Targeted Nano-enhanced Optical Delivery of opsin for dry-AMD therapy

干燥年龄相关性黄斑变性(AMD)的地理萎缩(GA)的特征是 光感受器，是导致约1500万人新视力丧失的主要原因。既没有治愈的方法 既不能阻止退化，也不能恢复视力损失。我们已经开发出可激活的环境光 多特征视蛋白(MCO-II)，可刺激视网膜神经节细胞(RGC)以恢复视力。 然而，将这种基因疗法临床翻译到GA患者将需要有针对性的视蛋白- 在不干扰剩余功能视网膜的情况下，将基因编码到萎缩的区域。因此，我们有 开发了一种基于近红外激光的体内靶向基因进入视网膜的有效方法。在这 纳米增强光学传输(NOD)方法，我们利用基于表面等离子体共振场 功能化金纳米棒(FGNR)瞬时穿孔细胞膜的增强作用 分子。在第一阶段，我们展示了MCO-II的体内靶向光学递送到变性的视网膜。 小鼠在多个波长使用点头。此外，我们还对连续波和连续波进行了比较研究。 基于纳秒脉冲激光的MCO-II质粒的NOD及优化激光参数的确定 视网膜的高效转染性。由于点头，没有观察到可检测到的眼损伤。此外， 体内注射MCO-II后视网膜免疫染色未见明显的细胞死亡。 电生理学研究表明，MCO-II致敏细胞可被光激活，从而在视觉上 诱发了大脑皮层活动。这个第二阶段提案的总体目标是开发组合NOD产品 用于安全地光敏变性视网膜中的RGC并刺激光敏的RGC 通过环境光进行视力康复。为了实现这一目标，我们有以下目标：(1)量化长期 NOD在光感受器缺失小鼠模型中的稳定性和安全性；(2)评价靶向视网膜的功能 在小鼠和大鼠模型中使用NOD辅助MCO-II递送对视网膜节细胞重新光敏后的区域；以及 (3)NOD-MCO-II载体在非人类体内的毒性、生物分布和有效性的GLP研究 灵长类(NHP)。该协作提案汇集了光学传输方面的互补专业知识， 光遗传学，眼科学，仪器，分子生物学，纳米材料，视网膜生物学和功能， 神经科学/行为学、电生理学、生物统计学和毒理学，以应对视网膜中的挑战 退化。NHP的安全性/有效性研究将在CRO设施进行。在完成 我们预计第二阶段将推进：(I)用于临床研究的NOD产品开发，(Ii)向FDA申请IND， 以及(Iii)与风险投资和Pharma公司合作实现商业化。这项提议的成功 将导致一种新的临床方法来治疗GA患者，通过传统的玻璃体内注射fGNRs 和MCO-II。基于NOD的不渗透外源材料(小分子， 蛋白质和基因)将有利于药物、疫苗和基因治疗。

Geographic atrophies (GA) in dry-age related macular degeneration (AMD) is characterized by degeneration of photoreceptors, and is the leading cause of new vision loss in ~15 million persons. There is neither a cure that can stop the degeneration nor a therapy to restore vision loss. We have developed ambient-light activatable multi-characteristic opsin (MCO-II) to allow stimulation of retinal ganglion cells (RGCs) for vision rehabilitation. However, clinical translation of such gene therapy to patients with GA will require targeted delivery of opsin- encoding genes into the atrophic regions without perturbing remaining functional retina. Therefore, we have developed a near-infrared laser based efficient method for in-vivo targeted gene delivery into retina. In this Nano-enhanced Optical Delivery (NOD) method, we utilize surface plasmon resonance based field enhancement by functionalized gold nanorods (fGNRs) to transiently perforate cell membrane to deliver the molecules. In the Phase I, we demonstrated targeted in-vivo optical delivery of MCO-II to degenerated retina in mice using NOD at multiple wavelengths. Further, we made comparative study of continuous wave (cw) and nanosecond pulsed laser based NOD of MCO-II plasmids and determined optimized laser parameters for efficient transfection of retina. No detectable ocular damage was observed due to NOD. Further, the immunostaining of retina after in-vivo NOD of MCO-II plasmids showed no noticeable cell death. Electrophysiology studies demonstrate that MCO-II sensitized cells are activatable by light, allowing visually evoked cortical activities. The overall goal of this Phase-II proposal is to develop the combination NOD product for photosensitizing RGCs in the degenerated retina in a safe manner and stimulating photosensitized RGCs by ambient light for vision rehabilitation. Towards this goal we have following aims: (1) Quantify long-term stability and safety of NOD in mice model lacking photoreceptors; (2) Evaluate functioning of targeted retinal regions after re-photosensitization of RGCs using NOD assisted MCO-II delivery in mice and rat models; and (3) GLP study of toxicity, biodistribution and efficacy of NOD-delivered MCO-II plasmids in non-human primates (NHPs). This collaborative proposal brings together complementary expertise in optical delivery, optogenetics, ophthalmology, instrument, molecular biology, nanomaterials, retina biology and function, neuroscience/behavior, electrophysiology, biostatistics, and toxicology to address the challenge in retinal degeneration. The safety/efficacy study in NHPs will be performed at CRO facility. Upon completion of the Phase II we envision to advance: (i) NOD product development for clinical studies, (ii) IND application to FDA, and (iii) partnering with venture capital and Pharma company for commercialization. Success of this proposal will lead to a new clinical approach for treating patients with GA by conventional intravitreal injection of fGNRs and MCO-II. The NOD based targeted delivery of impermeable exogenous materials (small molecules, proteins and genes) will benefit drug, vaccine and gene therapy.