Endothelial and neuronal engagement by AAV-BR1 alleviates cholesterol deposition in a mouse model of Niemann-Pick type C2

Endothelial and neuronal engagement by AAV-BR1 alleviates cholesterol deposition in a mouse model of Niemann-Pick type C2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Endothelial and neuronal engagement by AAV-BR1 alleviates cholesterol deposition in a mouse model of Niemann-Pick type C2 Charlotte Laurfelt Munch Rasmussen, Christian Würtz Heegaard, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4238772/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Patients with the genetic disorder Niemann-Pick type C2 disease (NP-C2) suffer from lysosomal accumulation of cholesterol causing both systemic and severe neurological symptoms. In a murine NP-C2 model, otherwise successful intravenous Niemann-Pick C2 protein (NPC2) replacement therapy fails to alleviate progressive neurodegeneration as infused NPC2 is unable to cross the blood-brain barrier (BBB). Genetic modification of brain endothelial cells (BECs) is thought to enable secretion of recombinant proteins thereby overcoming the restrictions of the BBB. We hypothesized that BBB-directed gene therapy using the AAV-BR1-NPC2 vector would transduce both BECs and neurons in a mouse model of NP-C2 ( Npc2 -/-). Methods Six weeks old Npc2 -/- mice were intravenously injected with the AAV-BR1-NPC2 vector. Post-mortem analyses included gene expression analyses, determination of NPC2 transduction in the CNS, and co-detection of cholesterol with NPC2 in neurons. Results The vector exerted tropism for BECs and neurons resulting in a widespread NPC2 distribution in the brain with a concomitant reduction of cholesterol in adjacent neurons, presumably not transduced by the vector. Conclusion The data suggests cross-correcting gene therapy to the brain via delivery of NPC2 from BECs and neurons. Figures Figure 1 Introduction Niemann-Pick (NP) type C disease (NP-C) is an autosomal recessive lysosomal storage disease caused by mutations in the Npc1 or Npc2 genes leading to neurodegeneration ( 1 ). Loss of function of either of the resulting NP-C proteins results in lysosomal accumulation of cholesterol leading to human diseases NP-C1 and NP-C2 ( 2 ). The glycoprotein NPC2 is found in lysosomes and secretory fluids, and secreted NPC2 is endocytosed via the ubiquitous mannose-6-phosphate receptor (M6PR) pathway ( 3 ). Transport of NPC2 from blood to the brain is restricted because of the blood-brain barrier (BBB) formed by brain endothelial cells (BECs) and because M6PR expression by BECs declines after birth ( 4 ). NP-C2 progresses with fatal outcomes, and most patients die between 10 and 25 years of age. Curative treatment of NP-C2 is unavailable, and the development of new treatments is highly warranted ( 5 ). Genetic modification using a capsid-modified adeno-associated virus vector (AAV-BR1), known for its robust tropism for BECs, enables recombinant NPC2 secretion in vitro ( 3 ), suggesting that NPC2 released from BECs also might access the intact brain. The therapeutic potential of AAV-BR1 was recently verified in mouse models of neurodegeneration, i.e. Incontinentia pigmenti, Sandhoff disease, and Allan-Herndon-Dudley syndrome ( 6 – 8 ), with successful treatment enabled by induced gene expression in BECs. We recently showed that systemic administration of AAV-BR1 encoding NPC2 in healthy mice somewhat surprisingly transduced both BECs and neurons ( 3 ), suggesting that AAV-BR1 passes the BBB and enables neuronal transduction. Accordingly, we hypothesized that BBB-directed gene therapy using the AAV-BR1-NPC2 vector would transduce both BECs and neurons in a mouse model of NP-C2 ( Npc2 -/-). The present study demonstrates that intravenous (IV) injection of the AAV-BR1-NPC2 vector in NPC2-deficient mice aged six weeks evokes the distribution of NPC2 in BECs and neurons and lowers neuronal cholesterol. Npc2 -/- mice exhibit normal growth patterns similar to wildtype (WT) littermates until 6 weeks of age at which a single IV dose of 1.6 x 10 11 vg of the AAV-BR1-NPC2 vector was administered to Npc2 -/- mice. At 12 weeks, untreated and AAV-BR1-NPC2-treated Npc2 -/- mice revealed growth retardation, similar to that reported previously ( 10 ). In the Npc2 -/- mice, the AAV-BR1-NPC2 vector accumulated in brain, lung, and lightly in spleen (Fig. 1A). We subsequently compared Npc2 gene expression in these organs with levels found in untreated Npc2 -/- and WT littermates using qPCR (Supplementary Table S1 ). High accumulation of AAV-BR1-NPC2 vector in the brain increased the Npc2 gene expression to a level not significantly different from that of Npc2+/+ mice. Only a slight increase in Npc2 gene expression was seen in the lung after AAV-BR1 treatment despite high vector accumulation (Fig. 1A). Npc2 gene expression was absent from the spleen (data not shown), suggesting unspecific uptake of the AAV-BR1 vector. Cerebral distribution of NPC2 after transduction with the AAV-BR1-NPC2 vector was evaluated using immunohistochemistry. Injecting the vector revealed NPC2-positive neurons, especially in the cerebral cortex and hippocampus (Fig. 1A), and dispersed in the striatum, thalamus, hypothalamus, mesencephalon, pons, medulla oblongata, and cerebellum (not shown). Neurons were intensively stained with a weaker, yet consistent, staining of several brain capillaries. NPC2-immunoreactive cells with morphology corresponding to astrocytes, microglia, or oligodendrocytes were not observed. NPC2 protein was undetectable in untreated Npc2 -/- mice. There were variations in transduction efficiency among the treated mice, which correlated with an increased number of NPC2-positive cells, and a higher therapeutic potential. Cholesterol accumulation in the cortex cerebri and hippocampus visualized using filipin, a fluorescent antibiotic binding specifically to unesterified cholesterol, was undetectable in WT mice while present in Npc2 -/- mice with reduction after AAV-BR1-NPC2-treatment (Fig. 1B). As reduced cholesterol appeared in areas with many NPC2-containing neurons after AAV-BR1-NPC2-treatment, we aimed for co-detection of filipin and NPC2 focusing on cortex cerebri and hippocampus (Fig. 1C). In cortex cerebri of AAV-BR1-NPC2-treated Npc2 -/- mice, superimposing immunolabeled images with filipin staining evidenced that NPC2 neurons contained cholesterol but to a much lesser extent than seen in untreated Npc2 -/- mice (Fig. 1C, left panel). Similarly, the high number of NPC2-positive neurons found in the CA3 region of the hippocampus in AAV-BR1-NPC2-treated Npc2 -/- mice was almost completely reversed concerning cholesterol accumulation (Fig. 1C, right panel). The number of NPC2-labeled neurons in AAV-BR1-NPC2-treated Npc2 -/- mice was lower than that of cholesterol-negative cells in both cerebral cortex and hippocampus, which suggests that secreted NPC2 from AAV-BR1-NPC2- transduced neurons alleviates cholesterol accumulation in non-transduced neighboring neurons corresponding to neuronal cross-correction ( 6 ). Collectively, our data showed that BBB-directed gene therapy using the AAV-BR1 vector led to the widespread appearance of NPC2 in BECs and neurons, which was accompanied by reduction in neuronal cholesterol in the cerebral cortex and hippocampus. The integrity of the BBB is often compromised during pathological conditions, but this is not expected in the Npc2 -/- mice. NPC2 replacement therapy in a mouse model of NP-C2 similar to that of the present study mended visceral cholesterol storage but failed to improve neurological symptoms after repeated intravenous injections with NPC2, stating the incapability of NPC2 to cross the BBB (c.f. 3). This was emphasized previously where improvement in the BBB integrity was observed after intravenous injections of the AAV-BR1-vector in mice suffering from Incontinentia pigmenti ( 6 , 8 ). There is, therefore, no reason to believe that the AAV-BR1 vector passes the BBB and transduces neurons due to compromised barrier integrity in the Npc2 -/- mice. However, the mechanism of this BBB passage remains unknown. Loss of BBB integrity seen in pathological conditions was not observed in Npc2 -/- mice ( 9 ). The predominant appearance of NPC2-positive cells was in the hippocampus consistent with studies using healthy mice ( 9 ). Compared to evolutionary higher regions, the hippocampal vessels differ by lesser neurovascular coupling between vascular networks and functions of BECs and pericytes, which may facilitate viral vector binding and capillary migration. The distribution pattern of NPC2 in the neurons was similar to our previous study investigating the AAV-BR1-NPC2 in healthy BALB/cJRj mice ( 9 ). Here the AAV-BR1 vector was designed to produce two separate proteins; enhanced green fluorescent protein (eGFP) and NPC2, where the eGFP accumulated intracellularly as an indicator of cellular transduction. However, also neuronal cells expressed eGFP, indicating that some of the systemically injected AAV-BR1 undergoes transport across the BBB leading to neuronal transduction ( 9 ). In our previous study in healthy mice ( 9 ), we further examined the possibility of AAV-BR1 vector transport across the blood-cerebrospinal fluid barrier (BCSFB) but found no evidence to support this. Furthermore, it seems unlikely that the widespread distribution of NPC2 throughout the brain, including the cerebral cortex, could be achieved through BCSFB transport. When examining the correlation between the distribution of NPC2 and cholesterol storage in both the cerebral cortex and hippocampus, a clear reduction in cholesterol accumulation was seen in NPC2-positive neurons but also neighboring NPC2-negative cells, emphasizing the possibility of cross-correction after BBB-directed gene therapy. The heterogeneous, albeit substantial, neuronal presence of NPC2, might be a reflection of cation-independent M6PR mediated uptake of the mannose 6-phosphate tagged NPC2 ( 3 ), as cation-independent M6PR also distributes to neurons quite heterogeneously in the brain with a predominantly high occurrence in deep layers of the cortex (e.g., pyramidal neurons in layer V), neurons of the hippocampus, striatum, selected nuclei in the thalamus, Purkinje cells of the cerebellum, the deep cerebellar nuclei, red nucleus, pontine nucleus, and motor neurons of the brainstem (c.f. 3), corresponding well to the areas where NPC2-positive neurons are observed. In conclusion, BBB-directed gene therapy using the AAV-BR1 vector corresponded with widespread neuronal expression of NPC2 and reduction of cholesterol accumulation. Cortex cerebri and hippocampus are severely affected by neurodegeneration in mouse models of NP-C and human NP-C and display intraneuronal lipid deposition, especially in large pyramidal neurons. The high transduction in cortex cerebri and hippocampus reduced cholesterol accumulation in both genetically modified (NPC2+) and non-transduced (NPC2-) neurons suggesting cross-correcting gene therapy via secretion of NPC2 from BECs and NPC2 + neurons. Abbreviations AAV-BR1, adeno-associated virus vector BR-1; BBB, blood-brain barrier; BCSFB, blood-cerebrospinal fluid barrier; BECs, brain endothelial cells; IV, intravenous; NP, Niemann-Pick; NP-C, Niemann-Pick type C disease; WT, wildtype Declarations Ethics approval and consent for publication The animal studies were performed according to the Danish Animal Experimentation Act (BEK no. 2028 of 14/12/2020) and the European directive (2010/63/EU) and carried out by licensed staff. The Danish Animal Experiments Inspectorate under the Ministry of Food, Fisheries, and Agriculture has approved all animal experiments and breeding of NPC2-deficient mice (license no. 2018-15-0201-01467 and 2019-15-0202-00056). Consent for publication N ot applicable Availability of data and materials All data generated and analyzed during this study are included in this published paper. All datasets are available from the corresponding author upon reasonable request. Competing interests JK is listed as an inventor on a patent on AAV-BR1, held by Boehringer Ingelheim International. Funding This work was funded by Fonden til Lægevidenskabens Fremme (CLMR), Direktør Emil C. Hertz og hustru Inger Hertz' Fond (CWH), Dagmar Marshalls Fond, Lundbeck Foundation (CWH), Research Initiative on Blood-Brain Barriers and Drug Delivery (Grant no. 2013-14113) (TM ), Hørslev-Fonden (ABL), Læge Sophus Carl Emil Friis og Hustru Olga Doris Friis Legat (ABL), Scleroseforeningen (Grant no. A41926) (TM ) and Svend Andersen Fonden (TM ). German Research Foundation (DFG, SFB1328 - A13, Grant no. 335447717) (JK). Author Contributions Conceptualization: CLMR, JK, AB, TM; Methodology: CLMR, CWH, EH, JK, AB; Investigation: CLMR, MST, EH, BL, LBT, AB; Resources: CLMR, CWH, EH, JK, MS, AB, TM; Writing – Original Draft: CLMR, AB; Writing – Review & Editing: CLMR, CWH, MST, EH, BL, JK, LBT, MS, AB, TM; Visualization; CLMR, AB; Supervision: MST, CWH, LBT, AB, TM; Funding Acquisition: CLMR, JK, AB, TM Acknowledgments The authors thank laboratory technicians Merete Fredsgaard, Hanne Krone Nielsen, Ditte Bech Laursen, and Louise Hvilshøj Madsen, Aalborg University, Denmark, and animal technicians Karina Lassen Holm and Dorte Hermansen, Aarhus University, Denmark, for excellent technical assistance during the study. Associate Professor Anders Olsen and Helene Halkjær Jensen, Department of Chemistry and Bioscience, Aalborg University are acknowledged for assistance with the use of the Olympus IX83 inverted microscope equipped with Yokogawa confocal CSU-W1 spinning disk. References Platt FM. Emptying the stores: lysosomal diseases and therapeutic strategies. Nat Rev Drug Discov. 2018;17(2):133–50. Li X, Saha P, Lib J, Blobel G, Pfeffer SR. Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2. Proc Natl Acad Sci U S A. 2016;113(36):10079–84. Hede E, Christiansen CB, Heegaard CW, Moos T, Burkhart A. Gene therapy to the blood-brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease. J Neurochem. 2021;156(3):290–308. Lin Y, Wang X, Rose KP, Dai M, Han J, Xin M, et al. miR-143 Regulates Lysosomal Enzyme Transport across the Blood-Brain Barrier and Transforms CNS Treatment for Mucopolysaccharidosis Type I. Mol Ther. 2020;28(10):2161–76. Muramatsu K, Muramatsu SI. Adeno-associated virus vector-based gene therapies for pediatric diseases. Pediatr Neonatol. 2023;64(Suppl 1):S3–9. Körbelin J, Dogbevia G, Michelfelder S, Ridder DA, Hunger A, Wenzel J, et al. A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases. EMBO Mol Med. 2016;8(6):609–25. Dogbevia G, Grasshoff H, Othman A, Penno A, Schwaninger M. Brain endothelial specific gene therapy improves experimental Sandhoff disease. J Cereb Blood Flow Metab. 2020;40(6):1338–50. Sundaram SM, Arrulo Pereira A, Müller-Fielitz H, Köpke H, De Angelis M, Müller TD, et al. Gene therapy targeting the blood–brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency. Brain. 2022;145(12):4264–74. Rasmussen CLM, Hede E, Routhe LJ, Körbelin J, Helgudottir SS, Thomsen LB, et al. A novel strategy for delivering Niemann-Pick type C2 proteins across the blood–brain barrier using the brain endothelial-specific AAV-BR1 virus. J Neurochem. 2023;164:6–28. Nielsen GK, Dagnaes-Hansen F, Holm IE, Meaney S, Symula D, Andersen NT, et al. Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease. PLoS ONE. 2011;6(11):e27287. Supplementary Files Rasmussenetal.SupplMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4238772","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291118379,"identity":"4bc5f729-b7e8-40c1-b6bd-1daf281fe39c","order_by":0,"name":"Charlotte Laurfelt Munch Rasmussen","email":"","orcid":"","institution":"Aalborg University: Aalborg Universitet","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"Laurfelt Munch","lastName":"Rasmussen","suffix":""},{"id":291118380,"identity":"b637dab0-019c-4f62-bb1e-568f89976597","order_by":1,"name":"Christian Würtz 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Universitet","correspondingAuthor":false,"prefix":"","firstName":"Annette","middleName":"","lastName":"Burkhart","suffix":""}],"badges":[],"createdAt":"2024-04-08 23:29:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4238772/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4238772/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55009849,"identity":"4e4ec108-5b6e-450e-8491-e0bf6856693b","added_by":"auto","created_at":"2024-04-19 19:16:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15988002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003cbr\u003e\nUpper left, \u003c/strong\u003eBiodistribution of AAV-BR1-NPC2 (viral genomes (vg)) in brain, lung, liver, and spleen, analyzed by quantitative qPCR at 12 weeks (n=3).\u003cbr\u003e\n\u003cstrong\u003eLower left, \u003c/strong\u003eRelative\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eNpc2\u003c/em\u003e gene expression in the brain and lung analyzed by RT-qPCR in \u003cem\u003eNpc2\u003c/em\u003e+/+ (n=3, black), \u003cem\u003eNpc2\u003c/em\u003e-/- (n=3, red), and AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice (n=3, green). \u003cem\u003eNpc2\u003c/em\u003e expression is significantly lower in the brains of untreated\u003cem\u003e Npc2\u003c/em\u003e-/- mice. \u003cem\u003eNpc2\u003c/em\u003e expression in the lungs of treated and untreated \u003cem\u003eNpc2\u003c/em\u003e-/- mice is significantly lower compared to wild-type littermates (Mean ± SD). Data are analyzed with a one-way ANOVA (F\u003csub\u003eCerebrum\u003c/sub\u003e[2,6] = 9.64, \u003cem\u003ep\u003c/em\u003e = 0.013, F\u003csub\u003eLung\u003c/sub\u003e[2,6] = 74.52, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) with Tukey's multiple comparisons test (* \u003cem\u003ep\u003c/em\u003e = 0.011, *** \u003cem\u003ep\u003c/em\u003e ≤ 0.0001).\u003cbr\u003e\n\u003cstrong\u003eUpper right, \u003c/strong\u003eImmunohistochemistry reveals NPC2-positive cells in the brain of \u003cem\u003eNpc2\u003c/em\u003e-/- mice after AAV-BR1-NPC2 gene therapy, particularly prominent in neurons of cortex cerebri (ctxc) and hippocampus (hp) (CA3 region). NPC2 is also seen in brain capillaries (arrows). Scale bars = 50 µm (ctxc and hp), 25 µm (lower row).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCholesterol accumulation (visualized as white elements in filipin staining) is virtually absent in cortex cerebri of \u003cem\u003eNpc2\u003c/em\u003e+/+ mice, clearly present in \u003cem\u003eNpc2\u003c/em\u003e-/- mice, and reduced when treated with AAV-BR1-NPC2. Scale bars = 50 µm and 20 µm (white boxes).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeurons (visualized with neuronal nuclei antigen (NeuN) immunolabeling (red)) also exhibit NPC2 immunoreactivity (green, arrows)) in cortex cerebri and hippocampus in AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice. Of note, AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice are virtually devoid of filipin labeling, clearly contrasted by the robust appearance in untreated \u003cem\u003eNpc2\u003c/em\u003e-/- mice. In \u003cem\u003eNpc2\u003c/em\u003e-/- mice, treatment reduction of cholesterol accumulation is not limited to NPC2 expressing cells, but also present in neighboring cells suggesting uptake of secreted NPC2 equivalent to cross-correction. Scale bar = 25µm.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4238772/v1/dbb9b7b6a614a99b0a18b506.png"},{"id":56894276,"identity":"bc488cf5-788a-4fa2-8c18-f300f1c7afcb","added_by":"auto","created_at":"2024-05-21 21:13:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19827025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4238772/v1/ba4e2b54-0c89-4773-9054-d936e6f3b0b9.pdf"},{"id":55009848,"identity":"11af6fe2-0c9b-403d-a675-7d19d088ad7f","added_by":"auto","created_at":"2024-04-19 19:16:23","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":43062,"visible":true,"origin":"","legend":"","description":"","filename":"Rasmussenetal.SupplMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4238772/v1/4c5b809581a214196066b818.docx"}],"financialInterests":"","formattedTitle":"Endothelial and neuronal engagement by AAV-BR1 alleviates cholesterol deposition in a mouse model of Niemann-Pick type C2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNiemann-Pick (NP) type C disease (NP-C) is an autosomal recessive lysosomal storage disease caused by mutations in the \u003cem\u003eNpc1\u003c/em\u003e or \u003cem\u003eNpc2\u003c/em\u003e genes leading to neurodegeneration (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Loss of function of either of the resulting NP-C proteins results in lysosomal accumulation of cholesterol leading to human diseases NP-C1 and NP-C2 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The glycoprotein NPC2 is found in lysosomes and secretory fluids, and secreted NPC2 is endocytosed via the ubiquitous mannose-6-phosphate receptor (M6PR) pathway (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Transport of NPC2 from blood to the brain is restricted because of the blood-brain barrier (BBB) formed by brain endothelial cells (BECs) and because M6PR expression by BECs declines after birth (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNP-C2 progresses with fatal outcomes, and most patients die between 10 and 25 years of age. Curative treatment of NP-C2 is unavailable, and the development of new treatments is highly warranted (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Genetic modification using a capsid-modified adeno-associated virus vector (AAV-BR1), known for its robust tropism for BECs, enables recombinant NPC2 secretion \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), suggesting that NPC2 released from BECs also might access the intact brain. The therapeutic potential of AAV-BR1 was recently verified in mouse models of neurodegeneration, i.e. Incontinentia pigmenti, Sandhoff disease, and Allan-Herndon-Dudley syndrome (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), with successful treatment enabled by induced gene expression in BECs. We recently showed that systemic administration of AAV-BR1 encoding NPC2 in healthy mice somewhat surprisingly transduced both BECs and neurons (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), suggesting that AAV-BR1 passes the BBB and enables neuronal transduction. Accordingly, we hypothesized that BBB-directed gene therapy using the AAV-BR1-NPC2 vector would transduce both BECs and neurons in a mouse model of NP-C2 (\u003cem\u003eNpc2\u003c/em\u003e-/-).\u003c/p\u003e \u003cp\u003eThe present study demonstrates that intravenous (IV) injection of the AAV-BR1-NPC2 vector in NPC2-deficient mice aged six weeks evokes the distribution of NPC2 in BECs and neurons and lowers neuronal cholesterol. \u003cem\u003eNpc2\u003c/em\u003e-/- mice exhibit normal growth patterns similar to wildtype (WT) littermates until 6 weeks of age at which a single IV dose of 1.6 x 10\u003csup\u003e11\u003c/sup\u003e vg of the AAV-BR1-NPC2 vector was administered to \u003cem\u003eNpc2\u003c/em\u003e-/- mice. At 12 weeks, untreated and AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice revealed growth retardation, similar to that reported previously (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eNpc2\u003c/em\u003e-/- mice, the AAV-BR1-NPC2 vector accumulated in brain, lung, and lightly in spleen (Fig.\u0026nbsp;1A). We subsequently compared \u003cem\u003eNpc2\u003c/em\u003e gene expression in these organs with levels found in untreated \u003cem\u003eNpc2\u003c/em\u003e-/- and WT littermates using qPCR (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). High accumulation of AAV-BR1-NPC2 vector in the brain increased the \u003cem\u003eNpc2\u003c/em\u003e gene expression to a level not significantly different from that of \u003cem\u003eNpc2+/+\u003c/em\u003e mice. Only a slight increase in \u003cem\u003eNpc2\u003c/em\u003e gene expression was seen in the lung after AAV-BR1 treatment despite high vector accumulation (Fig.\u0026nbsp;1A). \u003cem\u003eNpc2\u003c/em\u003e gene expression was absent from the spleen (data not shown), suggesting unspecific uptake of the AAV-BR1 vector. Cerebral distribution of NPC2 after transduction with the AAV-BR1-NPC2 vector was evaluated using immunohistochemistry. Injecting the vector revealed NPC2-positive neurons, especially in the cerebral cortex and hippocampus (Fig.\u0026nbsp;1A), and dispersed in the striatum, thalamus, hypothalamus, mesencephalon, pons, medulla oblongata, and cerebellum (not shown). Neurons were intensively stained with a weaker, yet consistent, staining of several brain capillaries. NPC2-immunoreactive cells with morphology corresponding to astrocytes, microglia, or oligodendrocytes were not observed. NPC2 protein was undetectable in untreated \u003cem\u003eNpc2\u003c/em\u003e-/- mice. There were variations in transduction efficiency among the treated mice, which correlated with an increased number of NPC2-positive cells, and a higher therapeutic potential.\u003c/p\u003e \u003cp\u003eCholesterol accumulation in the cortex cerebri and hippocampus visualized using filipin, a fluorescent antibiotic binding specifically to unesterified cholesterol, was undetectable in WT mice while present in \u003cem\u003eNpc2\u003c/em\u003e-/- mice with reduction after AAV-BR1-NPC2-treatment (Fig.\u0026nbsp;1B). As reduced cholesterol appeared in areas with many NPC2-containing neurons after AAV-BR1-NPC2-treatment, we aimed for co-detection of filipin and NPC2 focusing on cortex cerebri and hippocampus (Fig.\u0026nbsp;1C). In cortex cerebri of AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice, superimposing immunolabeled images with filipin staining evidenced that NPC2 neurons contained cholesterol but to a much lesser extent than seen in untreated \u003cem\u003eNpc2\u003c/em\u003e-/- mice (Fig.\u0026nbsp;1C, left panel). Similarly, the high number of NPC2-positive neurons found in the CA3 region of the hippocampus in AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice was almost completely reversed concerning cholesterol accumulation (Fig.\u0026nbsp;1C, right panel). The number of NPC2-labeled neurons in AAV-BR1-NPC2-treated \u003cem\u003eNpc2\u003c/em\u003e-/- mice was lower than that of cholesterol-negative cells in both cerebral cortex and hippocampus, which suggests that secreted NPC2 from AAV-BR1-NPC2- transduced neurons alleviates cholesterol accumulation in non-transduced neighboring neurons corresponding to neuronal cross-correction (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCollectively, our data showed that BBB-directed gene therapy using the AAV-BR1 vector led to the widespread appearance of NPC2 in BECs and neurons, which was accompanied by reduction in neuronal cholesterol in the cerebral cortex and hippocampus.\u003c/p\u003e \u003cp\u003eThe integrity of the BBB is often compromised during pathological conditions, but this is not expected in the \u003cem\u003eNpc2\u003c/em\u003e-/- mice. NPC2 replacement therapy in a mouse model of NP-C2 similar to that of the present study mended visceral cholesterol storage but failed to improve neurological symptoms after repeated intravenous injections with NPC2, stating the incapability of NPC2 to cross the BBB (c.f. 3). This was emphasized previously where improvement in the BBB integrity was observed after intravenous injections of the AAV-BR1-vector in mice suffering from Incontinentia pigmenti (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). There is, therefore, no reason to believe that the AAV-BR1 vector passes the BBB and transduces neurons due to compromised barrier integrity in the \u003cem\u003eNpc2\u003c/em\u003e-/- mice. However, the mechanism of this BBB passage remains unknown. Loss of BBB integrity seen in pathological conditions was not observed in \u003cem\u003eNpc2\u003c/em\u003e-/- mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The predominant appearance of NPC2-positive cells was in the hippocampus consistent with studies using healthy mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Compared to evolutionary higher regions, the hippocampal vessels differ by lesser neurovascular coupling between vascular networks and functions of BECs and pericytes, which may facilitate viral vector binding and capillary migration.\u003c/p\u003e \u003cp\u003eThe distribution pattern of NPC2 in the neurons was similar to our previous study investigating the AAV-BR1-NPC2 in healthy BALB/cJRj mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Here the AAV-BR1 vector was designed to produce two separate proteins; enhanced green fluorescent protein (eGFP) and NPC2, where the eGFP accumulated intracellularly as an indicator of cellular transduction. However, also neuronal cells expressed eGFP, indicating that some of the systemically injected AAV-BR1 undergoes transport across the BBB leading to neuronal transduction (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In our previous study in healthy mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), we further examined the possibility of AAV-BR1 vector transport across the blood-cerebrospinal fluid barrier (BCSFB) but found no evidence to support this. Furthermore, it seems unlikely that the widespread distribution of NPC2 throughout the brain, including the cerebral cortex, could be achieved through BCSFB transport.\u003c/p\u003e \u003cp\u003eWhen examining the correlation between the distribution of NPC2 and cholesterol storage in both the cerebral cortex and hippocampus, a clear reduction in cholesterol accumulation was seen in NPC2-positive neurons but also neighboring NPC2-negative cells, emphasizing the possibility of cross-correction after BBB-directed gene therapy. The heterogeneous, albeit substantial, neuronal presence of NPC2, might be a reflection of cation-independent M6PR mediated uptake of the mannose 6-phosphate tagged NPC2 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), as cation-independent M6PR also distributes to neurons quite heterogeneously in the brain with a predominantly high occurrence in deep layers of the cortex (e.g., pyramidal neurons in layer V), neurons of the hippocampus, striatum, selected nuclei in the thalamus, Purkinje cells of the cerebellum, the deep cerebellar nuclei, red nucleus, pontine nucleus, and motor neurons of the brainstem (c.f. 3), corresponding well to the areas where NPC2-positive neurons are observed.\u003c/p\u003e \u003cp\u003eIn conclusion, BBB-directed gene therapy using the AAV-BR1 vector corresponded with widespread neuronal expression of NPC2 and reduction of cholesterol accumulation. Cortex cerebri and hippocampus are severely affected by neurodegeneration in mouse models of NP-C and human NP-C and display intraneuronal lipid deposition, especially in large pyramidal neurons. The high transduction in cortex cerebri and hippocampus reduced cholesterol accumulation in both genetically modified (NPC2+) and non-transduced (NPC2-) neurons suggesting cross-correcting gene therapy via secretion of NPC2 from BECs and NPC2\u0026thinsp;+\u0026thinsp;neurons.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAV-BR1, adeno-associated virus vector BR-1; BBB, blood-brain barrier; BCSFB, blood-cerebrospinal fluid barrier; BECs, brain endothelial cells; IV, intravenous; NP, Niemann-Pick; NP-C, Niemann-Pick type C disease; WT, wildtype\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent for publication\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe animal studies were performed according to the Danish Animal Experimentation Act (BEK no. 2028 of 14/12/2020) and the European directive (2010/63/EU) and carried out by licensed staff. The Danish Animal Experiments Inspectorate under the Ministry of Food, Fisheries, and Agriculture has approved all animal experiments and breeding of NPC2-deficient mice (license no. 2018-15-0201-01467 and 2019-15-0202-00056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003cbr\u003e\u0026nbsp;N\u003c/strong\u003eot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eAll data generated and analyzed during this study are included in this published paper. All datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eJK is listed as an inventor on a patent on AAV-BR1, held by Boehringer Ingelheim International.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThis work was funded by Fonden til L\u0026aelig;gevidenskabens Fremme (CLMR), Direkt\u0026oslash;r Emil C. Hertz og hustru Inger Hertz\u0026apos; Fond (CWH), Dagmar Marshalls Fond, Lundbeck Foundation (CWH), Research Initiative on Blood-Brain Barriers and Drug Delivery (Grant no. 2013-14113) (TM ), H\u0026oslash;rslev-Fonden (ABL), L\u0026aelig;ge Sophus Carl Emil Friis og Hustru Olga Doris Friis Legat (ABL),\u0026nbsp;Scleroseforeningen (Grant no. A41926) (TM ) and\u0026nbsp;Svend Andersen Fonden (TM ).\u0026nbsp;German Research Foundation (DFG, SFB1328 - A13, Grant no.\u0026nbsp;335447717) (JK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eConceptualization: CLMR, JK, AB, TM; Methodology: CLMR, CWH, EH, JK, AB; Investigation: CLMR, MST, EH, BL, LBT, AB; Resources: CLMR, CWH, EH, JK, MS, AB, TM; Writing \u0026ndash; Original Draft: CLMR, AB; Writing \u0026ndash; Review \u0026amp; Editing: CLMR, CWH, MST, EH, BL, JK, LBT, MS, AB, TM; Visualization; CLMR, AB; Supervision: MST, CWH, LBT, AB, TM; Funding Acquisition: CLMR, JK, AB, TM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe authors thank laboratory technicians Merete Fredsgaard, Hanne Krone Nielsen, Ditte Bech Laursen, and Louise Hvilsh\u0026oslash;j Madsen, Aalborg University, Denmark, and animal technicians Karina Lassen Holm and Dorte Hermansen, Aarhus University, Denmark, for excellent technical assistance during the study. Associate Professor Anders Olsen and Helene Halkj\u0026aelig;r Jensen, Department of Chemistry and Bioscience, Aalborg University are acknowledged for assistance with the use of the Olympus IX83 inverted microscope equipped with Yokogawa confocal CSU-W1 spinning disk.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003ePlatt FM. Emptying the stores: lysosomal diseases and therapeutic strategies. Nat Rev Drug Discov. 2018;17(2):133\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi X, Saha P, Lib J, Blobel G, Pfeffer SR. Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2. Proc Natl Acad Sci U S A. 2016;113(36):10079\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHede E, Christiansen CB, Heegaard CW, Moos T, Burkhart A. Gene therapy to the blood-brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease. J Neurochem. 2021;156(3):290\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLin Y, Wang X, Rose KP, Dai M, Han J, Xin M, et al. miR-143 Regulates Lysosomal Enzyme Transport across the Blood-Brain Barrier and Transforms CNS Treatment for Mucopolysaccharidosis Type I. Mol Ther. 2020;28(10):2161\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMuramatsu K, Muramatsu SI. Adeno-associated virus vector-based gene therapies for pediatric diseases. Pediatr Neonatol. 2023;64(Suppl 1):S3\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eK\u0026ouml;rbelin J, Dogbevia G, Michelfelder S, Ridder DA, Hunger A, Wenzel J, et al. A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases. EMBO Mol Med. 2016;8(6):609\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDogbevia G, Grasshoff H, Othman A, Penno A, Schwaninger M. Brain endothelial specific gene therapy improves experimental Sandhoff disease. J Cereb Blood Flow Metab. 2020;40(6):1338\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSundaram SM, Arrulo Pereira A, M\u0026uuml;ller-Fielitz H, K\u0026ouml;pke H, De Angelis M, M\u0026uuml;ller TD, et al. Gene therapy targeting the blood\u0026ndash;brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency. Brain. 2022;145(12):4264\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRasmussen CLM, Hede E, Routhe LJ, K\u0026ouml;rbelin J, Helgudottir SS, Thomsen LB, et al. A novel strategy for delivering Niemann-Pick type C2 proteins across the blood\u0026ndash;brain barrier using the brain endothelial-specific AAV-BR1 virus. J Neurochem. 2023;164:6\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNielsen GK, Dagnaes-Hansen F, Holm IE, Meaney S, Symula D, Andersen NT, et al. Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease. PLoS ONE. 2011;6(11):e27287.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4238772/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4238772/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePatients with the genetic disorder Niemann-Pick type C2 disease (NP-C2) suffer from lysosomal accumulation of cholesterol causing both systemic and severe neurological symptoms. In a murine NP-C2 model, otherwise successful intravenous Niemann-Pick C2 protein (NPC2) replacement therapy fails to alleviate progressive neurodegeneration as infused NPC2 is unable to cross the blood-brain barrier (BBB). Genetic modification of brain endothelial cells (BECs) is thought to enable secretion of recombinant proteins thereby overcoming the restrictions of the BBB. We hypothesized that BBB-directed gene therapy using the AAV-BR1-NPC2 vector would transduce both BECs and neurons in a mouse model of NP-C2 (\u003cem\u003eNpc2\u003c/em\u003e-/-).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSix weeks old \u003cem\u003eNpc2\u003c/em\u003e-/- mice were intravenously injected with the AAV-BR1-NPC2 vector. Post-mortem analyses included gene expression analyses, determination of NPC2 transduction in the CNS, and co-detection of cholesterol with NPC2 in neurons.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe vector exerted tropism for BECs and neurons resulting in a widespread NPC2 distribution in the brain with a concomitant reduction of cholesterol in adjacent neurons, presumably not transduced by the vector.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe data suggests cross-correcting gene therapy to the brain via delivery of NPC2 from BECs and neurons.\u003c/p\u003e","manuscriptTitle":"Endothelial and neuronal engagement by AAV-BR1 alleviates cholesterol deposition in a mouse model of Niemann-Pick type C2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 19:16:18","doi":"10.21203/rs.3.rs-4238772/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"572f6fa1-2d35-44d7-8040-231ca5126754","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-21T21:04:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-19 19:16:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4238772","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4238772","identity":"rs-4238772","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}