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.

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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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