Pre-mRNA slice-regulatory peptide-conjugated antisense therapeutics for disease treatment

Abstract

The present disclosure relates generally to DG9 peptide-coupled splice modulation therapy for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy.

Description

Detailed Description of the Invention

[0001] This application claims priority to U.S. Provisional Application No. 63 / 303,577, filed January 27, 2022, the entirety of which is incorporated herein by reference. [Field of the Invention] The present invention relates generally to pre-mRNA splice regulatory peptide-conjugated antisense therapeutics for the treatment of disease, in particular to peptide-conjugated antisense therapeutics for the treatment of Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).

[0002] [background] Antisense therapeutics that modulate pre-messenger RNA splicing by either inducing exon splicing or inhibiting exon splicing function by binding to pre-messenger RNA have been approved for the treatment of a variety of diseases, such as Duchenne muscular dystrophy and spinal muscular atrophy.

[0003] Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive defect caused by mutations in the DMD gene that cause a deficiency of dystrophin in muscle. Dystrophin stabilizes the sarcolemma by cross-linking cytoskeletal actin to the extracellular matrix through the formation of a membrane-bound glycoprotein complex [1, 2]. Dystrophin deficiency causes progressive generalized muscle degeneration, loss of ambulation before teenage years, and cardiopulmonary insufficiency in the third decade, usually resulting in death [3]. DMD is present in 19.8 per 100,000 live male births and is the most common inherited neuromuscular disorder worldwide [4].

[0004] Most patients with DMD (approximately 70%) have large out-of-frame deletions [5]. Exon skipping-based therapies are based on the observation that, at least approximately 90% of the time, in-frame mutations in DMD cause a milder phenotype, such as that seen 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 mutations to in-frame mutations, generating a truncated but partially functional dystrophin. Four exon-skipping therapies have been approved by the U.S. FDA: eteplirsen / Exondys 51 (Sarepta)[7], golodirsen / Vyondys 53 (Sarepta)[8], viltolarsen / Viltepso (NS Pharma)[9], and casimersen / Amondys 45 (Sarepta)

[10] . All are phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides.

[0005] However, the mutation-specific nature of DMD limits the applicability of single-exon skipping. All of the above treatments can only treat a maximum of 8%–13% of all patients

[11] . Multi-exon skipping, especially exon 45–55 skipping, overcomes this problem. Exons 45–55 are a mutation hotspot in the DMD gene, harboring 66% of large (one or more exons) deletions and 15% of large duplications in patients (5). Exon 45–55 skipping could theoretically treat more than 40% of all DMD patients

[12] .

[0006] Exon 45-55 deletions are furthermore commonly associated with an asymptomatic to mild phenotype: in the Leiden DMD database, 90% of patients with exon 45-55 deletions have BMD, and in other databases and clinical studies, including UMD-TREAT-NMD, the deletion has been associated with BMD or asymptomatic individuals in all cases examined [12-15].

[0007] A skipping cocktail of exons 45–55 has been developed that restored dystrophin synthesis in the muscles of dystrophic mice

[16] . A PMO cocktail has been developed to skip human DMD exons 45–55 in immortalized patient myotubes and humanized DMD mice [13, 17]. They observed average skipping efficiencies of 27%–61% and 15%–22%, respectively, and treatments produced up to 14% of normal levels of dystrophin in vitro [13, 17]. However, this cocktail used one PMO per exon (except for exon 48, for which two are required) to skip exons 45–55. Because all PMOs must be present in the same nucleus to induce exon 45–55 skipping, the efficacy of this cocktail was reduced and off-target effects increased.

[0008] The efficacy of PMO-based exon skipping therapy is affected by the rapid clearance of PMO from the bloodstream, poor uptake into muscle, and inability to escape from endosomes [19, 20]. For example, eteplirsen restored only 0.93% of normal levels of dystrophin in patients after 180 weeks of treatment at doses of 30 mg / kg / week or 50 mg / kg / week [7].

[0009] Thus, there is a need for improved exon-skipping therapies for the treatment of Duchenne muscular dystrophy. Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease that affects motor neurons in the anterior horn of the spinal cord

[45] . SMA can cause progressive muscle weakness, respiratory distress, and even paralysis, and is one of the leading genetic causes of infant death

[46] . SMA is characterized by mutations in the survival of telomeric motor neuron 1 (SMN1) gene that lead to homozygous deletion

[47] . Complete deletion of the SMN protein is fetal lethal [48, 49]. Humans have a paralog of SMN1 called SMN2, and it is possible for patients to give birth [47, 50]. However, SMN2 cannot fully compensate for the deletion of SMN1 due to a C to T transition in exon 7, leading to its elimination.

[0010] While full-length SMN2 (FL-SMN2) transcripts can generate stable functional protein (~10%), transcripts lacking exon 7 (Δ7SMN2) are unstable and the protein generated is rapidly degraded (~90%)

[51] . Exclusion of SMN2 exon 7 is due to ISS-N1 (intronic splicing silencer N1) binding to the repressor protein hnRNPA1 (heterogeneous ribonucleoprotein A1) [52-54].

[0011] All SMA patients lack a functional SMN1 gene and therefore rely on the SMN2 gene to produce the SMN protein required for survival. Thus, multiple copies of SMN2 are potent genetic modifiers of SMA, with copy number inversely correlated with the severity of SMA [55, 56]. The inability of SMN2 to complement SMN1 deletion is responsible for preferential degeneration of motor neurons [51, 57]. Although the pathological hallmark of SMA is the degeneration of motor neurons, recent studies have highlighted defects in peripheral tissues [58–60]. Furthermore, in vivo mouse studies have reported bradycardia and dilated cardiomyopathy, which improved with restoration of SMN levels [61–63]. For reasons that are unclear, motor neurons are particularly susceptible to SMN deletion

[64] .

[0012] Currently, three SMA-modifying therapeutic agents have been approved by the U.S. Food and Drug Administration (FDA): nusinersen (brand name Spinraza), onasemnogene abeparvovec (brand name Zolgensma), and risdiplam (brand name Evrysdi). However, these three agents have several associated concerns. Onasemnogene abeparvovec is a gene therapy drug and is only approved for patients under the age of 2

[65] . Risdiplam is an orally available small molecule drug. However, there are safety concerns due to associated off-target effects on pre-mRNA splicing

[66] .

[0013] Nusinersen is a splice-switching antisense oligonucleotide (AO) with 2'-O-methoxyethyl (MOE) phosphorothioate chemistry. It regulates SMN2 splicing by targeting ISS-N1, promoting the inclusion of exon 7. Nusinersen does not cross the blood-brain barrier (BBB) ​​and therefore requires repeated intraspinal administration to ensure robust restoration of SMN protein primarily to central nervous system (CNS) tissues

[67] . Despite offering a safety profile, approximately one-third of treated patients suffer from scoliosis, persistent lumbar fluid leak, thrombocytopenia, and other coagulation disorders [68, 69].

[0014] To overcome the problems associated with intraspinal injections, several AO chemistries and modifications have been investigated, for example, phosphorodiamidate morpholino oligomers (PMOs), locked nucleic acids (LNAs), and constrained ethyl (c-Et)

[70] . The uncharged backbone, low protein binding affinity, and nuclease stability make PMO chemistry an ideal candidate, since several studies have shown that high doses of multiple administration can be achieved with minimal toxicity [70, 71]. However, the main pitfalls associated with PMOs are their rapid clearance from the bloodstream, poor uptake in tissues such as skeletal muscle, and low efficacy associated with endosomal trapping [72, 73]. To improve the delivery of PMOs to the nucleus of target tissues and reduce the dosage, numerous cell-penetrating peptide (CPP)-conjugated PMOs have been investigated. Some of these peptides facilitate the transport of PMOs through the BBB, thus ensuring the possibility of noninvasive administration for the treatment of neurological diseases

[74] . Hammond et al. showed that systemic administration of Pip6a, a peptide from the PMO-internalizing peptide (Pip) family, increased FL-SMN2 expression in peripheral and CNS tissues in adult mice

[71] . However, the localization and distribution of AO in the CNS is unknown. Furthermore, Pip peptides are known to be highly toxic

[74] .

[0015] Thus, there is a need for improved splice modulation therapies for the treatment of spinal muscular atrophy. [Summary of the Invention] The present invention provides pre-mRNA splice regulator peptide-conjugated antisense therapeutics for the treatment of disease.

[0016] In one embodiment of the invention, there is provided a conjugate comprising an antisense oligonucleotide capable of modulating exon splicing in a pre-mRNA linked to a cell penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids.

[0017] In another embodiment of the present invention, there is provided a conjugate comprising an antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently linked to a cell penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids.

[0018] In some embodiments, a conjugate capable of inducing exon skipping in human dystrophin has an antisense oligonucleotide that binds to a target within 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.

[0019] In some embodiments, the conjugate capable of inducing exon skipping in human dystrophin has an antisense oligonucleotide comprising, or consisting of, any one of the following oligonucleotides: 5'-AAAACGCCGCCATTTCTCAACAGATCTGTC-3', 5'-GACAACAGTTTGCCGCTGCCCAATGCCATC-3', 5'-AGTTGCTGCTCTTTTCCAGGTTCAAGTGGG-3', 5'-GTTTGAGAATTCCCTGGCGCAGGGGCAACT-3', 5'-CAATTTCTCCTTGTTTCTCAGGTAAAGCTC-3', 5'-CAGATGATTTAACTGCTCTTCAAGGTCTTC-3', 5'-ATCTCTTCCACATCCGGTTGTTTAGCTTGA-3', 5'-GTAAACGGTTTACCGCCTTCCACTCAGAGC-3', 5'-GTGTCACCAGAGTAACAGTCTGAGTAGGAG-3', 5'-GGTAATGAGTTCTTCCAACTGGGGACGCCT-3', 5'-CCTCCGGTTCTGAAGGTGTTCTTGTACTTC-3', 5'-GAGAAGTTTCAGGGCCAAGTCATTTGCCAC-3', and 5'-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3'.

[0020] According to another embodiment of the present invention, there is provided a first conjugate comprising a first antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently linked to a cell penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase: L-amino acids, lowercase: D-amino acids. and A second conjugate comprising a second antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently linked to a cell-penetrating peptide (CPP). A composition comprising:

[0021] In some embodiments, the composition further comprises at least one other conjugate comprising another antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently linked to a cell penetrating peptide (CPP).

[0022] In some embodiments, compositions capable of inducing multi-exon skipping in human dystrophin comprise peptide-conjugated antisense oligonucleotides targeted to exon 45, peptide-conjugated antisense oligonucleotides targeted to exon 46, peptide-conjugated antisense oligonucleotides targeted to exon 47, peptide-conjugated antisense oligonucleotides targeted to exon 48, peptide-conjugated antisense oligonucleotides targeted to exon 49, peptide-conjugated antisense oligonucleotides targeted to exon 50, peptide-conjugated antisense oligonucleotides targeted to exon 51, peptide-conjugated antisense oligonucleotides targeted to exon 52, peptide-conjugated antisense oligonucleotides targeted to exon 53, peptide-conjugated antisense oligonucleotides targeted to exon 54, and peptide-conjugated antisense oligonucleotides targeted to exon 55.

[0023] In some embodiments, compositions capable of inducing multi-exon skipping in human dystrophin include peptide-conjugated antisense oligonucleotides targeted to exon 45, peptide-conjugated antisense oligonucleotides targeted to exon 47, peptide-conjugated antisense oligonucleotides targeted to exon 49, peptide-conjugated antisense oligonucleotides targeted to exon 51, peptide-conjugated antisense oligonucleotides targeted to exon 53, and peptide-conjugated antisense oligonucleotides targeted to exon 55.

[0024] In some embodiments, a composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeted to exon 45, a peptide-conjugated antisense oligonucleotide targeted to exon 47, and a peptide-linked antisense oligonucleotide targeted to exon 53.

[0025] According to an embodiment of the present invention, there is provided a method of treating a subject having DMD comprising administering a therapeutically effective amount of one or more peptide conjugates of the present invention capable of inducing exon skipping in human dystrophin.

[0026] According to another aspect of the present invention, there is provided a conjugate capable of inducing exon inclusion in the human SMN2 gene, comprising an antisense oligonucleotide covalently linked to a cell penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids.

[0027] In some embodiments, a conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in the human SMN2 gene binds to the intronic splicing silencer N1 of the SMN2 pre-mRNA.

[0028] In some embodiments, a conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in the human SMN2 gene comprises an antisense oligonucleotide comprising, or consisting of, the sequence 5'-TCACTTTCATAATGCTGG-3', where thymine is optionally replaced with uracil, and optionally, where the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer.

[0029] According to another aspect of the present invention, there is provided a method of treating spinal muscular atrophy (SMA) in a subject, comprising administering a therapeutically effective amount of a conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in the human SMN2 gene of the present invention.

[0030] According to another aspect of the invention, there is provided a pharmaceutical composition comprising one or more peptide conjugates of the invention and a pharma- ceutically acceptable excipient. Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: FIG. [Brief description of the drawings]

[0031] [Figure 1-1] Figure 1 shows the testing of the minimized exon 45-55 skipping cocktail in immortalized patient myotubes. (A) DMD exons targeted by the "all" cocktail and derivatives are indicated by circles. (B) Culture scheme of PMO transfection in immortalized patient myotubes. (C) RT-PCR exon 45-55 skipping efficiency results for transfection of the PMO cocktail in KM155, KM571, 6594, and 6311 myotubes. Black (upper) arrows indicate native / unskipped bands, while blue (lower) arrows indicate exon 45-55 skipped bands. GAPDH is shown below as a control. Quantification is shown at the bottom. (D) Western blot of dystrophin in PMO-treated and non-treated (NT) KM571 myotubes using various antibodies (ab15277, DYS1, MANEX45A, MANEX4850E). Protein extracts were loaded at 40 μg for treated and NT samples and at the indicated percentages for wild type KM155 samples (WT). Desmin was detected as a loading control. (E) Quantification of DYS1 signal in (D), compared to the intensity of the 12.5% ​​WT band. (n=3 for C-E) Errors: SEM *p<0.05, **p<0.005, ***p<0.001 One-way ANOVA with Dunnett's test against mock, φp<0.05, φφφp<0.001 One-way ANOVA with Dunnett's test against "all". [Figure 1-2]Figure 1 shows the testing of the minimized exon 45-55 skipping cocktail in immortalized patient myotubes. (A) DMD exons targeted by the "all" cocktail and derivatives are indicated by circles. (B) Culture scheme of PMO transfection in immortalized patient myotubes. (C) RT-PCR exon 45-55 skipping efficiency results for transfection of the PMO cocktail in KM155, KM571, 6594, and 6311 myotubes. Black (upper) arrows indicate native / unskipped bands, while blue (lower) arrows indicate exon 45-55 skipped bands. GAPDH is shown below as a control. Quantification is shown at the bottom. (D) Western blot of dystrophin in PMO-treated and non-treated (NT) KM571 myotubes using various antibodies (ab15277, DYS1, MANEX45A, MANEX4850E). Protein extracts were loaded at 40 μg for treated and NT samples and at the indicated percentages for wild type KM155 samples (WT). Desmin was detected as a loading control. (E) Quantification of DYS1 signal in (D), compared to the intensity of the 12.5% ​​WT band. (n=3 for C-E) Errors: SEM *p<0.05, **p<0.005, ***p<0.001 One-way ANOVA with Dunnett's test against mock, φp<0.05, φφφp<0.001 One-way ANOVA with Dunnett's test against "all". [Figure 1-3]Figure 1 shows the testing of the minimized exon 45-55 skipping cocktail in immortalized patient myotubes. (A) DMD exons targeted by the "all" cocktail and derivatives are indicated by circles. (B) Culture scheme of PMO transfection in immortalized patient myotubes. (C) RT-PCR exon 45-55 skipping efficiency results for transfection of the PMO cocktail in KM155, KM571, 6594, and 6311 myotubes. Black (upper) arrows indicate native / unskipped bands, while blue (lower) arrows indicate exon 45-55 skipped bands. GAPDH is shown below as a control. Quantification is shown at the bottom. (D) Western blot of dystrophin in PMO-treated and non-treated (NT) KM571 myotubes using various antibodies (ab15277, DYS1, MANEX45A, MANEX4850E). Protein extracts were loaded at 40 μg for treated and NT samples and at the indicated percentages for wild type KM155 samples (WT). Desmin was detected as a loading control. (E) Quantification of DYS1 signal in (D), compared to the intensity of the 12.5% ​​WT band. (n=3 for C-E) Errors: SEM *p<0.05, **p<0.005, ***p<0.001 One-way ANOVA with Dunnett's test against mock, φp<0.05, φφφp<0.001 One-way ANOVA with Dunnett's test against "all". [Figure 2-1]Figure 2 shows single-dose exon 51 skipping treatment with DG9-PMO. (A) Male, 3-month-old hDMDdel52;mdx mice received a single retro-orbital injection (1x ro) of saline, 50 mg / kg PMO for exon 51 skipping, or equimolar 64 mg / kg DG9-PMO. Tissues were harvested 1 week later. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot for dystrophin (DYS1). Wild type (WT) is shown for reference. Protein extracts were loaded at 40 μg for saline-injected and treated muscles and at the indicated percentage for WT tibialis anterior samples. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (D) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and treatment conditions. Scale bar: 100 μm. (For B-D, n=3 / group) Errors: SEM *p<0.05, **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. TA / T, tibialis anterior; GAS / G, gastrocnemius; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Figure 2-2]Figure 2 shows single-dose exon 51 skipping treatment with DG9-PMO. (A) Male, 3-month-old hDMDdel52;mdx mice received a single retro-orbital injection (1x ro) of saline, 50 mg / kg PMO for exon 51 skipping, or equimolar 64 mg / kg DG9-PMO. Tissues were harvested 1 week later. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot for dystrophin (DYS1). Wild type (WT) is shown for reference. Protein extracts were loaded at 40 μg for saline-injected and treated muscles and at the indicated percentage for WT tibialis anterior samples. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (D) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and treatment conditions. Scale bar: 100 μm. (For B-D, n=3 / group) Errors: SEM *p<0.05, **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. TA / T, tibialis anterior; GAS / G, gastrocnemius; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Figure 3-1]Figure 3 shows repeated dose exon 51 skipping treatment with DG9-PMO. (A) Male, 2-month-old hDMDdel52;mdx mice received saline or 30 mg / kg PMO for exon 51 skipping by retro-orbital injection once a week for three times (3xro). Purple arrows indicate functional testing. Tissues were harvested after 2 weeks. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot of dystrophin (DYS1) in various skeletal muscles or (D) heart. Corresponding wild-type (WT) samples were used for reference. Protein extracts were loaded at 40 μg in saline-injected and treated muscles and at the indicated ratios in WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (For B-D, n=3 / group) Error: SEM *p<0.05, **p<0.005, ***p<0.001, unpaired two-tailed t-test for (B) to (D). (E) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and conditions. Scale bar: 100 μm. (n=3 / group) (F) Body weight of saline-treated and DG9-PMO-treated mice throughout the experiment. (G) Forelimb grip strength of saline-treated and DG9-PMO-treated mice. Normalized to body weight. % change from baseline is shown on the right. (H) As in (G), but for grip strength across all four limbs (for F–H, n=11–14, WT; n=4, saline; n=6, DG9-PMO). Errors: SEM*p<0.05, **p<0.005 One-way ANOVA with Tukey's test for (G) to (H). TA / T, tibialis anterior; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Figure 3-2]Figure 3 shows repeated dose exon 51 skipping treatment with DG9-PMO. (A) Male, 2-month-old hDMDdel52;mdx mice received saline or 30 mg / kg PMO for exon 51 skipping by retro-orbital injection once a week for three times (3xro). Purple arrows indicate functional testing. Tissues were harvested after 2 weeks. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot of dystrophin (DYS1) in various skeletal muscles or (D) heart. Corresponding wild-type (WT) samples were used for reference. Protein extracts were loaded at 40 μg in saline-injected and treated muscles and at the indicated ratios in WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (For B-D, n=3 / group) Error: SEM *p<0.05, **p<0.005, ***p<0.001, unpaired two-tailed t-test for (B) to (D). (E) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and conditions. Scale bar: 100 μm. (n=3 / group) (F) Body weight of saline-treated and DG9-PMO-treated mice throughout the experiment. (G) Forelimb grip strength of saline-treated and DG9-PMO-treated mice. Normalized to body weight. % change from baseline is shown on the right. (H) As in (G), but for grip strength across all four limbs (for F–H, n=11–14, WT; n=4, saline; n=6, DG9-PMO). Errors: SEM*p<0.05, **p<0.005 One-way ANOVA with Tukey's test for (G) to (H). TA / T, tibialis anterior; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Figure 3-3]Figure 3 shows repeated dose exon 51 skipping treatment with DG9-PMO. (A) Male, 2-month-old hDMDdel52;mdx mice received saline or 30 mg / kg PMO for exon 51 skipping by retro-orbital injection once a week for three times (3xro). Purple arrows indicate functional testing. Tissues were harvested after 2 weeks. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot of dystrophin (DYS1) in various skeletal muscles or (D) heart. Corresponding wild-type (WT) samples were used for reference. Protein extracts were loaded at 40 μg in saline-injected and treated muscles and at the indicated ratios in WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (For B-D, n=3 / group) Error: SEM *p<0.05, **p<0.005, ***p<0.001, unpaired two-tailed t-test for (B) to (D). (E) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and conditions. Scale bar: 100 μm. (n=3 / group) (F) Body weight of saline-treated and DG9-PMO-treated mice throughout the experiment. (G) Forelimb grip strength of saline-treated and DG9-PMO-treated mice. Normalized to body weight. % change from baseline is shown on the right. (H) As in (G), but for grip strength across all four limbs (for F–H, n=11–14, WT; n=4, saline; n=6, DG9-PMO). Errors: SEM*p<0.05, **p<0.005 One-way ANOVA with Tukey's test for (G) to (H). TA / T, tibialis anterior; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Diagram 3-4]Figure 3 shows repeated dose exon 51 skipping treatment with DG9-PMO. (A) Male, 2-month-old hDMDdel52;mdx mice received saline or 30 mg / kg PMO for exon 51 skipping by retro-orbital injection once a week for three times (3xro). Purple arrows indicate functional testing. Tissues were harvested after 2 weeks. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot of dystrophin (DYS1) in various skeletal muscles or (D) heart. Corresponding wild-type (WT) samples were used for reference. Protein extracts were loaded at 40 μg in saline-injected and treated muscles and at the indicated ratios in WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (For B-D, n=3 / group) Error: SEM *p<0.05, **p<0.005, ***p<0.001, unpaired two-tailed t-test for (B) to (D). (E) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and conditions. Scale bar: 100 μm. (n=3 / group) (F) Body weight of saline-treated and DG9-PMO-treated mice throughout the experiment. (G) Forelimb grip strength of saline-treated and DG9-PMO-treated mice. Normalized to body weight. % change from baseline is shown on the right. (H) As in (G), but for grip strength across all four limbs (for F–H, n=11–14, WT; n=4, saline; n=6, DG9-PMO). Errors: SEM*p<0.05, **p<0.005 One-way ANOVA with Tukey's test for (G) to (H). TA / T, tibialis anterior; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Figure 3-5]Figure 3 shows repeated dose exon 51 skipping treatment with DG9-PMO. (A) Male, 2-month-old hDMDdel52;mdx mice received saline or 30 mg / kg PMO for exon 51 skipping by retro-orbital injection once a week for three times (3xro). Purple arrows indicate functional testing. Tissues were harvested after 2 weeks. (B) RT-PCR exon 51 skipping efficiency results after treatment in various muscles. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot of dystrophin (DYS1) in various skeletal muscles or (D) heart. Corresponding wild-type (WT) samples were used for reference. Protein extracts were loaded at 40 μg in saline-injected and treated muscles and at the indicated ratios in WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 5% WT band. (For B-D, n=3 / group) Error: SEM *p<0.05, **p<0.005, ***p<0.001, unpaired two-tailed t-test for (B) to (D). (E) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue) in various muscles and conditions. Scale bar: 100 μm. (n=3 / group) (F) Body weight of saline-treated and DG9-PMO-treated mice throughout the experiment. (G) Forelimb grip strength of saline-treated and DG9-PMO-treated mice. Normalized to body weight. % change from baseline is shown on the right. (H) As in (G), but for grip strength across all four limbs (for F–H, n=11–14, WT; n=4, saline; n=6, DG9-PMO). Errors: SEM*p<0.05, **p<0.005 One-way ANOVA with Tukey's test for (G) to (H). TA / T, tibialis anterior; QUA / Q, quadriceps; DIA / D, diaphragm; HRT / H, heart. [Figure 4-1]Figure 4 shows local treatment with the minimized "block" DG9-PMO exon 45-55 skipping cocktail. (A) Male, 5-6 month old hDMDdel52;mdx mice were injected with the DG9 conjugate "block" cocktail at 5 μg / DG9-PMO (total dose 25 μg) in the right tibialis anterior muscle (R) and saline in the left tibialis anterior muscle (L). Tissues were harvested 1 week later. (B) RT-PCR exon 45-55 skipping efficiency results after treatment. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot for dystrophin (DYS1). Wild type (WT) and non-treated (NT) tibialis anterior muscle samples were used for reference. Protein extracts were loaded at 40 μg for NT, saline-treated, and DG9-PMO-treated muscles and at this indicated percentage for WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 1% WT band. (D) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue). Scale bar: 100 μm. (n=3, wild type; n=6, saline; n=6, DG9-PMO) Error bars: SEM *p<0.05, ***p<0.001 unpaired two-tailed t-test. [Figure 4-2]Figure 4 shows local treatment with the minimized "block" DG9-PMO exon 45-55 skipping cocktail. (A) Male, 5-6 month old hDMDdel52;mdx mice were injected with the DG9 conjugate "block" cocktail at 5 μg / DG9-PMO (total dose 25 μg) in the right tibialis anterior muscle (R) and saline in the left tibialis anterior muscle (L). Tissues were harvested 1 week later. (B) RT-PCR exon 45-55 skipping efficiency results after treatment. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot for dystrophin (DYS1). Wild type (WT) and non-treated (NT) tibialis anterior muscle samples were used for reference. Protein extracts were loaded at 40 μg for NT, saline-treated, and DG9-PMO-treated muscles and at this indicated percentage for WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 1% WT band. (D) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue). Scale bar: 100 μm. (n=3, wild type; n=6, saline; n=6, DG9-PMO) Error bars: SEM *p<0.05, ***p<0.001 unpaired two-tailed t-test. [Figure 4-3]Figure 4 shows local treatment with the minimized "block" DG9-PMO exon 45-55 skipping cocktail. (A) Male, 5-6 month old hDMDdel52;mdx mice were injected with the DG9 conjugate "block" cocktail at 5 μg / DG9-PMO (total dose 25 μg) in the right tibialis anterior muscle (R) and saline in the left tibialis anterior muscle (L). Tissues were harvested 1 week later. (B) RT-PCR exon 45-55 skipping efficiency results after treatment. Quantification is shown on the right. Gapdh is shown as a control. (C) Western blot for dystrophin (DYS1). Wild type (WT) and non-treated (NT) tibialis anterior muscle samples were used for reference. Protein extracts were loaded at 40 μg for NT, saline-treated, and DG9-PMO-treated muscles and at this indicated percentage for WT. Desmin and myosin heavy chain (MyHC) serve as loading controls. Quantification of dystrophin signal is shown relative to the intensity of the 1% WT band. (D) Representative immunofluorescence images of dystrophin (DYS1, green) and nuclei (DAPI, blue). Scale bar: 100 μm. (n=3, wild type; n=6, saline; n=6, DG9-PMO) Error bars: SEM *p<0.05, ***p<0.001 unpaired two-tailed t-test. [Diagram 5]Figure 5 details preliminary testing of the minimized exon 45-55 skipping cocktail. Minimized derivatives of the "all" exon 45-55 skipping PMO cocktail were generated and tested in immortalized myotubes. (A) Strategy #1 for minimization involved two consecutive rounds of removal of individual PMOs from the "all" cocktail. (B) Strategy #2 involved preparing an "all" cocktail derivative based on endogenous splicing of the exon 45-55 region in humans. RT-PCR exon 45-55 skipping efficiency results are shown in both (A) and (B). (n=3) Error bars: SEM *p<0.05, **p<0.005, ***p<0.001 one-way ANOVA with Dunnett's test for mock, φp<0.05, φφp<0.005, φφφp<0.001 one-way ANOVA with Dunnett's test for "all". NT, non-treated. [Figure 6-1]Figure 6 shows histological data of 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 harvested 1 week later for evaluation, sectioned, and stained using hematoxylin and eosin (HE). (A) Representative HE images of tibialis anterior and diaphragm from wild-type, saline-treated, PMO-treated, and DG9-PMO-treated mice. Scale bar: 100 μm. (B) Quantification of centrally nuclear fibers (CNFs) from HE images. Error bars: SEM. Minimum Feret's diameter quantification obtained from HE images of (C) tibialis anterior and (D) diaphragm. Frequency distributions are shown on the left, while quantification of individual fibers is shown on the right. Box edges, 25th and 75th percentiles; center line, median; whiskers, range. (n=3 / group for A-D, 719-854 fibers counted for tibialis anterior and 962-1,479 fibers counted for diaphragm per group) **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. [Figure 6-2]Figure 6 shows histological data of 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 harvested 1 week later for evaluation, sectioned, and stained using hematoxylin and eosin (HE). (A) Representative HE images of tibialis anterior and diaphragm from wild-type, saline-treated, PMO-treated, and DG9-PMO-treated mice. Scale bar: 100 μm. (B) Quantification of centrally nuclear fibers (CNFs) from HE images. Error bars: SEM. Minimum Feret's diameter quantification obtained from HE images of (C) tibialis anterior and (D) diaphragm. Frequency distributions are shown on the left, while quantification of individual fibers is shown on the right. Box edges, 25th and 75th percentiles; center line, median; whiskers, range. (n=3 / group for A-D, 719-854 fibers counted for tibialis anterior and 962-1,479 fibers counted for diaphragm per group) **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. [Figure 7-1]Figure 7 shows functional and histological data obtained from repeated dose exon 51 skipping treatment with DG9-PMO. Male, 2-month-old hDMDdel52;mdx mice were injected retro-orbitally with saline or 30 mg / kg DG9-PMO for exon 51 skipping once a week for 3 weeks for three doses. Functional tests were performed at baseline and 2 weeks after the final injection. (A) Results of the rotarod test show the mean fall time and peak fall time, as well as their respective % change values ​​from baseline. (B) Results of the Run-to-Exhaustion test show the total distance traveled on the treadmill. The % change from baseline is shown on the right. (n=8-11 wild type; n=2, saline; n=6, DG9-PMO) Error bars: SEM*p<0.05, unpaired two-tailed t-test. (C) Tissues were collected after post-treatment functional tests and stained using hematoxylin and eosin (HE). Representative HE images of tibialis anterior and diaphragm from wild-type, saline-treated, and DG9-PMO-treated mice are shown. Scale bar: 100 μm. (D) Quantification of centrally-nucleated fibers (CNFs) from HE images. Error bars: SIM. (E) Quantification of minimum Feret's diameter for tibialis anterior and (F) diaphragm as shown in Figure 6. (n=3 / group for C-F, 610-865 fibers were counted for tibialis anterior and 1,167-1,412 fibers for diaphragm per group) **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. [Figure 7-2]Figure 7 shows functional and histological data obtained from repeated dose exon 51 skipping treatment with DG9-PMO. Male, 2-month-old hDMDdel52;mdx mice were injected retro-orbitally with saline or 30 mg / kg DG9-PMO for exon 51 skipping once a week for 3 weeks for three doses. Functional tests were performed at baseline and 2 weeks after the final injection. (A) Results of the rotarod test show the mean fall time and peak fall time, as well as their respective % change values ​​from baseline. (B) Results of the Run-to-Exhaustion test show the total distance traveled on the treadmill. The % change from baseline is shown on the right. (n=8-11 wild type; n=2, saline; n=6, DG9-PMO) Error bars: SEM*p<0.05, unpaired two-tailed t-test. (C) Tissues were collected after post-treatment functional tests and stained using hematoxylin and eosin (HE). Representative HE images of tibialis anterior and diaphragm from wild-type, saline-treated, and DG9-PMO-treated mice are shown. Scale bar: 100 μm. (D) Quantification of centrally-nucleated fibers (CNFs) from HE images. Error bars: SIM. (E) Quantification of minimum Feret's diameter for tibialis anterior and (F) diaphragm as shown in Figure 6. (n=3 / group for C-F, 610-865 fibers were counted for tibialis anterior and 1,167-1,412 fibers for diaphragm per group) **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. [Figure 7-3]Figure 7 shows functional and histological data obtained from repeated dose exon 51 skipping treatment with DG9-PMO. Male, 2-month-old hDMDdel52;mdx mice were injected retro-orbitally with saline or 30 mg / kg DG9-PMO for exon 51 skipping once a week for 3 weeks for three doses. Functional tests were performed at baseline and 2 weeks after the final injection. (A) Results of the rotarod test show the mean fall time and peak fall time, as well as their respective % change values ​​from baseline. (B) Results of the Run-to-Exhaustion test show the total distance traveled on the treadmill. The % change from baseline is shown on the right. (n=8-11 wild type; n=2, saline; n=6, DG9-PMO) Error bars: SEM*p<0.05, unpaired two-tailed t-test. (C) Tissues were collected after post-treatment functional tests and stained using hematoxylin and eosin (HE). Representative HE images of tibialis anterior and diaphragm from wild-type, saline-treated, and DG9-PMO-treated mice are shown. Scale bar: 100 μm. (D) Quantification of centrally-nucleated fibers (CNFs) from HE images. Error bars: SIM. (E) Quantification of minimum Feret's diameter for tibialis anterior and (F) diaphragm as shown in Figure 6. (n=3 / group for C-F, 610-865 fibers were counted for tibialis anterior and 1,167-1,412 fibers for diaphragm per group) **p<0.005, ***p<0.001, one-way ANOVA with Tukey's test. [Figure 8] Figure 8 shows liver and kidney histology from single-dose and repeat-dose exon 51 skipping treatment studies. Representative hematoxylin and eosin staining images of wild-type and hDMDdel52;mdx liver and kidney from (A) single-dose and (B) repeat-dose experiments are shown. Scale bar: 100 μm. (n=3 / group). [Figure 9-1]Figure 9 shows that subcutaneous administration of DG9-PMO on postnatal day 0 extends survival and improves motor function in severe SMA mice. (A) Survival curves for heterozygous mice (Het), no treatment (NT), unconjugated-PMO (PMO), DG9-PMO, and MOE injected at either 40 mg / kg or 80 mg / kg doses at PD0. For the 40 mg / kg study, n=15 (Hets), n=22 (NT), n=49 (unconjugated-PMO), n=14 (DG9-PMO), n=29 (MOE). For the 80 mg / kg study, n=15 (Hets), n=22 (NT), n=7 (unconjugated PMO), n=6 (DG9-PMO), n=4 (MOE) (p≦0.0001, log-rank Mantel Cox test). (B) Body weight of mice at PD7 receiving either the 40 mg / kg or 80 mg / kg dose. Each dot (symbol) represents a newborn pup. (C) Hindlimb suspension assay (HLS). Mice were treated with 40 mg / kg AO on PD0. Scores are based on the position of the hindlimb when suspended from a Falcon tube. (D) Righting reflex test. Mice were treated with 40 mg / kg AO on PD0. The ability of mice to rear up on their forepaws was measured every other day between PD2 and PD20 (left). Mean righting latencies at PD6 and PD8 are also shown (right: box plots). Box edges, 25th and 75th percentiles; center line, median; whisker extent. (E) Forelimb grip strength measured at PD30 and PD60 in adult mice from the 40 mg / kg treatment group normalized to body weight. In B, D (box plots), and E, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. In C and D, two-way ANOVA in A and B followed by Sidak's multiple comparisons was performed. *p<0.03, **p<0.002, ***p<0.0002. *NT, #PMO, @DG9-PMO, and MOE. Error bars: SEM. [Figure 9-2]Figure 9 shows that subcutaneous administration of DG9-PMO on postnatal day 0 extends survival and improves motor function in severe SMA mice. (A) Survival curves for heterozygous mice (Het), no treatment (NT), unconjugated-PMO (PMO), DG9-PMO, and MOE injected at either 40 mg / kg or 80 mg / kg doses at PD0. For the 40 mg / kg study, n=15 (Hets), n=22 (NT), n=49 (unconjugated-PMO), n=14 (DG9-PMO), n=29 (MOE). For the 80 mg / kg study, n=15 (Hets), n=22 (NT), n=7 (unconjugated PMO), n=6 (DG9-PMO), n=4 (MOE) (p≦0.0001, log-rank Mantel Cox test). (B) Body weight of mice at PD7 receiving either the 40 mg / kg or 80 mg / kg dose. Each dot (symbol) represents a newborn pup. (C) Hindlimb suspension assay (HLS). Mice were treated with 40 mg / kg AO on PD0. Scores are based on the position of the hindlimb when suspended from a Falcon tube. (D) Righting reflex test. Mice were treated with 40 mg / kg AO on PD0. The ability of mice to rear up on their forepaws was measured every other day between PD2 and PD20 (left). Mean righting latencies at PD6 and PD8 are also shown (right: box plots). Box edges, 25th and 75th percentiles; center line, median; whisker extent. (E) Forelimb grip strength measured at PD30 and PD60 in adult mice from the 40 mg / kg treatment group normalized to body weight. In B, D (box plots), and E, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. In C and D, two-way ANOVA in A and B followed by Sidak's multiple comparisons was performed. *p<0.03, **p<0.002, ***p<0.0002. *NT, #PMO, @DG9-PMO, and MOE. Error bars: SEM. [Figure 9-3]Figure 9 shows that subcutaneous administration of DG9-PMO on postnatal day 0 extends survival and improves motor function in severe SMA mice. (A) Survival curves for heterozygous mice (Het), no treatment (NT), unconjugated-PMO (PMO), DG9-PMO, and MOE injected at either 40 mg / kg or 80 mg / kg doses at PD0. For the 40 mg / kg study, n=15 (Hets), n=22 (NT), n=49 (unconjugated-PMO), n=14 (DG9-PMO), n=29 (MOE). For the 80 mg / kg study, n=15 (Hets), n=22 (NT), n=7 (unconjugated PMO), n=6 (DG9-PMO), n=4 (MOE) (p≦0.0001, log-rank Mantel Cox test). (B) Body weight of mice at PD7 receiving either the 40 mg / kg or 80 mg / kg dose. Each dot (symbol) represents a newborn pup. (C) Hindlimb suspension assay (HLS). Mice were treated with 40 mg / kg AO on PD0. Scores are based on the position of the hindlimb when suspended from a Falcon tube. (D) Righting reflex test. Mice were treated with 40 mg / kg AO on PD0. The ability of mice to rear up on their forepaws was measured every other day between PD2 and PD20 (left). Mean righting latencies at PD6 and PD8 are also shown (right: box plots). Box edges, 25th and 75th percentiles; center line, median; whisker extent. (E) Forelimb grip strength measured at PD30 and PD60 in adult mice from the 40 mg / kg treatment group normalized to body weight. In B, D (box plots), and E, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. In C and D, two-way ANOVA in A and B followed by Sidak's multiple comparisons was performed. *p<0.03, **p<0.002, ***p<0.0002. *NT, #PMO, @DG9-PMO, and MOE. Error bars: SEM. [Figure 9-4]Figure 9 shows that subcutaneous administration of DG9-PMO on postnatal day 0 extends survival and improves motor function in severe SMA mice. (A) Survival curves for heterozygous mice (Het), no treatment (NT), unconjugated-PMO (PMO), DG9-PMO, and MOE injected at either 40 mg / kg or 80 mg / kg doses at PD0. For the 40 mg / kg study, n=15 (Hets), n=22 (NT), n=49 (unconjugated-PMO), n=14 (DG9-PMO), n=29 (MOE). For the 80 mg / kg study, n=15 (Hets), n=22 (NT), n=7 (unconjugated PMO), n=6 (DG9-PMO), n=4 (MOE) (p≦0.0001, log-rank Mantel Cox test). (B) Body weight of mice at PD7 receiving either the 40 mg / kg or 80 mg / kg dose. Each dot (symbol) represents a newborn pup. (C) Hindlimb suspension assay (HLS). Mice were treated with 40 mg / kg AO on PD0. Scores are based on the position of the hindlimb when suspended from a Falcon tube. (D) Righting reflex test. Mice were treated with 40 mg / kg AO on PD0. The ability of mice to rear up on their forepaws was measured every other day between PD2 and PD20 (left). Mean righting latencies at PD6 and PD8 are also shown (right: box plots). Box edges, 25th and 75th percentiles; center line, median; whisker extent. (E) Forelimb grip strength measured at PD30 and PD60 in adult mice from the 40 mg / kg treatment group normalized to body weight. In B, D (box plots), and E, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. In C and D, two-way ANOVA in A and B followed by Sidak's multiple comparisons was performed. *p<0.03, **p<0.002, ***p<0.0002. *NT, #PMO, @DG9-PMO, and MOE. Error bars: SEM. [Figure 10-1]FIG. 10 shows that 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 transcript (Δ7SMN2) measured by qPCR. (B) Representative images of Western blotting and quantification of SMN protein levels relative to β-tubulin. Heterozygous mice were used as a control for relative SMN expression and were set up to be injected with 1.40 mg / kg AO at PD0. Tissues were collected at PD7. One-way ANOVA followed by post hoc Tukey's test was performed for *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 10-2] FIG. 10 shows that 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 transcript (Δ7SMN2) measured by qPCR. (B) Representative images of Western blotting and quantification of SMN protein levels relative to β-tubulin. Heterozygous mice were used as a control for relative SMN expression and were set up to be injected with 1.40 mg / kg AO at PD0. Tissues were collected at PD7. One-way ANOVA followed by post hoc Tukey's test was performed for *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 11-1]FIG. 11 shows that DG9-PMO treatment improves respiratory function at postnatal day 7 in SMA mice. (A) Representative traces of whole-body plethysmograph recordings from P7 pups (n=6 each) in normoxia (left column) and hypoxia (11% O2, right column). (B) Respiratory frequency fR. (C) Tidal volume (VT) relative to mean (100%) of heterozygotes in normoxia. (D) Minute ventilation (VE) relative to mean (100%) of heterozygotes in normoxia. (E) Coefficient of variation (CV) of frequency. (F) Total apnea duration (seconds in 1 minute). For data (fR, VT, VE, CV in B-E) that passed the normality test (Shapiro-Wilk) and homogeneity of variance test (Brown-Forsythe), parametric statistics were used using a two-way repeated measures analysis of variance (ANOVA) followed by the Holm-Sidak method. Data did not pass the normality test, non-parametric statistics were used. (G) Correlation between respiratory frequency and body weight. Respiratory frequency was plotted against body weight in heterozygotes (n=10) with a correlation coefficient of 0.303 (p=0.394). For homozygotes (n=43) the correlation coefficient was 0.791 (p<0.001). AO at 40 mg / kg was injected at PD0. Comparisons of differences between normoxia or hypoxia were performed using Kruskal-Wallis one-way ANOVA based on ranks, followed by the Dunn method. Differences between hypoxia and normoxia were performed using signed-rank tests. p<0.05 was considered a statistically significant difference; p<0.05, p<0.01, p<0.001 when compared between the indicated groups (*NT, #PMO, @DG9-PMO, and MOE); $<0.05, $$<0.01, $$$<0.001 when compared to normoxia. [Figure 11-2]FIG. 11 shows that DG9-PMO treatment improves respiratory function at postnatal day 7 in SMA mice. (A) Representative traces of whole-body plethysmograph recordings from P7 pups (n=6 each) in normoxia (left column) and hypoxia (11% O2, right column). (B) Respiratory frequency fR. (C) Tidal volume (VT) relative to mean (100%) of heterozygotes in normoxia. (D) Minute ventilation (VE) relative to mean (100%) of heterozygotes in normoxia. (E) Coefficient of variation (CV) of frequency. (F) Total apnea duration (seconds in 1 minute). For data (fR, VT, VE, CV in B-E) that passed the normality test (Shapiro-Wilk) and homogeneity of variance test (Brown-Forsythe), parametric statistics were used using a two-way repeated measures analysis of variance (ANOVA) followed by the Holm-Sidak method. Data did not pass the normality test, non-parametric statistics were used. (G) Correlation between respiratory frequency and body weight. Respiratory frequency was plotted against body weight in heterozygotes (n=10) with a correlation coefficient of 0.303 (p=0.394). For homozygotes (n=43) the correlation coefficient was 0.791 (p<0.001). AO at 40 mg / kg was injected at PD0. Comparisons of differences between normoxia or hypoxia were performed using Kruskal-Wallis one-way ANOVA based on ranks, followed by the Dunn method. Differences between hypoxia and normoxia were performed using signed-rank tests. p<0.05 was considered a statistically significant difference; p<0.05, p<0.01, p<0.001 when compared between the indicated groups (*NT, #PMO, @DG9-PMO, and MOE); $<0.05, $$<0.01, $$$<0.001 when compared to normoxia. [Figure 12-1]Figure 12 shows that systemic administration of DG9-PMO ameliorates muscle pathology in SMA mice. (A) Representative images from H&E staining of quadriceps (top row), diaphragm (middle row), and intercostal muscles (bottom row) at PD7 in heterozygous, NT control, and treatment groups. Scale bar: 100 μm. (B) Frequency distribution (top) and quantification (bottom) of minimum Feret's diameter (μm) in individual muscle fibers. Box ends, 25th and 75th percentiles; center line, median; whiskers, range (n=3-7 / group). We measured 1292-1653 fibers for quadriceps, 917-1746 fibers for diaphragm, and 642-1127 fibers for intercostal muscles. (C) Centrally cored fibers (%) quantified from H&E images. AO at 40 mg / kg was injected at PD0. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 12-2] Figure 12 shows that systemic administration of DG9-PMO ameliorates muscle pathology in SMA mice. (A) Representative images from H&E staining of quadriceps (top row), diaphragm (middle row), and intercostal muscles (bottom row) at PD7 in heterozygous, NT control, and treatment groups. Scale bar: 100 μm. (B) Frequency distribution (top) and quantification (bottom) of minimum Feret's diameter (μm) in individual muscle fibers. Box ends, 25th and 75th percentiles; center line, median; whiskers, range (n=3-7 / group). We measured 1292-1653 fibers for quadriceps, 917-1746 fibers for diaphragm, and 642-1127 fibers for intercostal muscles. (C) Centrally cored fibers (%) quantified from H&E images. AO at 40 mg / kg was injected at PD0. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 12-3]Figure 12 shows that systemic administration of DG9-PMO ameliorates muscle pathology in SMA mice. (A) Representative images from H&E staining of quadriceps (top row), diaphragm (middle row), and intercostal muscles (bottom row) at PD7 in heterozygous, NT control, and treatment groups. Scale bar: 100 μm. (B) Frequency distribution (top) and quantification (bottom) of minimum Feret's diameter (μm) in individual muscle fibers. Box ends, 25th and 75th percentiles; center line, median; whiskers, range (n=3-7 / group). We measured 1292-1653 fibers for quadriceps, 917-1746 fibers for diaphragm, and 642-1127 fibers for intercostal muscles. (C) Centrally cored fibers (%) quantified from H&E images. AO at 40 mg / kg was injected at PD0. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 13-1]Figure 13 shows that DG9-PMO treatment results in improvements at the neuromuscular junction (NMJ). (A) Representative confocal images of neuromuscular junction (NMJ) staining in quadriceps and intercostal muscles collected at PD30. Scale bar: 100 μm. Postsynaptic endplates were stained using α-bungarotoxin (red, α-BTX), while neurofilaments (2H3) and synaptic vesicles (SV2) indicate 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 presynaptic nerve terminals at the endplate. White arrow: complete innervation. Yellow arrow: partial innervation. Blue arrow: denervation. White arrow: collapsed NMJ. (B) Innervation characteristics: complete innervation, partial innervation, denervation, and collapsed NMJs were quantified from at least 300–500 NMJs per group (n=3–7) and plotted as a percentage of total NMJs analyzed. (C) The number of collapsed vesicles (flat, not pretzel-shaped) was quantified (n=4–6 per group). In (B), two-way ANOVA followed by Sidak's multiple comparisons was used. *p<0.03; **p<0.002; ***p<0.0002. In c, statistics were performed using one-way ANOVA followed by post hoc Tukey's test. #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. AO at 40 mg / kg was injected at PD0. [Figure 13-2]Figure 13 shows that DG9-PMO treatment results in improvements at the neuromuscular junction (NMJ). (A) Representative confocal images of neuromuscular junction (NMJ) staining in quadriceps and intercostal muscles collected at PD30. Scale bar: 100 μm. Postsynaptic endplates were stained using α-bungarotoxin (red, α-BTX), while neurofilaments (2H3) and synaptic vesicles (SV2) indicate 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 presynaptic nerve terminals at the endplate. White arrow: complete innervation. Yellow arrow: partial innervation. Blue arrow: denervation. White arrow: collapsed NMJ. (B) Innervation characteristics: complete innervation, partial innervation, denervation, and collapsed NMJs were quantified from at least 300–500 NMJs per group (n=3–7) and plotted as a percentage of total NMJs analyzed. (C) The number of collapsed vesicles (flat, not pretzel-shaped) was quantified (n=4–6 per group). In (B), two-way ANOVA followed by Sidak's multiple comparisons was used. *p<0.03; **p<0.002; ***p<0.0002. In c, statistics were performed using one-way ANOVA followed by post hoc Tukey's test. #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. AO at 40 mg / kg was injected at PD0. [Figure 13-3]Figure 13 shows that DG9-PMO treatment results in improvements at the neuromuscular junction (NMJ). (A) Representative confocal images of neuromuscular junction (NMJ) staining in quadriceps and intercostal muscles collected at PD30. Scale bar: 100 μm. Postsynaptic endplates were stained using α-bungarotoxin (red, α-BTX), while neurofilaments (2H3) and synaptic vesicles (SV2) indicate 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 presynaptic nerve terminals at the endplate. White arrow: complete innervation. Yellow arrow: partial innervation. Blue arrow: denervation. White arrow: collapsed NMJ. (B) Innervation characteristics: complete innervation, partial innervation, denervation, and collapsed NMJs were quantified from at least 300–500 NMJs per group (n=3–7) and plotted as a percentage of total NMJs analyzed. (C) The number of collapsed vesicles (flat, not pretzel-shaped) was quantified (n=4–6 per group). In (B), two-way ANOVA followed by Sidak's multiple comparisons was used. *p<0.03; **p<0.002; ***p<0.0002. In c, statistics were performed using one-way ANOVA followed by post hoc Tukey's test. #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. AO at 40 mg / kg was injected at PD0. [Figure 14-1]Figure 14 shows that DG9 increases PMO uptake in target tissues after subcutaneous administration at PD0. (A) PMO concentrations (pM) were detected by ELISA using an avidin-biotin affinity system and compared in quadriceps, liver, heart, kidney, brain, and spinal cord (n=3-7 / group) between DG9-PMO and unconjugated PMO treatment at a dose of 40 mg / kg. *p<0.05, **p<0.01, ***p<0.001 unpaired two-tailed Student's t-test. Error bars: SEM. (B) Representative immunohistochemistry images from PD7 heart, quadriceps, brain, and spinal cord after subcutaneous administration of fluorescently tagged DG9-PMO at PD0. Green: fluorescein-DG9-PMO. Magenta: DAPI. DG9-PMO without fluorescent tag was used as a negative control. White arrows indicate DG9-PMO overlapping with nuclei (DAPI), n = 3. Scale bar: 50 μm. [Figure 14-2] Figure 14 shows that DG9 increases PMO uptake in target tissues after subcutaneous administration at PD0. (A) PMO concentrations (pM) were detected by ELISA using an avidin-biotin affinity system and compared in quadriceps, liver, heart, kidney, brain, and spinal cord (n=3-7 / group) between DG9-PMO and unconjugated PMO treatment at a dose of 40 mg / kg. *p<0.05, **p<0.01, ***p<0.001 unpaired two-tailed Student's t-test. Error bars: SEM. (B) Representative immunohistochemistry images from PD7 heart, quadriceps, brain, and spinal cord after subcutaneous administration of fluorescently tagged DG9-PMO at PD0. Green: fluorescein-DG9-PMO. Magenta: DAPI. DG9-PMO without fluorescent tag was used as a negative control. White arrows indicate DG9-PMO overlapping with nuclei (DAPI), n = 3. Scale bar: 50 μm. [Figure 15-1]Figure 15 shows that DG9-PMO crosses the blood-brain barrier and increases the expression of FL-SMN2 in a mild SMA model. (A) Representative immunohistochemistry images at PD7 from quadriceps, heart, brain, and spinal cord after subcutaneous administration of fluorescently tagged DG9-PMO (green) at PD5 in F0 mice (Smn- / - SMN2+ / +). Magenta: DAPI. n=3 / group. White arrows indicate DG9-PMO overlapping with nuclei (DAPI). Scale bar: 50 μm. (B) The concentration of PMO (pM) at PD7 was detected by ELISA using an avidin-biotin affinity system and compared between DG9-PMO treatment and unconjugated PMO treatment injected at 40 mg / kg at PD5 in a mild SMA model (n=3-6 / group). Statistics were performed using unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (C) Relative expression levels of full-length SMN2 (FL-SMN2) compared to the deleted SMN2 transcript (Δ7SMN2) in quadriceps, liver, heart, spleen, brain, and spinal cord. Saline, unconjugated PMO, and DG9-PMO were injected subcutaneously into PD5 mice (n=3–6 / group). Tissues were collected at PD7. In c, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 15-2]Figure 15 shows that DG9-PMO crosses the blood-brain barrier and increases the expression of FL-SMN2 in a mild SMA model. (A) Representative immunohistochemistry images at PD7 from quadriceps, heart, brain, and spinal cord after subcutaneous administration of fluorescently tagged DG9-PMO (green) at PD5 in F0 mice (Smn- / - SMN2+ / +). Magenta: DAPI. n=3 / group. White arrows indicate DG9-PMO overlapping with nuclei (DAPI). Scale bar: 50 μm. (B) The concentration of PMO (pM) at PD7 was detected by ELISA using an avidin-biotin affinity system and compared between DG9-PMO treatment and unconjugated PMO treatment injected at 40 mg / kg at PD5 in a mild SMA model (n=3-6 / group). Statistics were performed using unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (C) Relative expression levels of full-length SMN2 (FL-SMN2) compared to the deleted SMN2 transcript (Δ7SMN2) in quadriceps, liver, heart, spleen, brain, and spinal cord. Saline, unconjugated PMO, and DG9-PMO were injected subcutaneously into PD5 mice (n=3–6 / group). Tissues were collected at PD7. In c, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 16-1]FIG. 16 shows that 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 transcript (Δ7SMN2) measured by qPCR in quadriceps, liver, heart, brain, and spinal cord in NT mice and treated mice after 80 mg / kg AO treatment. (B) Representative images from Western blotting and quantification of SMN levels relative to β-tubulin. Heterozygous mice were used as controls with relative SMN expression set to 1. Tissues from 80 mg / kg treated mice were collected at PD7. In A-B, one-way ANOVA followed by post hoc Tukey's test was performed. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 16-2] FIG. 16 shows that 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 transcript (Δ7SMN2) measured by qPCR in quadriceps, liver, heart, brain, and spinal cord in NT mice and treated mice after 80 mg / kg AO treatment. (B) Representative images from Western blotting and quantification of SMN levels relative to β-tubulin. Heterozygous mice were used as controls with relative SMN expression set to 1. Tissues from 80 mg / kg treated mice were collected at PD7. In A-B, one-way ANOVA followed by post hoc Tukey's test was performed. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 17]Figure 17 shows that DG9-PMO maintains SMN levels at postnatal day 30 in SMA mice. Representative images from Western blotting and quantification of SMN levels. Tissues were collected at PD30 from the 40 mg / kg group and compared to β-tubulin. Heterozygous mice were used as controls with relative SMN expression set to 1. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 18] FIG. 18 shows muscle strength and motor function tests of DG9-PMO treated adult SMA mice. (A) Forelimb grip strength measured at PD30 in adult males and females. 40 mg / kg AO was injected at PD0. (B) Forelimb grip strength normalized to body weight. Measured at PD30 and PD60. 80 mg / kg AO was injected at PD0. (C) Rotarod test using acceleration profile. Measured from PD30 to PD35. Each mouse underwent three trials at 20 min intervals from each other. Maximum time on the beam was recorded. 40 mg / kg AO was injected at PD0. One-way ANOVA followed by post hoc Tukey's test was performed. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 19]FIG. 19 shows that DG9-PMO ameliorates muscle pathology at postnatal day 7 in SMA mice. Frequency distribution and quantification of cross-sectional area (CSA) (μm2) in individual muscle fibers from quadriceps, diaphragm, and intercostal muscles harvested at PD7. Box ends, 25th and 75th percentiles; centerline, median; whiskers, range (n=3-7 per group), approximately 1292-1653 fibers for quadriceps, 917-1746 fibers for diaphragm, and 642-1127 fibers for intercostal muscles. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 20-1] Figure 20 shows muscle pathology in DG9-PMO treated mice at postnatal day 30. (A) Representative images of H&E staining of quadriceps (top), diaphragm (middle), and intercostal muscles (bottom) at PD30 in heterozygous, NT control, and treated groups. 40 mg / kg AO was injected at PD0. Scale bar: 100 μm. (B) Frequency distribution of minimum Feret's diameter (μm) in individual myofibers from tissue harvested at PD30 and quantification shown at bottom. (C) Frequency distribution of cross-sectional area (CSA) (μm2) in individual myofibers and quantification shown at bottom. Box edges, 25th and 75th percentiles; centerline, median; whiskers, range (n=3-6 per group), approximately 960-1868 fibers for quadriceps, 983-1436 fibers for diaphragm, and 800-1802 fibers for intercostal muscles. 40 mg / kg AO was injected at PD0. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 20-2]Figure 20 shows muscle pathology in DG9-PMO treated mice at postnatal day 30. (A) Representative images of H&E staining of quadriceps (top), diaphragm (middle), and intercostal muscles (bottom) at PD30 in heterozygous, NT control, and treated groups. 40 mg / kg AO was injected at PD0. Scale bar: 100 μm. (B) Frequency distribution of minimum Feret's diameter (μm) in individual myofibers from tissue harvested at PD30 and quantification shown at bottom. (C) Frequency distribution of cross-sectional area (CSA) (μm2) in individual myofibers and quantification shown at bottom. Box edges, 25th and 75th percentiles; centerline, median; whiskers, range (n=3-6 per group), approximately 960-1868 fibers for quadriceps, 983-1436 fibers for diaphragm, and 800-1802 fibers for intercostal muscles. 40 mg / kg AO was injected at PD0. Statistics were performed using one-way ANOVA followed by post hoc Tukey's test. #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 21-1]Figure 21 shows that DG9-PMO treatment does not cause obvious immune responses and toxicity. (A) Representative images from immunostaining of CD68+ macrophages (green) and DAPI (blue) in quadriceps muscle at PD7. (B) Average CD68+ cells per section. Three to five sections from the quadriceps muscle of each mouse were used for analysis (n=3-5 / group). Scale bar: 100 μm. (C) Serum analysis for ALP, ALT, AST, total bilirubin, GGT, BUN, creatinine, CK, total protein, albumin, and globulin. Serum was collected at PD30 (n=2-7 / group). (D) Representative images of H and E staining of liver (upper row) and kidney (lower row) at PD30. No obvious morphological differences were observed between healthy heterozygous control and treated groups. Scale bar: 100 μm. 40 mg / kg AO was injected at PD0. One-way ANOVA followed by post hoc Tukey's test was performed. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 21-2] Figure 21 shows that DG9-PMO treatment does not cause obvious immune responses and toxicity. (A) Representative images from immunostaining of CD68+ macrophages (green) and DAPI (blue) in quadriceps muscle at PD7. (B) Average CD68+ cells per section. Three to five sections from the quadriceps muscle of each mouse were used for analysis (n=3-5 / group). Scale bar: 100 μm. (C) Serum analysis for ALP, ALT, AST, total bilirubin, GGT, BUN, creatinine, CK, total protein, albumin, and globulin. Serum was collected at PD30 (n=2-7 / group). (D) Representative images of H and E staining of liver (upper row) and kidney (lower row) at PD30. No obvious morphological differences were observed between healthy heterozygous control and treated groups. Scale bar: 100 μm. 40 mg / kg AO was injected at PD0. One-way ANOVA followed by post hoc Tukey's test was performed. *NT, #PMO, @DG9-PMO, and MOE. *p<0.05, **p<0.01, ***p<0.005. Error bars: SEM. [Figure 22] FIG. 22 shows DG9 PMO-induced cardiac-specific exon skipping in a transgenic zebrafish model. (A) Transgenic zebrafish lines are generated by flanking a dual fluorescent protein switch with Tol2 inverted terminal repeats (ITRs) using the Tol2 transposon system. Ubiquitous mRNA is generated using the carp beta-actin promoter, non-coding exons, and mini-intron 1 (β-act). The mRNA will be generated from two exons. The first exon contains the complete blue fluorescent protein (BFP) open reading frame with a stop codon. The second exon contains the red fluorescent protein (RFP) open reading frame with the stop codon, polyadenylation signal, and transcription terminator. This dual cistronic mRNA will normally generate only the functional BFP. However, in the presence of a PMO targeting the splice acceptor of BFP (derived from exon 2 of the carp beta-actin gene), the BFP exon is skipped, generating a shorter mRNA. RFP can be translated from this shorter mRNA. The switch from expression of BFP to RFP corresponds to PMO activity in the nucleus and indicates successful delivery to the cells. B. qPCR data shows the detection of RFP transcripts collected from DG9 PPMO-treated zebrafish groups (15 months old, approximately 8 per group, injected once with 25mg / Kg PPMO via the intravenous route). ΔCT plot of RFP transcripts: Of all organs examined, the heart was the only one that showed detection of RFP possible. C. Gel electrophoresis was performed on the qPCR products to visualize the RFP transcripts. The approximately 100bp band detected in the heart was sequenced and the results aligned with the desired product. [Figure 23-1]Figure 23 shows improved efficacy after DG9-PMO treatment compared to R6-PMO treatment in SMA mice. (A) Survival curves of heterozygous mice (Het), untreated (NT), unconjugated PMO (PMO), DG9-PMO, MOE, and R6G-PMO injected at a dose of 40 mg / kg on PD0. n=15 (Hets), n=22 (NT), n=49 (unconjugated PMO), n=14 (DG9-PMO), n=29 (MOE), and n=6 (R6G-PMO), (p≦0.0001, log-rank Mantel Cox test). (B) Representative images (left to right) of heterozygous mice, mice treated with 80 mg / kg or 40 mg / kg DG9-PMO, mice injected with unconjugated PMO (40 mg / kg), and saline-treated NT mice at PD7. (C) Body weight of mice at PD7 treated with 40 mg / kg or 80 mg / kg dose. Each point (symbol) represents a newborn. (D) Hindlimb suspension assay (HLS). Mice were treated with 40 mg / kg AO at PD0. Scores are based on the position of the hindlimb when suspended from a Falcon tube. (E) Righting reflex test. Mice were treated with 40 mg / kg AO at PD0. The ability of mice to rear up on their paws was measured every other day between PD2 and PD20 (left). The mean righting reflex times at PD6 and PD8 are also shown (right: box plots). Box ends, 25th and 75th percentiles; center line, median; whiskers, range. (F) Relative expression levels of full-length SMN2 (FL-SMN2) compared to the deleted SMN2 transcript (Δ7SMN2) measured by qPCR after 40 mg / kg ASO or PBS treatment. In C, E (box plots), and F, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. In D and E, two-way ANOVA followed by Sidak's multiple comparisons was performed. *p<0.03, **p<0.002, ***p<0.0002. *NT, #PMO, @DG9-PMO, and MOE, ^R6-PMO. Error bars: SEM. [Figure 23-2]Figure 23 shows improved efficacy after DG9-PMO treatment compared to R6-PMO treatment in SMA mice. (A) Survival curves of heterozygous mice (Het), untreated (NT), unconjugated PMO (PMO), DG9-PMO, MOE, and R6G-PMO injected at a dose of 40 mg / kg on PD0. n=15 (Hets), n=22 (NT), n=49 (unconjugated PMO), n=14 (DG9-PMO), n=29 (MOE), and n=6 (R6G-PMO), (p≦0.0001, log-rank Mantel Cox test). (B) Representative images (left to right) of heterozygous mice, mice treated with 80 mg / kg or 40 mg / kg DG9-PMO, mice injected with unconjugated PMO (40 mg / kg), and saline-treated NT mice at PD7. (C) Body weight of mice at PD7 treated with 40 mg / kg or 80 mg / kg dose. Each point (symbol) represents a newborn. (D) Hindlimb suspension assay (HLS). Mice were treated with 40 mg / kg AO at PD0. Scores are based on the position of the hindlimb when suspended from a Falcon tube. (E) Righting reflex test. Mice were treated with 40 mg / kg AO at PD0. The ability of mice to rear up on their paws was measured every other day between PD2 and PD20 (left). The mean righting reflex times at PD6 and PD8 are also shown (right: box plots). Box ends, 25th and 75th percentiles; center line, median; whiskers, range. (F) Relative expression levels of full-length SMN2 (FL-SMN2) compared to the deleted SMN2 transcript (Δ7SMN2) measured by qPCR after 40 mg / kg ASO or PBS treatment. In C, E (box plots), and F, one-way ANOVA followed by post hoc Tukey's test was performed. *p<0.05, **p<0.01, ***p<0.005. In D and E, two-way ANOVA followed by Sidak's multiple comparisons was performed. *p<0.03, **p<0.002, ***p<0.0002. *NT, #PMO, @DG9-PMO, and MOE, ^R6-PMO. Error bars: SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] [Detailed Description] The present invention provides pre-mRNA splicing modulating therapeutic agents, optionally suitable for systemic delivery, comprising antisense oligonucleotides conjugated to a cell-penetrating peptide derived from the protein transduction domain (PTD) in the human Hph-1 transcription factor.

[0033] The therapeutics are designed to promote either exon skipping or exon inclusion. The therapeutics include peptide-conjugated antisense therapeutics for the treatment of Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).

[0034] The DMD therapeutic agent promotes exon skipping. Optionally, the therapeutic agent comprises two or more conjugates and is configured to promote multi-exon skipping. Optionally, the therapeutic agent comprises two, three, four, five, six, seven, or more different conjugates, where each conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide derived from a protein transduction domain (PTD) in human Hph-1 transcription factor. In some embodiments, each different conjugate targets a different exon. In some embodiments, the different conjugates target the same exon.

[0035] SMA therapeutics promote exon inclusion. Conjugates The conjugate of the present invention comprises an antisense oligonucleotide capable of regulating exon splicing of pre-mRNA linked to a cell penetrating peptide (CPP). The cell penetrating peptide of the present invention, also called DG9, facilitates the delivery of the conjugated antisense oligonucleotide and has improved activity over the R6G cell penetrating peptide. In some embodiments, the conjugate of the present invention is for intravenous delivery.

[0036] The cell penetrating peptide of the present invention is derived from the protein transduction domain of the human HPH-1 transcription factor and comprises the following amino acid sequence: YARVRRRGPRGYARVRRRGPRR.

[0037] Optionally, in some embodiments, one or two amino acids are deleted or substituted. Optionally, an amino acid is substituted with a non-natural amino acid. In some embodiments, one or more L-amino acids are replaced with D-amino acids to improve serum stability. In some embodiments, 2, 3, 4, 5, or 6 L-amino acids are replaced with D-amino acids. Optionally, 2, 3, 4, 5, or 6 L-arginines are replaced with D-arginines.

[0038] In some embodiments, the cell penetrating peptide (CPP) has the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Here, capital letters represent L-amino acids and lower case letters represent D-amino acids.

[0039] In some embodiments, the CPP is conjugated to the 5' end of the oligonucleotide. In other embodiments, the CPP is conjugated to the 3' end of the oligonucleotide.

[0040] "Antisense oligonucleotide", "antisense therapeutic agent", "AO", "oligo", "oligomer", as used interchangeably herein, refer to a sequence of subunits, each having bases carried in a backbone subunit, 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 oligonucleotide may have exact sequence complementarity to the target sequence, or may have sufficient complementarity to selectively bind to the target sequence.

[0041] Antisense oligonucleotides are about 20 to about 50 nucleotides in length, containing at least 10, 12, 15, 17, or 20 contiguous nucleotides complementary to the target sequence.

[0042] In one embodiment, the antisense oligonucleotides are 20 to 30, or 24 to 28 nucleotides in length. In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide analog.

[0043] The terms "oligonucleotide analog" and "nucleotide analog" refer to any modified synthetic analogue of an oligonucleotide or nucleotide, respectively, known in the art.

[0044] Examples of oligonucleotide analogs 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 oligonucleotides.

[0045] In some embodiments, the antisense oligonucleotide comprises a morpholino subunit. In some embodiments, the antisense oligonucleotide is a morpholino antisense oligonucleotide.

[0046] In some embodiments, the antisense oligonucleotide comprises morpholino subunits linked together by phosphorus-containing linkages. In certain instances, the antisense oligonucleotide is a phosphoramidate morpholino antisense oligonucleotide or a phosphorodiamidate morpholino antisense oligonucleotide.

[0047] The term "morpholino antisense oligonucleotide" or "PMO" (phosphoroamidate morpholino oligonucleotide or phosphorodiamidate morpholino oligonucleotide) refers to an antisense oligonucleotide analogue composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing bonds, e.g., 1 to 3 atoms long, e.g., 2 atoms long, and e.g., uncharged or cationic, linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring has a purine or pyrimidine base pairing moiety effective to bind to a base of a polynucleotide by base-specific hydrogen bonding.

[0048] In some embodiments, the antisense oligonucleotides contain a phosphorus-containing intersubunit linkage connecting the morpholino nitrogen of one subunit and the 5' exocyclic carbon of an adjacent subunit.

[0049] Optionally, variations can be made in the intersubunit linkages so long as they do not interfere with binding or activity. For example, the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate). The 5' oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted with (optionally substituted) lower alkyl, or disubstituted.

[0050] Antisense oligonucleotides may be made by the well-known technique of solid phase synthesis. In some embodiments, the conjugate further comprises a label. The label provides a detectable signal, either directly or indirectly. Suitable labels are known in the art and include fluorescent or radioactive labels.

[0051] Methods for conjugating a CPP to an oligonucleotide are known in the art or the conjugates can be prepared commercially. Duchenne Muscular Dystrophy (DMD) Conjugates The present invention provides an exon-skipping peptide-conjugated antisense therapeutic for the treatment of Duchenne muscular dystrophy. Treatment of DMD with the peptide-conjugated antisense therapeutic restores partially functional dystrophin to DMD patients.

[0052] A "functional" dystrophin protein refers to a dystrophin protein that has sufficient biological activity to alleviate the progressive degradation of muscle tissue that is another hallmark of muscular dystrophy, when compared to a defective form of dystrophin protein present in patients with a muscle disease such as DMD.

[0053] In some embodiments, 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. Activity of dystrophin in in vitro muscle cultures can be measured according to myotube size, myofibrillar organization, contractile activity, and spontaneous clustering of acetylcholine receptors.

[0054] Thus, in some embodiments, peptide-conjugated antisense therapeutics of the invention are used to treat DMD by inducing exon skipping in human dystrophin pre-mRNA and restoring functional dystrophin protein expression.

[0055] In one embodiment, the therapeutic agent comprises one peptide-conjugated antisense oligonucleotide. In an alternative embodiment, the therapeutic agent comprises two or more peptide-conjugated antisense oligonucleotides, where each peptide-conjugated antisense oligonucleotide targets a different sequence in the dystrophin pre-mRNA.

[0056] As described herein, peptide-conjugated antisense therapeutics are provided that may be therapeutically effective for exon skipping therapy in 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 of the human dystrophin (DMD) gene.

[0057] In some embodiments, the peptide-conjugated antisense therapeutic comprises a plurality of peptide-conjugated antisense oligonucleotides and promotes skipping of exons 45-55.

[0058] In some embodiments, peptide-conjugated antisense therapeutic agents include peptide-conjugated antisense oligonucleotides targeted to exon 45, peptide-conjugated antisense oligonucleotides targeted to exon 46, peptide-conjugated antisense oligonucleotides targeted to exon 47, peptide-conjugated antisense oligonucleotides targeted to exon 48, peptide-conjugated antisense oligonucleotides targeted to exon 49, peptide-conjugated antisense oligonucleotides targeted to exon 50, peptide-conjugated antisense oligonucleotides targeted to exon 51, peptide-conjugated antisense oligonucleotides targeted to exon 52, peptide-conjugated antisense oligonucleotides targeted to exon 53, peptide-conjugated antisense oligonucleotides targeted to exon 54, and peptide-conjugated antisense oligonucleotides targeted to exon 55.

[0059] In other embodiments, peptide-conjugated antisense therapeutic agents include peptide-conjugated antisense oligonucleotides targeted to exon 45, peptide-conjugated antisense oligonucleotides targeted to exon 47, peptide-conjugated antisense oligonucleotides targeted to exon 49, peptide-conjugated antisense oligonucleotides targeted to exon 51, peptide-conjugated antisense oligonucleotides targeted to exon 53, and peptide-conjugated antisense oligonucleotides targeted to exon 55.

[0060] In yet other embodiments, the peptide-conjugated antisense therapeutic comprises a peptide-conjugated antisense oligonucleotide targeted to exon 45, a peptide-conjugated antisense oligonucleotide targeted to exon 47, and a peptide-conjugated antisense oligonucleotide targeted to exon 53.

[0061] In some embodiments, the antisense oligonucleotide in the peptide-conjugated antisense therapeutic 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. Optionally, the target sequence is proximal to the exon acceptor splice site of the pre-mRNA. In some embodiments, the target sequence is within 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.

[0062] In some embodiments, the antisense 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 the thymine bases in the sequence are optionally uracil.

[0063] [ka]

[0064] Optionally, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the conjugate enhances delivery to, uptake by, or retention by cardiac cells.

[0065] Spinal Muscular Atrophy (SMA) The present invention provides an exon inclusion peptide-conjugated antisense therapeutic for the treatment of spinal muscular atrophy. Treatment of SMA with the peptide-conjugated antisense therapeutic results in stable production of SMN2 to compensate for the loss of SMN1.

[0066] The peptide-conjugated antisense therapeutic comprises DG-9 conjugated to an antisense oligonucleotide that binds to the intron splicing silencer N1 of the SMN2 pre-mRNA. Optionally, the antisense oligonucleotide comprises or consists of the sequence 5'-TCACTTTCATAATGCTGG-3', where thymine is optionally replaced with uracil.

[0067] In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer. Pharmaceutically acceptable excipients In one aspect, a pharmaceutical composition is described comprising an antisense oligonucleotide of the invention or a conjugate thereof, further comprising one or more pharma- ceutically acceptable excipients.

[0068] In some embodiments, the pharmaceutical compositions are prepared using pharma- ceutically inert inorganic and / or organic excipients in a manner known in the art. The term "pharmacologically acceptable" refers to molecules and compositions that are physiologically tolerable and do not normally produce allergic or similar untoward reactions when administered to a patient.

[0069] In some embodiments, the pharmaceutical compositions may be formulated as pills, tablets, coated tablets, hard gelatin capsules, soft gelatin capsules, and / or suppositories, solutions and / or syrups, injectable solutions, microcapsules, implants, and / or rods, and the like.

[0070] In some embodiments, the pharmaceutical compositions may be formulated as injectable solutions. In some embodiments, pharma- ceutically acceptable excipients for preparing pills, tablets, coated tablets, and hard gelatin capsules may be selected from any of lactose, corn starch and / or its derivatives, talc, stearic acid and / or its salts, etc.

[0071] In some embodiments, pharma- ceutically 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, and the like.

[0072] In some embodiments, pharma- ceutically acceptable excipients for preparing solutions and / or syrups may be selected from water, sucrose, invert sugar, glucose, polyols, and the like.

[0073] In some embodiments, pharma- ceutically acceptable excipients for preparing injectable solutions may be selected from water, saline, alcohol, glycerol, polyols, vegetable oils, and the like.

[0074] In some embodiments, pharma- ceutically acceptable excipients for preparing microcapsules, implants and / or rods may be selected from mixed polymers such as, for example, glycolic acid and lactic acid.

[0075] In some embodiments, the pharmaceutical composition may include a liposomal formulation. In some embodiments, the pharmaceutical composition may optionally comprise two or more different antisense oligonucleotides, or conjugates thereof.

[0076] In some embodiments, the antisense oligonucleotides and / or conjugates may optionally be present in the pharmaceutical composition as physiologically acceptable salts.

[0077] Suitably, physiologically acceptable salts retain the desired biological activity of the antisense oligonucleotides and / or conjugates thereof and do not impart undesired toxicological effects. For antisense oligonucleotides, suitable examples of pharma- ceutically acceptable salts include: (a) salts formed with cations such as, for example, sodium, potassium, ammonium, magnesium, calcium, polyamines such as, for example, spermine and spermidine; (b) acid addition salts formed with inorganic acids such as, 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, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as, for example, chlorine, bromine, iodine, and the like.

[0078] In some embodiments, in addition to the active ingredient and excipients, the pharmaceutical composition may include additives such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, dyes, flavors or fragrances, thickeners, diluents or buffer substances, as well as solvents and / or solubilizers and / or agents for achieving a sustained release effect, salts for modifying osmotic pressure, coating agents and / or antioxidants, etc. Suitable additives may include Tris-HCl, acetates, phosphates, Tween 80, polysorbate 80, ascorbic acid, sodium metabisulfite, thimersol, benzyl alcohol, lactose, mannitol, etc.

[0079] Administration In some embodiments, the peptide-conjugated antisense oligonucleotides and / or pharmaceutical compositions are for topical, enteral or parenteral administration.

[0080] In some embodiments, the peptide-conjugated antisense oligonucleotides and / or pharmaceutical compositions may be for administration orally, transdermally, intravenously, intrathecally, intramuscularly, subcutaneously, intranasally, transmucosally, and the like.

[0081] In some embodiments, the antisense oligonucleotides and / or pharmaceutical compositions are for intramuscular administration. In some embodiments, the antisense oligonucleotides and / or pharmaceutical compositions are for intramuscular administration by injection.

[0082] "Effective amount" or "therapeutically effective amount" refers to the amount of antisense oligonucleotide administered to a subject, either in a single dose or as part of a series, that is effective to produce a desired physiological response or therapeutic effect in a subject.

[0083] In some embodiments, the antisense oligonucleotide or conjugate thereof is administered every day, 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.

[0084] In some embodiments, the antisense oligonucleotide or conjugate thereof may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

[0085] In order to obtain a better understanding of the invention described herein, the following examples are presented. It should be understood that these examples are for illustrative purposes only. Thus, the examples should not be construed as limiting the scope of the invention in any way.

[0086] Working Example Example 1: Peptide-conjugated PMOS for the treatment of Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholine oligomers (PMOs) converts out-of-frame mutations to in-frame mutations, generating partially functional dystrophin. Skipping of exons 45–55 is capable of treating 40%–47% of all patients and is associated with improved clinical outcomes. The development of peptide-conjugated PMOs for exon 45–55 skipping has been reported. Experiments with immortalized patient myotubes revealed that it is possible to skip exons 45–55 by targeting only five exons. Conjugation of DG9, a cell-permeable peptide, to PMOs improved single exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of the minimized exon 45–55 skipping DG9-PMO cocktail restored dystrophin production.

[0087] result Minimizing exon 45-55 skipping cocktail The number of PMOs required to skip DMD exons 45–55 was minimized using two strategies.

[0088] First, a PMO (or multiple PMOs) targeting an exon within the region was deleted from a previously developed exon 45-55 skipping cocktail

[13] (Table 1). The complete cocktail with PMOs targeting all exons within exons 45-55 is referred to as the "all" cocktail (Fig. 1a). When the "all" cocktail and its derivatives were transfected into healthy immortalized patient-derived KM155 myotubes (Fig. 1b), only the "all" cocktail skipped exons 45-55 significantly more than mock (p<0.005) (Fig. 5a). PMOs whose deletion results in a significant reduction in skipping were retained as part of the minimization cocktail, i.e., those targeting exons 45, 47, and 53. PMOs targeting exons 49, 51, and 55 were also retained. This minimized cocktail, termed the "Base" cocktail, underwent another series of minimized runs in exon 52 deletion immortalized patient-derived KM571 myotubes. The "Base" cocktail showed significant exon 45-55 skipping (p<0.05) compared to mock and its derivatives not targeting exons 47 (p<0.05) or 51 (p<0.05) (Figure 5a). The "Base" and "Base-51" cocktails showed the highest skipping efficiency in this batch (Figure 1a).

[0089] For the second strategy, we prepared a minimization cocktail based on endogenous splicing of DMD exons 45–55. The model proposes that exons 45–49, 50–52, and 53–55 are spliced ​​first, and then these groups are spliced ​​together to complete the exon 45–55 region (25). We designed derivative cocktails from the “all” cocktail with PMOs targeting terminal exons in these three groups. Transfection into healthy KM155 myotubes revealed that the three derivative cocktails skipped exons 45–55 significantly more than mock (p<0.05) (Figure 5b), and the skipping efficiency was not significantly different from the “all” cocktail. On this basis, we selected a cocktail targeting exons 45, 49, 50, 52, 53, and 55 (the “Block” cocktail) as well as a cocktail targeting exons 45, 50, and 55 (the “3-PMO” cocktail) for further experiments ( Fig. 1 a ).

[0090] The minimized exon skipping cocktail efficiently skips exons 45–55 in a variety of patient cells The "all", "base", "base-51", "blocked", and "3-PMO" exon 45-55 skipping cocktails were subsequently tested in the following immortalized patient-derived muscle cell lines: KM155 (healthy), KM571 (Δex52), 6594 (Δex48-50), and 6311 (Δex45-52) (Figure 1a). For each cell line, a mutation-matched cocktail version was used, removing the unnecessary PMO. Transfection into myotubes (Figure 1b) and RT-PCR analysis showed that the "all" cocktail significantly skipped exons 45-55 in all lines compared to mock (p<0.005 or p<0.001) (Figure 1c). The "Base-51", "Block", and "3-PMO" cocktails significantly induced exon 45-55 skipping in KM155, KM571, and 6594 myotubes compared to mock (at least p<0.05), with only the "Base" cocktail showing significant skipping in KM155 cells (p<0.001). In 6594 myotubes, the "3-PMO" cocktail induced significantly more skipping than the "All" cocktail. Interestingly, none of the minimized cocktails showed any exon 45-55 skipping in 6311 myotubes.

[0091] As the "Block" and "3-PMO" cocktails induced the highest levels of exon 45-55 skipping in this line, we assessed the ability of the "Block" and "3-PMO" cocktails to restore dystrophin in KM571 myotubes. Western blots using antibodies against the rod (DYS1, exons 26-30) and C-terminal domain (ab15277) in dystrophin detected dystrophin upon treatment with the "All" and "Block" cocktails, but not with the "3-PMO" cocktail (Fig. 1d). Quantification of DYS1 signal showed that the "Block" cocktail treatment restored wild-type levels of dystrophin by an average of 2.94%, slightly higher than the average of 2.33% dystrophin restored by the "All" cocktail and compared to the 0.10% dystrophin level in mock (p<0.001) (Fig. 1e). Western blots using MANEX45A (corresponding to exons 45 to 46) and MANEX4850E (corresponding to exons 48–50) antibodies did not detect dystrophin in treated myotubes (Fig. 1d), suggesting that the dystrophin induced after treatment is likely derived from exon 45–55 skipping transcripts.

[0092] A single intravenous treatment with DG9-PMO induces higher dystrophin production in skeletal muscle and heart than unconjugated PMO To evaluate DG9 as a conjugate for the minimized exon 45-55 skipping cocktail, its in vivo efficacy was assessed in single exon skipping applications. DG9 was conjugated to Ex51_Ac0, an exon 51-skipping PMO from the “all” cocktail that was up to 7-fold more effective at restoring dystrophin production in vitro than eteplisene [13, 26]. Three-month-old hDMDdel52;mdx mice received 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 were evaluated 1 week later (Figure 2a). DG9-PMO-treated mice had significantly higher levels of exon 51 skipping than saline- or PMO-treated groups for various skeletal muscles (at least p<0.05) and heart (p<0.001) (Fig. 2b). DG9-PMO induced an average of 2.2- to 12.3-fold higher skipping in skeletal muscles and an average of 14.4-fold higher skipping in heart compared to unconjugated PMO. DG9-PMO treatment significantly restored dystrophin production in gastrocnemius and quadriceps compared to saline (p<0.005) and PMO (p<0.05) treatments, reaching up to 3% of wild-type levels (Fig. 2c). In the heart, DG9-PMO restored an average of 2.5% of wild-type levels of dystrophin, significantly higher than saline or PMO groups (p<0.05). Compared with PMO-treated mice, mice receiving DG9-PMO had an average of 1.5- to 3.4- and 4.5-fold higher dystrophin protein levels in skeletal muscle and heart, respectively.

[0093] Extensive dystrophin-positive fibers were observed in the tibialis anterior muscle, diaphragm, and heart of DG9-PMO-treated mice, and few or no fibers were observed in saline- or PMO-treated mice (Fig. 2d). However, histological analysis showed no reduction in the percentage of centrally nucleated fibers (CNFs, a marker of cumulative muscle regeneration) in the tibialis anterior muscle and diaphragm after PMO or DG9-PMO treatment (Fig. 6a,b), nor did it improve myofiber size (Fig. 6a,c,d).

[0094] Repeated intravenous treatment with DG9-PMO improves dystrophin production, muscle function, and muscle fiber size in dystrophic mice To further understand the efficacy of DG9-PMO exon skipping therapy in improving dystrophic symptoms, we performed a repeat-dose treatment study. Two-month-old hDMDdel52;mdx mice received retro-orbital injections of saline or 30 mg / kg DG9-PMO once a week for 3 weeks. Functional assessments were performed at baseline and 2 weeks after the last injection, after which tissues were harvested (Figure 3a). Again, DG9-PMO treatment induced significantly higher levels of exon 51 skipping in skeletal muscle and heart compared to saline controls (55%-71% on average, p<0.001) (Figure 3b). This resulted in significant dystrophin production in various skeletal muscles ranging from an average of 2.8% to 3.9% compared to a range of 0.1% to 0.3% in saline-treated mice (at least p<0.05) (Figure 3c), and in the heart, an average of 7.7% dystrophin production compared to 0.5% in the saline group (p<0.001) (Figure 3d). Immunofluorescence confirmed the presence of widespread dystrophin-positive fibers in the tibialis anterior, diaphragm, and heart (Figure 3e).

[0095] Body weights between groups were not significantly different over the treatment period (Fig. 3f). Most strikingly, repeated treatment with DG9-PMO significantly improved forelimb (p<0.05) (Fig. 3g) and total limb (p<0.005) (Fig. 3h) grip strength in hDMDdel52;mdx mice, without any significant difference from wild-type controls. Nearly all mice improved forelimb / total limb grip strength from baseline, except for one mouse that showed a -0.3% difference in forelimb grip strength after treatment. This is in contrast to the saline control group, which showed no direction of change (Fig. 3g) or no change from baseline (Fig. 3h). Rotarod and treadmill tests showed similar improvements, notably treadmill distance traveled in treated mice significantly improved from baseline compared to saline control mice (Fig. 7a, b).

[0096] Histological analysis still showed no significant reduction in CNFs in the tibialis anterior and diaphragm (Fig. 7c, d). However, we observed a significant increase (p < 0.001) in fiber size with DG9-PMO treatment (Fig. 7e, f), with most fibers having a minimum Feret's diameter of 45-50 μm in the tibialis anterior and 25-30 μm in the diaphragm. This differs from saline control muscles, where most fibers had diameters of 30-35 μm and 20-25 μm, respectively. Qualitative histological analysis in the liver and kidney of single- and repeat-dose treated mice was performed, but did not reveal any evidence of toxicity due to PMO or DG9-PMO treatment (Fig. 8a, b).

[0097] Local treatment with a DG9-coupled minimized exon 45-55 skipping cocktail induces successful skipping and dystrophin production Finally, DG9 was conjugated to each PMO in the minimized "block" exon 45-55 skipping cocktail for in vivo testing. The "block" cocktail was chosen because it induced high levels of exon 45-55 skipping and restoration of dystrophin protein in KM571 myotubes (Fig. 1c-e). Thus, 5- to 6-month-old hDMDdel52;mdx mice were intramuscularly injected in the tibialis anterior muscle with either saline or the DG9-conjugated form aligned with the mutations in the "block" cocktail (5 DG9-PMO, 5 μg / DG9-PMO, 25 μg total dose) and assessed 1 week later (Fig. 4a). We observed significant exon 45-55 skipping compared to saline controls (p<0.001) (Fig. 4b). Western blots revealed that dystrophin restoration in DG9-PMO-treated muscles was, on average, 0.76% of wild-type levels, significantly higher than that in saline-treated muscles, which had 0.46% (p<0.05) (Figure 4c). As one leg of the same mouse was administered DG9-PMO and the contralateral leg administered saline, protein from a non-treated mouse was included to account for possible leakage between the legs. Non-treated tibialis anterior muscles had 0.12% of wild-type levels of dystrophin, lower than that of saline controls. We observed few scattered dystrophin-positive fibers in DG9-PMO-treated muscles, confirming dystrophin restoration, which was hardly observed in saline controls (Figure 4d).

[0098] Consideration We developed a minimized exon 45-55 skipping cocktail that induced restoration of dystrophin in immortalized patient cells and dystrophic hDMDdel52;mdx mice. For exon 52-deleted DMD transcripts, the number of PMOs used for exon 45-55 skipping was reduced from 11 in the "all" cocktail to 5 in the "block" cocktail (Fig. 1a), resulting in a greater than 50% reduction in PMO content. Most minimized cocktails significantly skipped exons 45-55 in KM571 (Δex52) and 6594 (Δex48-50) myotubes, whereas in 6311 myotubes (Δex45-52) it appears that all remaining exons must be targeted. The "block" cocktail restored dystrophin production to approximately 3% of wild-type, close to the amount seen with the "all" cocktail (Fig. 1e).

[0099] We demonstrated the efficacy of conjugating DG9 to PMO in single- and multi-exon skipping applications. Treatment with DG9-PMO resulted in higher single-exon skipping and dystrophin restoration levels in vivo compared to unconjugated PMO (Figures 2b-d), similar to results widely observed for other cell-penetrating peptides (20, 28). Repetitive treatments enhanced dystrophin restoration in all tissues tested, particularly in the heart, resulting in restoration of 2.5% to 7.7% of wild-type (Figures 2c, 3d).

[0100] The advantage of DG9 is its potentially better toxicity profile compared to other peptides. Peptide-conjugated PMOs have induced dose-dependent toxic effects in preclinical studies, including lethargy, weight loss, and kidney damage (20). This is related to the membrane-disruptive properties of cell-penetrating peptides, which are greatly influenced by the amino acid composition of the peptide (20, 29). In DG9, certain L-arginine residues are converted to D-arginine, which improves the viability of peptide-conjugated PMO-treated cells in vitro (30). DG9 further does not contain any 6-aminohexanoic acid residues, which have been associated with increased toxicity (30). Compared to DG9, previous peptides used primarily L-arginine and contained multiple 6-aminohexanoic acid residues, but were similar in length and overall arginine content (20). The above changes reduce the antisense activity of peptide-conjugated PMOs, but with the benefit of improved safety. For peptide-conjugated PMOs, efficacy must be balanced against safety, as too frequent or high doses can increase toxicity. (20, 29) No evidence of toxicity caused by DG9-PMO on the liver and kidney was evident (Figure 8).

[0101] On the topic of efficacy, although exon 51 skipping was highly induced by our DG9-PMO, the observed dystrophin restoration was generally lower than that achieved by other peptide-conjugated PMOs (20, 29). This could be due to differences in strategy, with the majority of peptide-conjugated PMO studies skipping exon 23 (20). Similar discrepancies between exon skipping and dystrophin restoration levels in vivo were observed by Aoki et al. (2010), who skipped exon 51 with PMO (31), and Aupy et al. (2020), who also skipped exon 51 but used adeno-associated virus-delivered U7 small nuclear RNA (32). It is possible that transcripts or proteins skipping exon 51 may be less stable than those skipping exon 23, leading to the observed reduced dystrophin levels. In vitro studies using various internally truncated dystrophin proteins revealed that exon 51-skipped dystrophin was mostly as stable as full-length dystrophin (33), supporting the hypothesis that transcript stability is reduced. Previous studies have shown that DMD transcript stability affects dystrophin protein production (34, 35), but it remains unclear whether this explains the differences in the cases of exon 23- and exon 51-skipped transcripts. Another factor would be the animal model used for testing. Humanized dystrophic mice, such as hDMDdel52;mdx, have only recently been developed, so it will be important to characterize the stability of human DMD transcripts and protein in these models and determine whether they affect the efficacy of the exon-skipping therapies tested.

[0102] Despite the relatively low activity, functional improvements were observed in mice that received repeated administration of DG9-PMO (Fig. 3g, h; Fig. 7a, b). Treated hDMDdel52;mdx mice had an average restored level of dystrophin in skeletal muscle that was 2.8%-3.9% of wild type (Fig. 3c). Concomitantly, fiber size was also improved, as seen in the tibialis anterior and diaphragm (Fig. 7c, e, f). This result supports the idea that not much dystrophin may be required to achieve functional benefits in vivo. Indeed, mdx mice with nonrandom X-chromosome inactivation (mdx-Xist Δhs Previous studies in mice have shown that as little as 3%–14% of normal levels of dystrophin were sufficient to improve performance in the hanging test and grip strength test to wild-type levels (36).

[0103] Concerning multi-exon skipping, it was promising that DG9 conjugates of the minimized “blocking” cocktail successfully skipped exons 45–55 with 9.5% efficiency, restoring dystrophin production in hDMDdel52;mdx mice at about 0.8% of wild-type levels on average (Fig. 4b, c). The “all” cocktail was previously shown to induce 15% exon 45–55 skipping efficiency when intramuscularly treated into different humanized DMD mouse models at a dose of 1.67 μg / PMO

[13] . It is clear that a higher dose of the “blocking” cocktail is required to induce skipping levels equivalent to the “all” cocktail in vivo. Since the human and mouse transcriptional target sequences for DG9-PMO in the “blocking” cocktail are 70%–93% identical, it is possible that the mouse Dmd transcripts expressed in hDMDdel52;mdx mice may sequester a portion of the administered DG9-PMO. Crossing the hDMDdel52 transgene into a mouse Dmd-null background

[37] may eliminate this possibility and obtain more representative efficacy results.

[0104] method Cell culture. All immortalized human muscle cells were kindly provided by the MRC Centre for Neuromuscular Disease Biobank (NHS Research Ethics Committee reference 06 / Q0406 / 33, HTA license number 12198) through Dr. Francesco Muntoni. The following immortalized patient-derived myoblast cell lines were used: KM155 (healthy), KM571 (DMDΔex52), 6594 (DMDΔex48–50), and 6311 (DMDΔex45–52). Information on these cell lines is summarized in Table 2. Myoblasts were grown in DMEM / F12 medium (HEPES-containing; Gibco) containing 20% ​​fetal bovine serum (Sigma), 1 vial of skeletal muscle growth supplement mix (Promocell), 50 U / mL penicillin, and 50 µg / mL streptomycin. Myoblasts were then plated onto collagen type 1-coated 12-well plates at 0.53 × 10 5 cells / cm 2 Cells were seeded at a density of 1000 x g / mL. Upon reaching 90% confluence, myoblasts were differentiated into myotubes by replacing the growth medium with differentiation medium (DMEM / F12 containing 2% horse serum [GE Healthcare], 1x ITS solution [Sigma], 50 U / mL penicillin, and 50 μg / mL streptomycin). All cells were cultured at 37 °C and 5% CO2.

[0105] PMO transfection. The PMOs used are summarized in Table 1 and were taken 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 myofibers on day 3 after differentiation using 6 μM Endoporter reagent (Gene Tools) in differentiation medium. Each PMO in the cocktail was transfected at a final concentration of 5 μM for all experiments. Cells were incubated in PMOs for 2 days, after which RNA and protein were harvested from the cells (Figure 1a). Random control 25-N (Gene Tools) was used for mock treatment. For non-treated samples, transfection was performed only without any PMO as described with Endoporter.

[0106] Animal treatment. Only male hDMDdel52;mdx mice (C57BL / 6J background) heterozygous for the hDMDdel52 transgene (2, 3) were used. For single exon skipping studies, DG9 (sequence N-YArVRRrGPRGYArVRRrGPRr-C; uppercase: L-amino acids, lowercase: D-amino acids) was conjugated to the 3' end of Ex51_Ac0 (Table 1), a human DMD exon 51 skipping PMO that we previously developed [1, 4]. We performed two experiments: single-dose and repeated-dose. For single-dose treatment, 3-month-old hDMDdel52;mdx mice were injected retro-orbitally with either phosphate-buffered saline (PBS), PMO (50 mg / kg, unconjugated Ex51_Ac0), or DG9-PMO (64 mg / kg, equimolar to 50 mg / kg PMO) and then euthanized 1 week later for tissue collection. For repeat-dose treatment, 2-month-old hDMDdel52;mdx mice received three retro-orbital injections of either PBS or DG9-PMO (30 mg / kg) once a week for 3 weeks. Body weights were recorded during the treatment period, and mice underwent mouse muscle function testing before the first injection and 2 weeks after the last injection, as described in Functional Testing. After functional testing, mice were euthanized for tissue collection. After dissection, tissues were mounted on cork with tragacanth gum and flash frozen in liquid nitrogen-cooled isopentane.

[0107] For multi-exon skipping, DG9 was conjugated to each PMO in a minimized "blocked" exon 45-55 skipping cocktail (Fig. 1a). This DG9-PMO cocktail (5 μg per DG9-PMO, total dose 25 μg) was then administered intramuscularly into the tibialis anterior muscle of 5- to 6-month-old hDMDdel52;mdx mice, followed by an injection of PBS into the contralateral leg. Mice were euthanized 1 week later for tissue collection as described above. All injections in the retro-orbital and intramuscular regions were performed under isoflurane anesthesia. Tissue from age-matched wild-type male C57BL / 6J mice was harvested as controls.

[0108] Functional testing. Forelimb and whole limb grip strength testing was performed by blinded personnel using a Chatillon DFE II grip strength meter (Columbus Instruments) according to TREAT-NMD SOP DMD_M.2.2.001. The average of the three most consistent measurements per mouse was used, and results were normalized to body weight. The rotarod test was performed using an AccuRotor 4-channel rotarod (Omnitech Electronics, Inc.). Mice were first placed on the rod rotating at a constant speed of 5 rpm. Once all mice were in position, the rotation was accelerated from 5 to 45 rpm over a period of 300 s [5]. Fall times were recorded automatically by the software. Three trials were performed with a 15-min interval between each trial, and the mean or peak fall time of these trials was used for analysis. Run-to-Exhaustion tests were performed on an Exer 3 / 6 animal treadmill (Columbus Instruments, Inc.) according to TREAT-NMD SOP DMD_M.2.1.003. Tests were performed using a 3-D CT scanner (Mitsubishi UFG Instruments). Mice ran on a treadmill using the following program: 5 m / min for 5 min, then the speed increased by 1 m / min every minute until exhaustion. Exhaustion was considered as the point at which the mouse did not return to the treadmill within 10 seconds of repeated gentle pushing. The maximum test duration was set at 15 min. All tests were performed at baseline (before receiving any treatment) and 2 weeks after receiving the final treatment. Age-matched wild-type male C57BL / 6J mice were used as controls.

[0109] RT-PCR and exon skipping assessment Total RNA was extracted from cells and 20 μm tissue sections using Trizol (Invitrogen) according to the manufacturer's instructions.

[0110] For exon 45-55 skipping analysis, the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq (Invitrogen) was used. Briefly, a solution of 200 ng of total RNA in 25 μL solution containing 1× reaction mix, 0.2 μM each of forward and reverse primers for DMD or GAPDH / Gapdh (Table 3), and 1 μL of SuperScript III RT / Platinum Taq was used as template. Reactions were performed under the following conditions: 1) 50°C, 5 min; 2) 94°C, 2 min; 3) 94°C, 15 sec; 60°C, 30 sec; 35 cycles of 68°C, 33-118 sec; 4) 68°C, 5 min; 5) 4°C, hold.

[0111] For exon 51 skipping analysis, cDNA was synthesized from 750-1000 ng of total RNA using SuperScript™ IV Reverse Transcriptase (Invitrogen) with 2.5 μM random hexamers (Invitrogen) in a 20 μL reaction according to the manufacturer's instructions. From this cDNA, 8 μL of cDNA was used for PCR in a 25 μL reaction using 1× GoTaq® Green Master Mix (Promega) and 0.3 μM of each forward and reverse primer for DMD or Gapdh (Table 3). Reactions were performed 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 solution with 1.5% agarose gel in 1x Tris-borate-EDTA buffer, and band intensities were quantified by ImageJ (NIH). The % of successful exon skipping was calculated using the following formula: (intensity of desired skip band / total intensity of non-skipped, intermediate, and desired skip bands) x 100.

[0112] Western blots. Total protein was extracted from cells using RIPA buffer (Sigma) supplemented with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Roche) according to our previously published protocol (6). Meanwhile, total protein was extracted from 20 μm tissue sections using high SDS lysis buffer containing 10% SDS in water, 70 mM Tris-HCl (pH 6.7), 5 mM EDTA (pH 8.0), 5% β-mercaptoethanol, and cOmplete protease inhibitor cocktail, further following our previous protocol (7). Protein was quantified using the Pierce™ BCA kit (Thermo Scientific) or Pierce™ Coomassie (Bradford) kit (Thermo Fisher), respectively.

[0113] In preparation for Western blotting, proteins were mixed with NuPAGE™ LDS Sample Buffer (Invitrogen, 1× final concentration) and NuPAGE™ Sample Reducing Agent (Invitrogen, 1× final concentration) and then heated at 70° C. for 10 min. SDS-PAGE was performed using pre-cast NuPAGE™ 3-8% Tris Acetate Midi Gels (Invitrogen) at 150 V for 75 min. Proteins were then transferred to a PVDF membrane (Millipore) using a semi-dry blotting system at 20 V for 70 min. Membranes were blocked with 2% ECL Prime Blocking Agent (GE Biosciences) at 4° C. with shaking. The gels were blocked overnight in 1:100 Fibroblast Growth Hormone (Healthcare) and the transferred gels were stained with PageBlue protein staining solution (Thermo Scientific) for 1 hour at room temperature to detect myosin heavy chain bands. After blocking, the membranes were cut and incubated with one of the following primary antibodies for 1 hour at room temperature: 1:200 NCL-DYS1 (Leica), 1:2,500 anti-dystrophin C-terminus (Abcam, ab15277), 1:100 MANEX45A (Developmental Studies Hybridoma Bank, DSHB), (8) 1:100 MANEX4850E (MDA Monoclonal Antibody Resource for Inherited Neuromuscular Disorders, Wolfson Centre). Centre) (8), or 1:4,000 anti-desmin (Abcam, ab8592).Membranes were then washed three times for 10 min each with PBS containing 0.05% Tween 20 (PBST) and incubated as appropriate with either anti-mouse IgG2a, anti-mouse IgG1, or anti-rabbit IgG(H+L) horseradish peroxidase-conjugated secondary antibodies (Invitrogen), all at 1:10,000 in PBST. Membranes were then similarly washed in PBST and detected with ECL Select detection reagents (GE Healthcare). The intensity of the DYS1 band was quantified using Image Lab Proprietary software, v.6.0.1 (Bio-Rad), and dystrophin levels were expressed relative to the intensity of the lowest concentration wild-type sample in the series.

[0114] Dystrophin immunofluorescence. Frozen muscle and heart samples were cut to a thickness of 7 μm and placed on poly-L-lysine-coated slides. After thawing for 30 min at room temperature, sections were blocked in PBS with 10% goat serum and 0.1% Triton X-100 for 2 h at room temperature. Sections were then incubated overnight at 4° C. with a solution of 1:50 NCL-DYS1 in blocking agent. The next day, sections were washed three times with PBS for 5 min each, followed by incubation with Alexa 488-labeled goat anti-mouse IgG2a secondary antibody (Life Technologies) for 30 min at room temperature. Sections were washed again with PBS and mounted with Vectashield HardSet Antifade mounting medium (Vector Laboratories) with DAPI. Samples were visualized for dystrophin and DAPI at 200x magnification using a Zeiss LSM710 confocal microscope by personnel blinded to the treatment conditions.

[0115] Histology. Frozen muscles were sectioned at 7 μm and placed on poly-L-lysine-coated slides. After thawing for 30 min at room temperature, slides were stained with Mayer's hematoxylin (Electron Microscopy Sciences) for 15 min, washed in running water for 15 min, and then stained with Eosin Y (Electron Microscopy Sciences) for 10 min. Sections were then dehydrated using an ethanol series (70%-90%-99%), cleared with xylene substitute, and mounted with Permount™ (Fisher Scientific). A blinded investigator visualized the samples at 200x magnification using an Optika B-290TB microscope under bright field and photographed three randomly selected fields per sample. The percentage of CNF was calculated by (#CNF / total number of fibers) × 100, and the number of fibers was counted manually using ImageJ. The average CNF percentage from all fields was obtained for each sample. The minimum Feret's diameter was quantified by blinded personnel in two steps. First, images were measured semi-automatically using an in-house developed ImageJ macro based on Open-CSAM (9). Because Open-CSAM was initially developed for immunofluorescence images, we had to extensively modify Open-CSAM for compatibility with hematoxylin and eosin stained images, which required the use of the Colour Deconvolution 2 plugin [10, 11]. Second, images with semi-automated measurements were manually curated to correct for fiber boundaries. Individual fiber measurements across the entire sample were included in the analysis. For both CNF and minimum Feret's diameter quantification, fibers bordering the edge of the image were not considered.

[0116] Statistical analysis. All statistical tests were performed using Prism v.9.0.1 (GraphPad Software). Unpaired two-tailed t-tests or one-way ANOVA with post-hoc Tukey or Dunnett multiple comparison tests were performed as appropriate. P values ​​<0.05 were considered statistically significant.

[0117] [Table 1]

[0118] [Table 2]

[0119] [Table 3]

[0120] [Table 4]

[0121] Example 2: Peptide-conjugated PMOs for the treatment of spinal muscular atrophy Spinal muscular atrophy (SMA) is the most common genetic cause of infant death resulting from mutations in the survival motor neuron 1 (SMN1) gene. Humans have an SMN1 paralog, SMN2. However, SMN2 cannot rescue the SMA phenotype because 90% of exon 7 in SMN2 mRNA is spliced ​​out by a single C-to-T nucleotide transition. The currently approved antisense oligonucleotide (AO) nusinersen (trademark Spinraza) targets SMN2 and induces SMN expression by promoting exon 7 inclusion. However, it is difficult to deliver AO to both the central nervous system (CNS) and tissues throughout the body. Current approaches are limited to using highly invasive intrathecal injections, primarily treating motor neurons, but recent studies indicate that SMA is a multi-organ disease and a systemic therapeutic approach is needed. To avoid intrathecal injections, we use a novel cell-penetrating peptide called DG9, which significantly increases the uptake of AO after a single subcutaneous administration. We demonstrated that DG9-conjugated AO (DG9-PMO) extends the lifespan of SMA mice and improves respiratory dysfunction, the most common cause of death in SMA. DG9-PMO treatment significantly increases full-length SMN2 expression and SMN protein levels in both peripheral and central nervous system tissues. Furthermore, treatment also improved skeletal muscle and neuromuscular junction pathology. Finally, we show that DG9-PMO can cross a fully developed blood-brain barrier and be distributed to the CNS after systemic injection.

[0122] [result] Subcutaneous administration of DG9-PMO enhances survival and increases SMN2 expression in severe SMA mice in a dose-dependent manner To investigate whether the DG9 peptide can enhance the efficiency of PMO, the DG9 peptide was conjugated to an 18-nt PMO with the same sequence as nusinersen. The DG9-conjugated PMO, DG9-unconjugated PMO, and 2'-O-methoxyethyl-RNA (MOE) were injected into SMA model mice. The Taiwanese SMA mouse model (Smn), which shows a severe phenotype and a median survival of 8 days (d), was used. - / - SMN2 Tg / - )[77,78] were used.

[0123] Mice were subcutaneously injected with AO or saline (NT) at two different doses - 40 mg / kg or 80 mg / kg - on postnatal day 0 (PD0). Survival was recorded until a humane endpoint was reached. For the 40 mg / kg dose, the median survival was 12 days (unconjugated PMO), 15.5 days (MOE), and increased to 58 days with DG9-PMO treatment (Figure 9a). For the higher dose of 80 mg / kg, the median survival increased to 57 days (unconjugated PMO), 121 days (DG9-PMO), and 64 days (MOE) (Figure 9a). This result indicates that DG9-PMO can extend the lifespan of SMA mice more effectively than unmodified PMO. For both doses, DG9-PMO treated mice weighed significantly more than NT, non-conjugated PMO treated littermates at postnatal day 7 (PD7) and age-matched heterozygous littermates (Hets) used as healthy controls (Smn + / - SMN2 Tg / - ), there was no significant difference in body weight and they display normal wild-type (WT)-like characteristics (Figure 9b). DG9-PMO treated mice closely resembled Het, whereas unconjugated PMO treated newborns displayed a weak phenotype similar to NT mice (Figure 16).

[0124] Next, FL-SMN2 expression was assessed using real-time quantitative PCR (RT-qPCR) at PD7 in comparison to Δ7SMN2 transcripts. At both doses, DG9-PMO treatment led to 4- to 30-fold higher FL-SMN2 expression than NT controls in both peripheral and CNS tissues (Figure 10a). Furthermore, it led to an approximately 5-fold increase in FL-SMN2 expression when compared to unconjugated PMO and MOE treatment in most tissues (Figure 10a, Figure 16a). These data indicate that DG9-PMO can induce FL-SMN2 expression more efficiently than unmodified PMO. These results were validated at the protein level using Western blotting, where DG9-PMO treatment increased SMN protein levels in both peripheral and CNS tissues (Figure 10b, Figure 16b). We further revealed that subcutaneous administration of DG9-PMO at 40 mg / kg led to sustained SMN expression levels at PD30 (Figure 17). DG9-PMO and MOE treatments had similar SMN levels in the study tissues. These results indicate that DG9-PMO is more effective than unconjugated PMO with a dose-dependent effect.

[0125] Subcutaneous administration of DG9-PMO improves motor function and muscle strength in SMA mice To evaluate the effect of systemic DG9-PMO treatment on muscle strength and motor neuron function, we compared the motor function of treated and NT mice at various time points using several functional tests. Hindlimb suspension (HLS) analysis and righting reflex tests were performed during the first few weeks of life. From PD6 onwards, NT mice were unable to extend their hindlimbs when suspended by their tails, resulting in a decline in HLS scores (Figure 9c). These mice also showed a decrease in tube latency. At PD12, DG9-PMO-treated neonates showed hindlimb strength comparable to Het, with significantly higher scores and longer tube latency than uncoupled PMO and NT mice (Figure 9c). In the righting reflex test, DG9-PMO mice took significantly less time to rise on their forepaws between PD6 and PD10, indicating improved muscle strength and coordination (Figure 9d). Unconjugated PMO and MOE treated mice that survived beyond PD12 performed better but still took longer than DG9-PMO mice. It should be noted that these results are slightly skewed as only the healthiest unconjugated PMO and MOE treated mice survived to these time points. This improvement was further reinforced by forelimb grip strength assessment at PD30 in DG9-PMO treated mice (Figure 9e). DG9-PMO treated mice demonstrated forelimb grip strength comparable to Het and significantly higher than unconjugated PMO and MOE treated mice (Figure 9e). We also evaluated the relationship between forelimb data at PD30 and sex. Surprisingly, no significant differences were observed between males across groups, but significant differences were observed in females (Figure 18a). At PD60, there was no significant difference between the performance evidenced by Het and treated mice (Figure 9e). Uncoupled PMO mice did not survive to PD60 and were excluded from this portion of the study. We observed similar results in the 80 mg / kg dose study at both PD30 and PD60 (Figure 18b). We further performed the rotarod test at PD30 for the 40 mg / kg group to assess overall muscle coordination and balance.Mice across all groups varied in their ability to coordinate and balance on a rotating beam, with no significant differences detected

[36] (Figure 18c).Based on conclusive findings from these functional tests, systemic injections of DG9-PMO improve motor function and muscle strength in SMA mice.

[0126] DG9-PMO treatment improves respiratory function in neonatal SMA mice The majority of SMA patients suffer from respiratory dysfunction and rely on external respiratory support. To evaluate the effect of DG9-PMO treatment on respiratory function, we performed whole-body plethysmography recordings under normoxic (21% O2) and hypoxic (11% O2) conditions at PD7 (Fig. 11a). Het mice did not exhibit a respiratory phenotype under normoxic conditions. On the other hand, NT mice showed slow and irregular breathing indicated by a high coefficient of variation (CoV, a measure of relative variation) in frequency, and prominent apneas (no change in airflow / pressure for a period equal to or greater than two complete respiratory cycles) (Fig. 11b, f). Half of the uncoupled PMO mice (n=14) showed parameters similar to those seen in NT mice, whereas the other half were similar to Het and showed increased respiratory frequency (fR, number of breaths per minute), minute ventilation (VE), and tidal volume (VT, amount of air entering and leaving the lungs during each respiratory cycle) when compared to NT controls (Figure 11b, d). Under the same normoxic conditions, the majority of DG9-PMO (n=11) and MOE (n=10) treated mice did not show any respiratory phenotype and were similar to Het mice (Figure 11b-f).

[0127] When switched to a hypoxic environment, Het, DG9-PMO, and MOE treated mice showed increased respiratory parameters-fR, VE, and VT compared to normoxia (Fig. 11b-d). Although hypoxia reduced the number of apneas, breathing was still slow and irregular as revealed by higher CoV in NT mice (Fig. 11e). Most of the uncoupled PMO neonates showed increased respiratory parameters and no effect on apneas relative to normoxia when compared to NT mice, but breathing was still slow, weak, and irregular when compared to Het (Fig. 11b-d). Correlation between severity of respiratory phenotype (reduced fR or VE) and weight loss was examined by using Pearson product-moment correlation t-test (Fig. 11g). We observed no correlation between fR and weight in Het but revealed a strong correlation in NT and treated mice (grouped as homozygotes). Due to the low CoV and the significant improvement in respiratory parameters, DG9-PMO treatment ameliorated the respiratory dysfunction seen in SMA mice.

[0128] DG9-PMO treatment ameliorates muscle pathology and neuromuscular junction (NMJ) properties in SMA mice Atrophic muscle tissue is a classic characteristic of SMA with reduced skeletal muscle fiber size. To determine the effect of treatment on muscle pathology, we evaluated the physiology and structure of muscle fibers in the two affected muscle groups, quadriceps and intercostal muscles, and the less affected diaphragm, at PD7 and PD30 [79, 80]. We quantified the cross-sectional area (CSA), minimum Feret's diameter, and centrally nucleated fibers of at least 500 muscle fibers per muscle for each treatment using hematoxylin and eosin (H&E) staining at PD7 and PD30 (Figures 12a, b, 19, and 20).

[0129] At PD7, myofiber ferret diameter and CSA were significantly larger after DG9-PMO treatment in all three muscle groups when compared to NT controls (Fig. 12b, Fig. 19). In the quadriceps, there was no significant difference between Het and DG9-PMO treated myofiber sizes. Meanwhile, uncoupled PMO and MOE quadriceps fibers were significantly smaller than DG9-PMO quadriceps fibers. In the diaphragm and intercostal muscles, all three treatment groups showed similar myofiber sizes (Fig. 12b, Fig. 19). Furthermore, DG9-PMO treatment led to a significant decrease in the percentage of centrally nucleated fibers, indicative of degenerated 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 muscle fibers and a higher proportion of centrally located nuclei compared to DG9-PMO in all three tissue types (FIG. 20c).

[0130] SMA mice typically begin to show neuropathological deficits around PD4–PD5, paralyzing nerves and disrupting neuromuscular junctions (NMJs) as the disease progresses

[37] . We assessed NMJ structure in quadriceps and intercostal muscles at PD30 to understand phenotypic recovery after injection at PD0 (Figure 13a). DG9-PMO treatment restored NMJ integrity, increased endplate size, reduced denervation, and showed a similar innervation pattern to Het (Figure 13a–c). Peripheral synapses in DG9-PMO-treated muscles revealed over 60% complete innervation, whereas uncoupled PMO and MOE treatment had closer to 40%–50% innervation (Figure 13b). Uncoupled PMO-treated mice showed smaller endplates and vesicle disruption, especially in the intercostal muscles (Figure 11c). These data show that DG9-PMO treatment ameliorates the NMJ phenotype in SMA mice, supporting our hypothesis that DG9-PMO treatment rescues muscle-neuron crosstalk by ameliorating muscle pathology and NMJ properties.

[0131] DG9 peptide increases PMO uptake in both systemic and CNS tissues after a single subcutaneous administration To determine the effect of the DG9 peptide in increasing PMO uptake, we assessed the biodistribution of unconjugated PMO and DG9-PMO in peripheral and CNS tissues

[82] . We compared the concentrations of DG9-PMO and unconjugated PMO in tissues of treated PD7 mice using an established hybridization-based ELISA assay specifically designed to detect PMO

[82] . Conjugation of DG9 to PMO resulted in significantly higher uptake in most tissues except liver, with a mean AO detection value of 12202 pM (quadriceps, (n = 5)) and 2 × 10 8 pM (liver (n = 5)), 8359 pM (brain (n = 5)), 13907 pM (spinal cord (n = 4)), 8886 pM (heart (n = 4)), 2 × 10 7 pM (kidney (n=6)) (FIG. 14a). Both unconjugated PMO and DG9-PMO showed high levels in the liver and kidney, which are responsible for the metabolism and elimination of PMO.

[0132] PMO uptake is mediated by a caveolin-dependent pathway in myotubes

[83] . Some CPP-PMOs are often trapped in endosomes, limiting the efficiency of splicing correction

[84] . To demonstrate the subcellular localization of DG9-PMO, we fluorescently tagged DG9-PMO. SMA mice were injected subcutaneously (40 mg / kg) at PD0, and immunohistochemistry (IHC) was performed to examine the localization of DG9-PMO in frozen tissue sections taken at PD7. Some DG9-PMO localized to cell nuclei in the heart and quadriceps, and to a lesser extent in CNS tissues (Figure 14b). This experiment shows that DG9 promotes PMO uptake in both peripheral and CNS tissues after a single subcutaneous administration, thereby globally increasing SMN levels and ameliorating the SMA phenotype.

[0133] DG9-PMO crosses the blood-brain barrier in a mild SMA model To examine whether DG9-PMO can cross the fully developed BBB, we used a milder SMA model (F0) (Smn - / - SMN2 + / + ) were injected with DG9-PMO or fluorescently tagged DG9-PMO at PD5. These mice have a normal life span and only show tail and ear necrosis at approximately 8-12 weeks of age. Tissues were harvested at PD7. First, we observed that fluorescently tagged DG9-PMO was localized in the nuclei of cells in both peripheral and CNS tissues (Figure 15a). This supports our hypothesis that DG9-PMO can cross the BBB. ELISA was performed to examine the biodistribution of unconjugated PMO and DG9-PMO. Despite subcutaneous administration at PD5, DG9 significantly increased PMO uptake in CNS and peripheral tissues (Figure 15b). Finally, we analyzed FL-SMN2 levels normalized to saline-injected mice (NT control) and observed increased FL-SMN2 expression in the spinal cord and other peripheral tissues (Figure 15c). These findings demonstrate that DG9-PMO is able to cross the fully developed BBB and ensure widespread distribution of AO in both peripheral and CNS tissues.

[0134] DG9-PMO treatment restores the SMA phenotype without apparent toxicity Peptides are usually presented as antigens and can trigger immune responses. Therefore, we investigated the susceptibility of DG9-PMO to trigger immune activation in the early neonatal stage by examining CD68-positive cells (CD68) that show circulating and tissue macrophages in quadriceps muscle sections. + ) at PD7 (Fig. 21a). NT control mice showed muscle atrophy in SMA, leading to the loss of CD68 + showed elevated levels of macrophages.

[0135] Unconjugated PMO and MOE treated muscles down-regulate CD68 when compared to Het +The number of macrophages was significantly higher in NT and DG9-PMO mice, whereas there was no significant difference in the number of macrophages in NT and DG9-PMO mice (Figure 21a). The apparent decrease in circulating macrophages after DG9-PMO treatment is likely due to an improvement in atrophied muscle that would compensate for any increase seen from the treatment itself.

[0136] To further elucidate possible long-term toxicity, serum from Het- and Het-treated mice was collected at PD30–PD35. Toxicity assessment was performed for the levels of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatinine, total bilirubin, total protein, albumin, globulin, and gamma-glutamyltransferase (GGT). All indices tested were comparable between groups, indicating no obvious toxic effects (Figure 21b). We further performed qualitative histological analysis of liver and kidney, which revealed no observable signs of toxicity (Figure 21c). These results highlight that DG9-PMO does not show obvious toxicity in mice and is not associated with immune dysfunction.

[0137] [Consideration] Duplication and mutation of the telomeric SMN1 gene leads to the formation of its paralogous centromeric SMN2 gene with a few nucleotide differences

[85] . SMN2 is a valid target for the treatment of SMA patients because it can generate small amounts of functional SMN. 95% of SMA patients have mutations in the SMN1 gene, making the SMN2 gene a candidate for AO therapy to treat almost all SMA patients.

[0138] We used CPPs conjugated to AOs (PMOs) because some of the studied CPPs can deliver biological cargos into cells and also cross the BBB [86,73]. Some peptides have also demonstrated neuroprotective effects, minimizing the risk of toxicity and side effects, making them suitable for clinical application

[87] . There are still some concerns associated with nusinersen, and therapies addressing invasive administration, immune responses, toxicity, and other adverse events are still needed for SMA treatment.

[0139] In this study, we evaluated the efficacy of a novel peptide, DG9, identified by screening several peptides in a zebrafish model system

[75] . To overcome the invasive intraspinal injection and ensure systemic recovery of SMN protein, we subcutaneously injected neonatal SMA mice with 40 mg / kg DG9-PMO, unconjugated PMO, MOE, or saline at PD0. DG9-PMO treatment significantly extended survival and increased body weight, FL-SMN2 expression, and SMN levels in both peripheral and CNS tissues.

[0140] DG9-PMO-treated newborn and adult mice showed significant improvements in muscle strength and coordination, as well as in functional tests. We further observed that treatment with a high dose of AO at 80 mg / kg dose-dependently increased survival and FL-SMN2 expression, and that DG9-PMO-treated mice maintained improved motor function even at PD60.

[0141] DG9 peptide led to sustained delivery of PMO to skeletal and respiratory muscles, resulting in a decrease in the number of myofibers with central nuclei and an increase in myofiber size. At PD30, uncoupled PMO mice had significantly smaller myofibers in both intercostal and quadriceps muscles, suggesting a possible decline in respiratory function and atrophied myofibers responsible for early death. Although SMA mice are born normally, by PD7 they will exhibit reduced respiratory rate and increased apnea frequency, which is usually due to inspiratory and expiratory muscle weakness

[88] . Our plethysmography recordings at PD7 under normoxic and hypoxic conditions demonstrated that breathing was weak, slow, and irregular in NT SMA mice. Although we did not perform statistics to compare treatments alone, half of the PMO mice showed a severe respiratory phenotype with irregular breathing and apnea when compared to Het, whereas most of the MOE mice showed normal respiratory function. The variability of results, especially in uncoupled PMO mice, suggests a deeper understanding of respiratory dysfunction in SMA that leads to sudden death. Under both normoxic and hypoxic conditions, DG9-PMO mice had a respiratory outcome similar to Het and significantly better than NT PD7 mice. These findings provide evidence for the efficacy of DG9-PMO in rescuing early respiratory dysfunction in SMA mice.

[0142] At PD30, DG9-PMO mice showed improved NMJ structure, fewer collapsed structures, and increased innervation patterns. In contrast, uncoupled PMO mice showed a higher number of denervated endplates and collapsed NMJs, suggesting a clear SMA phenotype. Although the association between NMJs and motor neurons is not well established, NMJ and motor neuron development are closely linked. NMJ defects are an early pathology of motor neuron disease and require SMN for proper innervation [81, 89, 90]. Our treatment improved NMJ innervation, which inversely correlates with muscle atrophy

[94] , thereby translating into improved performance in functional tests.

[0143] Both peripheral and CNS tissues demonstrated significantly higher uptake of PMO when conjugated to DG9 peptide compared to unconjugated PMO alone, providing evidence that the peptide distributes AO widely, even to the brain and spinal cord. Our mouse model has a median survival of only 8 days, so the exact efficiency of treatment at the late symptomatic stage cannot be fully elucidated. To demonstrate the ability of DG9 to cross a fully developed BBB in mice, we used a mild model (Smn) with a normal life span. - / - ;SMN2 Tg / Tg ) was used. We observed robust uptake of PMO after a single subcutaneous injection at PD5 and localization of the DG9 peptide in CNS tissues. However, from a therapeutic perspective, our findings need to be corroborated in detail in other mild SMA models with multiple copies of SMN2 that could serve as models for treating late-onset SMA types III-IV or symptomatic clinical stage I-II patients.

[0144] Toxicity associated with cationic peptide-conjugated PMOs is usually seen within 24 hours after administration. There were no obvious physiological differences in neonates who received subcutaneous injections of AO. Neonates were as active as healthy neonates. We furthermore found that CD68 expression in the quadriceps muscle at PD7 was significantly increased in the neonates. + The number of cells was quantified, which indicates macrophages activated during an immune response in the body upon AO administration. SMA mice suffer from immune dysregulation and have elevated numbers of immune cells. DG9-PMO and NT mice have similar CD68 + Although we had a significant increase in cell numbers, we believe that DG9-PMO treatment may induce an immune response, but at the same time, the improvement in muscle pathology and atrophy muscle reduced the total number of circulating macrophages. No obvious toxicity was observed in the liver or kidneys according to serum and histological analysis at PD30.

[0145] material and method Synthesis of DG9-PMO DG9 (sequence N-YArVRRrGPRGYArVRRrGPRr-C; uppercase: L-amino acids, lowercase: D-amino acids) was synthesized and covalently attached to the 3' end of the PMO. PMO targeting ISS-N1 intron 7 (5'-TCACTTTCATAATGCTGG-3') was purchased from Gene Tools LLC. 2'MOE was purchased from Eurogentec North America, USA.

[0146] Animal models All animal experiments were performed at the University of Alberta and approved by the University of Alberta Research Ethics Animal Care and Use Committee. SMA transgenic mice (JAX stock #005058 FVB.Cg-Tg(SMN2)2HungSmn1 tm1Hung / J(Smn - / - ;SMN2 Tg / Tg ), also known as Taiwanese mice, were purchased from Jackson Laboratory (Bar Harbor, Maine, USA).

[0147] Heterozygous mice for Smn1 (Smn1 + / - ;SMN2 - / - ) to Smn1Smn - / - ;SMN2 Tg / Tg The mice were crossed with homozygous mice to generate SMA mice (Smn - / - ;SMN2 Tg / - ) or heterozygous healthy controls (Smn1 + / - ;SMN2 Tg / - ) were obtained. SMA mice display a severe overt phenotype similar to SMA type I patients. Mice were genotyped using PCR analysis on genomic DNA isolated from tail biopsies using the Phire Tissue Direct PCR kit (ThermoFisher) as per manufacturer's instructions. DNA was amplified using primers listed in Table 5 using the following conditions mouse Smn: 98°C / 5 min → 98°C / 5 sec_58°C / 10_72°C / 10 sec x 35 cycles → 72°C / 1 min.

[0148] treatment Injections were performed using a 30-gauge Hamilton syringe. SMA neonates were injected subcutaneously with 40 mg / kg or 80 mg / kg AO at P0, while NT and heterozygous control mice were injected with saline per group (n=10–25). Tissues including quadriceps, liver, kidney, spleen, diaphragm, intercostal muscles, heart, brain, and spinal cord were harvested and snap-frozen in isopentane cooled on dry ice and then stored at -80°C.

[0149] Real-time quantitative PCR (RT-qPCR) Frozen tissues harvested at P7 were cut into 20 μm sections using a cryostat (Leica CM1950, Leica). RNA was extracted from these sections using TRIzol 10 reagent (Invitrogen). cDNA was synthesized from 50 ng / μl RNA using superscript IV reverse transcriptase (ThermoFisher) and oligo(dT) primers (ThermoFisher) according to the manufacturer's instructions. qPCR reactions were performed using SsoAdvanced Universal SYBR Green Supermix (BioRad) and the QuantStudio 3 Real-Time PCR System (Applied Biosystems). Relative gene expression of full-length SMN2 versus the deleted SMN2 transcript lacking exon 7 was normalized to the NT control sample and analyzed using the ΔΔCt method.

[0150] Western blotting 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). Protein concentration was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). For SDS-PAGE, 5–10 μg protein per well was loaded onto NuPAGE Novex 4–12% Bis-Tris Midi protein gels (Life Technologies). The wells were run in a 150V Western Blotting Detection Kit (GE Healthcare) for 60 min at 150V per well, followed by semi-dry transfer at 20V for 30 min. Polyvinylidene difluoride (PVDF) membranes were blocked overnight with a solution of 5% skim milk and 0.05% Tween 20 in PBST (PBST). The membranes were incubated with mouse purified anti-SMN antibody (BD Biosciences) (1:10,000) for 1 h at room temperature (RT) under agitation. The membranes were washed three times with PBST (10 min each wash) and incubated with HRP-conjugated goat anti-mouse (IgGH+L) for 1 h at RT under agitation. Bands were detected using an Amersham ECL Select Western Blotting Detection Kit (GE Healthcare) and imaged using a ChemiDoc Touch Imaging System. For β-tubulin, membranes were incubated with stripping buffer (15 g glycine, 1 g SDS, 10 ml Tween 20, pH 2.2) for 10 min at RT, followed by two washes with PBS (10 min each) and two washes with Tris-buffered saline and 0.05% Tween 20 (TBST) (5 min each).Similar to the primary antibody protocol, the membrane was then blocked overnight and incubated the next day with β-tubulin rabbit antibody (Abcam ab6046, 1:5000) for 1 h at room temperature under agitation. The secondary antibody used was HRP-conjugated goat anti-rabbit (IgG H+L) tubulin (BioRad, 1:10,000). Bands were visualized as described above.

[0151] Enzyme-linked immunosorbent assay (ELISA) ELISA was performed as previously described [83,96]. Briefly, proteins were extracted from frozen tissue sections (approximately 20 μm) using RIPA buffer (Sigma) with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Sigma). Probes (Integrated DNA Technologies) were designed complementary to the PMO sequence with phosphorothioate backbones at the 5' and 3' ends. The 5' and 3' ends were labeled with digoxigenin and biotin, respectively. Tissue lysates (0.02 mg / ml protein concentration) were pretreated with 2.5 mg / mL trypsin containing 10 mM CaCl2 at 37°C overnight to digest the DG9 peptide. Probes were added to the samples and hybridized for 30 min at 37°C. After probe-PMO hybridization, the hybridized samples were transferred to Pierce NeutrAvidin coated 96-well plates, black (Thermo Fisher Scientific). Avidin-biotin interaction between the plate and the probe was then allowed by adding avidin-biotin to the plate (Promega Scientific). Unhybridized probes were digested using micrococcal nuclease enzyme (New England Biolabs) at 0.1 gel units / μl. Anti-digoxigenin antibody conjugated with alkaline phosphatase (1:5000, Roche Applied Sciences) was then added. 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).

[0152] Histology and immunohistochemistry H and E staining Seven-micrometer cryosections of quadriceps, diaphragm, and intercostal muscles were stained with Meyer's H&E reagent (electron microscopy reagent)

[97] . Centrally nucleated fibers and myofiber size were randomly selected, 500–800 per muscle per treatment. Cross-sectional area and minimum Fetter's diameter were quantified using Image J. All H and E analyses were performed in a blinded manner.

[0153] NMJ staining NMJ staining was performed as previously described

[98] . Briefly, quadriceps and intercostal muscles were fixed with 4% paraformaldehyde (PFA) for 2 h. Muscles were quickly washed with PBS and blocked with 5% goat serum and 2% Triton-X in PBS for 1 h at RT. They were then incubated overnight at 4°C with primary mouse monoclonal antibodies anti-neurofilament 2H3 (1:100) and anti-synaptophysin (1:500) (DSHB, Iowa). Muscles were then incubated in the dark with Alexafluor 488-goat anti-mouse IgG1 (1:1000) (Life Technologies) and Alexafluor 594-bungarotoxin (1:5000) (ThermoFisher Scientific). At least 300 NMJs were Z-stacked and visualized for analysis of denervation and cross-sectional area using a confocal microscope (Zeiss LSM710) and Zeiss Zen software. Synaptic area was quantified using ImageJ. All analyses were performed in a blinded manner.

[0154] Macrophage detection 7 μm frozen sections of quadriceps muscle were fixed with 4% PFA. Sections were incubated with rat anti-mouse CD68 antibody (Bio-Rad, MCA1957T). CD68 +Cell numbers were counted at 20x magnification at random intervals from at least five sections per sample and averaged.

[0155] Functional testing Righting reflex test Mice were tested for their ability to spontaneously right themselves from P2 to weaning age P20. Neonates were placed on their backs and the time it took them to return to a normal position and place all four paws on the ground was recorded. Maximum recording time was 60 seconds. Each neonate underwent three trials with at least a 10 minute rest period between trials. Data are expressed as the mean time ± SEM to complete the righting reflex test.

[0156] Hindlimb suspension analysis (tube test) This test was performed on P2-P12 neonatal mice as described in Treat NMD protocol SMA_M.2.2.001. Briefly, neonatal mice were suspended by their hindlimbs from a tube. Mice were scored based on hindlimb position and the latency to fall was recorded with a cutoff of 30 seconds. Each neonatal mouse was tested three times with a 15 minute break between each test. The average score was recorded by an observer blinded to the test.

[0157] Forelimb grip strength This assay was performed as described in Treat NMD protocol SMA_M.2.1.002. Briefly, mice were placed on the wire mesh of an automated grip dynamometer (Columbus Instruments) so that only the front paws could grip the metal grid. The mouse was steadily pulled with the help of the tail to completely release the front paws from the grid. P30 and P60 mice were used. Each mouse underwent three trials.

[0158] Rotarod test The rotarod test (AccuScan Instruments) was performed on mice between P30 and P40 using a 300 s acceleration profile as previously described

[99] .

[0159] toxicology Blood was collected from mice (n=4-10 per group) around P30-P40 during the dissection procedure. Blood was left at RT for 30 min and centrifuged at 2000rcf. The resulting supernatant (serum) was transferred to a new 1.5ml tube and stored at -20°C. Serum samples were analyzed by Idexxx Bioanalytics. 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.

[0160] Whole-body plethysmography recording Measurements were performed in a cylindrical, transparent, Plexiglas whole-body plethysmograph with one inlet and two outlet ports for continuous supply of fresh room air and removal of exhaled carbon dioxide [100,101]. The volumes of the plethysmograph were 10 ml (inner diameter: 1.9 cm, length: 3.5 cm for P7 mice weighing less than 2.5 g), 30 ml (inner diameter: 2.6 cm, length: 5.6 cm for P7 mice weighing more than 2.5 g), and 80 ml (inner diameter: 3.8 cm, length: 7 cm for P30) with flow rates of 15 ml / min, 45 ml / min, and 120 ml / min, respectively, for measurements of respiratory parameters. Gases were mixed in a gas mixer (GSM-3, CWE InC, USA), delivered from compressed pure oxygen and pure nitrogen canisters, and monitored using a gas regulator (Porter Instrument Company, USA) at 0–20 ml / min.

[0161] Hypoxic challenge (11% oxygen concentration for 5 min) was performed by switching the inlet gas from normoxia (21% oxygen concentration, balanced by nitrogen) to hypoxia (11% oxygen concentration, balanced by nitrogen) without physical handling of the animals and with continuous monitoring of plethysmographic recordings. Gas exchange was verified using a gas analyzer (model: ML206, ADInstruments) and took approximately 1 min to complete. For P7, the plethysmograph was contained within an incubator (Isolette, model C-86, Air-Shields / Drager Medical, USA) and the ambient temperature was maintained at approximately the incubator temperature of 32°C. For P30, plethysmographs were recorded at room temperature of approximately 22°C. Pressure changes were detected using a pressure transducer (model DP103, Validyne, USA), a signal conditioner (CD-15, Validyne), and recorded with data acquisition software (Axoscope) via an analog-digital board (Digidata 1322A). Signals were high-pass filtered (0.01 kHz) with a sampling rate of 1 kHz. Respiratory frequency and tidal volume (VT) were measured in blood pressure settings using Labchart 8 (AD Instruments Inc., USA). A threshold level for bursts was set, which were then automatically detected, thus calculating frequency (from cycle duration) and tidal volume (maximum pressure minus minimum pressure).

[0162] It should be noted that our plethysmograph is effective in studying respiratory frequency (fR) and detecting apnea. Apnea is defined as the absence of airflow (pressure change) for a period equal to or greater than two complete respiratory cycles. Our whole-body plethysmography system provides semiquantitative measurements of tidal volume (VT, mL / g) and minute ventilation, from which we report changes compared to wild-type normoxia (Ren et al., 2009, 2015). The coefficient of variation of frequency, a measure of relative variation, is the ratio of the standard deviation to the mean (average). The smaller the ratio, the more regular the breathing. The experiments were performed from 10:00 am to 5:00 pm. After the experiments, the animals were returned to the animal facility.

[0163] Respiratory parameters were calculated from continuous plethysmographic recordings averaged over 1 min. Respiratory parameters VT and VE were reported relative to the mean of heterozygotes in normoxia (100%). The nature of hypothesis testing was two-sided. We first performed normality tests (Shapiro-Wilk) and homogeneity of variance tests (Brown-Forsythe).

[0164] For data that passed both tests (Figure 6B-E: fR, VT, VE, and CV), parametric statistics were used with a two-way repeated measures analysis of variance (ANOVA), followed by the Holm-Sidak method (two factors: different treatments and different conditions). p<0.05 was considered a statistically significant difference, n refers to the number of animals, and animals were used as the unit of analysis for statistical tests. For data that failed either the normality test or the equal variance test (total apnea duration, Figure 6F), nonparametric statistics were applied. Comparisons of differences between normoxia or hypoxia were performed with a Kruskal-Wallis one-way ANOVA on ranks, followed by the Dunn method. Differences between hypoxia and normoxia were performed with a signed-rank test. To examine whether the severity of the respiratory phenotype (reduced respiratory frequency or VE) correlated with the loss of body weight, we used a Pearson product-moment correlation t test (Figure 6G). For whole-body plethysmography recordings, data are expressed as mean ± SD, or as the 1st quartile 25%, median 50%, and 3rd quartile 75% (Sigmaplot 11 Systat Software Inc., USA).

[0165] statistical analysis All statistical analyses for all data (except respiratory analyses) were performed using GraphPad Prism 9 software. One-way ANOVA with Tukey's test for multiple comparisons or log-rank Mantel-Cox test for survival analyses were used where appropriate. No statistical power calculations were performed prior to the study. Sample sizes were based on our previous experience with these experimental protocols.

[0166] [Table 5]

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[0178] The embodiments described herein are intended to be examples only. Changes, modifications, and variations may be made to the specific embodiments by those skilled in the art. The scope of the claims should not be limited by the specific embodiments described herein, but should be interpreted in a manner consistent with the specification as a whole.

[0179] All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0180] The invention being thus described, it will be apparent that the same may be modified in various ways, which modifications are not to be regarded as departures from the spirit and scope of the invention, and all such modifications that would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

1. A conjugate comprising an antisense oligonucleotide capable of modulating exon splicing in pre-mRNA linked to a cell penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids.

2. 2. The conjugate of claim 1, wherein the antisense oligonucleotide is capable of modulating exon splicing of human dystrophin pre-mRNA or SMN2 pre-mRNA.

3. A conjugate comprising an antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently linked to a cell-penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids.

4. 4. The conjugate of claim 3, wherein the antisense oligonucleotide binds to a target within 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.

5. 4. The conjugate of claim 3, wherein the antisense oligonucleotide comprises or consists of any one of the following oligonucleotides: 5'-AAAACGCCGCCATTTCTCAACAGATCTGTC-3', 5'-GACAACAGTTTGCCGCTGCCCAAATGCCATC-3', 5'-AGTTGCTGCTCTTTTCCAGGTTCAAGTGGG-3', 5'-GTTTGAGAATTCCCTGGCGCAGGGGCAACT-3', 5'-CAATTTCTCCTTGTTTCTCAGGTAAAGCTC-3', 5'-CAGATGATTTAACTGCTCTTCAAGGTCTTC-3', 5'-ATCTCTTCCACATCCGGTTGTTTAGCTTGA-3', 5'-GTAAACGGTTTACCGCCTTCCACTCAGAGC-3', 5'-GTGTCACCAGAGTAACAGTCTGAGTAGGAG-3', 5'-GGTAATGAGTTCTTCCAACTGGGGACGCCT-3', 5'-CCTCCGGTTCTGAAGGTGTTCTTGTACTTC-3', 5′-GAGAAGTTTCAGGGCCAAGTCATTTGCCAC-3′, and 5′-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3′.

6. a first conjugate comprising a first antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently linked to a cell-penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids. and a second conjugate comprising a second antisense oligonucleotide capable of inducing exon skipping in human dystrophin, covalently linked to a cell-penetrating peptide (CPP); composition.

7. 7. The composition of claim 6, further comprising at least one other conjugate comprising another antisense oligonucleotide capable of inducing exon skipping in human dystrophin, covalently linked to a cell-penetrating peptide (CPP).

8. 8. The composition of claim 7, comprising a peptide-conjugated antisense oligonucleotide targeted to exon 45, a peptide-conjugated antisense oligonucleotide targeted to exon 46, a peptide-conjugated antisense oligonucleotide targeted to exon 47, a peptide-conjugated antisense oligonucleotide targeted to exon 48, a peptide-conjugated antisense oligonucleotide targeted to exon 49, a peptide-conjugated antisense oligonucleotide targeted to exon 50, a peptide-conjugated antisense oligonucleotide targeted to exon 51, a peptide-conjugated antisense oligonucleotide targeted to exon 52, a peptide-conjugated antisense oligonucleotide targeted to exon 53, a peptide-conjugated antisense oligonucleotide targeted to exon 54, and a peptide-conjugated antisense oligonucleotide targeted to exon 55.

9. 8. The composition of claim 7, comprising a peptide-conjugated antisense oligonucleotide targeted to exon 45, a peptide-conjugated antisense oligonucleotide targeted to exon 47, a peptide-conjugated antisense oligonucleotide targeted to exon 49, a peptide-conjugated antisense oligonucleotide targeted to exon 51, a peptide-conjugated antisense oligonucleotide targeted to exon 53, and a peptide-conjugated antisense oligonucleotide targeted to exon 55.

10. 8. The composition of claim 7, comprising a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47, and a peptide-linked antisense oligonucleotide targeting exon 53.

11. 8. The composition of claim 7 comprising: A peptide-conjugated antisense oligonucleotide having the sequence 5'-GACAACAGTTTGCCGCTGCCCAAATGCCATC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-AGTTGCTGCTCTTTTCCAGGTTCAAGTGGG-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTTTGAGAATTCCCTGGCGCAGGGGCAACT-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-CAATTTCTCCTTGTTTCTCAGGTAAAGCTC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-CAGATGATTTAACTGCTCTTCAAGGTCTTC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-ATCTCTTCCACATCCGGTTGTTTAGCTTGA-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTAAACGGTTTACCGCCTTCCACTCAGAGC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTGTCACCAGAGTAACAGTCTGAGTAGGAG-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GGTAATGAGTTCTTTCCAACTGGGGACGCCT-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-CCTCCGGTTCTGAAGGTGTTCTTGTACTTC-3' A peptide-conjugated antisense oligonucleotide having the following sequence: 5'-GAGAAGTTTCAGGGCCAAGTCATTTGCCAC-3', and A peptide-conjugated antisense oligonucleotide having the sequence 5'-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3' wherein one or more thymines in the sequence are optionally substituted with uracil.

12. 8. The composition of claim 7 comprising: A peptide-conjugated antisense oligonucleotide having the sequence 5'-GACAACAGTTTGCCGCTGCCCAAATGCCATC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTTTGAGAATTCCCTGGCGCAGGGGCAACT-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-ATCTCTTCCACATCCGGTTGTTTAGCTTGA-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTGTCACCAGAGTAACAGTCTGAGTAGGAG-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-CCTCCGGTTCTGAAGGTGTTCTTGTACTTC-3', and A peptide-conjugated antisense oligonucleotide having the sequence 5'-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3' wherein one or more thymines in the sequence are optionally substituted with uracil.

13. 8. The composition of claim 7 comprising: A peptide-conjugated antisense oligonucleotide having the sequence 5'-GACAACAGTTTGCCGCTGCCCAAATGCCATC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTTTGAGAATTCCCTGGCGCAGGGGCAACT-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-ATCTCTTCCACATCCGGTTGTTTAGCTTGA-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-CCTCCGGTTCTGAAGGTGTTCTTGTACTTC-3', and A peptide-conjugated antisense oligonucleotide having the sequence 5'-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3' wherein one or more thymines in the sequence are optionally substituted with uracil.

14. 8. The composition of claim 7 comprising: A peptide-conjugated antisense oligonucleotide having the sequence 5'-GACAACAGTTTGCCGCTGCCCAAATGCCATC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-ATCTCTTCCACATCCGGTTGTTTAGCTTGA-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTAAACGGTTTACCGCCTTCCACTCAGAGC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GGTAATGAGTTCTTTCCAACTGGGGACGCCT-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-CCTCCGGTTCTGAAGGTGTTCTTGTACTTC-3', and A peptide-conjugated antisense oligonucleotide having the sequence 5'-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3' wherein one or more thymines in the sequence are optionally substituted with uracil.

15. 8. The composition of claim 7 comprising: A peptide-conjugated antisense oligonucleotide having the sequence 5'-GACAACAGTTTGCCGCTGCCCAAATGCCATC-3', A peptide-conjugated antisense oligonucleotide having the sequence 5'-GTAAACGGTTTACCGCCTTCCACTCAGAGC-3', and A peptide-conjugated antisense oligonucleotide having the sequence 5'-TCTTCCAAAGCAGCCTCTCGCTCACTCACC-3' wherein one or more thymines in the sequence are optionally substituted with uracil.

16. A pharmaceutical composition comprising a peptide conjugate according to any one of claims 1 to 5 or a composition according to any one of claims 6 to 15, and a pharmaceutically acceptable excipient.

17. A pharmaceutical composition for treating a subject with DMD, comprising a peptide conjugate described in any one of claims 1 to 5 or a composition described in any one of claims 6 to 15, wherein the treatment comprises administering to the subject a therapeutically effective amount of the peptide conjugate or the composition.

18. 18. The method of claim 17, wherein the subject is a human.

19. A conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in the human SMN2 gene covalently linked to a cell-penetrating peptide (CPP) comprising the following amino acid sequence: YArVRRrGPRGYArVRRrGPRr; Uppercase letters: L-amino acids, lowercase letters: D-amino acids.

20. 20. The conjugate of claim 19, wherein the antisense binds to the intronic splicing silencer N1 of the SMN2 pre-mRNA.

21. 20. The conjugate of claim 19, wherein the antisense oligonucleotide comprises or consists of the sequence 5'-TCACTTTCATAATGCTGG-3', wherein the thymine is optionally substituted with uracil.

22. The conjugate of any one of claims 19 to 21, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer.

23. 22. A pharmaceutical composition for treating spinal muscular atrophy (SMA) in a subject, comprising the conjugate of any one of claims 19 to 21, wherein said treatment comprises administering to said subject a therapeutically effective amount of said conjugate.

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