Construction and Characterization of intelligent Phage-Assembled Gene Expression systems (iPhAGEs).

智能噬菌体组装基因表达系统 (iPhAGE) 的构建和表征。

• 批准号: RGPIN-2018-05370
• 负责人: Slavcev, Roderick
• 金额: $ 4.08万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=762149
• 关键词: Construction; Characterization; intelligent; Phage; Assembled

Background: Bacteriophages form the world's largest reservoir of genetic information, offering innumerable biotechnological applications. The proposed research program aims to exploit phages and phage genetic elements in the construction of systems to engineer highly efficient, efficacious, and safe therapeutic production platforms for transgene delivery applications. Current modalities of transgene delivery have been less successful than envisaged due to: cellular toxicity, poor transgene expression efficiency in target cells, extensive vector degradation, and inefficient/non-scale-able vector production. We have previously engineered a production platform for the generation of DNA minivectors that exploits phage-encoded genetic elements in their efficient production. Our technology employs a genetic system from phage PY54 to produce linear covalently-closed (LCC) DNA minivectors, or “DNA Ministrings”. These stable, linear, double-stranded DNA constructs carry only the eukaryotic promoter, gene of interest, intron and polyA sequence, and nuclear translocation enhancing sequences. DNA ministrings offer more efficient cellular trafficking and transfection compared to conventional plasmid vectors and integration of a DNA ministring into a cell's chromosome (bacterial or mammalian) is a highly lethal event that protects against vector integration events that may result in mutations or silencing of adjacent genes. This program proposes novel strategies for the construction and characterization of intelligent Phage Assembled Gene Expression systems (iPhAGEs) bacteriophage-based DNA delivery platforms that are efficient, inexpensive, scale-able, temperature-stable and easy to manipulate. To pioneer the iPhAGEs endeavour we aim to produce and package DNA ministrings using the single-stranded DNA (ssDNA) filamentous phage, M13, as well as a lytic Lambda phage, both of which offer the capacity for phage display and "intelligent" targeting. These platforms are expected to maximize transgene delivery efficiency, simplify purification, and provide the option of intelligent targeting.Significance of the Research: This research program seeks to establish game-changing, robust, and powerful one-step genetic processes to generate stable and efficient production of novel DNA delivery vectors. The knowledge gained from this work will extend to maximize efficiency, efficacy, and safety of transgene delivery toward a number of downstream transgene delivery applications in agriculture and mammalian medicine. Furthermore, display phages are highly amenable to the surface expression of genetically fused peptides on the capsid, easily facilitating targeting of cell surface receptors, reporter tagging, or linkage with effector peptides. Such highly efficient systems will serve to greatly expand the genetic engineering and molecular biology toolbox.

背景：噬菌体是世界上最大的遗传信息库，提供了无数的生物技术应用。拟议的研究计划旨在利用噬菌体和噬菌体遗传元件构建系统，为转基因递送应用设计高效，有效和安全的治疗生产平台。目前的转基因递送模式比设想的要不成功，这是由于：细胞毒性、靶细胞中转基因表达效率差、载体广泛降解和载体生产效率低/不可规模化。我们之前已经设计了一个生产平台，用于生产DNA微载体，该微载体在其有效生产中利用噬菌体编码的遗传元件。我们的技术采用来自噬菌体PY 54的遗传系统来产生线性共价闭合（LCC）DNA微载体或“DNA微串”。这些稳定的线性双链DNA构建体仅携带真核启动子、目的基因、内含子和polyA序列以及核转位增强序列。与常规质粒载体相比，DNA微串提供更有效的细胞运输和转染，并且DNA微串整合到细胞的染色体（细菌或哺乳动物）中是高度致死的事件，其防止可能导致相邻基因突变或沉默的载体整合事件。该计划提出了构建和表征智能噬菌体组装基因表达系统（iPhAGEs）基于噬菌体的DNA递送平台的新策略，这些平台高效，廉价，可扩展，温度稳定且易于操作。为了开拓iPhAGEs的努力，我们的目标是使用单链DNA（ssDNA）丝状噬菌体M13以及裂解性λ噬菌体来产生和包装DNA微串，这两者都提供噬菌体展示和“智能”靶向的能力。这些平台有望最大限度地提高转基因的传递效率，简化纯化，并提供智能targeting.Significance的研究：这项研究计划旨在建立改变游戏规则，强大的，强大的一步遗传过程，以产生稳定和有效的生产新的DNA传递载体。从这项工作中获得的知识将扩展到最大限度地提高转基因递送的效率，功效和安全性，并将其应用于农业和哺乳动物医学中的许多下游转基因递送应用。 此外，展示噬菌体非常适合基因融合肽在衣壳上的表面表达，很容易促进细胞表面受体的靶向、报告基因标记或与效应肽的连接。这种高效的系统将大大扩展遗传工程和分子生物学工具箱。