The adenovirus vector platform remains one of the most efficient toolboxes for generation of transfer vehicles used in gene therapy and virotherapy to treat tumors, as well as vaccines to protect from infectious diseases. The adenovirus genome and capsids can be modified using highly efficient techniques, and vectors can be produced at high titers, which facilitates their rapid adaptation to current needs and disease applications. Over recent years, the adenovirus vector platform has been in the center of attention for vaccine development against the ongoing coronavirus SARS-CoV-2/COVID-19 pandemic. The worldwide deployment of these vaccines has greatly deepened the knowledge on virus-host interactions and highlighted the need to further improve the effectiveness and safety not only of adenovirus-based vaccines but also of gene therapy and oncolytic virotherapy vectors. Based on the current evidence, we discuss here how adenoviral vectors can be further improved by intelligent molecular design. This review covers the full spectrum of state-of-the-art strategies to avoid vector-induced side effects ranging from the vectorization of non-canonical adenovirus types to novel genome engineering techniques.

In this work, we are reporting that “Shock and Kill”, a therapeutic approach designed to eliminate latent HIV from cell reservoirs, is extrapolatable to cancer therapy. This is based on the observation that malignant cells express a spectrum of human endogenous retroviral elements (HERVs) which can be transcriptionally boosted by HDAC inhibitors. The endoretroviral gene HERV-V2 codes for an envelope protein, which resembles syncytins. It is significantly overexpressed upon exposure to HDAC inhibitors and can be effectively targeted by simultaneous application of TLR7/8 agonists, triggering intrinsic apoptosis. We demonstrated that this synergistic cytotoxic effect was accompanied by the functional disruption of the TLR7/8-NFκB, Akt/PKB, and Ras-MEK-ERK signalling pathways. CRISPR/Cas9 ablation of TLR7 and HERV-V1/V2 curtailed apoptosis significantly, proving the pivotal role of these elements in driving cell death. The effectiveness of this new approach was confirmed in ovarian tumour xenograft studies, revealing a promising avenue for future cancer therapies.

The adenoviral vector based AstraZeneca and Janssen COVID vaccines have been associated with rare cases of thrombosis, a condition which depends on adenovirus binding to the blood protein Platelet Factor 4 (PF4). In order to identify adenoviruses with low or absent affinity for PF4, we screened dozens of types from various adenovirus species, and Adenovirus type 5 (Ad5) derived vectors carrying genetic or chemical modifications of different hexon hyper-variable regions (HVR). For this purpose, we established an armamentarium of techniques including ELISA-qPCR and Aggregate Pull-Down (APD), which enabled fast and sensitive assessments of virus-protein interactions. Unlike most tested serotypes, Ad34 did not bind to PF4. Likewise, the deletion or shielding of the HVR1 loop of Ad5 seemingly ablated its PF4 binding. Therefore, we showed that PF4 binds to adenovirus hexon through interactions dependent on HVR1, and identified vectors that may avoid or decrease the risk of thrombosis and represent safer candidates for vaccine or gene therapy vector development.

Adenoviruses are non-enveloped linear double-stranded DNA viruses with over 100 types in humans. Adenovirus vectors have gained tremendous attention as gene delivery vehicles, as vaccine vectors and as oncolytic viruses. Although various methods have been used to generate adenoviral vectors, the vector-producing process remains technically challenging regarding efficacious genome modification. Based on our previously reported adenoviral genome modification streamline via linear–circular homologous recombination, we further develop an HEHR (combining Homing Endonucleases and Homologous Recombination) method to engineer adenoviral genomes more efficiently. I-PpoI, a rare endonuclease encoded by a group I intron, was introduced into the previously described ccdB counter-selection marker. We found that the I-PpoI pre-treatment of counter-selection containing parental plasmid increased the homologous recombination efficiency up to 100%. The flanking of the counter-selection marker with either single or double I-PpoI sites showed enhanced efficacy. In addition, we constructed a third counter-selection marker flanked by an alternative restriction enzyme: AbsI, which could be applied in case the I-PpoI site already existed in the transgene cassette that was previously inserted in the adenovirus genome. Together, HEHR can be applied for seamless sequence replacements, deletions and insertions. The advantages of HEHR in seamless mutagenesis will facilitate rational design of adenoviral vectors for diverse purposes.