Identification of beta-cell-inducing small RNAs by random shRNA selection

描述（由申请人提供）：通过表达Ngn3、Pdx1和MafA，分泌胰岛素的内分泌β细胞可以从小鼠胰腺的外分泌细胞中获得，这一发现可能会给糖尿病治疗带来革命性的变化。这个提议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，这可能会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，以避免对载体整合或引入基因的需要。然而，这些方法都不完全令人满意。我们最近开发了一种新技术，它有可能解决上述问题。该技术是一个shrna表达文库，在核苷酸水平上完全随机。在此，我们建议使用这个文库来鉴定可以重编程或提高重编程效率的shRNA序列，从胰腺的外分泌细胞中重编程内分泌β细胞。小rna对基因表达的调节可以通过从DNA载体中表达shrna，或通过外源添加预合成的sirna到细胞中来完成。外源性sirna的优势在于，它们的作用很容易通过重复添加来维持，而且很容易逆转——因为没有载体整合，人们可以简单地停止添加它们，让它们降解。因此，从我们的随机shRNA编码库中鉴定出的有效序列，经过随机诱变和重新筛选优化，可以用作预合成的sirna，从而避免了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导入细胞。

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.