{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T20:52:28Z","timestamp":1783803148020,"version":"3.55.0"},"reference-count":41,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,3,30]],"date-time":"2021-03-30T00:00:00Z","timestamp":1617062400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100001116","name":"Foundation Fighting Blindness","doi-asserted-by":"publisher","award":["TA-GT-0715-0665-LUMC"],"award-info":[{"award-number":["TA-GT-0715-0665-LUMC"]}],"id":[{"id":"10.13039\/100001116","id-type":"DOI","asserted-by":"publisher"}]},{"name":"the Netherlands Organization for Health Research and Development","award":["ZonMw grant 43200004"],"award-info":[{"award-number":["ZonMw grant 43200004"]}]},{"DOI":"10.13039\/501100005040","name":"Bontius Stichting","doi-asserted-by":"publisher","award":["Bontius 23122019"],"award-info":[{"award-number":["Bontius 23122019"]}],"id":[{"id":"10.13039\/501100005040","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Dutch blindness funds","award":["Uitzicht 2013-13"],"award-info":[{"award-number":["Uitzicht 2013-13"]}]},{"name":"Dutch blindness funds","award":["Uitzicht 2018-6"],"award-info":[{"award-number":["Uitzicht 2018-6"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in M\u00fcller glial cells while CRB2 was observed at the subapical region in both photoreceptors and M\u00fcller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly M\u00fcller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect M\u00fcller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in M\u00fcller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.<\/jats:p>","DOI":"10.3390\/ijms22073563","type":"journal-article","created":{"date-parts":[[2021,3,30]],"date-time":"2021-03-30T09:57:04Z","timestamp":1617098224000},"page":"3563","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Defining Phenotype, Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in Crb1"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9756-8898","authenticated-orcid":false,"given":"Nanda","family":"Boon","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9776-5701","authenticated-orcid":false,"given":"C. Henrique","family":"Alves","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aat A.","family":"Mulder","sequence":"additional","affiliation":[{"name":"Department of Cell &amp; Chemical Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlotte A.","family":"Andriessen","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9594-1123","authenticated-orcid":false,"given":"Thilo M.","family":"Buck","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9940-4264","authenticated-orcid":false,"given":"Peter M. J.","family":"Quinn","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rogier M.","family":"Vos","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abraham J.","family":"Koster","sequence":"additional","affiliation":[{"name":"Department of Cell &amp; Chemical Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8342-1094","authenticated-orcid":false,"given":"Carolina R.","family":"Jost","sequence":"additional","affiliation":[{"name":"Department of Cell &amp; Chemical Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0099-460X","authenticated-orcid":false,"given":"Jan","family":"Wijnholds","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands"},{"name":"Netherlands Institute for Neuroscience, Institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), Meibergdreef 47, 1105 BA Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1038\/13848","article-title":"Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)","volume":"23","author":"Payne","year":"1999","journal-title":"Nat. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1083\/jcb.200109010","article-title":"The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of crumbs and discs lost","volume":"157","author":"Roh","year":"2002","journal-title":"J. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4169","DOI":"10.1242\/jcs.01301","article-title":"Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure","volume":"117","author":"Kantardzhieva","year":"2004","journal-title":"J. Cell Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1101\/cshperspect.a027961","article-title":"The Crumbs3 polarity protein","volume":"10","author":"Margolis","year":"2018","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"564","DOI":"10.1523\/JNEUROSCI.3496-06.2007","article-title":"A single amino acid substitution (Cys249Trp) in Crb1 causes retinal degeneration and deregulates expression of pituitary tumor transforming gene Pttg1","volume":"27","author":"Meuleman","year":"2007","journal-title":"J. Neurosci."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Pellissier, L.P., Alves, C.H., Quinn, P.M., Vos, R.M., Tanimoto, N., Lundvig, D.M.S., Dudok, J.J., Hooibrink, B., Richard, F., and Beck, S.C. (2013). Targeted ablation of Crb1 and Crb2 in retinal progenitor cells mimics leber congenital amaurosis. PLoS Genet.","DOI":"10.1371\/journal.pgen.1003976"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3384","DOI":"10.1093\/hmg\/ddu048","article-title":"Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa","volume":"23","author":"Alves","year":"2014","journal-title":"Hum. Mol. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1093\/hmg\/ddy337","article-title":"Loss of CRB2 in M\u00fcller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype","volume":"28","author":"Quinn","year":"2019","journal-title":"Hum. Mol. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3137","DOI":"10.1093\/hmg\/ddy194","article-title":"CRB2 in immature photoreceptors determines the superior-inferior symmetry of the developing retina to maintain retinal structure and function","volume":"27","author":"Quinn","year":"2018","journal-title":"Hum. Mol. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"6093","DOI":"10.1523\/JNEUROSCI.3412-14.2015","article-title":"A new CRB1 rat mutation links m\u00fcller glial cells to retinal telangiectasia","volume":"35","author":"Zhao","year":"2015","journal-title":"J. Neurosci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1038\/s41573-019-0012-9","article-title":"Adeno-associated virus vector as a platform for gene therapy delivery","volume":"18","author":"Wang","year":"2019","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/hmg\/ddv062","article-title":"Gene therapy into photoreceptors and M\u00fcller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models","volume":"24","author":"Pellissier","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1016\/j.omtm.2020.12.012","article-title":"AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse mode","volume":"20","author":"Buck","year":"2021","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4611","DOI":"10.1167\/iovs.04-0541","article-title":"Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system","volume":"45","author":"Prusky","year":"2004","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1017\/S0952523805225166","article-title":"Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system","volume":"22","author":"Douglas","year":"2005","journal-title":"Vis. Neurosci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1016\/0014-4835(78)90022-2","article-title":"Glutamine synthetase in the developing rat retina: An immunohistochemical study","volume":"27","author":"Riepe","year":"1978","journal-title":"Exp. Eye Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2179","DOI":"10.1093\/hmg\/ddg232","article-title":"CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina","volume":"12","author":"Mehalow","year":"2003","journal-title":"Hum. Mol. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"van de Pavert, S.A., Sanz sanz, A., Aartsen, W.M., Vos, R.M., Versteeg, I., Beck, S.C., Klooster, J., Seeliger, M.W., and Wijnholds, J. (2007). Crb1 is a determinant of retinal apical muller glia cell features. Glia, 1486\u20131497.","DOI":"10.1002\/glia.20561"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1016\/j.stemcr.2019.03.002","article-title":"Human iPSC-derived retinas recapitulate the fetal CRB1 CRB2 complex formation and demonstrate that photoreceptors and m\u00fcller glia are targets of AAV5","volume":"12","author":"Quinn","year":"2019","journal-title":"Stem Cell Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"6232","DOI":"10.1167\/iovs.12-9569","article-title":"Discordant anatomical, electrophysiological, and visual behavioral profiles of retinal degeneration in rat models of retinal degenerative disease","volume":"53","author":"McGill","year":"2012","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-020-17009-7","article-title":"Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease","volume":"11","author":"Ray","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fnins.2020.00860","article-title":"Research models and gene augmentation therapy for CRB1 retinal dystrophies","volume":"14","author":"Boon","year":"2020","journal-title":"Front. Neurosci."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Berger, A., Cavallero, S., Dominguez, E., Barbe, P., Simonutti, M., Sahel, J.A., Sennlaub, F., Raoul, W., Paques, M., and Bemelmans, A.P. (2014). Spectral-domain optical coherence tomography of the rodent eye: Highlighting layers of the outer retina using signal averaging and comparison with histology. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0096494"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6898","DOI":"10.1167\/iovs.11-7701","article-title":"Human CRB1-associated retinal degeneration: Comparison with the rd8 Crb1-mutant mouse model","volume":"52","author":"Aleman","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1378","DOI":"10.1167\/iovs.16-20363","article-title":"Long-term characterization of retinal degeneration in royal college of surgeons rats using spectral-domain optical coherence tomography","volume":"58","author":"Ryals","year":"2017","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2659","DOI":"10.1093\/hmg\/ddl194","article-title":"Pals1\/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized M\u00fcller glia cells","volume":"15","author":"Aartsen","year":"2006","journal-title":"Hum. Mol. Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3759","DOI":"10.1093\/hmg\/ddu089","article-title":"CRB2 acts as a modifying factor of CRB1-related retinal dystrophies in mice","volume":"23","author":"Pellissier","year":"2014","journal-title":"Hum. Mol. Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"175","DOI":"10.3389\/fnins.2017.00175","article-title":"The CRB1 complex: Following the trail of crumbs to a feasible gene therapy strategy","volume":"11","author":"Quinn","year":"2017","journal-title":"Front. Neurosci."},{"key":"ref_29","unstructured":"Han, I.C., Cheng, J.L., Burnight, E., Ralston, C.L., Fick, J.L., Thomsen, G.J., Tovar, E.F., Russell, S., Sohn, E.H., and Mullins, R.F. (2020). Retinal tropism and transduction of adeno-associated virus (AAV) varies by serotype and route of delivery (intravitreal, subretinal or suprachoroidal) in rats. Hum. Gene Ther., 1\u201329."},{"key":"ref_30","first-page":"1","article-title":"Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina","volume":"8","author":"Lipinski","year":"2013","journal-title":"PLoS ONE"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Klimczak, R.R., Koerber, J.T., Dalkara, D., Flannery, J.G., and Schaffer, D.V. (2009). A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat M\u00fcller cells. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0007467"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"571","DOI":"10.1089\/hum.2009.194","article-title":"Changes in adeno-associated virus-mediated gene delivery in retinal degeneration","volume":"21","author":"Kolstad","year":"2010","journal-title":"Hum. Gene Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/S1525-0016(03)00144-8","article-title":"AAV-mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa","volume":"8","author":"Smith","year":"2003","journal-title":"Mol. Ther."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5326","DOI":"10.1167\/iovs.16-19701","article-title":"A novel method combining vitreous aspiration and intravitreal AAV2\/8 injection results in retina-wide transduction in adult mice","volume":"57","author":"Segelken","year":"2016","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1093\/hmg\/ddu424","article-title":"The severity of retinal pathology in homozygous Crb1rd8\/rd8 mice is dependent on additional genetic factors","volume":"24","author":"Luhmann","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"6006","DOI":"10.1038\/ncomms7006","article-title":"Gene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179","volume":"6","author":"Nishiguchi","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/srep10731","article-title":"Electroretinographic assessment of rod- and cone-mediated bipolar cell pathways using flicker stimuli in mice","volume":"5","author":"Tanimoto","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1007\/s10633-016-9534-1","article-title":"Differentiation of murine models of \u201cnegative ERG\u201d by single and repetitive light stimuli","volume":"132","author":"Tanimoto","year":"2016","journal-title":"Doc. Ophthalmol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1007\/978-1-4939-7522-8_10","article-title":"AAV gene augmentation therapy for CRB1-associated retinitis pigmentosa","volume":"Volume 1715","author":"Alves","year":"2018","journal-title":"Methods in Molecular Biology (Clifton, N.J.)"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"8356","DOI":"10.1167\/iovs.11-7575","article-title":"Ultrastructural localization and expression of TRPM1 in the human retina","volume":"52","author":"Klooster","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1083\/jcb.201201140","article-title":"Virtual nanoscopy: Generation of ultra-large high resolution electron microscopy maps","volume":"198","author":"Faas","year":"2012","journal-title":"J. Cell Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/7\/3563\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:33:16Z","timestamp":1760362396000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/7\/3563"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,30]]},"references-count":41,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,4]]}},"alternative-id":["ijms22073563"],"URL":"https:\/\/doi.org\/10.3390\/ijms22073563","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,30]]}}}