SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies.
SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies.
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DOI：
10.1021/acssynbio.2c00244
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发表时间：
2022-10
影响因子：
4.7
通讯作者：
Sahan B. W. Liyanagedera;Joshua Williams;Joseph P. Wheatley;A. Biketova;Muhammad Hasan;Antonia P. Sagona;K. Purdy;R. J. Puxty;T. Fehér;V. Kulkarni
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作者：
Sahan B. W. Liyanagedera;Joshua Williams;Joseph P. Wheatley;A. Biketova;Muhammad Hasan;Antonia P. Sagona;K. Purdy;R. J. Puxty;T. Fehér;V. Kulkarni
在过去的十年中，多药耐药病原体的出现成为全球死亡的主要原因，重新点燃了人们对噬菌体治疗领域的兴趣。噬菌体基因工程的现代进展已经实现了几个有用的结果，包括宿主范围的改变，组成性裂解生长和噬菌体复制的控制。然而，转基因生物的许可证发放过程缓慢，显然阻碍了新的工程变异体的快速治疗应用，这些变异体是对抗在大流行病过程中出现的突变病原体所必需的。作为这个问题的解决方案，我们提出了SpyPhage系统，其中“支架”噬菌体被工程化以在其衣壳头上并入SpyTag部分，从而能够用SpyCatcher融合的治疗性蛋白对其表面进行快速合成后修饰。作为概念证明，通过CRISPR/Cas促进的噬菌体工程和全基因组组装，我们将SpyTag衣壳融合物靶向K1 F，这是一种靶向致病性菌株大肠杆菌K1的噬菌体。我们首次展示了无细胞组装和装饰的噬菌体表面与两种可选的融合蛋白，SpyCatcher-mCherry-EGF和SpyCatcher-mCherry-Rck，这两种蛋白都有利于膀胱上皮细胞系的内吞摄取。总的来说，我们的工作提出了一种无细胞噬菌体生产管道，用于产生具有单一潜在“支架”基因型的多个表型不同的噬菌体。这些基因组可以成为下一代噬菌体疗法的基础，其中许多噬菌体变体的基于知识的工程将在不使用活细菌或需要重复许可新的遗传改变的情况下快速实现。
The past decade has seen the emergence of multidrug resistant pathogens as a leading cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including host range alterations, constitutive lytic growth, and control over phage replication. However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to this problem, we propose the SpyPhage system where a "scaffold" bacteriophage is engineered to incorporate a SpyTag moiety on its capsid head to enable rapid postsynthetic modification of their surfaces with SpyCatcher-fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and whole genome assembly, we targeted a SpyTag capsid fusion to K1F, a phage targeting the pathogenic strain Escherichia coli K1. We demonstrate for the first time the cell-free assembly and decoration of the phage surface with two alternative fusion proteins, SpyCatcher-mCherry-EGF and SpyCatcher-mCherry-Rck, both of which facilitate the endocytotic uptake of the phages by a urinary bladder epithelial cell line. Overall, our work presents a cell-free phage production pipeline for the generation of multiple phenotypically distinct phages with a single underlying "scaffold" genotype. These phages could become the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations.