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

背景：噬菌体构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件构建系统，以设计高效，有效和安全的治疗生产平台，用于转基因递送应用。由于细胞毒性、靶细胞中较差的转基因表达效率、广泛的载体降解以及低效/不可规模化的载体生产，目前的转基因传递方式没有设想的那么成功。我们之前设计了一个生产平台，用于生成DNA微型载体，利用噬菌体编码的遗传元素在其有效生产中。我们的技术采用噬菌体PY54的遗传系统来生产线性共价封闭（LCC） DNA微载体，或“DNA服务”。这些稳定的线性双链DNA结构只携带真核启动子、感兴趣的基因、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片段提供了更有效的细胞运输和转染，并且DNA片段整合到细胞染色体（细菌或哺乳动物）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。该项目提出了构建和表征智能噬菌体组装基因表达系统（iPhAGEs）的新策略，该系统基于噬菌体的DNA传递平台高效、廉价、可扩展、温度稳定且易于操作。作为iPhAGEs的先驱，我们的目标是使用单链DNA （ssDNA）丝状噬菌体M13和裂解型Lambda噬菌体生产和包装DNA服务，这两种噬菌体都具有噬菌体展示和“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，并提供智能靶向选择。研究意义：该研究项目旨在建立改变游戏规则的、稳健的、强大的一步遗传过程，以产生稳定有效的新型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传递的效率、功效和安全性，并用于农业和哺乳动物医学中的下游转基因传递应用。此外，展示噬菌体高度适应衣壳上基因融合肽的表面表达，易于靶向细胞表面受体、报告基因标记或与效应肽连接。这种高效的系统将极大地扩展基因工程和分子生物学的工具箱。

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.