Mechanosensitive Channel based Pressure-Modulating Gene Therapy for Glaucoma Treatment

青光眼是一种难以治疗的疾病，并且所导致的视神经损伤是不可逆的。所有人口统计数据 易患青光眼，尤其是老年人、非洲裔美国人、西班牙裔美国人、糖尿病患者和 有青光眼病史。大约有300万美国人患有青光眼， 每年损失30亿美元的税收，福利支出和医疗补贴，以支持这些 公民非常需要青光眼的长期治疗，因为青光眼引起的视力丧失是非常严重的。 美国第二大致盲原因目前可用的药理学和外科手术 青光眼的治疗具有显著的局限性和副作用，其包括全身反应， 药物、患者不依从、眼睛感染、手术器械故障和眼睛损伤。初级 开角型青光眼（POAG）是常见的，其特征在于通过眼内分泌系统的房水引流不良。 传统的外流途径。在这里，我们提出了一个新的委员会， 机械敏感通道（EMC）作为压力调节剂在昏迷眼TM受损中的作用。使用 这项正在申请专利的技术，我们已经证明了EMC是：（i）在培养的TM细胞中的功能，（ii） 在TM细胞中成功地体内转导;和（iii）有效降低眼内炎小鼠模型中的IOP， 无（i）非靶向表达（通过QPCR测量）;（ii）眼损伤（通过OCT评估）的高血压; (iii)血浆中的炎性细胞因子（通过ELISA检测）;或（iv）免疫细胞应答和（v）活力丧失 长期表达EMC的靶细胞。EMC是细菌伸展的工程版本- 激活的通道，它直接感受细胞膜脂双层中的张力， 瞬时打开其大的非特异性孔用于反式/旁胞吞作用。在其压力阈值下，EMC可以释放 TM细胞中的液体通过SC被清除。此外，细胞体积和大小的减少将 扩大细胞间隙并促进房水的细胞旁流动。EMC可作为理想的排水系统 这是因为它是一个相对较小的同源寡聚通道，不需要任何相关的 蛋白质或能量来源来组装和发挥作用。我们已经确定了激活的最佳EMC变体 在生理相关的压力下使用压力钳。这项研究的目标是进一步 开发基于自互补AAV载体的工程化机械敏感EMC产品 通道（scEMC）用于POAG的基因治疗。这一SBIR项目第二阶段的目标将得以实现 通过三个具体目标。目的1：量化scEMC在野生型大鼠中的长期稳定性和安全性; 目的2：在POAG啮齿动物模型中评价scEMC启用的长期降IOP疗效，目的3：GLP 前房注射scEMC在野生型NHP中的毒性和生物分布的研究。的发展 通过该提案的成功，安全有效的青光眼单剂量长效治疗将提高 许多美国人的生活。

Glaucoma is recalcitrant to treatment and the resulting optic nerve damage is irreversible. All demographics are susceptible to glaucoma, especially the elderly, African-Americans, Hispanics, diabetics and families with a history of glaucoma. About three million Americans have glaucoma and the US government loses an estimated 3 billion USD each year in lost tax revenue, benefit payouts and healthcare subsidies needed to support these citizens. There is great need for a long-lasting treatment for glaucoma, because vision loss due to glaucoma is the second leading cause of blindness in America. The currently available pharmacological and surgical treatments for glaucoma have significant limitations and side-effects, which include, systemic reactions to medications, patient non-compliance, eye infections, surgical device failure, and damage to the eye. Primary Open Angle Glaucoma (POAG) is common and is characterized by poor drainage of aqueous humor through the conventional outflow pathway. Here, we put forth a novel commission for virally delivered Engineered Mechanosensitive Channel (EMC) as a pressure modulator in the impaired TM of glaucomatous eyes. Using this patent pending technology, we have demonstrated that EMC is: (i) functional in cultured TM cells, (ii) successfully transduced in vivo in TM cells; and (iii) effective in lowering the IOP in a mouse model of ocular hypertension without (i) non-targeted expression (measured by QPCR); (ii) ocular damage (assessed by OCT); (iii) inflammatory cytokines in plasma (detected by ELISA); or (iv) immune cell response and (v) loss of viability of targeted cells expressing EMC over long period. EMC is an engineered version of the bacterial stretch- activated channel, which directly senses tension in the membrane lipid bilayer of cells and in response, transiently opens its large non-specific pore for trans/paracytosis. At its pressure threshold, EMC can release fluid from TM cells to be cleared through SC. Additionally, the resulting decrease in cell volume and size will widen intercellular spaces and facilitate the paracellular flow of aqueous humor. EMC acts as an ideal drainage valve for TM cells, because it is a relatively small homo-oligomeric channel and does not need any associated proteins or energy sources to assemble and function. We have identified optimal EMC variant that is activated at physiologically relevant pressures using pressure clamp. The goal of the proposed research is to further develop the product based on the self-complementary AAV carried engineered mechanosensitive EMC channel (scEMC) for gene therapy of POAG. The objective of this phase-II SBIR project will be accomplished through three specific aims. Aim 1: Quantify long-term stability of scEMC and safety of scEMC in wild type rats; Aim 2: Evaluate scEMC-enabled long-term IOP lowering Efficacy in rodent models of POAG and Aim 3: GLP Study of toxicity and Biodistribution of intracamerally-injected scEMC in wild-type NHPs. The development of a safe effective single-dose long-lasting treatment for glaucoma via the success of this proposal would improve the lives of many Americans.