the saline or PMO groups (p ⁇ 0.05). Compared to PMO-treated mice, mice that received DG9-PM0 had 1.5 to 3.4- fold and 4.5-fold higher dystrophin protein levels on average in the skeletal muscles and heart, respectively.
• FIGs. 7c, d Histological analysis still did not show any significant reductions in CNFs in the tibialis anterior and diaphragm. However, a significant increase in fiber size with DG9-PMO treatment (p ⁇ 0.001) was observed (FIGs. 7e,f), with most fibers having a minimum Feret’s diameter of 45-50 pm in the tibialis anterior and 25-30 pm in the diaphragm. This is in contrast to most fibers having diameters of 30-35 pm and 20-25 pm in saline control muscles, respectively.
• DG9 was conjugated to each PMO of the minimized “block” exons 45-55 skipping cocktail for in vivo testing.
• the “block” cocktail was chosen as it induced high levels of exons 45-55 skipping and dystrophin protein restoration in KM571 myotubes (FIGs. 1c-e).
• 5- to 6-month- old hDMDdel52;mdx mice were intramuscularly injected in the tibialis anterior with either saline or the mutation-tailored DG9-conjugated version of the “block” cocktail (five DG9-PMOs, 5 pg/DG9-PMO, 25 pg total dose), and assessed a week later (FIG. 4a).
• a minimized exons 45-55 skipping cocktail that induces dystrophin restoration in immortalized patient cells and dystrophic hDMDdel52;mdx mice was developed.
• the number of PMOs used for exons 45-55 skipping was reduced from 11 in the “all” to 5 in the “block” cocktail (FIG. 1a), more than a 50% decrease in PMO content.
• Most minimized cocktails significantly skipped exons 45-55 in KM571 (Aex52) and 6594 (Aex48-50) myotubes, apparently all remaining exons have to be targeted in 6311 myotubes (Aex45-52).
• the “block” cocktail restored dystrophin production to ⁇ 3% of wild-type levels, near the amount seen with the “all” cocktail (FIG. 1e).
• DG9 is in its potentially better toxicity profile compared to other peptides.
• Peptide-conjugated PMOs have induced dose-dependent toxic effects in pre-clinical studies, including lethargy, weight loss, and kidney damage (20). This is linked to the membrane-disruptive properties of cell-penetrating peptides, which are largely influenced by their amino acid compositions (20, 29).
• Certain L-arginine residues in DG9 were converted to D-arginine, as this improves the viability of peptide-conjugated PMO-treated cells in vitro (30).
• DG9 also does not contain any 6-aminohexanoic acid residues, which have been associated with increased toxicity (30).
• Exon 51-skipped transcripts or proteins may be less stable than their exon 23-skipped counterparts, leading to the reduced dystrophin levels observed.
• An in vitro study using various internally truncated dystrophin proteins revealed that exon 51-skipped dystrophins were mostly as stable as full-length dystrophin (33), favoring a hypothesis of decreased transcript stability. While previous work has shown that DMD transcript stability has an impact on dystrophin protein production (34, 35), it remains to be determined if this explains the differences in the case of exon 23- and exon 51-skipped transcripts. Another factor would be the animal model used for testing.
• mice given repeated doses of DG9-PMO (FIG. 3g, h, FIGs. 7a, b) were observed.
• mice may have sequestered some of the administered DG9- PMOs. Crossing the hDMDdel52 transgene over to a mouse Dmd-null background [37] would eliminate this possibility, and may yield more representative results of efficacy.
• Myoblasts were grown in DMEM/F12 medium (with HEPES; Gibco) containing 20% fetal bovine serum (Sigma), 1 vial of skeletal muscle growth supplement mix (Promocell), 50 U/rnL penicillin, and 50 pg/mL streptomycin. Myoblasts were then seeded into collagen type 1-coated 12-well plates, at a density of 0.53 x 10 5 cells/cm 2 . Once 90% confluent, myoblasts were differentiated into myotubes by replacing the growth medium with differentiation medium (DMEM/F12 containing 2% horse serum [GE Healthcare], 1 x ITS solution [Sigma], 50 U/rnL penicillin, and 50 pg/mL streptomycin). All cells were incubated at 37°C, 5% CO2.
• differentiation medium DMEM/F12 containing 2% horse serum [GE Healthcare], 1 x ITS solution [Sigma], 50 U/rnL penicillin, and 50 pg/mL streptomycin. All cells
• PMO transfection The PMOs used are summarized in Table 1 and were derived from cocktail set no. 3 in our previous publication [1], Prior to transfection, PMOs (Gene Tools) were heated at 65°C for 15 min to remove aggregates. PMOs were then transfected into muscle fibers at 3 days post-differentiation using 6 pM Endoporter reagent (Gene Tools) in differentiation medium. Each PMO in a cocktail was transfected at a final 5 pM concentration for all experiments. Cells were incubated in PMOs for 2 days, after which they were harvested for RNA and protein (FIG. 1a). Random control 25-N (Gene Tools) was used for mock treatment. For non-treated samples, transfection was done as described with Endoporter, only without any PMO.
• hDMDdel52;mdx mice were retro-orbital ly injected with either phosphate-buffered saline (PBS), PMO (50 mg/kg, unconjugated Ex51_AcO), or DG9-PMO (64 mg/kg, equimolar to 50 mg/kg PMO) and then euthanized a week later for tissue collection.
• PBS phosphate-buffered saline
• PMO 50 mg/kg, unconjugated Ex51_AcO
• DG9-PMO 64 mg/kg, equimolar to 50 mg/kg PMO
• mice were subjected to muscle function tests before the first injection and 2 weeks after the last injection as described in Functional testing. Mice were euthanized after the post-function tests for tissue collection. Upon dissection, tissues were mounted in tragacanth gum on corks and snap-frozen in liquid nitrogen- cooled isopentane.
• DG9 was conjugated to each PMO of the minimized “block” exons 45-55 skipping cocktail (FIG. 1a).
• This DG9-PMO cocktail (5 pg per DG9-PMO, total dose of 25 pg) was then administered intramuscularly into the tibialis anterior of 5- to 6- month-old hDMDdel52;mdx mice; the contralateral leg was injected with PBS. Mice were euthanized a week later for tissue collection as described above. All injections, retro-orbital and intramuscular, were conducted under isoflurane anesthesia. Tissues from age-matched wild-type male C57BL/6J mice were collected as controls.
• Forelimb and total limb grip tests were conducted by blinded personnel according to TREAT-NMD SOP DMD_M.2.2.001 using the Chatillon DFE II grip strength meter (Columbus Instruments). The average of the three most consistent readings was used per mouse, and results were normalized to body weight.
• the rotarod test was performed using the AccuRotor 4-channel rotarod (Omnitech Electronics, Inc.). Mice were first placed on a rod rotating at a steady speed of 5 rpm. Once all mice were in place, rotation was accelerated from 5 to 45 rpm over a span of 300 s [5], Fall times were automatically recorded by the software.
• the SuperScriptTM III One-Step RT-PCR system with PlatinumTM Taq (Invitrogen) was used. Briefly, 200 ng of total RNA was used as template in a 25- pL solution containing 1 x reaction mix, 0.2 pM each of forward and reverse primers for DMD or GAPDH/Gapdh (Table 3), and 1 pL of SuperScript III RT/Platinum Taq.
• the reaction was run under the following conditions: 1) 50°C, 5 min, 2) 94°C, 2 min, 3) 35 cycles of 94°C, 15 s; 60°C, 30 s; 68°C, 33-118 s, 4) 68°C, 5 min, 5) 4°C, hold.
• cDNA was synthesized from 750-1000 ng of total RNA using SuperScriptTM IV Reverse Transcriptase (Invitrogen) with 2.5 pM random hexamers (Invitrogen) in a 20-pL reaction following manufacturer’s instructions. From this, 8 pL of cDNA was used for PCR with 1 x GoTaq® Green Master Mix (Promega) and 0.3 pM each of forward and reverse primers for DMD or Gapdh (Table 3) in a 25-pL reaction.
• SuperScriptTM IV Reverse Transcriptase Invitrogen
• 2.5 pM random hexamers Invitrogen
• 8 pL of cDNA was used for PCR with 1 x GoTaq® Green Master Mix (Promega) and 0.3 pM each of forward and reverse primers for DMD or Gapdh (Table 3) in a 25-pL reaction.
• the reaction was run as follows: 1) 95°C, 2 min, 2) 40 cycles of 95°C, 30 s; 60°C, 30 s; 72°C, 35 s, 3) 72°C, 5 min, and 4) 4°C, hold. All PCR products (exons 45-55 or exon 51 skipping) were run in 1.5% agarose gels in 1 x tris-borate- EDTA buffer, and band intensities were quantified by Image J (NIH). The % of successful exon skipping was calculated using the following formula: (intensity of desired skipped band I total intensity of unskipped, intermediate, and desired skipped bands) x 100.
• proteins were mixed with NuPAGETM LDS Sample Buffer (Invitrogen; 1 * final concentration) and NuPAGETM Sample Reducing Agent (Invitrogen; 1 * final concentration), then heated at 70°C for 10 min. SDS-PAGE was performed using pre-cast NuPAGETM 3-8% Tris-Acetate Midi gels (Invitrogen), run at 150 V for 75 min. Proteins were then transferred onto a PVDF membrane (Millipore) using a semi- dry blotting system at 20 V for 70 min.
• NuPAGETM LDS Sample Buffer Invitrogen; 1 * final concentration
• NuPAGETM Sample Reducing Agent Invitrogen; 1 * final concentration
• membranes were cut and incubated in one of the following primary antibodies for 1 hr at room temperature: 1 :200 NCL-DYS1 (Leica), 1 :2,500 anti-dystrophin C-terminal (Abeam, ab15277), 1 :100 MANEX45A (Developmental Studies Hybridoma Bank; DSHB),(8) 1 :100 MANEX4850E (MDA Monoclonal Antibody Resource, Wolfson Centre for Inherited Neuromuscular Disease) (8), or 1 :4,000 anti-desmin (Abeam, ab8592) in blocking agent.
• 1 :200 NCL-DYS1 Leica
• 1 :2,500 anti-dystrophin C-terminal Abeam, ab15277
• MANEX45A Developmental Studies Hybridoma Bank; DSHB
• MANEX4850E MDA Monoclonal Antibody Resource, Wolfson Centre for Inherited Neuromuscular Disease
• (8) or 1 :4,000 anti-desmin (Abeam,
• the membranes were then washed thrice for 10 min each with PBS containing 0.05% Tween 20 (PBST), before incubating in either anti-mouse lgG2a, anti- mouse lgG1 , or anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibodies (Invitrogen) as appropriate, all 1 :10,000 in PBST.
• Membranes were then similarly washed in PBST, and detected with ECL Select Detection Reagent (GE Healthcare). DYS1 band intensities were quantified using Image LabTM software, v.6.0.1 (Bio-Rad), and dystrophin levels were expressed relative to the intensity of the least concentrated wild-type sample in a series.
• Dystrophin immunofluorescence Frozen muscle and heart samples were sectioned at 7-pm thickness and placed on poly-L-lysine-coated slides. After thawing at room temperature for 30 min, sections were blocked for 2 hr in PBS with 10% goat serum and 0.1 % Triton X-100 at room temperature. Sections were then incubated with 1 :50 NCL-DYS1 in the blocking agent overnight at 4°C. The following day, sections were washed thrice with PBS for 5 min each and subsequently incubated with Alexa 488-conjugated goat anti-mouse lgG2a secondary antibody (Life Technologies) for 30 min at room temperature.
• Sections were washed again with PBS, and mounted with Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Laboratories). Samples were visualized for dystrophin and DAPI using a Zeiss LSM 710 confocal microscope at 200x magnification, by personnel blinded to the treatment condition. Histology. Frozen muscles were sectioned at 7-pm and placed on poly-L- lysine-coated slides. After thawing at room temperature for 30 min, slides were stained with Mayer’s hematoxylin (Electron Microscopy Sciences) for 15 min, washed with running tap water for 15 min, and then stained with eosin Y (Electron Microscopy Sciences) for 10 min.
• DAPI Vectashield HardSet Antifade Mounting Medium with DAPI
• Sections were then dehydrated with an ethanol series (70%-90%-99%), cleared with a xylene substitute, and mounted with PermountTM (Fisher Scientific).
• Blinded personnel visualized the samples under brightfield using the Optika B-290TB microscope at 200* magnification, taking three randomly chosen fields of view per sample.
• CNF percentage was calculated by (# CNFs I total # fibers) x 100, with fibers counted manually using Image J. The average CNF percentage from all fields of view was taken per sample.
• Minimal Feret’s diameters were quantified by blinded personnel in two steps. First, images were semi- automatically measured using an in-house developed Image J macro based on Open-CSAM (9).
• SMA Spinal muscular atrophy
• CNS central nervous system
• T o examine whether DG9 peptide can increase the efficacy of PMO
• DG9 peptide was conjugated to an 18-mer PMO with a sequence identical to nusinersen.
• the DG9-conjugated PMO and unconjugated-PMO and 2'-O-methoxyethyl-RNA (MOE) were injected into SMA model mice.
• MOE 2'-O-methoxyethyl-RNA
• mice were subcutanteously injected with the AOs or saline (NT) administered at postnatal day 0 (PDO) at two different doses-40 mg/kg or 80 mg/kg. The survival was recorded until a humane endpoint was reached. For the 40 mg/kg doses, the median survival was 12 d (unconjugated- PMO), 15.5 d (MOE), and increased to 58 d for DG9-PMO treatment (FIG. 9a). The median survival incremented to 57 d (unconjugated-PMO), 121 d (DG9-PMO) and 64 d (MOE) for the higher dose of 80 mg/kg (FIG. 9a).
• DG9-PMO can increase life-span in the SMA mice more effectively than the unmodified PMO.
• DG9-PMO treated mice were significantly heavier than the NT, unconjugated-PMO treated littermates at postnatal day 7 (PD7), with no significant difference in weights when compared to the age-matched heterozygous littermates (Hets) (Smn +/_ SMN2 Tg/ j that were used as healthy controls exhibit normal wild-type (WT) like-characteristics (FIG. 9b).
• the DG9-PMO treated mice looked very similar to the Hets, while the unconjugated PMO-treated neonates looked exhibited a weak phenotype-similar to the NT mice (FIG. 16)
• the FL-SMN2 expression was evaluated relative to A7 SMN2 transcripts using real-time quantitative PCR (RT-qPCR) at PD7.
• RT-qPCR real-time quantitative PCR
• DG9-PM0 treatment led to a 4-to-30-fold higher FL-SMN2 expression than NT control in both peripheral and CNS tissues (FIG. 10a). It also led to a ⁇ 5-fold increase in FL-SMN2 expression when compared to unconjugated-PMO and MOE treatments in majority of the tissues (FIG. 10a, FIG. 16a).
• mice had a decreasing HLS score as they could not extend their hindlimbs when suspended by the tail (FIG. 9c). These mice also exhibited a reduced latency on the tube.
• DG9-PMO treated neonates exhibited hindlimb strength comparable to the Hets, with a significantly higher score and greater latency on the tube than the unconjugated-PMO and NT mice (FIG. 9c).
• DG9-PMO-treated mice exhibited a forelimb grip force comparable to the Hets, and significantly higher than the unconjugated-PMO and MOE-treated mice (FIG. 9e).
• mice When switched to a hypoxic environment, the Hets, DG9-PMO, and MOE-treated mice exhibited an increase in the respiratory parameters- fR VE, and VT relative to normoxia (FIG. 11 b-d). Even though hypoxia reduced the number of apneas, breathing was still slow, and irregular as seen by the high CoV in NT mice (FIG. 11e). Most of the unconjugated-PMO neonates exhibited an increase in the respiratory parameters relative to normoxia with no effects on apnea when compared to NT mice, but still had slow, weak, and irregular breathing when compared to the Hets (FIG. 11b-d).
• DG9-PMO treatment improves muscle pathology and neuromuscular junction (NMJ) characteristics in SMA mice.
• Atrophic musculature is a classical characteristic feature of SMA with diminished skeletal muscle fiber size.
• the feret’s diameter and CSA of the myofibers were significantly larger in all three muscle groups following DG9-PMO treatment when compared to the NT control (FIG. 12b, FIG. 19).
• the quadriceps muscle there was no significant difference between the Hets and the DG9-PMO treated myofiber size.
• unconjugated-PMO and MOE quadriceps myofibers were significantly smaller than those of DG9-PMO.
• all three treatment groups exhibited a similar myofiber size (FIG. 12b, FIG. 19).
• DG9- PMO treatment did lead to a significant decrease in the percent of centrally nucleated fibers, indicative of degenerative myofibers in atrophic myofibers.
• FIG. 12c The effect of DG9-PMO persisted at PD30 (FIG. 20).
• Unconjugated-PMO and MOE treated mice had significantly smaller myofibers and a higher percent of central nuclei compared to DG9-PMO in all three tissue types (FIG. 20c).
• DG9 peptide increases the uptake of PMO in both systemic and CNS tissues following a single subcutaneous administration
• DG9-PM0 penetrates the blood-brain barrier in a mild SMA model
• Peptides can typically pose as antigens, leading to immune reactions. Therefore, we examined the susceptibility of DG9-PM0 to cause immune activation at an early neonatal stage, by looking at CD68 positive cells (CD68 + ), indicative of circulating and tissue macrophages in the quadriceps muscle sections at PD7 (FIG. 21a). The NT control mice exhibited elevated levels of CD68 + macrophages owing to the atrophic musculature in SMA.
• the unconjugated-PMO and MOE-treated muscle had a significantly higher number of CD68 + macrophages when compared to the Hets, while NT and DG9-PM0 mice had no significant difference (FIG. 21a).
• the apparent reduction in circulating macrophages following DG9-PM0 treatment is likely due to amelioration of the atrophic musculature which would compensate for any elevation seen from the treatment itself.
• serum from the Hets and treated mice at PD 30-35 was collected.
• a toxicological evaluation was performed on the levels of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatinine, total bilirubin, total protein, albumin, globulin, and gamma-glutamyl transferase (GGT). All examined indicators were comparable between the groups, suggesting no apparent toxic effects (FIG. 21 b).
• SMN2 can produce a small amount of functional SMN, it is a viable target for therapy in SMA patients. 95% of SMA patients have mutations in the SMN1 gene, making SMN2 gene a candidate for AO therapy to treat almost all the SMA patients.
• DG9-PM0 treated neonatal and adult mice showed robust improvement in muscle strength and coordination, with significant improvement in the functional tests.
• the DG9 peptide led to sustained delivery of PMO to the skeletal and respiratory muscles that, in turn, reduced the number of myofibers with central nuclei and increased the myofiber size.
• the unconjugated-PMO mice had significantly smaller myofibers in both the intercostal and quadriceps muscles, hinting at a possibility of compromised breathing, and atrophic myofibers being the cause of early mortality.
• DG9- PMO mice Under both normoxic and hypoxic conditions, DG9- PMO mice were similar to the Hets, with significantly better respiratory outcomes than NT PD7 mice. These findings provide evidence for the efficiency of DG9-PM0 in rescuing early respiratory dysfunction in SMA mice.
• Toxicity associated with cationic peptide conjugated PMOs is typically seen within 24 hours of administration. No apparent physiological differences in the neonates receiving subcutaneous injections of the AOs. They were active, similar to their healthy counterparts. We also quantified the number of CD68 + cells in quadriceps muscle at PD7, that are indicative of macrophages activated during an immune response in the body upon administration of AOs. SMA mice suffer from immune dysregulation and have an elevated number of immune cells. While DG9-PM0 and NT mice had similar numbers of CD68 + cells, we believe that although DG9-PM0 treatment might evoke an immune response, the simultaneous improvement in muscle pathology and amelioration of the atrophic muscles reduced the total number of circulating macrophages. No apparent toxicity in the liver or kidney as per the serum and histology analyses at PD30 was observed.
• DG9 (sequence N-YArVRRrGPRGYArVRRrGPRr-C; uppercase: L-amino acids, lowercase: D- amino acids) was synthesized and covalently conjugated to the 3’ end of the PMO.
• the PMO targeting ISS-N1 intron 7 (5’-TCACTTTCATAATGCTGG-3’) was purchased from Gene Tools LLC.
• the 2’MOE was purchased from Eurogentec North America, USA.
• SMA transgenic mice JMA stock #005058 FVB.Cg-Tg(SMN2)2HungSmn1 tm1Hun 9/j (Smn- Z - ; SMN2 T 9 /T 9), also known as the Taiwanese mice
• Jackson Laboratory Bar Harbor, ME, USA
• mice for Smn1 (Smn1 +/_ ; SMN2 _/ were crossed with mice homozygous for Smn1 Smn _/ - ; SMN2 T 9 /T 9 to obtain the SMA mice (Smn _/ - ; SMN2 T 9 Z -) or the heterozygous healthy control (Smn1 +/ - ; SMN2 T 9 Z -).
• the SMA mice display a severe overt phenotype similar to SMA type I patients.
• the mice were genotyped using a PCR assay on genomic DNA isolated from tail biopsies using the Phire Tissue Direct PCR kit (ThermoFisher) as per the manufacturer’s instructions.
• the DNA was amplified using the primers described in Table 5 using the condition Mouse Smn: 98°C/5 min 98°C/5 s_58°C/10 _72°C/10 s x 35 cycles 72°C/1 min.
• Injections were carried out using a 30-gauge Hamilton syringe.
• Tissues including the quadricep muscle, liver, kidney, spleen, diaphragm, intercostal muscle, heart, brain and spinal cord were collected and snap frozen in dry-ice cooled isopentane, and then subsequently stored at -80°C
• RT-qPCR Real-time quantitative PCR
• Total protein was extracted from frozen tissues harvested at P7 or P30 by using RIPA buffer (Sigma) with complete, Mini, EDTA-free protease inhibitor cocktail (Sigma). The protein concentration was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). For SDS- PAGE, 5-10 pg of protein per well was run per well in NuPAGE Novex 4-12% Bis- Tris Midi protein gels (Life Technologies) at 150 V for 60 minutes, followed by semi-dry transfer at 20 V for 30 minutes. The polyvinylidene fluoride (PVDF) membrane was blocked overnight with 5% skim milk and 0.05% Tween20 in PBS (PBST).
• PVDF polyvinylidene fluoride
• the membrane was incubated with a mouse purified anti- SMN antibody (BD Biosciences) (1 : 10,000) for one hour at room temperature (RT) under agitation.
• the membrane was washed three times with PBST (10 minutes each wash) and incubated with HRP conjugated goat anti-mouse (IgG H+L) at RT for 1 hour under agitation.
• the bands were detected using the Amersham ECL Select Western Blotting Detection Kit (GE Healthcare) and visualized by ChemiDoc Touch Imaging system (BioRad).
• the membrane was incubated with the stripping buffer (15 g glycine, 1 g SDS, 10 ml Tween20 at pH 2.2) for 10 minutes at RT, followed by two washes in PBS (10 minutes each) and two washes in tris-buffered saline and 0.05% Tween20 (TBST) (5 minutes each). Similar to the primary antibody protocol, the membrane was subsequently blocked overnight and incubated with p-tubulin rabbit antibody (Abeam ab6046, 1 :5000) at room temperature for 1 hour under agitation the next day. The secondary antibody used was HRP conjugated goat anti-rabbit (IgG H+L) for Tubulin (BioRad), 1 : 10,000). The bands were visualized as mentioned previously.
• the stripping buffer 15 g glycine, 1 g SDS, 10 ml Tween20 at pH 2.2
• ELISA was performed as described previously [83,96], In brief, protein was extracted from frozen tissue sections ( ⁇ 20 pm) using RIPA buffer (Sigma) with complete, Mini, EDTA-free protease inhibitor cocktail (Sigma).
• the probes (Integrated DNA Technologies) were designed complementary to the PMO sequence with phosphorothioated backbones at the 5’ and 3’ ends. The 5’ and 3’ ends were labelled with digoxigenin and biotin, respectively.
• the tissue lysates (0.02 mg/ml protein concentration) were pre-treated with 2.5 mg/mL trypsin containing 10 mM CaCl2 at 37 °C overnight to digest the DG9 peptide. The probes were added to the samples and allowed to hybridize at 37°C for 30 minutes.
• the hybridized samples were added to Pierce NeutrAvidin Coated 96-Well Plates, Black (Thermo Fisher Scientific) to allow avidin-biotin interaction between the plate and the probes.
• the unhybridized probes were digested using micrococcal nuclease enzyme at 0.1 gel unit/pl (New England Biolabs). This was followed by the addition of anti-digoxigenin antibody conjugated with alkaline phosphatase (1 :5000, Roche Applied Sciences).
• Attophos AP Fluorescent Substrate Promega was added to the PMO/DG9-PMO probes and fluorescence was detected at 444 nm excitation and 555 nm emission by using a monochromator SpectraMax M3 plate reader (Molecular Devices).
• NMJ staining was carried out as previously described [98], Briefly, the quadricep and the intercostal muscle were fixed in 4 % paraformaldehyde (PFA) for 2 hours. The muscles were washed quicky with PBS and blocked with 5 % goat serum and 2 % Triton- X in PBS for 1 hour at RT. This was followed by an overnight incubation at 4 °C with the primary mouse monoclonal antibodies anti-neurofilament 2H3 (1 :100) and anti-synaptophysin (1 :500) (DSHB, Iowa).
• PFA paraformaldehyde
• mice This spontaneous ability of the mice to right themselves up was tested from P2 until the weaning stage P20.
• the neonates were placed on their backs and the time taken to reposition and place all four paws on the ground was noted.
• the recording time was a maximum of 60 s.
• Each neonate underwent three trials, with a resting period of at least 10 minutes between trials.
• the data is expressed as the mean time to complete the righting reflex test ⁇ SEM.
• mice were suspended on their hindlimbs from a tube. They were scored based on the position of the hindlimbs and their latency to fall was recorded with a 30 second cut-off. Each neonatal pup underwent three trials, with a 15-minute break between each trial. The average score was noted down by the observer who was blinded to this test.
• This assay was conducted as described in Treat NMD protocol SMA_M.2.1.002.
• the mouse is placed on the wire mesh of an automated grip strength meter (Columbus Instruments) such that only the front paws are allowed to grip the metal grid.
• the mouse is steadily pulled with the help of its tail, such that it lets go of the grid completely.
• P30 and P60 mice were used. Each mouse underwent three trials.
• the evaluation was performed on a standard set of toxicity markers: glucose, total bilirubin, blood urea nitrogen (BUN), creatine kinase (CK), creatinine, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), globulin, albumin, and total protein.
• BUN blood urea nitrogen
• CK creatine kinase
• ALP alkaline phosphatase
• ALT alanine transaminase
• AST aspartate aminotransferase
• GTT gamma-glutamyl transferase
• the plethysmograph of volumes were 10 ml (inner diameter: 1.9 cm, length: 3.5 cm for P7 mice with body weight less than 2.5 g), 30 ml (inner diameter: 2.6 cm, length: 5.6 cm for P7 mice with body weight more than 2.5 g) and 80 ml (inner diameter: 3.8 cm, length: 7 cm for P30) for measures of respiratory parameters with a flow rate of 15, 45, 120 ml/min, respectively.
• the gas was mixed with gas mixer (GSM- 3, CWE InC, USA), delivered from compressed pure oxygen and pure nitrogen cannisters, being monitored using 0-200 ml/min gas regulators (Porter Instrument Company, USA).
• hypoxic challenge (11 % of oxygen for 5 min) was performed with continuous monitoring of plethysmographic recordings without physical handling of the animal by switching inflow gas from normoxia (21% of oxygen, balanced by nitrogen) to hypoxia (11 % of oxygen, balanced by nitrogen). It took about 1 min to finish gas exchange, confirmed with gas analyzer (Model: ML206, ADInstruments).
• gas analyzer Model: ML206, ADInstruments.
• the plethysmograph was contained within an infant incubator (Isolette, model C-86; Air-Shields/Drager Medical, USA) to maintain the ambient temperature at the approximate nest temperature of 32°C.
• the plethysmograph was recorded at room temperature of around 22°C.
• Pressure changes were detected with a pressure transducer (model DP 103; Validyne, USA), signal conditioner (CD-15; Validyne), recorded with data acquisition software (Axoscope) via analog-digital board (Digidata 1322A). Signals were high pass filtered (0.01 kHz), with a sampling rate at 1 kHz. Respiratory frequency and tidal volume (VT) was measured with blood pressure settings using Labchart 8 (AD Instruments Inc., USA). Threshold levels for bursts were set, and bursts were then automatically detected so that frequency (calculated from cycle duration) and tidal volume (calculated from maximal pressure minus minimal pressure).
• apnea is defined as the absence of airflow (pressure changes) for a period equivalent or greater than two complete respiratory cycles.
• Our whole-body plethysmographic system provided semiquantitative measurements of tidal volume (VT, mL/g) and minute ventilation, from which we report changes relative to the wildtype normoxia (Ren et al., 2009, 2015).
• the coefficient of variation of frequency is the ratio of the standard deviation to the mean (average). The smaller the ratio, the more regular the breathing. The experiments were conducted between 10 am and 5 pm. Animals were sent back to the animal facility after experiments.
• Respiratory parameters were calculated over an average of 1 min of continuous plethysmography recordings.
• the respiratory parameters VT and VE were reported relative to the mean of heterozygotes in normoxia (100%). The nature of the hypothesis testing is two- tailed. We first ran the normality test (Shapiro- Wilk) and equal variance test (Brown-Forsythe).
• hypoxia was conducted with a signed rank test.
• severity of respiratory phenotype decrease of respiratory frequency or VE
• body weight we used the Pearson product moment correlation t test (FIG. 6G).
• FOG. 6G Pearson product moment correlation t test
• Dystrophin the protein product of the Duchenne muscular dystrophy locus. Cell 51(6) :919-928.
• Moulton HM Moulton JD (2010) Morpholinos and their peptide conjugates: Therapeutic promise and challenge for Duchenne muscular dystrophy. Biochim Biophys Acta - Biomembr 1798(12):2296-2303.

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