48. High-Throughput Cloning and Tagging of an Engineered Adenovirus-Library Empowers Vector Diversity for Broad Applications

Abstract

Adenoviral vectors are the most widely used vectors in gene therapy, largely owing to their high efficacy in gene delivery and the ability to transduce a broad variety of cell types. Although more than 60 human adenoviruses types have been identified, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types (Ad2 and 5 from species C) and their genetic modification. Predefined tissue tropism and preexisting immunity considerably limit the application of currently used vectors, making the developing of alternative AdV s mandatory. However, generation of AdVs other than species C was hampered by the lack of convenient cloning methods, for traditional approaches were limited by the low efficiency and complicate procedure.We believe that the engineered adenovirus-library is going to provide spacious novel view to the adenovirus field. As a toolkit, it will bring the AdVs from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology studies.Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here we established a seamless recombineering pipeline for high-throughput cloning and tagging of adenoviral genomes. We amplified and purified wild type adenoviruses that represent all seven human adenovirus species. Using the recombineering pipeline we generated an engineered adenovirus-library including around half of the currently available adenovirus types. To prove integrity of the cloned adenovirus genomes we performed rescue experiments in respective cell lines permissive for adenovirus infection and we optimized rescue condition by testing different molecular forms of the adenoviral DNA (linear, circular, precise release of the DNA molecule). A limited number of selected types from each species will be tagged with a P2A peptide-mediated bicistronic-expression cassette providing a Turbo Green fluorescent protein as in vitro marker and a NanoLuc luciferase for in vivo studies. These tagged adenoviral vectors will be evaluated in cultured cell lines and mouse model. A summary of the human adenoviral vector-library which can be used for further genetic modification and tagging based on the recombineering technology is shown in figure 1.We believe that the engineered adenovirus-library is going to provide spacious novel view to the adenovirus field. As a toolkit, it will bring the AdVs from mono- to multitypes and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology studies. View Large Image | Download PowerPoint Slide