Targeting Retroviral DNA Integration: Role of Host Cell Proteins

描述（由申请人提供）： 摘要逆转录病毒载体广泛应用于基因治疗。然而，最近的基因治疗试验表明，逆转录病毒载体整合到宿主细胞基因中可能导致癌症的发展。因此，整合位点选择的控制对于使用这些载体的基因治疗方法的成功结果至关重要。这项拨款提案是基于一项新的工作，其中涉及宿主细胞转录因子和组蛋白修饰蛋白在逆转录病毒载体的整合位点选择，并侧重于开发新的方法，以靶向整合到预定的染色体区域。最近发表了支持这些蛋白质在靶向中的作用的结果，并在该提案中提出了新的初步数据。这些数据表明，基于转录因子的合成蛋白可用于控制整合位点的选择。组蛋白修饰剂的结果是新颖的，尚未发表。在目的1中，采用遗传和分子生物学方法来确定在高细胞内量的合成蛋白存在下转录单位内外的整合速率。建议瞬时表达由异染色质蛋白1 α（HP-1染色质结合结构域和转录因子3 '-末端将）组成的合成基因，并使用最近完成的人类基因组序列和新的基于PCR和ChIP的方法来确定基因内外整合的频率。类似地，将构建另一种基于转录因子的基因，其中HP-1染色质结合结构域将被另一种转录因子的DNA结合结构域取代。上述方法将用于确定这种新的融合基因是否靶向整合到特定的DNA序列。在目的2A和2B中，将确定组蛋白修饰蛋白及其活性是否靶向远离基因的整合。在最后一个子目标2C中提出的实验旨在通过同时表达基于转录因子的蛋白和组蛋白修饰剂蛋白来最大化靶向异染色质的整合效率。这些和相关实验的结果将对使用逆转录病毒载体的基因治疗方法具有重要意义，因为它们将导致新的治疗方法，该方法将降低由于整合到人类基因组的不期望区域中而导致癌症发展的危险。最后，我们的研究结果也应该显着提高我们的理解的作用，染色质修饰的整合位点选择的逆转录病毒载体。逆转录病毒被广泛用作载体，在称为整合的过程中将治疗基因递送到靶细胞中。在某些情况下，整合可能发生在患者DNA的不期望区域，并且因此患者可能发展成癌症。在这项资助申请中，我们提出了两种独立的方法，它们应该将整合目标定位在远离人类DNA潜在危险区域的地方，并导致整合到不可能发生癌症的区域。

DESCRIPTION (provided by applicant): ABSTRACT Retroviral vectors are widely used in gene therapy. However, a recent gene therapy trial demonstrated that integration of a retroviral vector into host cell genes may lead to development of cancer. Therefore, control of integration site selection is critical to a successful outcome of gene therapy approaches, which use these vectors. This grant proposal is based on a new work, which implicates a host cell transcription factor and a histone modifier protein in integration site selection by retroviral vectors, and focuses on the development of new methods to target integration to predetermined chromosomal regions. Results supporting a role of these proteins in targeting were published very recently, and new preliminary data are presented in this proposal. These data suggest that transcription factor-based synthetic proteins can be used to control integration site selection. The histone modifier results are novel and not yet published. In Aim 1, genetic and molecular biology approaches are employed to determine the integration rate in and outside transcription units in the presence of high intracellular amounts of the synthetic proteins. It is proposed that a synthetic gene consisting of the heterochromatin protein 1 alpha (HP-1 chromatin-binding domain and the transcription factor 3'-terminus will ) be transiently expressed and the frequency of integration inside and outside genes will be determined using the recently completed human genome sequence and novel Alu-PCR- and ChIP-based methods. Similarly, another transcription factor-based gene will be constructed, where the HP-1 chromatin-binding domain will be replaced by a DNA-binding domain of another transcription factor. The above mentioned methods will be used to determine if this novel fusion gene targets integration to a specific DNA sequence. In the Aims 2A and 2B, it will be determined whether the histone modifier protein and its activity targets integration away from genes. Experiments, which are proposed in the last sub-Aim, 2C, aim to maximize the efficiency of the integration targeting to heterochromatin by simultaneous expression of transcription factor- based and histone modifier proteins. Results from these and related experiments will have important implications for gene therapy approaches using retroviral vectors, since they will lead to novel therapeutic approaches that will decrease the danger of cancer development due to integration in undesirable regions of the human genome. Finally, our results should also significantly enhance our understanding of the role of chromatin modifications in integration site selection by retroviral vectors. NARRATIVE Retroviruses are widely used as vehicles to accomplish the delivery of therapeutic genes into target cells, in a process called integration. In some cases, integration may happen in an undesirable region of a patient's DNA, and as a result the patient may develop cancer. In this grant application, we propose two independent approaches, which should target integration away from potentially dangerous regions of human DNA, and lead to integration in regions that are safe from the possibility of cancer development.