Gene silencing by optoinjection and RNA interface

说明书(申请人提供)：RNA干扰(RNAi)是一种使用dsRNA模板由细胞内酶复合体介导的特异性基因沉默方法，由于与旧的反义方法相比具有重要的优点，目前引起了极大的兴趣。最近的报告吹捧RNAi在基础和应用研究中的效用，这些研究旨在确定基因产物的功能和验证药物靶点，以及治疗方法。然而，由于核酸不容易通过活的哺乳动物细胞膜，需要强大的技术将试剂输送到靶细胞，以充分实现RNAi介导的基因沉默的潜力。我们开发了一种新的基于激光的细胞处理系统，称为LEAP(激光启用的分析和处理)，用于高速细胞成像和基于激光的操作。LEAP以每秒10[5]的速度为细胞成像，并以每秒10[3]的速度对特定细胞进行激光照射，从而导致各种细胞操作，如细胞死亡(即细胞纯化)或光注射(即细胞可逆的透过性以进行转基因)。在第一阶段，LEAP被用于展示成功地将基于RNA和DNA的试剂光注射到细胞中以实现RNAi。重要的是，光注射在细胞存活率和转染率方面优于常用的基于脂质的转染法。第二阶段的总体目标现在是显著提高这一能力，并展示LEAP用于实现基于RNAi的大规模功能基因组研究。具体地说，第二阶段的目标是：(I)继续优化针对RNAi的光注射，包括困难的细胞类型；(Ii)评估光注射对细胞生理的影响并与现有方法进行比较；以及(Iii)在LEAP上实施功能基因组分析。第二阶段将展示LEAP的广泛用途，通过在各种细胞类型中通过光注射siRNA来影响各种细胞过程来原位基因沉默。光注射与其他转基因方法相比的具体好处(即利弊)将通过各种方法来确定，以证明对正在进行转基因的细胞的生理影响。由此产生的数据将引起广泛的兴趣，并将通过评估使用各种转基因方法后的细胞反应而使科学界受益。有了来自第二阶段的这些数据，第三阶段的商业化努力将能够促进采用光注射方法，以实现RNAi技术在推进功能基因组学努力方面的潜力。

DESCRIPTION (provided by applicant): RNA interference (RNAi), a specific gene silencing method mediated by an intracellular enzyme complex using a dsRNA template, is of great current interest due to important advantages over older antisense methods. Recent reports have touted the utility of RNAi for basic and applied research aimed at determining functions of gene products and validating drug targets, as well as for therapeutic approaches. However, because nucleic acids do not readily pass through living mammalian cell membranes, robust techniques to deliver reagents into target cells are needed to fully realize the potential of RNAi-mediated gene silencing. We have developed a novel laser-based cell processing system, called LEAP (Laser-Enabled Analysis and Processing), for high-speed cell imaging and laser-based manipulations. LEAP images cells at >10[5] per second, and laser-irradiates specific cells at >10[3] per second leading to various cell manipulations such as cell death (i.e., for cell purification) or optoinjection (i.e., reversible permeabilization of cells for transfection). During Phase I, LEAP was used to demonstrate successful optoinjection of RNA- and DNA-based reagents into cells for achieving RNAi. Importantly, optoinjection compared favorably to commonly-used lipid-based transfection methods with respect to cell viability and transfection efficiency. The overall goal of Phase II is now to significantly advance this capability and to demonstrate the use of LEAP for enabling large-scale RNAi-based functional genomics studies. Specifically, the Phase II aims are to: (i) continue optimization of optoinjection for RNAi including difficult cell types; (ii) evaluate impact of optoinjection on cell physiology and compare to existing methods; and (iii) implement functional genomics assays on LEAP. Phase II will demonstrate broad utility of LEAP for in situ gene silencing by optoinjection of siRNA in a variety of cell types to affect a variety of cellular processes. The specific benefits (i.e., pros and cons) of optoinjection versus other transfection methods will be established through a variety of approaches to demonstrate the physiological impact on cells undergoing transfection. The resulting data will be of broad interest and will benefit the scientific community by evaluating cell responses following the use of various transfection methods. With such data from Phase II, a Phase III commercialization effort would be enabled to promote adoption of the optoinjection approach to realize the potential that RNAi technology holds for advancing functional genomics efforts.