An Adenoviral Vector as a Versatile Tool for Delivery and Expression of miRNAs.

Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously.

Genetic and Chemical Capsid Modifications of Adenovirus Vectors to Modulate Vector-Host Interactions.

Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector-host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector-host interactions of Ad-based vectors.

Capsid and Genome Modification Strategies to Reduce the Immunogenicity of Adenoviral Vectors.

Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.