The Flexible, Versatile and Fast GreGT Platform of Fully Deleted Helper-Virus Independent Adenoviral Vectors

Abstract

Gene transfer (GT) vectors have diverse applications. They have been used to restore cellular activities by reconstituting normal cellular functions, by delivering therapeutic compounds, and by priming immune responses as genetic vaccines. Viruses, natures gene delivery vehicles, have formed the basis of most GT vectors. The biology of the underlying virus may, however, hamper their use. For instance, retroviral vectors with their ability to integrate into the genome, may cause malignant transformations. Vectors designed as minor variants of the relatively benign, yet complex adenovirus (Ad) excite vigorous immune responses, thus limiting their therapeutic effects. Deleting Ad vectors of all endogenous Ad genes brought their beneficial features to the front, such as their ability to transduce cells of many types with high efficiency and to deliver large genetic payloads. Earlier production schemes of fully deleted Ad (fdAd) vectors depended on helper virus constructs to deliver the vector production information in trans. They suffered from contaminations with the helper virus or the recombination of replication competent adenoviruses (RCA). We previously developed a novel transfection-based helper virus-independent Ad vector encapsidation technology that avoided these pitfalls. It was built on a vector genome and a vector packaging module. We have now optimized our approach into the GreGT plug-and-play platform so that a new vector can be delivered in about four weeks. The GreGT system is built upon a set of base vector genome modules that can be quickly loaded with a new application, and a set of packaging modules that allow their encapsidation into capsids of different Ad serotypes. As both components can be freely combined, the GreGT platform is endowed with high degrees of flexibility and versatility. Finally, the deletion of all endogenous Ad genes from the vector genome limits the interference by anti-Ad immune responses. It also increases the genetic payload capacity to levels unique to this system.