Selective peripheral neuromodulation through organ-specific AAV-mediated gene transfer

 描述（由申请人提供）：外周神经调节已被用于控制慢性疼痛和盆腔器官功能障碍等疾病。 这种疗法的扩展受到对外周神经回路的不完全理解的挑战，外周神经回路包括支配多个器官并携带传入和传出外周神经元的轴突的外周神经节和神经的复杂网络。 通过遗传工程化受体和通道（例如通道视紫红质和DREADD（设计药物独家激活的设计受体））操纵神经活性的方法为开发通过病毒载体介导的细胞特异性基因转移进行靶向神经调节的工具创造了机会。 使用细胞特异性启动子和组合载体系统靶向表达光遗传学或药物遗传学转基因，为外周神经调节的神经解剖学挑战提供了一种潜在的解决方案。 这项研究计划的长期目标是通过使用腺相关病毒（AAV）载体将神经调节转基因细胞特异性递送至外周神经节来开发外周神经调节策略。 基于我们对AAV载体生物分布的研究，本申请的目的是提供转基因表达位点特异性靶向传入和传出系统的概念验证。 我们将解决的核心假设，细胞特异性靶向的神经调节转基因的外周神经元的AAV载体，使器官特异性功能干预。 为了解决该项目的具体目标，我们将开发用于DREADD细胞特异性靶向的AAV载体，从神经解剖学上验证其受限的生物分布，并使用行为，体外和体内神经生理学以及体内成像分析来证明转基因的神经调节功能。 目的1：测试用于器官特异性神经调节的策略的可行性， 通过AAV介导的基因转移的传入系统。 我们将测试以下假设：使用携带抑制性DREADD（hM 4Di）的基因的Cre依赖性载体和AAV-Cre载体靶向结肠神经支配的感觉神经元的组合AAV载体将使得能够对传入活动进行器官特异性控制。 目标2：测试a的可行性 通过AAV介导基因转移进行传出自主神经系统的器官特异性神经调节的策略。 我们将测试这样的假设，即靶向副交感神经膀胱节后神经元的细胞特异性AAV载体，使用携带胆碱乙酰转移酶（ChaT）启动子控制下的兴奋性DREADD（hM 3Dq）基因的载体，将促进脊髓损伤的啮齿动物模型中的膀胱排空。 所提出的策略和矢量工具将使周围神经回路的功能解剖，适用于多个器官和跨物种和动物模型。 这些方法可以扩展到光遗传神经调节，与闭环平台整合，并可能随着AAV介导的基因转移的临床翻译进展而开发下一代神经调节疗法。

 DESCRIPTION (provided by applicant): Peripheral neuromodulation has been pursued to control conditions such as chronic pain and dysfunctions of pelvic organs. The expansion of such therapies is challenged by incomplete understanding of peripheral neural circuits, which comprise complex networks of peripheral ganglia and nerves that innervate multiple organs and carry axons of afferent as well as efferent peripheral neurons. Methods for manipulation of neural activity through genetically engineered receptors and channels such as channelrhodopsins and DREADDs (Designer Receptor Exclusively Activated by Designed Drug) create an opportunity for the development of tools for targeted neuromodulation through viral vector-mediated cell-specific gene transfer. The use of cell-specific promoters and combinatorial vector systems for targeted expression of optogenetic or pharmacogenetic transgenes presents a potential solution to the neuroanatomical challenges of peripheral neuromodulation. The long-term objective of this research program is to develop strategies for peripheral neuromodulation through cell-specific delivery of neuromodulatory transgenes to peripheral ganglia using adeno-associated viral (AAV) vectors. Based on our work on the biodistribution of AAV vectors, the objective of this application is to provide proof-of-concept fo site-specific targeting of transgene expression to afferent and efferent systems. We will address the central hypothesis that cell-specific targeting of a neuromodulatory transgene to peripheral neurons by AAV vectors enables organ-specific functional interventions. To address the Specific Aims of the project, we will develop AAV vectors for cell-specific targeting of DREADD, validate neuroanatomically their restricted biodistribution, and demonstrate the neuromodulatory function of the transgenes using behavioral, in vitro and in vivo neurophysiological, and in vivo imaging analyses. Aim 1: Test the feasibility of a strategy for organ-specific neuromodulation of afferent systems through AAV-mediated gene transfer. We will test the hypothesis that combinatorial AAV vector targeting of colon-innervating sensory neurons, using a Cre- dependent vector that carries the gene for the inhibitory DREADD (hM4Di) and an AAV-Cre vector, will enable organ-specific control of afferent activity. Aim 2: Test the feasibility of a strategy for organ-specific neuromodulation of efferent autonomic systems through AAV-mediated gene transfer. We will test the hypothesis that cell-specific AAV vector targeting to parasympathetic bladder post-ganglionic neurons, using a vector that carries the gene for the excitatory DREADD (hM3Dq) under the control of the cholineacetyl transferase (ChaT) promoter, will promote bladder emptying in a rodent model of spinal cord injury. The proposed strategies and vector tools will enable functional dissection of peripheral neural circuits, applicable to multiple organs and across species and animal models. These approaches can be extended to optogenetic neuromodulation, to integration with closed-loop platforms, and potentially to development of next generation neuromodulation therapies as the clinical translation of AAV-mediated gene transfer advances.