优化无调性基因1诱导的前庭毛细胞再生。
Optimizing atoh1-induced vestibular hair cell regeneration.
作者信息
Staecker Hinrich, Schlecker Christina, Kraft Shannon, Praetorius Mark, Hsu Chi, Brough Douglas E
机构信息
Department of Otolaryngology, Head and Neck Surgery, University of Kansas School of Medicine, Kansas City, Kansas.
出版信息
Laryngoscope. 2014 Oct;124 Suppl 5(Suppl 5):S1-S12. doi: 10.1002/lary.24775. Epub 2014 Jun 17.
OBJECTIVES/HYPOTHESIS: Determine the optimal design characteristics of an adenoviral (Ad) vector to deliver atoh1 and induce regeneration of vestibular hair cells.
STUDY DESIGN
Evaluation of a mouse model of intralabyrinthine gene delivery. Tissue culture of mouse and human macular organs.
METHODS
Macular organs from adult C57Bl/6 mice were treated with binding modified and alternate adenovectors expressing green fluorescent protein (gfp) or luciferase (L). Expression of marker genes was determined over time to determine vector transfection efficiency. The inner ear of adult mice was then injected with modified vectors. Expression of gfp and distribution of vector DNA was followed. Hearing and balance function was evaluated in normal animals to ensure safety of the novel vector designs. An optimized vector was identified and tested for its ability to induce hair cell regeneration in a mouse vestibulopathy model. Finally, this vector was tested for its ability to induce hair cell regeneration in human tissue.
RESULTS
Ad5 serotype-based vectors were identified as having a variety of different binding capacities for inner ear tissue. This makes it difficult to limit the dose of vector due to entry into nontargeted cells. Screening of rare adenovector serotypes demonstrated that Ad-based vectors were ideally suited for delivery to supporting cells; therefore, they were useful for hair cell regeneration studies. Utilization of an Ad28-based vector to deliver atoh1 to a mouse model of vestibular loss resulted in significant functional recovery of balance. This vector was also capable of transfecting human macular organs and inducing regeneration of human vestibular hair cells in vitro.
CONCLUSIONS
Improvement in vector design can lead to more specific cell-based delivery and reduction of nonspecific delivery of the trans gene, leading to the development of optimized molecular therapeutics to induce hair cell regeneration.
LEVEL OF EVIDENCE
N/A. Laryngoscope 124:S1-S12, 2014.
目的/假设：确定腺病毒（Ad）载体的最佳设计特征，以递送atoh1并诱导前庭毛细胞再生。
研究设计
对内耳基因递送小鼠模型进行评估。对小鼠和人类黄斑器官进行组织培养。
方法
用表达绿色荧光蛋白（gfp）或荧光素酶（L）的结合修饰型和替代腺病毒载体处理成年C57Bl/6小鼠的黄斑器官。随时间测定标记基因的表达，以确定载体转染效率。然后向成年小鼠内耳注射修饰载体。追踪gfp的表达和载体DNA的分布。在正常动物中评估听力和平衡功能，以确保新型载体设计的安全性。鉴定出一种优化载体，并在小鼠前庭病变模型中测试其诱导毛细胞再生的能力。最后，测试该载体在人体组织中诱导毛细胞再生的能力。
结果
基于Ad5血清型的载体被确定对内耳组织具有多种不同的结合能力。这使得由于进入非靶向细胞而难以限制载体剂量。对稀有腺病毒载体血清型的筛选表明，基于Ad的载体非常适合递送至支持细胞；因此，它们可用于毛细胞再生研究。利用基于Ad28的载体将atoh1递送至前庭丧失小鼠模型，导致平衡功能显著恢复。该载体还能够转染人类黄斑器官并在体外诱导人类前庭毛细胞再生。
结论
载体设计的改进可导致更具特异性的基于细胞的递送，并减少转基因的非特异性递送，从而开发出优化的分子疗法以诱导毛细胞再生。
证据水平
无。《喉镜》2014年第124卷：S1 - S12。
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