双 AAV 介导的人类奥托费林表达的恢复动力学。

IF 3.5 3区 医学 Q2 NEUROSCIENCES Frontiers in Molecular Neuroscience Pub Date : 2024-06-17 eCollection Date: 2024-01-01 DOI:10.3389/fnmol.2024.1376128
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
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引用次数: 0

摘要

基于双重腺相关病毒(AAV)的方法已在临床前解决了奥托费林(OTOF)致聋缺陷的问题。然而,使用双杂交 AAV 方法进行转导、mRNA 重组和蛋白质表达的时机尚未确定。在此,我们建立了一种体内外试验,以确定双 AAV 介导的 OTOF 在小鼠胞顶部毛细胞中的表达动力学。我们使用了两种不同的重组载体,其中一种包含在毛细胞特异性 Myo15 启动子控制下的 OTOF 的 5' 部分,另一种包含 OTOF 的 3' 部分。我们探索了小鼠胞顶部毛细胞中 Myo15 启动子的特异性,在 OTOF 缺失的小鼠模型中确定了 DB-OTO 的体内外剂量反应特性,并证明了 AAV1 在胞顶部毛细胞中的耐受性。此外,我们还确定了 5' 与 3' 载体一一对应比例的偏差对重组 OTOF 的影响很小。最后,我们确定了重组 OTOF mRNA 和蛋白表达量在体外 14 到 21 天达到高峰,恢复时间与体内模型相当。这些发现证明了体内外模型系统对探索表达动力学的实用性,并确定了体内外双 AAV 介导的 OTOF 表达的恢复时间。
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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.

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来源期刊
CiteScore
5.70
自引率
2.10%
发文量
669
审稿时长
14 weeks
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.
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