Enhanced gene delivery for CNS and sensory disorders

项目摘要/摘要。用腺相关病毒(AAV)载体进行基因治疗在 失明和血友病等疾病的人体临床试验。然而，基因治疗还处于早期阶段 解决遗传性听力损失(IHL)。感觉细胞，称为“毛细胞”，驻留在内耳，调节听力和 平衡，是基因治疗纠正IHL的主要靶点。AAV载体是目前最有前景的载体 为内耳提供基因治疗的载体，尽管将基因输送到毛细胞的效率并不理想。主要的长线- 这项建议的长期目标是为听力和平衡障碍的治疗开发一个有效的基因传递平台。 我们已经开发了一种由内源性纳米颗粒相关的AAV载体组成的混合基因递送载体 我们称之为exosome，我们称之为exo-AAV。外源AAV1基因加成疗法的最新研究进展 在小鼠模型中实现了史无前例的内耳毛细胞基因传递和部分听力恢复和平衡 耳聋涉及毛细胞立体纤毛(Tmhs)基因的四胺基膜蛋白(Tmhs)。目前的提案旨在 在提高我们的基因传递效率和向临床相关的大型动物模型转化方面。我们有 最近发现AAV9变异体AAV9-PHP.B在注射到新生小鼠体内后强健地转导毛细胞 和非人灵长类(NHP)。通过AAV将基因输送到NHP的内耳以前从未被证明过，而且 将是迈向临床试验的关键一步。我们将测试是否可以提高AAV9-PHP.B的转导效率 甚至通过将其整合到外体中(exo-AAV9-PHP.B)。我们的具体目标是(1)改善基因传递 AAV9-PHP.B和exo-AAV9-PHP.B对耳蜗毛细胞的作用；(2)探讨外源AAV的作用机制 细胞转导和AAV9-PHP.B毛细胞转导；(3)开发外源AAV平台，使工业 用于临床的放大/制造。对于目标1，我们有初步数据显示AAV9-PHP.B介导 在啮齿动物和NHP体内将基因高效地传递到耳蜗毛细胞。在目标1a中，我们将直接比较外源基因 几种剂量的AAV9-PHP.B和标准AAV9-PHP.B，用于给小鼠和NHP的毛细胞输送。在目标1b中，我们测试 外源AAV9-PHP.B和编码TMHs的AAV9-PHP.B对TMHs听力和平衡功能障碍的挽救作用 基因敲除老鼠。在目标1c中，我们将比较外源AAV9-PHP.B和AAV9-PHP.B对NHP毛细胞的基因传递。在……里面 目的2通过阻断特定细胞，研究外源AAV转导毛细胞的机制。 过程以及AAV衣壳突变体，以辨别AAV衣壳或外体对基因转移的特定贡献 组件。在目标3中，我们将利用外切体膜的特性来允许阴离子交换柱 层析，一种工业可扩展的方法，用于纯化外源AAV。我们还在与一个细胞合作 和基因治疗合同研究机构，在悬浮细胞中开发可规模生产的外源AAV。这些 是将该技术转化为临床的重要步骤。这项研究很重要，因为它解决了一个 医疗需要以及对社会的经济负担，治疗遗传性耳聋。此外，所获得的数据 在这项工作中，将对其他可以用外源AAV基因治疗系统治疗的疾病具有重要意义。

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.