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

背景：噬菌体形成了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素构建系统，为转基因应用设计高效、高效和安全的治疗性生产平台。目前的转基因传递方式没有想象的那么成功，原因是：细胞毒性、转基因在靶细胞中的表达效率低、载体的广泛降解以及低效/非规模化的载体生产。我们之前已经设计了一个生产平台，用于生成DNA微型载体，该载体在高效生产中利用噬菌体编码的遗传元件。我们的技术采用了来自噬菌体PY54的遗传系统来产生线性共价闭合(LCC)DNA微型载体，或“DNA微型串”。这些稳定的线性双链DNA结构只携带真核启动子、目的基因、内含子和PolyA序列以及核易位增强序列。与传统的质粒载体相比，DNA服务提供了更有效的细胞运输和转染，并且DNA服务到细胞染色体(细菌或哺乳动物)的整合是一种高度致命的事件，可以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(IPhone AGEs)基于噬菌体的DNA传递平台的新策略，该平台高效、廉价、可扩展、温度稳定且易于操作。为了开创iPhone AGE的先河，我们的目标是使用单链DNA(SsDNA)丝状噬菌体M13以及裂解的Lambda噬菌体来生产和包装DNA服务，这两种噬菌体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传递效率，简化纯化，并提供智能靶向的选择。研究意义：本研究计划旨在建立改变游戏规则的、健壮的、强大的一步遗传过程，以产生稳定和高效的新型DNA传递载体的生产。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传递的效率、有效性和安全性，使其应用于农业和哺乳动物医学中的一些下游转基因传递应用。此外，展示噬菌体对衣壳上基因融合多肽的表面表达具有高度的适应性，易于靶向细胞表面受体、报告标记或与效应肽的连接。这种高效的系统将极大地扩展基因工程和分子生物学工具箱。

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.