Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue.

Recombinant adenoviruses are widely used in clinical and laboratory applications. Despite the wide variety of available sero- and genotypes, only a fraction is utilized in vivo. As adenoviruses are a large group of viruses, displaying many different tropisms, immune epitopes, and replication characteristics, the merits of translating these natural benefits into vector applications are apparent. This translation, however, proves difficult, since while research has investigated the application of these viruses, there are no universally applicable rules in vector design for non-classical adenovirus types. In this paper, we describe a generalized workflow that allows vectorization, rescue, and cloning of all adenoviral species to enable the rapid development of new vector variants. We show this using human and simian adenoviruses, further modifying a selection of them to investigate their gene transfer potential and build potential vector candidates for future applications.

Combining CRISPR-Cas-mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries.

While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 104 unique rAds per cm2 of transfected cell culture.