Abstract
Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide–PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.
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References
Theadom A, Rodrigues M, Roxburgh R et al (2014) Prevalence of muscular dystrophies: a systematic literature review. Neuroepidemiology 43:259–268
Brook JD, McCurrach ME, Harley HG et al (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 68:799–808
Mahadevan M, Tsilfidis C, Sabourin L et al (1992) Myotonic dystrophy mutation: an unstable ctg repeat in the 3′ untranslated region of the gene. Science 255(5049):1253–1255
Fu Y-H, Pizzuni A, Fenwick RG et al (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science (New York, N.Y.) 255(5049):1256–1258
Taneja KL, McCurrach M, Schalling M et al (1995) Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128:995–1002
Davis BM, Mccurrach ME, Taneja KL et al (1997) Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc Natl Acad Sci 94:7388–7393
Lee K, Li M, Manchanda M et al (2013) Compound loss of muscleblind-like function in myotonic dystrophy. EMBO Mol Med 5:1887–1900
Miller JW, Urbinati CR, Teng-Umnuay P et al (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 19:4439–4448
Wang ET, Treacy D, Eichinger K et al (2019) Transcriptome alterations in myotonic dystrophy skeletal muscle and heart. Hum Mol Genet 28:1312–1321
Mankodi A, Takahashi MP, Jiang H et al (2002) Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol Cell 10:35–44
Charizanis K, Lee KY, Batra R et al (2012) Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy. Neuron 75:437–450
Fugier C, Klein AF, Hammer C et al (2011) Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy. Nat Med 17:720–725
Freyermuth F, Rau F, Kokunai Y et al (2016) Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy. Nat Commun 7:25
López-Martínez A, Soblechero-Martín P, De-La-puente-ovejero L et al (2020) An overview of alternative splicing defects implicated in myotonic dystrophy type I. Genes 11:1109
Porter B, Drug development pipeline for myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), https://www.myotonic.org/sites/default/files/pages/files/Myotonic-Dystrophy-Drug-Development-Pipeline-as-of-Feb-01-2021.pdf
Reddy K, Jenquin JR, Cleary JD, et al (2019) Mitigating RNA toxicity in myotonic dystrophy using small molecules. www.mdpi.com/journal/ijms
López-Morató M, Brook JD, Wojciechowska M (2018) Small molecules which improve pathogenesis of myotonic dystrophy type:1. www.frontiersin.org
Roberts TC, Langer R, Wood MJA (2020) Advances in oligonucleotide drug delivery. Nat Rev Drug Discov 19:673–694.
Summerton J, Weller D (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 7:187–195
Hudziak RM, Barofsky E, Barofsky DF et al (1996) Resistance of Morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 6:267–272
FDA grants accelerated approval to first drug for Duchenne muscular dystrophy | FDA. https://www.fda.gov/news-events/press-announcements/fda-grants-accelerated-approval-first-drug-duchenne-muscular-dystrophy
Aartsma-Rus A, Krieg AM (2017) FDA approves eteplirsen for duchenne muscular dystrophy: the next chapter in the eteplirsen saga. Nucleic Acid Ther 27:1–3. /pmc/articles/PMC5312460/
Wheeler TM, Leger AJ, Pandey SK et al (2012) Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature 488:111–117
Lee JE, Bennett CF, Cooper TA (2012) RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1. Proc Natl Acad Sci U S A 109:4221–4226
Jauvin D, Chrétien J, Pandey SK et al (2017) Targeting DMPK with antisense oligonucleotide improves muscle strength in myotonic dystrophy type 1. Mice 7:465–474
Yadava RS, Yu Q, Mandal M et al (2020) Systemic therapy in an RNA toxicity mouse model with an antisense oligonucleotide therapy targeting a non-CUG sequence within the DMPK 3′UTR RNA. Hum Mol Genet 29:1440–1453
Klein AF, Varela MA, Arandel L et al (2019) Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice. J Clin Investig 129:4739–4744
Mulders SAM, van den Broek WJAA, Wheeler TM et al (2009) Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy. Proc Natl Acad Sci U S A 106:13915–13920
Wheeler TM, Sobczak K, Lueck JD et al (2009) Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA. Science 325:336–339
Wojtkowiak-Szlachcic A, Taylor K, Stepniak-Konieczna E et al (2015) Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy. Nucleic Acids Res 43:3318–3331
Nakamori M, Sobczak K, Puwanant A et al (2013) Splicing biomarkers of disease severity in myotonic dystrophy. Ann Neurol 74:862–872
Pandey SK, Wheeler TM, Justice SL et al (2015) Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1s. J Pharmacol Exp Ther 355:329–340
Ionis Pharmaceuticals Reports DMPKRx Phase 1/2 Trial Results. https://us8.campaign-archive.com/?u=8f5969cac3271759ce78c8354&id=8cc67ae9b8&e=cd1f4d18fe
Betts C, Saleh AF, Arzumanov AA et al (2012) Pip6-PMO, a new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment. Mol Ther Nucl Acids 1:e38
Yin H, Moulton HM, Seow Y et al (2008) Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet 17:3909–3918
Gao X, Zhao J, Han G et al (2014) Effective dystrophin restoration by a novel muscle-homing peptide-morpholino conjugate in dystrophin-deficient mdx mice. In: Molecular therapy. Nature Publishing Group, pp 1333–1341
Leger AJ, Mosquea LM, Clayton NP et al (2013) Systemic delivery of a peptide-linked morpholino oligonucleotide neutralizes mutant RNA toxicity in a mouse model of myotonic dystrophy. Nucleic Acid Ther 23:109–117
Hammond SM, Hazell G, Shabanpoor F et al (2016) Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy. Proc Natl Acad Sci USA 113:10962
Mankodi A, Logigian E, Callahan L et al (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289:1769–1772
Vaidya VS, Ozer JS, Dieterle F et al (2010) Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies. Nat Biotechnol 28:478–485
Arandel L, Espinoza MP, Matloka M et al (2017) Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds. Dis Model Mech 10:487–497
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. https://www.nature.com/articles/nmeth.2089
Behrendt R, White P, Offer J (2016) Advances in Fmoc solid-phase peptide synthesis. J Pep Sci 22:4–27
Collins JM, Porter KA, Singh SK et al (2014) High-efficiency solid phase peptide synthesis (he -Spps). Org Lett 16:940
Ferreira T, Rasband W (2012) ImageJ User Guide ImageJ User Guide IJ 1.46r
Burki U, Keane J, Blain A et al (2015) Development and application of an ultrasensitive hybridization-based ELISA method for the determination of peptide-conjugated phosphorodiamidate morpholino oligonucleotides. Nucleic Acid Ther 25:275
Acknowledgments
This work was supported by Vinnova, NNF Center for Biosustainability, WCPR Wallenberg Center for Protein Research, and the Knut and Alice Wallenberg foundation.
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Stoodley, J., Miraz, D.S., Jad, Y., Fischer, M., Wood, M.J.A., Varela, M.A. (2023). Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy. In: Maruyama, R., Yokota, T. (eds) Muscular Dystrophy Therapeutics. Methods in Molecular Biology, vol 2587. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2772-3_13
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DOI: https://doi.org/10.1007/978-1-0716-2772-3_13
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