Gene silencing by optoinjection and RNA interface

通过光注射和 RNA 界面进行基因沉默

• 批准号: 6940674
• 负责人: MANFRED R KOLLER
• 金额: $ 63.79万
• 依托单位: CYNTELLECT, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-11 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6940674
• 关键词: RNA interference; cell proliferation; cell type; electroporation; functional /structural genomics; gene induction /repression; gene targeting; hematopoietic stem cells; high throughput technology; injection /infusion; lasers; nucleic acid hybridization; nucleic acid purification; reagent /indicator; small interfering RNA; transfection

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.

描述（由申请人提供）：RNA干扰（RNAi）是一种使用dsRNA模板由细胞内酶复合物介导的特异性基因沉默方法，由于其相对于较旧的反义方法具有重要优势，因此目前备受关注。最近的报道吹捧了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，以及用于治疗方法。然而，由于核酸不容易穿过活的哺乳动物细胞膜，因此需要将试剂递送到靶细胞中的稳健技术来充分实现RNAi介导的基因沉默的潜力。我们已经开发了一种新型的基于激光的细胞处理系统，称为LEAP（激光使能分析和处理），用于高速细胞成像和基于激光的操作。LEAP以每秒>10[5]的速度对细胞进行成像，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，例如细胞死亡（即，用于细胞纯化）或光注射（即，用于转染的细胞的可逆透化）。在第一阶段，LEAP被用于证明成功的基于RNA和DNA的试剂的光注射到细胞中以实现RNAi。重要的是，光注射在细胞活力和转染效率方面优于常用的基于脂质的转染方法。第二阶段的总体目标是显着提高这一能力，并证明LEAP用于大规模基于RNAi的功能基因组学研究。具体来说，第二阶段的目标是：（i）继续优化包括困难细胞类型在内的RNAi的光注射;（ii）评估光注射对细胞生理学的影响，并与现有方法进行比较;以及（iii）在LEAP上实施功能基因组学测定。II期将证明LEAP通过在各种细胞类型中光注射siRNA以影响各种细胞过程而用于原位基因沉默的广泛用途。具体好处（即，将通过各种方法建立光注射相对于其它转染方法的优点和缺点），以证明对经历转染的细胞的生理影响。由此产生的数据将具有广泛的兴趣，并将通过评估使用各种转染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，第三阶段的商业化工作将能够促进光注入方法的采用，以实现RNAi技术在推进功能基因组学方面的潜力。