Gene correction of the CLN3 c.175G>A variant in patient-derived induced pluripotent stem cells prevents pathological changes in retinal organoids

Xiao Zhang, Dan Zhang, Jennifer A Thompson, Shang-Chih Chen, Zhiqin Huang, Luke Jennings, Terri L McLaren, Tina M Lamey, John N De Roach, Fred K Chen, Samuel McLenachan, Xiao Zhang, Dan Zhang, Jennifer A Thompson, Shang-Chih Chen, Zhiqin Huang, Luke Jennings, Terri L McLaren, Tina M Lamey, John N De Roach, Fred K Chen, Samuel McLenachan

Abstract

Background: Mutations in CLN3 cause Batten disease, however non-syndromic CLN3 disease, characterized by retinal-specific degeneration, has been also described. Here, we characterized an induced pluripotent stem cell (iPSC)-derived disease model derived from a patient with non-syndromic CLN3-associated retinopathy.

Methods: Patient-iPSC, carrying the 1 kb-deletion and c.175G>A variants in CLN3, coisogenic iPSC, in which the c.175G>A variant was corrected, and control iPSC were differentiated into neural retinal organoids (NRO) and cardiomyocytes. CLN3 transcripts were analyzed by Sanger sequencing. Gene expression was characterized by qPCR and western blotting. NRO were characterized by immunostaining and electron microscopy.

Results: Novel CLN3 transcripts were detected in adult human retina and control-NRO. The major transcript detected in patient-NRO displayed skipping of exons 2 and 4-9. Accumulation of subunit-C of mitochondrial ATPase (SCMAS) protein was demonstrated in patient-derived cells. Photoreceptor progenitor cells in patient-NRO displayed accumulation of peroxisomes and vacuolization of inner segments. Correction of the c.175G>A variant restored CLN3 mRNA and protein expression and prevented SCMAS and inner segment vacuolization.

Conclusion: Our results demonstrate the expression of novel CLN3 transcripts in human retinal tissues. The c.175G>A variant alters splicing of the CLN3 pre-mRNA, leading to features consistent with CLN3 deficiency, which were prevented by gene correction.

Trial registration: ClinicalTrials.gov NCT03872479.

Keywords: Batten disease; CLN3; induced pluripotent stem cells; retinal organoid; retinopathy.

Conflict of interest statement

The authors declare no conflict of interest.

© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.

Figures

FIGURE 1
FIGURE 1
Characterization of CLN3 transcripts. (a,b) CLN3 transcripts were amplified from the cDNA samples of adult human retina (Ret) and 16‐week NRO derived from control iPSC (Con) and LEIi004‐A patient iPSC (P). PCR reactions were performed using primers targeting exons 1–15 (a). Numbered bands were purified, cloned, and sequenced. Exon structures of sequenced CLN3 transcripts are shown in (b). Positions of DNA size markers are indicated. (c) CLN3 transcripts were amplified from the cDNA samples of 10‐week NRO derived from LEIi004‐A patient iPSC (P), LEIi004‐A‐1 gene corrected patient iPSC (GC) and control iPSC (Con). PCR reactions were performed using primers targeting exons 1–15, 9–15, and 9–13
FIGURE 2
FIGURE 2
Protein samples from undifferentiated iPSC, 4‐week CM, and 5‐week NRO cultures were analysed by western blotting using an anti‐CLN3 antibody and an anti‐GAPDH antibody. LEIi004‐A patient (P) iPSC and CM expressed lower levels of CLN3 protein than gene‐corrected LEIi004‐A‐1 patient (GC) iPSC and CM. LEIi004‐A‐1 iPSC expressed similar levels of CLN3 protein as a control iPSC line (Con). Similar levels of CLN3 protein expression were detected in NRO from LEIi004‐A and LEIi004‐A‐1
FIGURE 3
FIGURE 3
5‐week NRO derived from control iPSC (a) LEIi004‐A patient iPSC (b) and LEIi004‐A‐1 (c) gene corrected patient iPSC were analysed by transmission electron microscopy. Left panels show developing photoreceptor inner segments on the organoid surface. Right panels show cell bodies in the developing outer nuclear layer. Mitochondria (M), lysosomes (L), peroxisomes (P), vacuoles (V), and cell nuclei (N) are indicated
FIGURE 4
FIGURE 4
Protein samples from undifferentiated iPSC, 4‐week cardiomyocyte (CM), and 5‐week NRO cultures were analysed by western blotting using an anti‐SCMAS antibody and an anti‐GAPDH antibody (upper panels). Densitometry analysis demonstrated a significant increase in SCMAS protein in LEIi004‐A patient (P) iPSC, CM, and NRO compared with gene‐corrected LEIi004‐A‐1 patient (GC) iPSC, CM, and NRO (lower panels)

References

    1. Burkovetskaya, M. , Karpuk, N. , Xiong, J. , Bosch, M. , Boska, M. D. , Takeuchi, H. , Suzumura, A. , & Kielian, T. (2014). Evidence for aberrant astrocyte hemichannel activity in juvenile neuronal ceroid lipofuscinosis (JNCL). PLoS ONE, 9(4), e95023. 10.1371/journal.pone.0095023
    1. Cao, Y. , Staropoli, J. F. , Biswas, S. , Espinola, J. A. , MacDonald, M. E. , Lee, J. M. , & Cotman, S. L. (2011). Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells. PLoS ONE, 6(2), e17118. 10.1371/journal.pone.0017118
    1. Chen, F. K. , Zhang, X. , Eintracht, J. , Zhang, D. , Arunachalam, S. , Thompson, J. A. , & McLenachan, S. (2019). Clinical and molecular characterization of non‐syndromic retinal dystrophy due to c.175G>A mutation in ceroid lipofuscinosis neuronal 3 (CLN3). Documenta Ophthalmologica, 138(1), 55–70. 10.1007/s10633-018-9665-7
    1. Cotman, S. L. , & Staropoli, J. F. (2012). The juvenile Batten disease protein, CLN3, and its role in regulating anterograde and retrograde post‐Golgi trafficking. Clinical Lipidology, 7(1), 79–91. 10.2217/clp.11.70
    1. Cotman, S. L. , Vrbanac, V. , Lebel, L. A. , Lee, R. L. , Johnson, K. A. , Donahue, L. R. , & MacDonald, M. E. (2002). Cln3(Delta ex7/8) knock‐in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth. Human Molecular Genetics, 11(22), 2709–2721. 10.1093/hmg/11.22.2709
    1. de los Reyes, E. , Dyken, P. R. , Phillips, P. , Brodsky, M. , Bates, S. , Glasier, C. , & Mrak, R. E. (2004). Profound infantile neuroretinal dysfunction in a heterozygote for the CLN3 genetic defect. Journal of Child Neurology, 19(1), 42–46. 10.1177/08830738040190010703
    1. Getty, A. L. , & Pearce, D. A. (2011). Interactions of the proteins of neuronal ceroid lipofuscinosis: Clues to function. Cellular and Molecular Life Sciences, 68(3), 453–474. 10.1007/s00018-010-0468-6
    1. Haskell, R. E. , Carr, C. J. , Pearce, D. A. , Bennett, M. J. , & Davidson, B. L. (2000). Batten disease: Evaluation of CLN3 mutations on protein localization and function. Human Molecular Genetics, 9(5), 735–744. 10.1093/hmg/9.5.735
    1. Jarvela, I. , Autti, T. , Lamminranta, S. , Aberg, L. , Raininko, R. , & Santavuori, P. (1997). Clinical and magnetic resonance imaging findings in batten disease: Analysis of the major mutation (1.02‐kb deletion). Annals of Neurology, 42(5), 799–802. 10.1002/ana.410420517
    1. Jarvela, I. , Sainio, M. , Rantamaki, T. , Olkkonen, V. M. , Carpen, O. , Peltonen, L. , & Jalanko, A. (1998). Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease. Human Molecular Genetics, 7(1), 85–90. 10.1093/hmg/7.1.85
    1. Katz, M. L. , Gao, C. L. , Prabhakaram, M. , Shibuya, H. , Liu, P. C. , & Johnson, G. S. (1997). Immunochemical localization of the Batten disease (CLN3) protein in retina. Investigative Ophthalmology & Visual Science, 38(11), 2375–2386.
    1. Kitzmuller, C. , Haines, R. L. , Codlin, S. , Cutler, D. F. , & Mole, S. E. (2008). A function retained by the common mutant CLN3 protein is responsible for the late onset of juvenile neuronal ceroid lipofuscinosis. Human Molecular Genetics, 17(2), 303–312. 10.1093/hmg/ddm306
    1. Ku, C. A. , Hull, S. , Arno, G. , Vincent, A. , Carss, K. , Kayton, R. , & Pennesi, M. E. (2017). Detailed clinical phenotype and molecular genetic findings in CLN3‐associated isolated retinal degeneration. JAMA Ophthalmology, 135(7), 749–760. 10.1001/jamaophthalmol.2017.1401
    1. Lerner, T. J. , Boustany, R.‐M. , Anderson, J. W. , D'Arigo, K. L. , Schlumpf, K. , Buckler, A. J. , Gusella, J. F. , & Haines, J. L. (1995). Isolation of a novel gene underlying batten‐disease, Cln3. Cell, 82(6), 949–957. 10.1016/0092-8674(95)90274-0
    1. Lojewski, X. , Staropoli, J. F. , Biswas‐Legrand, S. , Simas, A. M. , Haliw, L. , Selig, M. K. , Coppel, S. H. , Goss, K. A. , Petcherski, A. , Chandrachud, U. , Sheridan, S. D. , Lucente, D. , Sims, K. B. , Gusella, J. F. , Sondhi, D. , Crystal, R. G. , Reinhardt, P. , Sterneckert, J. , Schöler, H. , … Cotman, S. L. (2014). Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects of TPP1 and CLN3 mutations on the endocytic pathway. Human Molecular Genetics, 23(8), 2005–2022. 10.1093/hmg/ddt596
    1. Luiro, K. , Kopra, O. , Lehtovirta, M. , & Jalanko, A. (2001). CLN3 protein is targeted to neuronal synapses but excluded from synaptic vesicles: New clues to Batten disease. Human Molecular Genetics, 10(19), 2123–2131. 10.1093/hmg/10.19.2123
    1. Luiro, K. , Yliannala, K. , Ahtiainen, L. , Maunu, H. , Jarvela, I. , Kyttala, A. , & Jalanko, A. (2004). Interconnections of CLN3, Hook1 and Rab proteins link Batten disease to defects in the endocytic pathway. Human Molecular Genetics, 13(23), 3017–3027. 10.1093/hmg/ddh321
    1. Mao, D. , Che, J. , Han, S. , Zhao, H. , Zhu, Y. , & Zhu, H. (2015). RNAi‐mediated knockdown of the CLN3 gene inhibits proliferation and promotes apoptosis in drug‐resistant ovarian cancer cells. Molecular Medicine Reports, 12(5), 6635–6641. 10.3892/mmr.2015.4238
    1. Margraf, L. R. , Boriack, R. L. , Routheut, A. A. J. , Cuppen, I. , Alhilali, L. , Bennett, C. J. , & Bennett, M. J. (1999). Tissue expression and subcellular localization of CLN3, the Batten disease protein. Molecular Genetics and Metabolism, 66(4), 283–289. 10.1006/mgme.1999.2830
    1. Mellough, C. B. , Collin, J. , Khazim, M. , White, K. , Sernagor, E. , Steel, D. H. , & Lako, M. (2015). IGF‐1 signaling plays an important role in the formation of three‐dimensional laminated neural retina and other ocular structures from human embryonic stem cells. Stem Cells, 33(8), 2416–2430. 10.1002/stem.2023
    1. Mitchison, H. M. , Hofmann, S. L. , Becerra, C. H. , Munroe, P. B. , Lake, B. D. , Crow, Y. J. , & O'Rawe, A. M. (1998). Mutations in the palmitoyl‐protein thioesterase gene (PPT; CLN1) causing juvenile neuronal ceroid lipofuscinosis with granular osmiophilic deposits. Human Molecular Genetics, 7(2), 291–297. 10.1093/hmg/7.2.291
    1. Mole, S. E. , Williams, R. E. , & Goebel, H. H. (2005). Correlations between genotype, ultrastructural morphology and clinical phenotype in the neuronal ceroid lipofuscinoses. Neurogenetics, 6(3), 107–126. 10.1007/s10048-005-0218-3
    1. Nugent, T. , Mole, S. E. , & Jones, D. T. (2008). The transmembrane topology of Batten disease protein CLN3 determined by consensus computational prediction constrained by experimental data. FEBS Letters, 582(7), 1019–1024. 10.1016/j.febslet.2008.02.049
    1. Oetjen, S. , Kuhl, D. , & Hermey, G. (2016). Revisiting the neuronal localization and trafficking of CLN3 in juvenile neuronal ceroid lipofuscinosis. Journal of Neurochemistry, 139(3), 456–470. 10.1111/jnc.13744
    1. Persaud‐Sawin, D. A. , McNamara, J. O. , Rylova, S. , Vandongen, A. , & Boustany, R. M. N. (2004). A galactosylceramide binding domain is involved in trafficking of CLN3 from Golgi to rafts via recycling endosomes. Pediatric Research, 56(3), 449–463. 10.1203/01.Pdr.0000136152.54638.95
    1. Persaud‐Sawin, D. A. N. W. , VanDongen, A. , & Boustany, R. M. N. (2002). Motifs within the CLN3 protein: Modulation of cell growth rates and apoptosis. Human Molecular Genetics, 11(18), 2129–2142. 10.1093/hmg/11.18.2129
    1. Puranam, K. L. , Guo, W. X. , Qian, W. H. , Nikbakht, K. , & Boustany, R. M. (1999). CLN3 defines a novel antiapoptotic pathway operative in neurodegeneration and mediated by ceramide. Molecular Genetics and Metabolism, 66(4), 294–308. 10.1006/mgme.1999.2834
    1. Rapola, J. (1993). Neuronal ceroid‐lipofuscinoses in childhood. Perspectives in Pediatric Pathology, 17, 7–44.
    1. Ratajczak, E. , Petcherski, A. , Ramos‐Moreno, J. , & Ruonala, M. O. (2014). FRET‐assisted determination of CLN3 membrane topology. PLoS ONE, 9(7), e102593. 10.1371/journal.pone.0102593
    1. Ryazantsev, S. , Yu, W. H. , Zhao, H. Z. , Neufeld, E. F. , & Ohmi, K. (2007). Lysosomal accumulation of SCMAS (subunit c of mitochondrial ATP synthase) in neurons of the mouse model of mucopolysaccharidosis III B. Molecular Genetics and Metabolism, 90(4), 393–401. 10.1016/j.ymgme.2006.11.006
    1. Santavuori, P. (1988). Neuronal ceroid‐lipofuscinoses in childhood. Brain and Development, 10(2), 80–83.
    1. Santavuori, P. , Lauronen, L. , Kirveskari, K. , Aberg, L. , & Sainio, K. (2000). Neuronal ceroid lipofuscinoses in childhood. Supplements to Clinical neurophysiology, 53, 443–451.
    1. Stefanidakis, M. , Maeder, M. , Bounoutas, G. , Yudkoff, C. , Chao, H. , Haskett, S. , & Jiang, H. (2018). Efficient in vivo editing of CEP290 IVS26 by EDIT‐101 as a novel therapeutic for treatment of Leber Congenital Amaurosis 10. Investigative Ophthalmology & Visual Science, 59(9), 385.
    1. Wang, F. , Wang, H. , Tuan, H.‐F. , Nguyen, D. H. , Sun, V. , Keser, V. , Bowne, S. J. , Sullivan, L. S. , Luo, H. , Zhao, L. , Wang, X. , Zaneveld, J. E. , Salvo, J. S. , Siddiqui, S. , Mao, L. , Wheaton, D. K. , Birch, D. G. , Branham, K. E. , Heckenlively, J. R. , … Chen, R. (2014). Next generation sequencing‐based molecular diagnosis of retinitis pigmentosa: Identification of a novel genotype‐phenotype correlation and clinical refinements. Human Genetics, 133(3), 331–345. 10.1007/s00439-013-1381-5
    1. Xiong, J. , & Kielian, T. (2013). Microglia in juvenile neuronal ceroid lipofuscinosis are primed toward a pro‐inflammatory phenotype. Journal of Neurochemistry, 127(2), 245–258. 10.1111/jnc.12385
    1. Zhang, X. , Zhang, D. , Chen, S.‐C. , Lamey, T. , Thompson, J. A. , McLaren, T. , De Roach, J. N. , Chen, F. K. , & McLenachan, S. (2018). Generation of an induced pluripotent stem cell line from a patient with non‐syndromic CLN3‐associated retinal degeneration and a coisogenic control line. Stem Cell Research, 29, 245–249. 10.1016/j.scr.2018.04.014

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel