Targeting retroviral and virus-like particles for gene and protein delivery

靶向逆转录病毒和病毒样颗粒进行基因和蛋白质传递

• 批准号: 10002252
• 负责人: MONICA J ROTH
• 金额: $ 63.03万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10002252
• 关键词: Address; Area; Award; Biochemical; Bioinformatics; C-terminal; Cells; Charge; Complex; CpG Islands; DNA; Disease; Gammaretrovirus; Gene Delivery; Gene Proteins; Genes; Goals; Human Genome; Laboratories; Link; Mitosis; Mitotic Chromosome; Modeling; Murine leukemia virus; Mus; N-terminal; National Institute of General Medical Sciences; Nucleosomes; Oncogenic; Pathogenesis; Peptides; Process; Promoter Regions; Property; Protein Family; Proteins; Research; Site; Structure; System; Tail; Testing; Transcription Initiation Site; Viral; Viral Pathogenesis; Viral Physiology; Virus; Virus-like particle; base; experimental study; gag Gene Products; integration site; laboratory experiment; next generation sequencing; novel; particle; preference; research study; structured data; targeted delivery; vector; viral leukemia; viral nanoparticle

Abstract This MIRA application combines three NIGMS awards R01GM070837, 1R01GM110639, and 5R01GM108487, studying the tethering and integration the murine leukemia virus (MLV) preintegrative complex (PIC). Gammaretroviruses require cells to undergo mitosis for integration and preferentially integrate within active promoters regions, proximal to transcriptional start sites (TSS) and CpG islands. The requirement for mitosis limits the use of these vectors to dividing cells. The site of integration is linked to the oncogenic potential of the virus and thus its pathogenesis and use as a gene delivery vector. Studies are focused on three targeted areas. The p12 protein is required for tethering the PIC to mitotic chromosomes. Recent analysis of MLV p12 tethering properties has revealed a novel mechanism of suppression of tethering localizing in the N-terminus of the p12 protein. A model is proposed that incorporates an orientation reversal from the precursor Gag protein to that found in the PIC that transitions through an intermediate in which the tethering domain is inhibited by N- and C-terminal charge interactions. Experiment testing this model will involve biochemical and structural approaches, including NMR analysis of the p12 tethering domain. Our collaborative research has identified the host BET family of proteins, specifically the Brd 2,3, and 4 proteins as interactors with the MLV IN. The interaction between the MLV IN C- terminal tail peptide and the ET domain influences the integration into TSS and CpG islands. Studies proposed build on this newly defined viral-host interaction. Building on the solution structure of the MLV IN CTD from our laboratory, experiments are aimed at defining the structural data of larger complexes including the MLV IN CTD:Brd3 ET complex, and the domains of IN bound to DNA. The ability of the MLV IN protein to compete with known ET domain interactors will be studied. Next-generation sequencing and bioinformatics will be used to probe a panel of IN proteins for their target-site preferences, both locally at the site of integration as well as globally within the human genome. The recognition of the target DNA within the nucleosome structure is examined. These studies will be combined with two emerging areas in our laboratory. The first develops MLV based virus-like particles (VLP) for the delivery of proteins, rather than genes into cells. The second area of research develops a modified Env system that allows entry through scFvs and monobodies as targeting moieties. This system could be generalized and expanded to address a wide range of applications and diseases.

抽象的 该 MIRA 应用程序结合了三个 NIGMS 奖项 R01GM070837、1R01GM110639、 和 5R01GM108487，研究鼠白血病病毒 (MLV) 的束缚和整合 预整合复合体（PIC）。 γ逆转录病毒需要细胞进行有丝分裂 整合并优先整合在活性启动子区域内，接近 转录起始位点 (TSS) 和 CpG 岛。有丝分裂的要求限制了其使用 这些载体用于分裂细胞。整合位点与致癌潜力有关 病毒及其发病机制和作为基因递送载体的用途。 研究集中在三个目标领域。 p12 蛋白是束缚 PIC 至有丝分裂染色体。最近对 MLV p12 系链特性的分析揭示了 抑制 p12 蛋白 N 末端束缚的新机制。一个 提出的模型结合了从前体 Gag 蛋白到 在 PIC 中发现，通过中间体进行转变，其中束缚结构域是 受 N 端和 C 端电荷相互作用的抑制。测试该模型的实验将涉及 生化和结构方法，包括 p12 束缚域的 NMR 分析。 我们的合作研究已经确定了宿主 BET 蛋白家族，特别是 Brd 2,3 和 4 蛋白作为 MLV IN 的相互作用物。 MLV IN C-之间的相互作用 末端尾肽和 ET 结构域影响 TSS 和 CpG 岛的整合。 拟议的研究建立在这种新定义的病毒与宿主相互作用的基础上。以解决方案为基础 我们实验室的 MLV IN CTD 结构，实验旨在定义 较大复合物的结构数据，包括 MLV IN CTD:Brd3 ET 复合物，以及 IN 结构域与 DNA 结合。 MLV IN 蛋白与已知 ET 竞争的能力 将研究域交互器。将使用下一代测序和生物信息学 探测一组 IN 蛋白的靶位点偏好，均位于本地 整合以及全球范围内的人类基因组。目标DNA的识别 检查核小体结构内的情况。 这些研究将与我们实验室的两个新兴领域相结合。第一个 开发基于 MLV 的病毒样颗粒 (VLP)，用于将蛋白质而不是基因传递到 细胞。研究的第二个领域开发了一种改进的环境系统，允许通过 scFv 和单体作为靶向部分。该系统可以推广和扩展 解决广泛的应用和疾病。