Identification of Beta-Cell-Inducing Small RNAs by Random shRNA Selection

通过随机 shRNA 选择鉴定β细胞诱导小 RNA

• 批准号: 8063051
• 负责人: ROBERT B WILSON
• 金额: $ 23.76万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-16 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063051
• 关键词: Address; Back; Beta Cell; Biological; Biological Assay; Cell Differentiation process; Cell Line; Cells; DNA; Development; Diabetes Mellitus; Endocrine; Gene Expression; Gene Targeting; Genes; Genome; Genomics; Green Fluorescent Proteins; Insulin; Libraries; Messenger RNA; Methods; MicroRNAs; Microarray Analysis; Modeling; Molecular Profiling; Mus; Mutagenesis; Nucleotides; Pancreas; Parents; Protocols documentation; Public Health; RNA; RNA Interference; RNA Sequences; Randomized; Recovery; Reporter; Resistance; Retroviridae; Risk; Screening procedure; Seeds; Small RNA; Sorting - Cell Movement; Stem cells; Technology; Testing; Therapeutic; Transfection; Viral Vector; antibiotic G 418; base; design; embryonic stem cell; improved; new technology; novel therapeutics; protein expression; public health relevance; tissue culture; tool; tumor; vector

DESCRIPTION (provided by applicant): The finding that insulin-secreting, endocrine beta cells can be derived from exocrine cells of the mouse pancreas by expressing Ngn3, Pdx1, and MafA could revolutionize diabetes therapeutics. The primary problem - which this proposal seeks to address - is that the viral vectors used to deliver the genes integrate permanently into the genome, which could interfere with the function of derived cells, or cause tumors. In the induced-stem-cell field, several methods have been tested to obviate the need for vector integration, or the introduction of genes. However, none of these methods is entirely satisfactory. We recently developed a novel technology that has the potential to address the problems described above. This technology is an shRNA-expressing library that is completely random at the nucleotide level. Herein, we propose to use this library to identify shRNA sequences that can reprogram, or increase the efficiency of reprogramming of, endocrine beta cells from exocrine cells of the pancreas. Modulation of gene expression by small RNAs can be accomplished by expression of shRNAs from DNA vectors, or by adding pre-synthesized siRNAs to cells exogenously. The advantage of exogenous siRNAs is that their effects are easy to sustain by repeated addition, and are easily reversible - because there is no vector integration, one can simply stop adding them and allow them to degrade. Thus, effective sequences identified from our random shRNA- encoding library, and optimized by random mutagenesis and re-screening, could be used as pre-synthesized siRNAs, thereby obviating the risks associated with vector integration. In addition, siRNAs are easy to synthesize, and easy to introduce into cells using well-established transfection protocols. PUBLIC HEALTH RELEVANCE: This proposal describes an approach to develop novel therapeutics and biologic tools using an shRNA- expressing library that is completely random at the nucleotide level. This approach has implications for the development of diabetes therapeutics and stem-cell-based therapeutics, and is highly relevant to public health.

描述（由申请人提供）：通过表达Ngn 3、Pdx 1和MafA，可以从小鼠胰腺的外分泌细胞中衍生出胰岛素分泌、内分泌β细胞的发现可能会彻底改变糖尿病治疗方法。该提案试图解决的主要问题是，用于传递基因的病毒载体永久地整合到基因组中，这可能干扰衍生细胞的功能或导致肿瘤。在诱导干细胞领域，已经测试了几种方法来消除载体整合或基因导入的需要。然而，这些方法中没有一种是完全令人满意的。我们最近开发了一种新技术，有可能解决上述问题。该技术是一种在核苷酸水平上完全随机的shRNA表达文库。在此，我们建议使用该文库来鉴定可以重新编程或提高来自胰腺外分泌细胞的内分泌β细胞重新编程效率的shRNA序列。小RNA对基因表达的调节可以通过从DNA载体表达shRNA或通过将预合成的siRNA外源性地添加到细胞中来实现。外源siRNA的优点是它们的作用容易通过重复添加来维持，并且容易可逆-因为没有载体整合，人们可以简单地停止添加它们并允许它们降解。因此，从我们的随机shRNA编码文库中鉴定并通过随机诱变和重新筛选优化的有效序列可以用作预合成的siRNA，从而避免与载体整合相关的风险。此外，siRNA易于合成，并且易于使用完善的转染方案引入细胞中。 公共卫生相关性：该提案描述了一种使用在核苷酸水平上完全随机的shRNA表达文库来开发新的治疗和生物工具的方法。这种方法对糖尿病治疗和基于干细胞的治疗的发展具有影响，并且与公共卫生高度相关。