Jonathan B Sellon, Kathy S So, Andrew D'Arcangelo, Sarah Cancelarich, Meghan C Drummond, Peter G Slade, Ning Pan, Tyler M Gibson, Tian Yang, Joseph C Burns, Adam T Palermo, Lars Becker
{"title":"双 AAV 介导的人类奥托费林表达的恢复动力学。","authors":"Jonathan B Sellon, Kathy S So, Andrew D'Arcangelo, Sarah Cancelarich, Meghan C Drummond, Peter G Slade, Ning Pan, Tyler M Gibson, Tian Yang, Joseph C Burns, Adam T Palermo, Lars Becker","doi":"10.3389/fnmol.2024.1376128","DOIUrl":null,"url":null,"abstract":"<p><p>Deafness-causing deficiencies in <i>otoferlin</i> (<i>OTOF</i>) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an <i>ex vivo</i> assay to determine the kinetics of dual-AAV mediated expression of <i>OTOF</i> in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of <i>OTOF</i> under the control of the hair cell-specific <i>Myo15</i> promoter, and the other the 3' portion of <i>OTOF</i>. We explored specificity of the <i>Myo15</i> promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO <i>ex vivo</i> in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined <i>OTOF</i>. Finally, we established a plateau in quantity of recombined <i>OTOF</i> mRNA and protein expression by 14 to 21 days <i>ex vivo</i> with comparable recovery timing to that <i>in vivo</i> model. These findings demonstrate the utility of an <i>ex vivo</i> model system for exploring expression kinetics and establish <i>in vivo</i> and <i>ex vivo</i> recovery timing of dual AAV-mediated <i>OTOF</i> expression.</p>","PeriodicalId":12630,"journal":{"name":"Frontiers in Molecular Neuroscience","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11215969/pdf/","citationCount":"0","resultStr":"{\"title\":\"Recovery kinetics of dual AAV-mediated human otoferlin expression.\",\"authors\":\"Jonathan B Sellon, Kathy S So, Andrew D'Arcangelo, Sarah Cancelarich, Meghan C Drummond, Peter G Slade, Ning Pan, Tyler M Gibson, Tian Yang, Joseph C Burns, Adam T Palermo, Lars Becker\",\"doi\":\"10.3389/fnmol.2024.1376128\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Deafness-causing deficiencies in <i>otoferlin</i> (<i>OTOF</i>) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an <i>ex vivo</i> assay to determine the kinetics of dual-AAV mediated expression of <i>OTOF</i> in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of <i>OTOF</i> under the control of the hair cell-specific <i>Myo15</i> promoter, and the other the 3' portion of <i>OTOF</i>. We explored specificity of the <i>Myo15</i> promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO <i>ex vivo</i> in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined <i>OTOF</i>. Finally, we established a plateau in quantity of recombined <i>OTOF</i> mRNA and protein expression by 14 to 21 days <i>ex vivo</i> with comparable recovery timing to that <i>in vivo</i> model. These findings demonstrate the utility of an <i>ex vivo</i> model system for exploring expression kinetics and establish <i>in vivo</i> and <i>ex vivo</i> recovery timing of dual AAV-mediated <i>OTOF</i> expression.</p>\",\"PeriodicalId\":12630,\"journal\":{\"name\":\"Frontiers in Molecular Neuroscience\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-06-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11215969/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Molecular Neuroscience\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3389/fnmol.2024.1376128\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"NEUROSCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Molecular Neuroscience","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3389/fnmol.2024.1376128","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"NEUROSCIENCES","Score":null,"Total":0}
Recovery kinetics of dual AAV-mediated human otoferlin expression.
Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.
期刊介绍:
Frontiers in Molecular Neuroscience is a first-tier electronic journal devoted to identifying key molecules, as well as their functions and interactions, that underlie the structure, design and function of the brain across all levels. The scope of our journal encompasses synaptic and cellular proteins, coding and non-coding RNA, and molecular mechanisms regulating cellular and dendritic RNA translation. In recent years, a plethora of new cellular and synaptic players have been identified from reduced systems, such as neuronal cultures, but the relevance of these molecules in terms of cellular and synaptic function and plasticity in the living brain and its circuits has not been validated. The effects of spine growth and density observed using gene products identified from in vitro work are frequently not reproduced in vivo. Our journal is particularly interested in studies on genetically engineered model organisms (C. elegans, Drosophila, mouse), in which alterations in key molecules underlying cellular and synaptic function and plasticity produce defined anatomical, physiological and behavioral changes. In the mouse, genetic alterations limited to particular neural circuits (olfactory bulb, motor cortex, cortical layers, hippocampal subfields, cerebellum), preferably regulated in time and on demand, are of special interest, as they sidestep potential compensatory developmental effects.