Development of a protein-driven gene targeting technology

描述(申请人提供)：该项目旨在通过提高核传递、基因靶向的效率和安全性并扩大其应用来改变基因和基因组在细胞内精确操纵的方式。基因打靶是一种随意修改内源DNA序列的遗传技术，通过将突变的DNA序列改变为野生型拷贝或反之亦然，而不会将其从活细胞染色体中的自然背景中移除。因此，基因打靶不仅是对基因、蛋白质和复杂生物系统进行功能分析的基本过程，而且可能用于预防和治疗由特定DNA改变引起的人类遗传病的分子治疗。基因打靶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，这是遵循自然同源重组的步骤。不幸的是，同源重组只有在极少数生物体和细胞中才是活跃和有效的，而且，它只能在细胞周期的有限时期发生。诱导DNA损伤，如靶点双链断裂，可以部分克服包括人类细胞在内的许多细胞类型中的低重组频率。然而，由于靶向区域断裂的诱导和非靶标断裂的发生而导致的不必要的突变和重排的强烈威胁严重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复合体进入细胞核，以非规范的方式促进同源配对和同源链交换的反应，而不是遵循同源重组的步骤，从而使基因靶向在所有可以被外源DNA转化、转导或转导的细胞中都是有效和无损伤的。我们假设，在那些通常直接或间接与染色体DNA相互作用的蛋白质中，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。我们开发和测试这一假说的具体目标是：目标1)测试不同的DNA靶向分子设计，并确定最有效的设计。目的2)制备含有位点特异性DNA结合蛋白的DNA-蛋白质复合体，以驱动DNA模板分子到达靶点。我们将DNA靶向分子与已知在所选靶点附近以序列特异性方式结合DNA的蛋白质复合。目的3)确定一组蛋白质，当与DNA模板分子结合时，可以[模块地]促进染色体基因靶向。这将通过以下途径实现：A)使用酵母细胞提取物产生的cDNA文库筛选酵母细胞中的基因靶向促进蛋白，以及B)使用从人类癌症干细胞的细胞提取物产生的cDNA文库直接在对损伤修复敏感的人类细胞中筛选基因靶向促进蛋白。[c)测试已确定的GTP促进基因靶向的模块化能力。]D)测试蛋白质驱动的基因打靶后细胞的基因组稳定性。 公共卫生相关性：通过基因打靶对所需基因进行精确的原位修饰，可以实现与特定遗传缺陷相关的疾病的最佳治疗/预防以及研究基因功能的最佳方法/S。然而，基因打靶大多是一个低效的过程，特别是在人类细胞中。DNA双链断裂(DSB)被认为能有效地刺激同源靶向。然而，DSB诱导酶的切割特异性是一个主要问题，因为即使是很少的非靶标DSB也容易产生突变和染色体重排，从而导致癌症。我们的目标是开发一种模块化、无癌和高效的基因打靶方法，其中DNA靶向分子由一种促进核传递并促进链交换反应的蛋白质直接驱动到其基因组靶向，而不会导致DNA损伤。这是一项探索性研究，所获得的结果将为未来的R01报告提供基础，重点是蛋白质引导的基因靶向机制，以及基因和基因组修饰和基因治疗的更广泛应用。

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.