[en] Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.
Disciplines :
Pediatrics Neurology
Author, co-author :
Domenger, Claire
Allais, Marine
Francois, Virginie
Leger, Adrien
Lecomte, Emilie
Montus, Marie
Servais, Laurent ; Université de Liège - ULiège > Département des sciences cliniques > Neuropédiatrie
Voit, Thomas
Moullier, Philippe
Audic, Yann
Le Guiner, Caroline
Language :
English
Title :
RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect.
Publication date :
2018
Journal title :
Molecular Therapy: Nucleic Acids
ISSN :
2162-2531
Publisher :
Elsevier
Volume :
10
Pages :
277-291
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
Copyright (c) 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Dominski, Z., Kole, R., Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 90 (1993), 8673–8677.
Ward, A.J., Cooper, T.A., The pathobiology of splicing. J. Pathol. 220 (2010), 152–163.
Chan, J.H.P., Lim, S., Wong, W.S.F., Antisense oligonucleotides: from design to therapeutic application. Clin. Exp. Pharmacol. Physiol. 33 (2006), 533–540.
Saleh, A.F., Arzumanov, A.A., Gait, M.J., Overview of alternative oligonucleotide chemistries for exon skipping. Methods Mol. Biol. 867 (2012), 365–378.
Mendell, J.R., Rodino-Klapac, L.R., Sahenk, Z., Roush, K., Bird, L., Lowes, L.P., Alfano, L., Gomez, A.M., Lewis, S., Kota, J., et al., Eteplirsen Study Group. Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann. Neurol. 74 (2013), 637–647.
Voit, T., Topaloglu, H., Straub, V., Muntoni, F., Deconinck, N., Campion, G., De Kimpe, S.J., Eagle, M., Guglieri, M., Hood, S., et al. Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study. Lancet Neurol. 13 (2014), 987–996.
Niks, E.H., Aartsma-Rus, A., Exon skipping: a first in class strategy for Duchenne muscular dystrophy. Expert Opin. Biol. Ther. 17 (2017), 225–236.
Gorman, L., Suter, D., Emerick, V., Schümperli, D., Kole, R., Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proc. Natl. Acad. Sci. USA 95 (1998), 4929–4934.
Goyenvalle, A., Vulin, A., Fougerousse, F., Leturcq, F., Kaplan, J.C., Garcia, L., Danos, O., Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306 (2004), 1796–1799.
Denti, M.A., Rosa, A., D'Antona, G., Sthandier, O., De Angelis, F.G., Nicoletti, C., Allocca, M., Pansarasa, O., Parente, V., Musarò, A., et al. Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model. Proc. Natl. Acad. Sci. USA 103 (2006), 3758–3763.
Asparuhova, M.B., Marti, G., Liu, S., Serhan, F., Trono, D., Schümperli, D., Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping. J. Gene Med. 9 (2007), 323–334.
Geib, T., Hertel, K.J., Restoration of full-length SMN promoted by adenoviral vectors expressing RNA antisense oligonucleotides embedded in U7 snRNAs. PLoS ONE, 4, 2009, e8204.
Gedicke-Hornung, C., Behrens-Gawlik, V., Reischmann, S., Geertz, B., Stimpel, D., Weinberger, F., Schlossarek, S., Précigout, G., Braren, I., Eschenhagen, T., et al. Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice. EMBO Mol. Med. 5 (2013), 1128–1145.
Kiss, T., Biogenesis of small nuclear RNPs. J. Cell Sci. 117 (2004), 5949–5951.
Mowry, K.L., Steitz, J.A., Identification of the human U7 snRNP as one of several factors involved in the 3′ end maturation of histone premessenger RNA's. Science 238 (1987), 1682–1687.
Grimm, C., Stefanovic, B., Schümperli, D., The low abundance of U7 snRNA is partly determined by its Sm binding site. EMBO J. 12 (1993), 1229–1238.
Basner-Tschakarjan, E., Bijjiga, E., Martino, A.T., Pre-clinical assessment of immune responses to adeno-associated virus (AAV) vectors. Front. Immunol., 5, 2014, 28.
Nathwani, A.C., Reiss, U.M., Tuddenham, E.G.D., Rosales, C., Chowdary, P., McIntosh, J., Della Peruta, M., Lheriteau, E., Patel, N., Raj, D., et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N. Engl. J. Med. 371 (2014), 1994–2004.
Bish, L.T., Sleeper, M.M., Forbes, S.C., Wang, B., Reynolds, C., Singletary, G.E., Trafny, D., Morine, K.J., Sanmiguel, J., Cecchini, S., et al. Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping. Mol. Ther. 20 (2012), 580–589.
Vulin, A., Barthélémy, I., Goyenvalle, A., Thibaud, J.-L., Beley, C., Griffith, G., Benchaouir, R., le Hir, M., Unterfinger, Y., Lorain, S., et al. Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping. Mol. Ther. 20 (2012), 2120–2133.
Le Guiner, C., Montus, M., Servais, L., Cherel, Y., Francois, V., Thibaud, J.-L., Wary, C., Matot, B., Larcher, T., Guigand, L., et al. Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients. Mol. Ther. 22 (2014), 1923–1935.
Mendell, J.R., Shilling, C., Leslie, N.D., Flanigan, K.M., al-Dahhak, R., Gastier-Foster, J., Kneile, K., Dunn, D.M., Duval, B., Aoyagi, A., et al. Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann. Neurol. 71 (2012), 304–313.
Bushby, K., Finkel, R., Birnkrant, D.J., Case, L.E., Clemens, P.R., Cripe, L., Kaul, A., Kinnett, K., McDonald, C., Pandya, S., et al., DMD Care Considerations Working Group. Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 9 (2010), 77–93.
Hoffman, E.P., Brown, R.H. Jr., Kunkel, L.M., Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51 (1987), 919–928.
Popplewell, L.J., Adkin, C., Arechavala-Gomeza, V., Aartsma-Rus, A., de Winter, C.L., Wilton, S.D., Morgan, J.E., Muntoni, F., Graham, I.R., Dickson, G., Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: implications for future clinical trials. Neuromuscul. Disord. 20 (2010), 102–110.
Gupta, A., Meng, X., Zhu, L.J., Lawson, N.D., Wolfe, S.A., Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res. 39 (2011), 381–392.
Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., Sander, J.D., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31 (2013), 822–826.
Mussolino, C., Morbitzer, R., Lütge, F., Dannemann, N., Lahaye, T., Cathomen, T., A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39 (2011), 9283–9293.
Kamola, P.J., Kitson, J.D.A., Turner, G., Maratou, K., Eriksson, S., Panjwani, A., Warnock, L.C., Douillard Guilloux, G.A., Moores, K., Koppe, E.L., et al. In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization. Nucleic Acids Res. 43 (2015), 8638–8650.
Obad, S., dos Santos, C.O., Petri, A., Heidenblad, M., Broom, O., Ruse, C., Fu, C., Lindow, M., Stenvang, J., Straarup, E.M., et al. Silencing of microRNA families by seed-targeting tiny LNAs. Nat. Genet. 43 (2011), 371–378.
Schmid, F., Hiller, T., Korner, G., Glaus, E., Berger, W., Neidhardt, J., A gene therapeutic approach to correct splice defects with modified U1 and U6 snRNPs. Hum. Gene Ther. 24 (2013), 97–104.
Guillouzo, A., Corlu, A., Aninat, C., Glaise, D., Morel, F., Guguen-Guillouzo, C., The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Biol. Interact. 168 (2007), 66–73.
Glushakova, L.G., Lisankie, M.J., Eruslanov, E.B., Ojano-Dirain, C., Zolotukhin, I., Liu, C., Srivastava, A., Stacpoole, P.W., AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells. Mol. Genet. Metab. 98 (2009), 289–299.
Bar, S., Barnea, E., Levy, Z., Neuman, S., Yaffe, D., Nudel, U., A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution. Biochem. J. 272 (1990), 557–560.
Summerton, J.E., Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity. Curr. Top. Med. Chem. 7 (2007), 651–660.
Aartsma-Rus, A., De Winter, C.L., Janson, A.A.M., Kaman, W.E., Van Ommen, G.-J.B., Den Dunnen, J.T., Van Deutekom, J.C., Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites. Oligonucleotides 15 (2005), 284–297.
Gabriel, R., Lombardo, A., Arens, A., Miller, J.C., Genovese, P., Kaeppel, C., Nowrouzi, A., Bartholomae, C.C., Wang, J., Friedman, G., et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29 (2011), 816–823.
Webster, C., Blau, H.M., Accelerated age-related decline in replicative life-span of Duchenne muscular dystrophy myoblasts: implications for cell and gene therapy. Somat. Cell Mol. Genet. 16 (1990), 557–565.
Aartsma-Rus, A., Janson, A.A., Kaman, W.E., Bremmer-Bout, M., den Dunnen, J.T., Baas, F., van Ommen, G.J., van Deutekom, J.C., Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum. Mol. Genet. 12 (2003), 907–914.
Stilwell, J.L., Samulski, R.J., Role of viral vectors and virion shells in cellular gene expression. Mol. Ther. 9 (2004), 337–346.
McCaffrey, A.P., Fawcett, P., Nakai, H., McCaffrey, R.L., Ehrhardt, A., Pham, T.-T.T., Pandey, K., Xu, H., Feuss, S., Storm, T.A., Kay, M.A., The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Mol. Ther. 16 (2008), 931–941.
Snøve, O. Jr., Holen, T., Many commonly used siRNAs risk off-target activity. Biochem. Biophys. Res. Commun. 319 (2004), 256–263.
Dapas, M., Kandpal, M., Bi, Y., Davuluri, R.V., Comparative evaluation of isoform-level gene expression estimation algorithms for RNA-seq and exon-array platforms. Brief. Bioinform. 18 (2017), 260–269.
Maier, T., Güell, M., Serrano, L., Correlation of mRNA and protein in complex biological samples. FEBS Lett. 583 (2009), 3966–3973.
Roberts, T.C., Johansson, H.J., McClorey, G., Godfrey, C., Blomberg, K.E.M., Coursindel, T., Gait, M.J., Smith, C.I., Lehtiö, J., El Andaloussi, S., Wood, M.J., Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration. Hum. Mol. Genet. 24 (2015), 6756–6768.
Vandamme, C., Adjali, O., Mingozzi, F., Unraveling the complex story of immune responses to AAV vectors trial after trial. Hum. Gene Ther. 28 (2017), 1061–1074.
Anders, S., Pyl, P.T., Huber, W., HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31 (2015), 166–169.
Noiret, M., Méreau, A., Angrand, G., Bervas, M., Gautier-Courteille, C., Legagneux, V., Deschamps, S., Lerivray, H., Viet, J., Hardy, S., et al. Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis. Dev. Biol. 426 (2017), 449–459.