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，最近的一项基因治疗试验表明，将逆转录病毒载体整合到宿主细胞基因中可能会导致癌症的发生。因此，对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。这项赠款建议是基于一项新的工作，该工作涉及通过逆转录病毒载体选择整合位点的宿主细胞转录因子和组蛋白修饰蛋白，并专注于开发新的方法来针对预定的染色体区域进行整合。最近发表了支持这些蛋白质在靶向中的作用的结果，并在这项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以用于控制整合位点的选择。组蛋白修饰物的结果是新颖的，尚未发表。在目标1中，采用遗传和分子生物学方法来确定在细胞内合成蛋白质数量较高的情况下转录单元内外的整合率。有人建议瞬时表达由异染色质蛋白1α(HP-1染色质结合区和转录因子3‘末端)组成的合成基因，并利用最近完成的人类基因组序列和基于Alu-PCR和芯片的新方法来确定基因内外整合的频率。同样，将构建另一个基于转录因子的基因，其中HP-1染色质结合域将被另一个转录因子的DNA结合域取代。上述方法将被用来确定该新型融合基因是否靶向整合到特定的DNA序列。在AIMS 2A和2B中，将确定组蛋白修饰蛋白及其活性是否针对远离基因的整合。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蛋白来最大化针对异染色质的整合效率。这些实验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，将降低由于整合在人类基因组的不良区域而导致的癌症发展的危险。最后，我们的结果还应该显著提高我们对染色质修饰在逆转录病毒载体整合位点选择中的作用的理解。叙述性逆转录病毒被广泛用作载体，在一个称为整合的过程中，完成将治疗基因输送到靶细胞中。在某些情况下，整合可能发生在患者DNA的不希望看到的区域，因此患者可能会患上癌症。在这项拨款申请中，我们提出了两种独立的方法，这两种方法应该针对远离人类DNA潜在危险区域的整合，并导致整合到远离癌症发展可能性的区域。

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.