Hostname: page-component-78c5997874-xbtfd Total loading time: 0 Render date: 2024-11-11T14:58:10.118Z Has data issue: false hasContentIssue false
  • English
  • Français

Recent advancements in exon-skipping therapies using antisense oligonucleotides and genome editing for the treatment of various muscular dystrophies

Published online by Cambridge University Press:  02 October 2019

Jaeho Hwang  and
Toshifumi Yokota Open the ORCID record for Toshifumi Yokota [Opens in a new window]
Jaeho Hwang
Affiliation:
Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton Canada
Toshifumi Yokota*
Affiliation:
Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton Canada Muscular Dystrophy Canada Research Chair, Edmonton Canada
*
Author for correspondence: Toshifumi Yokota, E-mail: toshifumi.yokota@ualberta.ca
Get access Rights & Permissions [Opens in a new window]

Abstract

Muscular dystrophy is a group of genetic disorders characterised by degeneration of muscles. Different forms of muscular dystrophy can show varying phenotypes with a wide range of age, severity and location of muscle deterioration. Many palliative care options are available for muscular dystrophy patients, but no curative treatment is available. Exon-skipping therapy aims to induce skipping of exons with disease-causing mutations and/or nearby exons to restore the reading frame, which results in an internally truncated, partially functional protein. In antisense-mediated exon-skipping synthetic antisense oligonucleotide binds to pre-mRNA to induce exon skipping. Recent advances in exon skipping have yielded promising results; the US Food and Drug Administration (FDA) approved eteplirsen (Exondys51) as the first exon-skipping drug for the treatment of Duchenne muscular dystrophy, and in vivo exon skipping has been demonstrated in animal models of dysferlinopathy, limb-girdle muscular dystrophy type 2C and congenital muscular dystrophy type 1A. Novel methods that induce exon skipping utilizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are also being developed where splice site mutations are created within the genome to induce exon skipping. Challenges remain as exon-skipping agents can have deleterious non-specific effects and different in-frame deletions show phenotypic variance. This article reviews the state of the art of exon skipping for treating muscular dystrophy and discusses challenges and future prospects.

Keywords

Antisense oligonucleotideCRISPRDuchenne muscular dystrophydysferlinopathyeteplirsenexon skippinggenome editingGolodirsenlimb-girdle muscular dystrophy type 2CNS-065/NCNP-01 (viltolarsen)splice modulation
Type
Invited Review
Information
Expert Reviews in Molecular Medicine , Volume 21 , 2019 , e5
Copyright
Copyright © Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

1.Shieh, PB (2013) Muscular dystrophies and other genetic myopathies. Neurologic Clinics 31, 1009–1029.Google Scholar
2.Beroud, C et al. (2007) Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy. Human Mutation 28, 196–202.Google Scholar
3.Mercuri, E and Muntoni, F (2013) Muscular dystrophy: new challenges and review of the current clinical trials. Current Opinion in Pediatrics 25, 701–707.Google Scholar
4.Yiu, EM and Kornberg, AJ (2015) Duchenne muscular dystrophy. Journal of Paediatrics and Child Health 51, 759–764.Google Scholar
5.Wyatt, EJ et al. (2018) Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers. JCI Insight 3, e99357.Google Scholar
6.Aartsma-Rus, A et al. (2010) Therapeutic exon skipping for dysferlinopathies? European Journal of Human Genetics 18, 889–894.Google Scholar
7.Aartsma-Rus, A et al. (2003) Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Human Molecular Genetics 12, 907–914.Google Scholar
8.Aoki, Y et al. (2013) Highly efficient in vivo delivery of PMO into regenerating myotubes and rescue in laminin-alpha2 chain-null congenital muscular dystrophy mice. Human Molecular Genetics 22, 4914–4928.Google Scholar
9.Aartsma-Rus, A et al. (2002) Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromuscular Disorders 12, S71–S77.Google Scholar
10.Matsuo, M et al. (1991) Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe. Journal of Clinical Investigation 87, 2127–2131.Google Scholar
11.Dunckley, MG et al. (1998) Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Human Molecular Genetics 7, 1083–1090.Google Scholar
12.Lu, QL et al. (2005) Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proceedings of the National Academy of Sciences of the USA 102, 198–203.Google Scholar
13.Goyenvalle, A et al. (2015) Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers. Nature Medicine 21, 270–275.Google Scholar
14.Watakabe, A et al. (1993) The role of exon sequences in splice site selection. Genes & Development 7, 407–418.Google Scholar
15.Mann, CJ et al. (2001) Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proceedings of the National Academy of Sciences of the USA 98, 42–47.Google Scholar
16.Annemieke Aartsma-Rus, et al. (2010) Exonic sequences provide better targets for antisense oligonucleotides than splice site sequences in the modulation of Duchenne muscular dystrophy splicing. Oligonucleotides 20, 70–77.Google Scholar
17.Echigoya, Y et al. (2017) Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy. Proceedings of the National Academy of Sciences of the USA 114, 4213–4218.Google Scholar
18.Echigoya, Y et al. (2015) In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy. PLoS ONE 10, e0120058.Google Scholar
19.Bladen, CL et al. (2015) The TREAT-NMD DMD global database: analysis of more than 7000 Duchenne muscular dystrophy mutations. Human Mutation 36, 395–402.Google Scholar
20.Shimizu-Motohashi, Y et al. (2019) Restoring dystrophin expression in Duchenne muscular dystrophy: current status of therapeutic approaches. Journal of Personalized Medicine 9, 1.Google Scholar
21.Stein, CA (2016) Eteplirsen approved for Duchenne muscular dystrophy: the FDA faces a difficult choice. Molecular Therapy 24, 1884–1885.Google Scholar
22.Long, C et al. (2018) Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing. Science Advances 4, eaap9004.Google Scholar
23.Gapinske, M et al. (2018) CRISPR-SKIP: programmable gene splicing with single base editors. Genome Biology 19, 107.Google Scholar
24.Mou, H et al. (2017) CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion. Genome Biology 18, 108.Google Scholar
25.Amoasii, L et al. (2018) Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 86–91.Google Scholar
26.Pramono, ZAD et al. (1996) Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence. Biochemical and Biophysical Research Communications 226, 5.Google Scholar
27.Malcher, J et al. (2018) Exon skipping in a Dysf-missense mutant mouse model. Molecular Therapy Nucleic Acids 13, 198–207.Google Scholar
28.Mendell, JR et al. (2012) Evidence-based path to newborn screening for Duchenne muscular dystrophy. Annals of Neurology 71, 304–313.Google Scholar
29.Eagle, M et al. (2002) Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation. Neuromuscular Disorders 12, 926–929.Google Scholar
30.Beggs, AH et al. (1991) Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies. Human Genetics 49, 54–67.Google Scholar
31Monaco, AP et al. (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2, 90–95.Google Scholar
32.Juan-Mateu, J et al. (2015) DMD mutations in 576 dystrophinopathy families: a step forward in genotype-phenotype correlations. PLoS ONE 10, e0135189.Google Scholar
33.US Food Drug Administration Briefing Document (2016) Peripheral and Central Nervous System: Drugs Advisory Committee Meeting. NDA 20648 https://www.fda.gov/media/97306/downloadGoogle Scholar
34.Komaki, H et al. (2018) Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy. Science Translational Medicine 10, eaan0713.Google Scholar
35.Nakamura, A et al. (2017) Comparison of the phenotypes of patients harboring in-frame deletions starting at exon 45 in the Duchenne muscular dystrophy gene indicates potential for the development of exon skipping therapy. Journal of Human Genetics 62, 459–463.Google Scholar
36.Echigoya, Y et al. (2015) Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45–55 with a cocktail of vivo-morpholinos in mdx52 mice. Molecular Therapy Nucleic Acids 4, e225.Google Scholar
37.Lee, J et al. (2018) Antisense PMO cocktails effectively skip dystrophin exons 45–55 in myotubes transdifferentiated from DMD patient fibroblasts. PLoS ONE 13, e0197084.Google Scholar
38.Aoki, Y et al. (2012) Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery. Proceedings of the National Academy of Sciences of the USA 109, 13763–8.Google Scholar
39.Moore, SA et al. (2006) Limb-girdle muscular dystrophy in the United States. Journal of Neuropathology Experimental Neurology 65, 995–1003.Google Scholar
40.Illa, I et al. (2001) Distal anterior compartment myopathy: a dysferlin mutation causing a new muscular dystrophy phenotype. Annals of Neurology 49, 5.Google Scholar
41.Nguyen, K et al. (2007) Phenotypic study in 40 patients with dysferlin gene mutations. Archives of Neurology 64, 1176–1182.Google Scholar
42.Vandre, DD et al. (2007) Dysferlin is expressed in human placenta but does not associate with caveolin. Biology of Reproduction 77, 533–542.Google Scholar
43.Llanga, T et al. (2017) Structure-based designed nano-dysferlin significantly improves dysferlinopathy in BLA/J mice. Molecular Therapy 25, 2150–2162.Google Scholar
44.Krahn, M et al. (2008) Partial functionality of a mini-dysferlin molecule identified in a patient affected with moderately severe primary dysferlinopathy. Neuromuscular Disorders 18, 1.Google Scholar
45.Krahn, M et al. (2010) A naturally occuring human minidysferlin protein repairs sarcolemmal lesions in a mouse model of dysferlinopathy. Science Translational Medicine 2, 50–69.Google Scholar
46.Barthelemy, F et al. (2015) Exon 32 skipping of dysferlin rescues membrane repair in patients’ cells. Journal of Neuromuscular Diseases 2, 281–290.Google Scholar
47.Lee, JJA et al. (2018) Identification of novel antisense-mediated exon skipping targets in DYSF for therapeutic treatment of dysferlinopathy. Molecular Therapy Nucleic Acids 13, 596–604.Google Scholar
48.Fayssoil, A et al. (2016) Natural history of cardiac and respiratory involvement, prognosis and predictive factors for long-term survival in adult patients with limb girdle muscular dystrophies type 2C and 2D. PLoS ONE 11, e0153095.Google Scholar
49.Gao, QQ et al. (2015) Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping. Journal of Clinical Investigation 125, 4186–4195.Google Scholar
50.Durbeej, M (2015) Laminin-alpha2 chain-deficient congenital muscular dystrophy: pathophysiology and development of treatment. Current Topics in Membranes 76, 31–60.Google Scholar
51.Sanes, JR (2003) The basement membrane/basal lamina of skeletal muscle. Journal of Biological Chemistry 278, 12601–4.Google Scholar
52.Le Doan, T, Chavany, C and Helene, C (1989) Antisense oligonucleotides as potential antiviral and anticancer agents. Bulletin du Cancer 76, 849–852.Google Scholar
53.Bennett, CF (2019) Therapeutic antisense oligonucleotides are coming of age. Annual Review of Medicine 70, 307–321.Google Scholar
54.Sardone, V et al. (2017) Antisense oligonucleotide-based therapy for neuromuscular disease. Molecules 22, E563.Google Scholar
55.Manoharan, M (2002) Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense & Nucleic Acid Drug Development 12, 103–128.Google Scholar
56.Branda, RF et al. (1993) Immune stimulation by an antisense oligomer complementary to the reu gene of HIV-I*. Biochemical Pharmacology 45, 2037–2043.Google Scholar
57.Voit, T et al. (2014) Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study. Lancet Neurology 13, 987–996.Google Scholar
58.Summerton, JE (2007) Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity. Current Topics in Medicinal Chemistry 7, 651–660.Google Scholar
59.Yokota, T et al. (2009) Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Annals of Neurology 65, 667–676.Google Scholar
60.McArdle, A, Edwards, RHT and Jackson, MJ (1994) Time course of changes in plasma membrane permeability in the dystrophin-deficient mdx mouse. Muscle and Nerve 17, 1378–1384.Google Scholar
61.Moulton, HM and Moulton, JD (2010) Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy. Biochimica et Biophysica Acta 1798, 2296–2303.Google Scholar
62.Hoffman, EP et al. (2011) Restoring dystrophin expression in Duchenne muscular dystrophy muscle progress in exon skipping and stop codon read through. American Journal of Pathology 179, 12–22.Google Scholar
63.Ozawa, E, Hagiwara, Y and Yoshida, M (1999) Creatine kinase, cell membrane and Duchenne muscular dystrophy. Molecular and Cellular Biochemistry 190, 143–151.Google Scholar
64.Novak, JS et al. (2017) Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle. Nature Communications 8, 941.Google Scholar
65.Carver, MP et al. (2016) Toxicological characterization of exon skipping phosphorodiamidate morpholino oligomers (PMOs) in non-human primates. Journal of Neuromuscular Diseases 3, 13.Google Scholar
66.Copolovici, DM et al. (2014) Cell-penetrating peptides: design, synthesis, and applications. ACS Nano 8, 1972–1994.Google Scholar
67.O'Donovan, L et al. (2015) Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy. Nucleic Acid Therapeutics 25, 1–10.Google Scholar
68.Koren, E and Torchilin, VP (2012) Cell-penetrating peptides: breaking through to the other side. Trends in Molecular Medicine 18, 385–393.Google Scholar
69.Wu, B et al. (2012) Long-term rescue of dystrophin expression and improvement in muscle pathology and function in dystrophic mdx mice by peptide-conjugated morpholino. American Journal of Pathology 181, 392–400.Google Scholar
70.Ye, J et al. (2016) CPP-assisted intracellular drug delivery, what is next? International Journal of Molecular Sciences 17, 1892.Google Scholar
71.Morcos, PA, Li, Y and Jiang, S (2008) Vivo-morpholinos: a non-peptide transporter delivers morpholinos into a wide array of mouse tissues. Biotechniques 45, 613–614, 616, 618 passim.Google Scholar
72.Ferguson, DP, Dangott, LJ and Lightfoot, JT (2014) Lessons learned from vivo-morpholinos: how to avoid vivo-morpholino toxicity. Biotechniques 56, 251–256.Google Scholar
73.Renneberg, D and Leumann, CJ (2002) Watson-crick base-pairing properties of tricyclo-DNA. American Chemical Society 124, 5993–6002.Google Scholar
74.Relizani, K et al. (2017) Efficacy and safety profile of tricyclo-DNA antisense oligonucleotides in Duchenne muscular dystrophy mouse model. Molecular Therapy Nucleic Acids 8, 144–157.Google Scholar
75.Renneberg, D et al. (2002) Antisense properties of tricyclo-DNA. Nucleic Acids Research 30, 2751–2757.Google Scholar
76.Aartsma-Rus, A et al. (2009) Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms. Molecular Therapy 17, 548–553.Google Scholar
77.Pramono, ZA et al. (2012) A prospective study in the rational design of efficient antisense oligonucleotides for exon skipping in the DMD gene. Human Gene Therapy 23, 781–790.Google Scholar
78.Malueka, RG et al. (2012) Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers. BioMed Central Genetics 13, 23.Google Scholar
79.Wee, KB et al. (2008) Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides. PLoS ONE 3, e1844.Google Scholar
80.Stadler, MB et al. (2006) Inference of splicing regulatory activities by sequence neighborhood analysis. PLoS Genetics 2, e191.Google Scholar
81.Stein, CA (1996) Phosphorothioate antisense oligodeoxynucleotides: questions of specificity. Tibtech 14, 147–149.Google Scholar
82.Hache, M et al. (2016) Intrathecal injections in children with spinal muscular atrophy: nusinersen clinical trial experience. Journal of Child Neurology 31, 899–906.Google Scholar
83.Wu, B et al. (2009) Octa-guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice. Molecular Therapy 17, 864–871.Google Scholar
84.Goemans, N et al. (2018) A randomized placebo-controlled phase 3 trial of an antisense oligonucleotide, drisapersen, in Duchenne muscular dystrophy. Neuromuscular Disorders 28, 4–15.Google Scholar
85.van Meer, L et al. (2016) Injection site reactions after subcutaneous oligonucleotide therapy. British Journal of Clinical Pharmacology 82, 340–351.Google Scholar
86.Riebesehl, BU (2015) Drug delivery with organic solvents or colloidal dispersed systems. In The Practice of Medicinal Chemistry(4th ed.), Academic Press, pp. 699–722.Google Scholar
87.Roy, B et al. (2018) CRISPR/cascade 9-mediated genome editing-challenges and opportunities. Frontiers in Genetics 9, 240.Google Scholar
88.Bengtsson, NE et al. (2017) Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nature Communications 8, 14454.Google Scholar
89.Hale, CR et al. (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956.Google Scholar
90.Kapahnke, M, Banning, A and Tikkanen, R (2016) Random splicing of several exons caused by a single base change in the target exon of CRISPR/Cas9 mediated gene knockout. Cells 5, 45.Google Scholar
91.Amoasii, L et al. (2017) Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy. Science Translational Medicine 9, aan8081.Google Scholar
92.Amoasii, L et al. (2018) Erratum for the research article: ‘single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy’. Science Translational Medicine 10, aat0240.Google Scholar
93.Komor, AC et al. (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424.Google Scholar
94.Gerhke, JM, Cervantes, OR, Clement, MK, Pinello, L and Joung, JK (2018) High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations. bioRxiv. https://doi.org/10.1101/273938.Google Scholar
95.Ma, H et al. (2014) Pol III promoters to express small RNAs: delineation of transcription initiation. Molecular Therapy Nucleic Acids 3, e161.Google Scholar
96.Swarts, DC and Jinek, M (2018) Cas9 versus Cas12a/Cpf1: structure-function comparisons and implications for genome editing. Wiley Interdisciplinary Reviews. RNA 9, e1481.Google Scholar
97.Kim, E et al. (2017) In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun 8, 14500.Google Scholar
98.Friedland, AE et al. (2015) Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biology 16, 257.Google Scholar
99.Moreno-Mateos, MA et al. (2015) CRISPRscan: designing highly efficient sgRNRNAs for CRISPR-Cas9 targeting in vivo. Nature Methods 12, 982–988.Google Scholar
100.Yin, H et al. (2016) Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nature Biotechnology 34, 328–333.Google Scholar
101.Mingozzi, F and High, KA (2011) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nature Reviews Genetics 12, 341–355.Google Scholar
102.Kattenhorn, LM et al. (2016) Adeno-associated virus gene therapy for liver disease. Human Gene Therapy 27, 947–961.Google Scholar
103.Grimm, D et al. (2006) Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype. Journal of Virology 80, 426–439.Google Scholar
104.Xie, J et al. (2011) MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Molecular Therapy 19, 526–535.Google Scholar
105.Li, W et al. (2008) Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Molecular Therapy 16, 1252–1260.Google Scholar
106.Asokan, A, Schaffer, DV and Samulski, RJ (2012) The AAV vector toolkit: poised at the clinical crossroads. Molecular Therapy 20, 699–708.Google Scholar
107.Abordo-Adesida, E et al. (2005) Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses. Human Gene Therapy 16, 741–751.Google Scholar
108.Naldini, L et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267.Google Scholar
109.Schambach, A et al. (2013) Biosafety features of lentiviral vectors. Human Gene Therapy 24, 132–142.Google Scholar
110.Kuruvilla, J, Sasmita, AO and Ling, AP (2018) Therapeutic potential of combined viral transduction and CRISPR/Cas9 gene editing in treating neurodegenerative diseases. Neurological Sciences 39, 1827–1835.Google Scholar
111.Palfi, S et al. (2014) Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial. The Lancet 383, 1138–1146.Google Scholar
112.Mehier-Humbert, S and Guy, RH (2005) Physical methods for gene transfer: improving the kinetics of gene delivery into cells. Advanced Drug Delivery Reviews 57, 733–753.Google Scholar
113.Yin, H et al. (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology 32, 551–553.Google Scholar
114.Vaughan, EE, DeGiulio, JV and Dean, DA (2006) Intracellular trafficking of plasmids for gene therapy: mechanisms of cytoplasmic movement and nuclear import. Current Gene Therapy 6, 671–681.Google Scholar
115.Miller, JB et al. (2017) Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angewandte Chemie (International ed. in English) 56, 1059–1063.Google Scholar
116.Aslesh, T, Maruyama, R and Yokota, T (2018) Skipping multiple exons to treat DMD – promises and challenges. Biomedicines 6, 12.Google Scholar
117.Doench, JG et al. (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology 34, 184–191.Google Scholar
118.Geary, RS et al. (2015) Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Advanced Drug Delivery Reviews 87, 46–51.Google Scholar
119.Lim, KR, Maruyama, R and Yokota, T (2017) Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug design, development and therapy 11, 533.Google Scholar
120.Aartsma-Rus, A et al. (2014) Translational and regulatory challenges for exon skipping therapies. Human Gene Therapy 10, 885–892.Google Scholar
121.Kyrychenko, V et al. (2017) Functional correction of dystrophin actin binding domain mutations by genome editing. JCI Insight 2.Google Scholar
122.Colella, P, Ronzitti, G and Mingozzi, F (2017) Emerging issues in AAV-mediated in vivo gene therapy. Molecular Therapy Methods & Cilnical Development 8, 87–104.Google Scholar
123.Moss, ML, Sklair-Tavron, L and Nudelman, R (2008) Drug insight: tumor necrosis factor-converting enzyme as a pharmaceutical target for rheumatoid arthritis. Nature Clinical Practice. Rheumatology 4, 300–309.Google Scholar
124.Shieh, PB (2018) Emerging strategies in the treatment of Duchenne muscular dystrophy. Neurotherapeutics 15, 840–848.Google Scholar
125.Cazzella, V et al. (2012) Exon 45 skipping through U1-snRNA antisense molecules recovers the Dys-nNOS pathway and muscle differentiation in human DMD myoblasts. Molecular Therapy 20, 11.Google Scholar
126.Nelson, CE et al. (2016) In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407.Google Scholar
127.Heckl, D et al. (2014) Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nature Biotechnology 32, 941–946.Google Scholar
128.Yokota, T et al. (2012) Exon skipping for nonsense mutations in Duchenne muscular dystrophy: too many mutations, too few patients? Expert Opinion on Biological Therapy 12, 1141–1152.Google Scholar
129.Wein, N et al. (2017) Efficient skipping of single exon duplications in DMD patient-derived cell lines using an antisense oligonucleotide approach. Journal of Neuromuscular Diseases 4, 199–207.Google Scholar
130.Aoki, Y et al. (2010) In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Molecular Therapy 18, 1995–2005.Google Scholar