Enhanced gene delivery for CNS and sensory disorders

项目总结/摘要。使用腺相关病毒（AAV）载体的基因治疗在癌症治疗中显示出巨大的前景。 用于治疗失明和血友病等疾病的人体临床试验。然而，基因治疗仍处于早期阶段， 遗传性听力损失（IHL）被称为“毛细胞”的感觉细胞位于内耳中并调节听觉和听觉。 平衡，并代表了基因治疗的主要目标，以纠正国际人道主义法。AAV载体是目前最有前途的 虽然基因递送到毛细胞的效率是次优的，但是用于内耳的基因治疗的载体。最长的- 本项目的长期目标是开发一种有效的基因传递平台，用于治疗听力和平衡障碍。 我们已经开发了一种由AAV载体和内源性纳米颗粒组成的杂交基因递送载体 叫做外泌体，我们称之为exo-AAV我们最近的手稿使用基因添加疗法与exo-AAV 1 在小鼠模型中实现了前所未有的基因传递到内耳毛细胞，并部分挽救了听力和平衡 毛细胞静纤毛四跨膜蛋白（tetraspan membrane protein of hair cell stereocilia，Tmhs）基因参与耳聋的发生。目前的提案旨在 在提高我们的基因传递效率和翻译到临床相关的大型动物模型。我们有 最近发现，AAV 9变体AAV 9-PHP.B在注射到新生小鼠中后可稳健地转导毛细胞 非人类灵长类动物（NHP）以前从未证实过通过AAV将基因递送至NHP内耳， 将是迈向临床试验的关键一步我们将测试我们是否能提高AAV 9-PHP.B的转导效率 甚至进一步通过其掺入外来体（exo-AAV 9-PHP.B）。我们的具体目标是（1）改善基因递送 AAV 9-PHP.B和exo-AAV 9-PHP.B对耳蜗毛细胞的作用;（2）探讨exo-AAV增强的机制 B转导毛细胞;（3）开发exo-AAV平台， 用于临床用途的规模扩大/生产。对于目的1，我们有初步数据显示AAV 9-PHP.B介导了 在啮齿类动物和NHP中，将基因有效递送至耳蜗的毛细胞。在Aim 1a中，我们将直接比较exo- AAV 9-PHP.B和标准AAV 9-PHP.B以几个剂量递送至小鼠和NHP中的毛细胞。在目标1b中，我们测试 exo-AAV 9-PHP.B和编码Tmhs的AAV 9-PHP.B拯救Tmhs中的听力和平衡功能障碍的能力 敲除小鼠。在目标Ic中，我们将比较外切-AAV 9-PHP.B和AAV 9-PHP.B在基因递送至NHP中的毛细胞方面的情况。在 目的2：探讨exo-AAV转导毛细胞的机制， 过程以及AAV衣壳突变体，以辨别AAV衣壳或外泌体对基因转移的特异性贡献 件.在目标3中，我们将利用外泌体膜的特性， 在一个实施方案中，使用层析（一种工业规模化的方法）纯化exo-AAV。我们还与一个小组合作 和基因治疗合同研究组织开发悬浮细胞中exo-AAV的可扩展生产。这些 是该技术临床转化的重要步骤。这项研究很重要，因为它解决了一个未得到满足的问题。 医疗需求以及对社会的经济负担，遗传性听力损失的治疗。此外，获得的数据 在工作期间，对于其他适合用exo-AAV基因治疗系统治疗的疾病将是重要的。

PROJECT SUMMARY/ABSTRACT. Gene therapy using adeno-associated virus (AAV) vectors has shown great promise in human clinical trials for diseases such as blindness and hemophilia. However, gene therapy is in its early phases to address inherited hearing loss (IHL). Sensory cells called “hair cells”, reside in the inner ear and mediate hearing and balance, and represent a major target for gene therapy to correct IHL. AAV vectors are currently the most promising vector for gene therapy to the inner ear, although gene delivery efficiency to hair cells is suboptimal. The major long- term goal of this proposal is to develop an effective gene delivery platform for therapy of hearing and balance disorders. We have developed a hybrid gene delivery vector comprised of AAV vectors associated with endogenous nanoparticles called exosomes, which we have termed exo-AAV. Our recent manuscript using gene addition therapy with exo-AAV1 achieved unprecedented gene delivery to inner ear hair cells and partial rescue of hearing and balance in a mouse model of deafness involving the tetraspan membrane protein of hair cell stereocilia (Tmhs) gene. The current proposal is aimed at enhancing our gene delivery efficiency and translation towards clinically relevant large animal models. We have recently discovered that an AAV9 variant, AAV9-PHP.B, robustly transduces hair cells after injection into neonatal mice and non-human primates (NHP). Gene delivery to the inner ear of NHP by AAV has never be demonstrated before and will be a crucial step towards clinical trials. We will test whether we can improve AAV9-PHP.B’s transduction efficiency even further via its incorporation into exosomes (exo-AAV9-PHP.B). Our specific aims are (1) To improve gene delivery to cochlear hair cells using AAV9-PHP.B and exo-AAV9-PHP.B; (2) Investigate mechanisms of enhanced exo-AAV transduction of cells and AAV9-PHP.B transduction of hair cells; (3) To develop the exo-AAV platform to allow industrial scale-up/manufacturing for clinical use. For aim 1, we have preliminary data showing that AAV9-PHP.B mediates efficient gene delivery to hair cells of the cochlea in vivo in rodents and NHP. In Aim 1a we will directly compare exo- AAV9-PHP.B and standard AAV9-PHP.B at several doses for delivery to hair cells in mice and in NHPs. In Aim 1b we test the ability of exo-AAV9-PHP.B and AAV9-PHP.B encoding Tmhs to rescue hearing and balance dysfunction in the Tmhs knockout mouse. In Aim 1 c we will compare exo-AAV9-PHP.B and AAV9-PHP.B at gene delivery to hair cells in NHPs. In Aim 2, we will investigate mechanism of transduction of hair cells by exo-AAV using blockade of specific cellular processes as well as AAV capsid mutants to discern specific contributions to gene transfer by the AAV capsid or exosome components. In aim 3, we will use properties of the exosome membrane to allow anion-exchange column chromatography, an industrial scalable approach, for the purification of exo-AAV. We are also collaborating with a cell and gene therapy contract research organization to develop scalable production of exo-AAV in suspension cells. These are important steps towards clinical translation of the technology. This research is important as it addresses an unmet medical need as well as financial burden to society, treatment of inherited hearing loss. Furthermore, the data obtained during the work will be important for other diseases amenable to treatment with the exo-AAV gene therapy system.