Rescue of channelopathy induced anosmias

描述(由申请人提供)：建议的K99/R00申请包含了一个全面的研究和培训计划，用于研究嗅觉系统中的离子通道和通道病变。离子通道在包括嗅觉神经元在内的许多细胞类型的兴奋性调节中起着关键作用。这些蛋白质调节离子在细胞膜上的移动。在OSNs中，离子通道负责对气味刺激做出反应，使细胞去极化，初始化动作电位和突触传递。通道病是一类人类遗传性疾病，其离子通道功能被破坏，导致多个器官系统的缺陷。众所周知，离子通道的中断会导致癫痫、心律失常、失明、耳聋和疼痛敏感性的改变。人类患者的通道病可能是由于离子通道基因的功能丧失和功能获得突变所致。最近有研究表明，通道病会导致人类嗅觉障碍。在小鼠模型中，几种不同的离子通道的缺失也会导致嗅觉障碍，这表明嗅觉信号可以在多个步骤中受到影响。这项拟议的研究将分析基因治疗在这些信号传递的不同阶段纠正经络病引起的嗅觉功能缺陷的能力。这项提案的目标1将使用两个在环核苷酸门控(CNG)通道亚基中具有靶向缺失的小鼠品系。CNGA2和CNGB1是嗅觉CNG通道的关键亚基，它们的缺失会导致嗅觉丧失。使用腺病毒，我将把缺失基因的功能性拷贝传递给突变的OSN，以测试恢复无嗅觉动物嗅觉功能的能力。嗅觉的恢复将用电生理和行为方法进行分析。目的2研究基因治疗对OSNs由于钠通道α亚单位Nav1.7(由SCN9A编码)丢失引起的突触传递功能障碍的纠正能力。本研究以嗅觉特异性SCN9A缺失小鼠为研究对象，使用腺病毒在OSNs中表达异位SCN9A，并分析其对突触传递的恢复作用。此外，这个目的将通过腺病毒介导的OSN中已识别的突变钠通道的表达来分析SCN9A功能获得突变的影响。这将测试过度兴奋对嗅觉功能的影响。最后，目标3将分析电压门控钙通道(VGCC)的两个α亚基Cav1.3和Cav2.2在嗅觉功能中的作用。VGCC的突变，包括CACNA1D(编码Cav1.3)，是影响感觉功能的经络病的基础，因此是嗅觉障碍的潜在原因。了解这些VGCC在嗅觉系统中的功能将有助于在无嗅觉患者中识别新的突变。总之，这三个目标的结果将为嗅觉信号的机制和基因治疗纠正离子通道功能缺陷的能力提供新的见解。这项拟议研究的结果将对帮助开发治疗因经络病变而导致的嗅觉障碍患者具有重要意义。此外，这些结果可能为开发其他感官缺陷的治疗方法提供洞察力，如视力和听力损失。

DESCRIPTION (provided by applicant): The proposed K99/R00 application incorporates a comprehensive research and training plan for studying ion channels and channelopathies in the olfactory system. Ion channels are critical for regulating excitability in many cell types includin olfactory sensory neurons (OSNs). These proteins regulate the movement of ions across the cellular membrane. In OSNs ion channels are responsible for depolarizing the cell in response to odor stimulation, initializing an action potential and synaptic transmission. Channelopathies are a class of human genetic disorders in which ion channel function is disrupted leading to defects in multiple organ systems. Disruptions in ion channels are known to cause epilepsy, cardiac arrhythmias, blindness, deafness and alterations in pain sensitivity. Channelopathies in human patients can result from both loss-of- function and gain-of-function mutations in ion channel genes. It has recently been shown that channelopathies can result in anosmia in humans. Deletions of several different ion channels in mouse models also causes anosmia, indicating that olfactory signaling can be affected at multiple steps. The proposed research will analyze the ability of gene therapy to correct channelopathy induced defects in olfactory function at these different stages in signaling. Aim 1 of this proposal will use two mouse strains with targeted deletions in cyclic nucleotide gated (CNG) channel subunits. CNGA2 and CNGB1 are critical subunits of the olfactory CNG channel necessary for odor detection and their loss leads to anosmia. Using adenovirus, I will deliver functional copies of the missing gene to mutant OSNs to test the ability to restore olfactory function to anosmic animals. Restoration of the sense of smell will be analyzed with electrophysiological and behavioral methods. Aim 2 will investigate the ability of gene therapy to correct channelopathy induced defects in synaptic transmission due to loss of the sodium channel alpha-subunit Nav1.7 (encoded by Scn9a) in OSNs. Using olfactory specific Scn9a null mice, I will use adenovirus to express ectopic Scn9a in OSNs and analyze the restoration of synaptic transmission. In addition this aim will analyze the effects of gain-of-function mutations in Scn9a through adenovirus-mediated expression of identified mutant sodium channels in OSNs. This will test the effect of hyper-excitability on olfactory function. Finally Aim 3 will analyze the role of two voltage gated calcium channel (VGCC) alpha-subunits, Cav1.3 and Cav2.2, in olfactory function. Mutations in VGCCs, including CACNA1D (encoding Cav1.3), underlie channelopathy disorders affecting sensory function and are therefore potential causes of anosmia. An understanding of the function of these VGCCs in the olfactory system will help to direct the identification of novel mutations in anosmic patients. Together the results from these 3 aims will provide new insight into the mechanisms of olfactory signaling and the ability of gene therapy to correct defects in ion channel function. The results from the proposed research will be important for helping to develop therapies for patients with anosmia due to channelopathies. In addition these results may provide insight into developing treatments for other sensory defects such as vision and hearing loss.