Development of a protein-driven gene targeting technology

项目描述（申请人提供）：本项目旨在通过提高基因靶向的核传递、效率和安全性以及扩大其应用范围，改变基因和基因组在细胞内精确操作的方式。基因靶向是一种随意修改内源性DNA序列的遗传技术，通过将突变DNA序列改变为野生型副本或反之亦然，而不将其从活细胞染色体中的自然环境中移除。因此，基因靶向不仅是基因、蛋白质和复杂生物系统功能分析的基本过程，而且在预防和治疗由特定DNA改变引起的人类遗传疾病的分子治疗中也具有潜在的作用。基因靶向的基础是遗传信息的原位交换，在目前的基因组修饰方法中，这种交换遵循自然同源重组的步骤。不幸的是，同源重组仅在极少数生物体和细胞中具有活性和效率，而且只能在细胞周期的有限时期内发生。诱导DNA损伤，如靶向位点的双链断裂，可以部分克服许多细胞类型（包括人类细胞）的低重组频率。然而，由于诱导靶区断裂和脱靶断裂的发生而产生的不需要的突变和重排的强烈威胁极大地限制了基因治疗的应用。事实上，双链断裂被认为是癌症的主要原因之一。本研究的目的是促进新的蛋白质-DNA复合物向细胞核的传递，以非规范模式促进同源配对和同源链交换的反应，而不遵循同源重组的步骤，从而在所有可被外源DNA转化、转染或转导的细胞中实现高效和无损伤的基因靶向。我们推测，在那些通常直接或间接与染色体DNA相互作用的蛋白质中，有几种蛋白质如果与DNA靶向分子结合，可以促进该分子的递送和靶向效率。我们发展和测试这一假设的具体目标是：目标1)测试不同的DNA靶向分子设计，并确定最有效的一种。目的2)生成含有位点特异性DNA结合蛋白的DNA-蛋白复合物，驱动DNA模板分子到达靶向位点。我们将复杂的DNA靶向分子与蛋白质已知结合DNA的序列特定的方式在一个选定的靶向位点附近。目标3)确定一组蛋白质，当与DNA模板分子结合时，可以[模块化地]促进染色体基因靶向。这将通过A)使用酵母细胞提取物生成的cDNA文库筛选酵母细胞中靶向促进蛋白的基因，以及B)使用从人类癌症干细胞提取的细胞文库生成的cDNA文库筛选人类细胞中直接致敏的促进蛋白基因来实现。[C]测试已鉴定的gtp促进基因靶向的模块化能力。D)蛋白驱动基因靶向后细胞基因组稳定性测试。

DESCRIPTION (provided by applicant): This project aims to transform the way in which genes and genomes can be precisely manipulated inside cells by improving the nuclear delivery, efficiency and safety of gene targeting and expanding its applications. Gene targeting is a genetic technique to modify endogenous DNA sequence at will, by changing a mutant DNA sequence into a wild-type copy or vice versa, without removing it from its natural context in the chromosome in a living cell. Gene targeting is therefore a fundamental process not only for functional analysis of genes, proteins and complex biological systems, but potentially also in molecular therapy for the prevention and cure of human genetic diseases originating from specific DNA alterations. The basis of gene targeting is the in situ exchange of genetic information, which in current approaches of genome modification follows the steps of natural homologous recombination. Unfortunately, homologous recombination is active and efficient only in an extremely small set of organisms and cells, where, moreover, it can occur only in a restricted period of the cell cycle. The induction of DNA damage such as double-strand breaks at the targeting locus can partially overcome the low recombination frequencies in many cell types, including human cells. However, the strong threat of unwanted mutations and rearrangements due to both the induction of a break at the targeting region and the occurrence of off-target breaks highly limits applications in gene therapy. In fact double-strand breaks are regarded as one of the primary causes of cancer. The goal of this study is to promote the delivery of novel protein-DNA complexes to the nucleus to promote the reactions of homologous pairing and homologous strand exchange in a non canonical mode without following the steps of homologous recombination, thereby making gene targeting efficient and damage-free in all cells that can be transformed, transfected, or transduced by exogenous DNA. We hypothesize that several proteins among those that normally directly or indirectly interact with chromosomal DNA, if bound to a DNA targeting molecule, can promote delivery and targeting efficiency of this molecule. Our Specific Aims to develop and test this hypothesis are: Aim 1) Test different designs for the DNA targeting molecules and identify the most effective one. Aim 2) Generate DNA-protein complexes containing a site-specific DNA binding protein to drive the DNA template molecule to the targeting locus. We will complex DNA targeting molecules with proteins that are known to bind the DNA in a sequence specific manner in the vicinity of a chosen targeting locus. Aim 3) Identify a set of proteins that, when bound to a DNA template molecule, can [modularly] boost chromosomal gene targeting. This will be achieved via A) screening for gene targeting promoting proteins in yeast cells using a cDNA library generated from yeast cell extract, and via B) screening for gene targeting promoting proteins directly in human cells sensitized to damage repair using a cDNA library generated from cell extract of human cancer stem cells. [C) Testing the identified GTPs for their modular capacity to promote gene targeting.] D) Testing the genomic stability of cells following protein- driven gene targeting. PUBLIC HEALTH RELEVANCE: The best cure/prevention of diseases associated with specific genetic defects as well as the best approach to study gene function can be achieved by precise in situ modification of the desired gene/s, via gene targeting. However, gene targeting is mostly an inefficient process, especially in human cells. DNA double-strand breaks (DSBs) are known to efficiently stimulate homologous targeting. However, cleavage specificity of DSB- inducing enzymes is a major problem since even few off-target DSBs are prone to generate mutations and chromosome rearrangements, which can lead to cancer. Our goal is to develop a modular, cancer-free and efficient gene targeting approach where the DNA targeting molecule is directly driven to its genomic target by a protein that facilitates nuclear delivery and promotes the reaction of strand exchange without inducing DNA damage. This is an exploratory study and the results obtained will provide a basis for a future R01 submission focused on mechanisms of gene targeting guided by proteins and on broader application for gene and genome modification and gene therapy.