Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease
AbstractCLN3 Batten disease is an autosomal recessive, neurodegenerative, lysosomal storage disease caused by mutations in CLN3, which encodes a lysosomal membrane protein1,2,3. There are no disease-modifying treatments for this disease that affects up to 1 in 25,000 births, has an onset of symptoms in early childhood and typically is fatal by 20–30 years of life4,5,6,7. Most patients with CLN3 Batten have a deletion encompassing exons 7 and 8 (CLN3∆ex7/8), creating a reading frameshift7,8. Here we demonstrate that mice with this deletion can be effectively treated using an antisense oligonucleotide (ASO) that induces exon skipping to restore the open reading frame. A single treatment of neonatal mice with an exon 5-targeted ASO-induced robust exon skipping for more than a year, improved motor coordination, reduced histopathology in Cln3∆ex7/8 mice and increased survival in a new mouse model of the disease. ASOs also induced exon skipping in cell lines derived from patients with CLN3 Batten disease. Our findings demonstrate the utility of ASO-based reading-frame correction as an approach to treat CLN3 Batten disease and broaden the therapeutic landscape for ASOs in the treatment of other diseases using a similar strategy.
Data availabilityThe authors declare that all data supporting the findings of this study are available within the paper and its extended data and supplementary information files. Source data are provided with this paper.References1.Kyttala, A., Ihrke, G., Vesa, J., Schell, M. J. & Luzio, J. P. Two motifs target Batten disease protein CLN3 to lysosomes in transfected nonneuronal and neuronal cells. Mol. Biol. Cell 15, 1313–1323 (2004).PubMed
PubMed Central
Google Scholar
2.The International Batten Disease Consortium Isolation of a novel gene underlying Batten disease, CLN3. Cell 82, 949–957 (1995).
Google Scholar
3.Cárcel-Trullols, J., Kovács, A. D. & Pearce, D. A. Cell biology of the NCL proteins: what they do and don’t do. Biochim. Biophys. Acta 1852, 2242–2255 (2015).PubMed
Google Scholar
4.Cialone, J. et al. Females experience a more severe disease course in Batten disease. J. Inherit. Metab. Dis. 35, 549–555 (2012).PubMed
Google Scholar
5.Gardiner, R. M. Clinical features and molecular genetic basis of the neuronal ceroid lipofuscinoses. Adv. Neurol. 89, 211–215 (2002).PubMed
Google Scholar
6.Johnson, T. B. et al. Therapeutic landscape for Batten disease: current treatments and future prospects. Nat. Rev. Neurol. 15, 161–178 (2019).PubMed
PubMed Central
Google Scholar
7.Sleat, D. E., Gedvilaite, E., Zhang, Y., Lobel, P. & Xing, J. Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis. Gene 593, 284–291 (2016).CAS
PubMed
PubMed Central
Google Scholar
8.Mole, S. E. & Cotman, S. L. Genetics of the neuronal ceroid lipofuscinoses (Batten disease). Biochim. Biophys. Acta 1852, 2237–2241 (2015).CAS
PubMed
PubMed Central
Google Scholar
9.Kousi, M., Lehesjoki, A. E. & Mole, S. E. Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses. Hum. Mutat. 33, 42–63 (2012).CAS
PubMed
Google Scholar
10.Wang, F. et al. Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements. Hum. Genet. 133, 331–345 (2014).CAS
PubMed
Google Scholar
11.Cao, Y. et al. Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis. J. Biol. Chem. 281, 20483–20493 (2006).CAS
PubMed
Google Scholar
12.Chang, J. W., Choi, H., Cotman, S. L. & Jung, Y. K. Lithium rescues the impaired autophagy process in CbCln3(Δex7/8/Δex7/8) cerebellar cells and reduces neuronal vulnerability to cell death via IMPase inhibition. J. Neurochem. 116, 659–668 (2011).CAS
PubMed
PubMed Central
Google Scholar
13.Chandrachud, U. et al. Unbiased cell-based screening in a neuronal cell model of Batten disease highlights an interaction between Ca2+ homeostasis, autophagy, and CLN3 protein function. J. Biol. Chem. 290, 14361–14380 (2015).CAS
PubMed
PubMed Central
Google Scholar
14.Vidal-Donet, J. M., Cárcel-Trullols, J., Casanova, B., Aguado, C. & Knecht, E. Alterations in ROS activity and lysosomal pH account for distinct patterns of macroautophagy in LINCL and JNCL fibroblasts. PLoS One 8, e55526 (2013).CAS
PubMed
PubMed Central
Google Scholar
15.Yasa, S. et al. CLN3 regulates endosomal function by modulating Rab7A-effector interactions. J. Cell Sci. 133, jcs234047 (2020).CAS
PubMed
Google Scholar
16.Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).CAS
Google Scholar
17.Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).PubMed
PubMed Central
Google Scholar
18.Havens, M. A. & Hastings, M. L. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 44, 6549–6563 (2016).PubMed
PubMed Central
Google Scholar
19.Cotman, S. L. et al. Cln3(Δex7/8) knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth. Hum. Mol. Genet. 11, 2709–2721 (2002).CAS
PubMed
Google Scholar
20.Tyynela, J., Cooper, J. D., Khan, M. N., Shemilts, S. J. & Haltia, M. Hippocampal pathology in the human neuronal ceroid-lipofuscinoses: distinct patterns of storage deposition, neurodegeneration and glial activation. Brain Pathol. 14, 349–357 (2004).PubMed
Google Scholar
21.Osorio, N. S. et al. Neurodevelopmental delay in the Cln3Δex7/8 mouse model for Batten disease. Genes Brain Behav. 8, 337–345 (2009).CAS
PubMed
PubMed Central
Google Scholar
22.Pontikis, C. C. et al. Late onset neurodegeneration in the Cln3
-/- mouse model of juvenile neuronal ceroid lipofuscinosis is preceded by low level glial activation. Brain Res. 1023, 231–242 (2004).CAS
PubMed
Google Scholar
23.Burkovetskaya, M. et al. Evidence for aberrant astrocyte hemichannel activity in juvenile neuronal ceroid lipofuscinosis (JNCL). PLoS One 9, e95023 (2014).PubMed
PubMed Central
Google Scholar
24.Sondhi, D. et al. Partial correction of the CNS lysosomal storage defect in a mouse model of juvenile neuronal ceroid lipofuscinosis by neonatal CNS administration of an adeno-associated virus serotype rh.10 vector expressing the human CLN3 gene. Hum. Gene Ther. 25, 223–239 (2014).CAS
PubMed
Google Scholar
25.Aldrich, A. et al. Efficacy of phosphodiesterase-4 inhibitors in juvenile Batten disease (CLN3). Ann. Neurol. 80, 909–923 (2016).CAS
PubMed
PubMed Central
Google Scholar
26.Kovács, A. D. & Pearce, D. A. Finding the most appropriate mouse model of juvenile CLN3 (Batten) disease for therapeutic studies: the importance of genetic background and gender. Dis. Model Mech. 8, 351–361 (2015).PubMed
PubMed Central
Google Scholar
27.Wang, X., Huang, T., Bu, G. & Xu, H. Dysregulation of protein trafficking in neurodegeneration. Mol. Neurodegener. 9, 31 (2014).CAS
PubMed
PubMed Central
Google Scholar
28.Yang, D. S. et al. Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits. Brain 137, 3300–3318 (2014).PubMed
PubMed Central
Google Scholar
29.Nilsson, P. et al. Aβ secretion and plaque formation depend on autophagy. Cell Rep. 5, 61–69 (2013).CAS
PubMed
Google Scholar
30.Wisniewski, K. E., Kida, E., Gordon-Majszak, W. & Saitoh, T. Altered amyloid β-protein precursor processing in brains of patients with neuronal ceroid lipofuscinosis. Neurosci. Lett. 120, 94–96 (1990).CAS
PubMed
Google Scholar
31.Wisniewski, K. E., Gordon-Krajcer, W. & Kida, E. Abnormal processing of carboxy-terminal fragment of beta precursor protein (βPP) in neuronal ceroid-lipofuscinosis (NCL) cases. J. Inherit. Metab. Dis. 16, 312–316 (1993).CAS
PubMed
Google Scholar
32.D’Andrea, M. R. et al. Lipofuscin and Aβ42 exhibit distinct distribution patterns in normal and Alzheimer’s disease brains. Neurosci. Lett. 323, 45–49 (2002).PubMed
Google Scholar
33.Maulik, M. et al. Mutant human APP exacerbates pathology in a mouse model of NPC and its reversal by a β-cyclodextrin. Hum. Mol. Genet. 21, 4857–4875 (2012).CAS
PubMed
Google Scholar
34.Chishti, M. A. et al. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J. Biol. Chem. 276, 21562–21570 (2001).CAS
PubMed
Google Scholar
35.Darras, B. T. et al. An integrated safety analysis of infants and children with symptomatic spinal muscular atrophy (SMA) treated with Nnusinersen in seven clinical trials. CNS Drugs 33, 919–932 (2019).CAS
PubMed
PubMed Central
Google Scholar
36.McClorey, G. & Wood, M. J. An overview of the clinical application of antisense oligonucleotides for RNA-targeting therapies. Curr. Opin. Pharm. 24, 52–58 (2015).CAS
Google Scholar
37.Foust, K. D. et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat. Biotechnol. 28, 271–274 (2010).CAS
PubMed
PubMed Central
Google Scholar
38.Dangouloff, T. & Servais, L. Clinical evidence supporting early treatment of patients with spinal muscular atrophy: current perspectives. Ther. Clin. Risk Manag. 15, 1153–1161 (2019).CAS
PubMed
PubMed Central
Google Scholar
39.De Vivo, D. C. et al. Nusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: interim efficacy and safety results from the Phase 2 NURTURE study. Neuromuscul. Disord. 29, 842–856 (2019).PubMed
PubMed Central
Google Scholar
40.Ke, Q. et al. Progress in treatment and newborn screening for Duchenne muscular dystrophy and spinal muscular atrophy. World J. Pediatr. 15, 219–225 (2019).PubMed
Google Scholar
41.Viklund, H. & Elofsson, A. OCTOPUS: improving topology prediction by two-track ANN-based preference scores and an extended topological grammar. Bioinformatics 24, 1662–1668 (2008).CAS
PubMed
Google Scholar
42.Reynolds, S. M., Kall, L., Riffle, M. E., Bilmes, J. A. & Noble, W. S. Transmembrane topology and signal peptide prediction using dynamic bayesian networks. PLoS Comput. Biol. 4, e1000213 (2008).PubMed
PubMed Central
Google Scholar
43.Kall, L., Krogh, A. & Sonnhammer, E. L. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 338, 1027–1036 (2004).CAS
PubMed
Google Scholar
44.Kall, L., Krogh, A. & Sonnhammer, E. L. An HMM posterior decoder for sequence feature prediction that includes homology information. Bioinformatics 21, i251–i257 (2005).PubMed
Google Scholar
45.Bernsel, A. et al. Prediction of membrane-protein topology from first principles. Proc. Natl Acad. Sci. USA 105, 7177–7181 (2008).CAS
PubMed
Google Scholar
46.Viklund, H., Bernsel, A., Skwark, M. & Elofsson, A. SPOCTOPUS: a combined predictor of signal peptides and membrane protein topology. Bioinformatics 24, 2928–2929 (2008).CAS
PubMed
Google Scholar
47.Tsirigos, K. D., Peters, C., Shu, N., Kall, L. & Elofsson, A. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res. 43, W401–W407 (2015).CAS
PubMed
PubMed Central
Google Scholar
48.Nugent, T., Mole, S. E. & Jones, D. T. The transmembrane topology of Batten disease protein CLN3 determined by consensus computational prediction constrained by experimental data. FEBS Lett. 582, 1019–1024 (2008).CAS
PubMed
Google Scholar
49.Perland, E. et al. Characteristics of 29 novel atypical solute carriers of major facilitator superfamily type: evolutionary conservation, predicted structure and neuronal co-expression. Open Biol. 7, 170142 (2017).PubMed
PubMed Central
Google Scholar
50.Yang, J. & Zhang, Y. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 43, W174–W181 (2015).CAS
PubMed
PubMed Central
Google Scholar
51.Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738 (2010).CAS
PubMed
PubMed Central
Google Scholar
52.Omasits, U., Ahrens, C. H., Muller, S. & Wollscheid, B. Protter: interactive protein feature visualization and integration with experimental proteomic data. Bioinformatics 30, 884–886 (2014).CAS
PubMed
Google Scholar
53.Chresta, C. M. et al. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 70, 288–298 (2010).CAS
PubMed
Google Scholar
54.Swayze, E. E. et al. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Res. 35, 687–700 (2007).CAS
PubMed
Google Scholar
55.Rigo, F. et al. Pharmacology of a central nervous system delivered 2′-O-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates. J. Pharm. Exp. Ther. 350, 46–55 (2014).
Google Scholar
56.Baker, B. F. et al. 2′-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem. 272, 11994–12000 (1997).CAS
PubMed
Google Scholar
57.Hinrich, A. J. et al. Therapeutic correction of ApoER2 splicing in Alzheimer’s disease mice using antisense oligonucleotides. EMBO Mol. Med. 8, 328–345 (2016).CAS
PubMed
PubMed Central
Google Scholar
58.Hua, Y. & Krainer, A. R. Antisense-mediated exon inclusion. Methods Mol. Biol. 867, 307–323 (2012).CAS
PubMed
PubMed Central
Google Scholar
59.Kovács, A. D. et al. Temporary inhibition of AMPA receptors induces a prolonged improvement of motor performance in a mouse model of juvenile Batten disease. Neuropharmacology 60, 405–409 (2011).PubMed
Google Scholar
Download referencesAcknowledgementsThe authors thank Forbes Porter and An Dang Do (NIH/NICHD) and Beverly Davidson (Children’s Hospital of Philadelphia) for human fibroblast cell lines and David Pearce (Sanford), David Mueller (RFUMS) and Susan Cotman (Harvard) for helpful discussions and other reagents. The authors also thank Maria Ruiz and Cecilia Reyes for technical support. This work was supported by NIH grant NS113233, the Batten Disease Support and Research Alliance and the ForeBatten Research Foundation. Quantitation of radioactive PCR products was performed on an instrument in the shared Proteomics facility at RFUMS obtained with NIH grant S10 OD010662.Author informationAffiliationsCenter for Genetic Diseases, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USAJessica L. Centa, Francine M. Jodelka, Anthony J. Hinrich & Michelle L. HastingsSchool of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USAJessica L. Centa & Michelle L. HastingsPediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD, USATyler B. Johnson & Jill M. WeimerIonis Pharmaceuticals, Carlsbad, CA, USAJoseph Ochaba, Michaela Jackson & Frank RigoCellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USADominik M. DuelliDepartment of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USAJill M. WeimerContributionsJ.L.C., F.M.J. and M.L.H. designed and carried out mouse behavioral analysis; J.L.C., A.J.H. and M.L.H. designed and performed in vitro ASO experiments; J.L.C. and A.J.H. performed immunofluorescent analysis of ASO distribution; T.B.J and J.M.W. carried out immunohistochemistry for SCMAS accumulation and GFAP; J.O. and F.R. designed, performed and analyzed data from experiments to measure autophagy and lysosomal volume; M.J. and F.R. designed and performed adult ICV treatments. J.M.W., F.R., D.M.D and M.L.H. provided critical materials and reagents. J.L.C. and M.L.H. wrote the manuscript. All authors discussed the results and contributed to the preparation of the manuscript.Corresponding authorCorrespondence to
Michelle L. Hastings.Ethics declarations
Competing interests
J.O, M.J. and F.R. are employees of Ionis Pharmaceuticals. T.B.J. and J.M.W. are currently employees of Amicus Therapeutics. D.M.D. is currently employed by Abbott Molecular. M.L.H. and F.R. are inventors on patents on ASOs filed by RFUMS and Ionis Pharmaceuticals and may be entitled to benefits from licensing of the associated intellectual property.
Additional informationPeer review information Kate Gao was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Modeling of CLN3 protein structure.a, A query of the structures of CLN3 using different prediction algorithms, generated models of human CLN3. The table shows the protein prediction servers with their predicted number of transmembrane domains (TMDs) for CLN3 (WT), CLN3Δex7/8 (Δ78), and CLN3Δex5/7/8 (Δ578). The variability of TMDs amongst the different predictions demonstrates the uncertainty associated with such modeling approaches and cautions that definitive conclusions about CLN3 structures must await high resolution information on the structure from cryo-EM or X-ray crystallographic studies. b, Homology model of the CLN3 structure based on the structure of the equilibrative nucleoside transporter (ENT1) encoded by SLC29A1, as a template, generated by I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). c, Membrane topology map of CLN3, CLN3Δex7/8 (Δ78), and CLN3Δex5/7/8 (Δ578) showing the numbered TMDs as predicted by TOPCONS and rendered using Protter (http://wlab.ethz.ch/protter). The exons corresponding to the amino acid sequence are shown in alternating green and white with exon 5-encoded amino acids highlighted in blue.Extended Data Fig. 2 Autophagic flux deficits associated with CLN3Δex7/8 but not CLN3Δex5/7/8 expression.a, Schematic representation of measuring autophagic flux using GFP-RFP-LC3B. To compare the effects of CLN3 protein isoforms on autophagic flux we used a tandem acid-stable red and acid-labile green fluorescent protein (RFP, GFP) sensor fused to the autophagosome-associated protein, microtubule-associated protein light chain 3 beta (GFP-RFP-LC3B) in HEK-293 cells. The GFP signal is quenched in acidic condition and thereby the dual-labeled RFP-GFP-LC3B provides a means to differentiate between autophagosomes (red and green=yellow fluorescent LC3B) and acidic autolysosomes (red only) and assess the relative abundance of the vesicles during autophagy. LC3BII localizes to the membrane of the autophagosome, which fuses with lysosomes to form acidic autolysosomes. This acidic environment causes quenching of the GFP making the autolysosome appear red. The contents, including RFP-LC3B and p62 are then degraded by lysosomal enzymes. b, HEK-293 cells were transfected with CLN3 WT, CLN3Δex7/8 (Δ78), or CLN3Δex5/7/8 (Δ578) expression plasmids. After 44 h, the cells were transfected with the tandem reporter GFP-RFP-LC3 to examine autophagic flux. Cells were treated with DMSO (-) or to induce autophagy, AZD8055 for 20 h then imaged. Shown are representative confocal microcopy images of the formation of autophagosomes (yellow in overlay images) and autolysosomes (red in overlay images) as quantitated in Fig. 1d, n = 10 cells/group. Scale bar, 5 µm.Extended Data Fig. 3 Lysosome assessment in cells expressing CLN3, CLN3Δex7/8, or CLN3Δex5/7/8.a, Representative confocal microscopy images of HEK-293 cells expressing CLN3 WT, CLN3Δex7/8, or CLN3Δex5/7/8 and incubated with Lysotracker (red) to visualize lysosomes. Scale bar, 10 µm. b, The number of lysosomes per CLN3 WT, CLN3Δex7/8, or CLN3Δex5/7/8 expressing cells plotted as mean ± s.e.m. n = 20 cells per group. WT CLN3 vs. CLN3Δex7/8, P = 0.797; CLN3Δex7/8 vs. CLN3Δex5/7/8, P = 0.9447; WT CLN3 vs. CLN3Δex5/7/8, P = 0.9447. One way ANOVA, Tukey’s multiple comparisons test; Non-significant (ns).
Source data
Extended Data Fig. 4 Normalization of autophagy measurements with CLN3Δex5/7/8 expression.a, During the initiation of autophagy, LC3I undergoes lipidation and phosphatidylethanolamine (PE) conjugation to form LC3II which is engulfed in a newly formed phagophore with other cargo such as p62, which subsequently forms the autophagosome. Fusion with lysosomes results in formation of the autolysosomes followed by degradation and recycling of resident contents. b, Western blot of protein lysate from HEK-293 cells transfected with CLN3 WT, CLN3Δex7/8 (Δ78), or CLN3Δex5/7/8 (Δ578) expression plasmids in the presence or absence of amino acids. Proteins were separated by SDS-PAGE and probed for LC3B, p62, and phospho-S6K, a marker of autophagy inhibition. α-tubulin was used as a loading control for each sample. Size markers (kilodaltons) are shown at right of gel images. c, Quantitation of p62 abundance relative to α-tubulin in b. Bars are mean ± s.e.m; n = 3 individually treated tissue culture wells per group. WT: fed vs. starvation, P
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