Enhanced intratympanic delivery of therapeutics to treat and prevent hearing loss using nanovesicles in the porcine model

项目摘要 我的长期目标是帮助开发和提供再生疗法，用于临床治疗 听力损失不幸的是，目前还没有高通量、非破坏性的治疗方法。 递送到耳蜗/内耳中。本建议的目标，实现这一目标的下一步， 长期目标是使用相关的大型动物模型（猪）来i）开发离体模型系统， 模仿人类圆窗膜（RWM），跟踪治疗相关物质通过 RWM，和ii）创建一种方法，以增强治疗剂进入内耳的鼓室内递送， 纳米囊泡，包括细胞外囊泡（外来体）。我们的初步研究支持的核心假设是， 数据，是离体猪RWM模型可以用来确定运输的最佳条件， 外泌体（或其他纳米囊泡）以通过RWM将货物递送到内耳中，从而促进内分泌。 治疗剂的高效递送。提出这一建议的理由是， 提供一个框架，从而可以测试新的治疗剂库，用于非手术递送到内部， 耳，在一个大型动物模型中，该模型在大小、生理学和 遗传相似性（常见耳聋基因的氨基酸序列）。以下具体目标 将在K99（目标1和2）和R 00阶段进行：目标1）进一步验证离体猪RWM 模型，以证明药物渗透性等同于人体组织报告的渗透性;目的2）鉴定和 评估外泌体和其他纳米囊泡在体外增强穿过猪RWM的货物运输;目的3） 评价通过纳米囊泡穿过猪RWM的体内转运。在第一个目标下，我们将测量 具有已知递送效率的治疗剂的渗透性和一些有前景的治疗相关材料， 初步可行性验证的离体RWM模型，并将其与人体组织报告的值进行比较。 对于第二个目的，纳米囊泡包括：RWM外泌体（成功分离并表征为纳米囊泡）。 初步结果）、间充质干细胞外泌体（金标准）和脂质体（FDA批准）， 将使用离体RWM模型评估装载有前景的治疗剂的细胞的转运效率。 最后，对于最后一个目标，一旦建立了与通过RWM的最佳运输相关联的参数， 在离体模型中，我们将通过猪鼓膜注射载药纳米囊泡 并测量它们穿过RWM的通道和体内耳蜗细胞的摄取。K99建成后， 预期结果为1。提供一个安全和可平移的平台，以测试治疗剂进入 内耳，和2.关于纳米囊泡作为有前途的治疗剂的新型非手术运输的效率的数据 到耳朵。这些结果预计将产生积极的影响，因为它们可以改善药物筛选， 这可能会促进递送新的再生疗法来治疗和预防听力损失。

PROJECT SUMMARY My long-term goal is to help develop and deliver regeneration therapeutics useful for the clinical treatment of hearing loss. Unfortunately, at this time there is no high throughput, non-destructive method of therapeutic delivery into the cochlea/inner ear. The objectives of this proposal, the next step toward the attainment of this long-term goal, are to use a relevant large animal model (the pig) to i) develop an ex-vivo model system, which mimics human round window membrane (RWM), to track the passage of therapy-related substances through the RWM, and ii) create a method to enhance the intratympanic delivery of therapeutics into the inner ear using nanovesicles including extracellular vesicles (exosomes). The central hypothesis, supported by our preliminary data, is that an ex-vivo porcine RWM model can be used to determine optimal conditions for the transport of exosomes (or other nanovesicles) to deliver cargo through the RWM into the inner ear, thereby facilitating the high-efficiency delivery of therapeutics. The rationale for this proposal is that its successful completion is likely to offer a framework whereby a new pool of therapeutics can be tested for non-surgical delivery into the inner ear, in a large animal model that has the preclinical advantage over rodents in terms of size, physiology, and genetic similarity (amino-acid sequences of common deafness genes) to humans. The following specific aims will be pursued during K99 (aim 1 and 2) and R00 phase: Aim 1) To further validate the ex-vivo porcine RWM model to demonstrate drug permeability equivalent to that reported for human tissue; Aim 2) To identify and evaluate exosomes and other nanovesicles enhancing cargo transport across porcine RWM in-vitro; Aim 3) To evaluate in-vivo transport across porcine RWM by nanovesicles. Under the first aim, we will measure the permeability of therapeutics with known delivery efficiencies and some promising therapy related materials using a preliminary viability-verified ex-vivo RWM model and compare those with values reported for human tissue. For the second aim, nanovesicles including: RWM exosomes (successfully isolated and characterized as a preliminary result), mesenchymal stem cell exosomes (gold standard), and liposomes (FDA approved), that are loaded with promising therapeutics will be evaluated for the efficiency of transport using the ex-vivo RWM model. Finally, for the last aim, once the parameters associated with optimal transport through the RWM are established with the ex-vivo model, we will inject therapeutic-loaded nanovesicles through the porcine tympanic membrane and measure their passage across RWM and uptake by cochlear cells in vivo. Upon completion of the K99, the expected outcomes are 1. Availability of a safe and translatable platform to test transport of therapeutics into the inner ear, and 2. Data on the efficiency of nanovesicles as novel nonsurgical transport of promising therapeutics to the ear. These results are expected to have a positive impact because they could improve drug screening for delivery and is likely to boost delivering novel regenerative therapeutics to treat and prevent hearing loss.