RNA interference-mediated inhibition of adenoviruses

Human adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients such as HIV-positive individuals or solid organ and particularly allogenic stem cell transplant recipients. Among the most severe forms of adenovirus infections of transplant recipients hemorrhagic cystitis, nephritis, pneumonia, hepatitis, enterocolitis and disseminated disease have been described and in disseminated disease mortality rates can be almost as high as 80% among hematopoietic stem cell transplant recipient. Constant monitoring of immunocompromised patients with state of the art diagnosis methods can identify patients at high risk. However, while progress that has been made for example in the treatment of cytomegalovirus infections, adenovirus therapy is still unsatisfactory. A number of potential anti-viral drugs has been investigated for their capacity to condemn adenovirus infections. However, responses are limited and treatment still cannot guarantee the prevention of fatal outcomes. Therefore, given the fact that numbers of solid organ and hematopoietic cell transplantations are constantly rising and that still no satisfactory anti-adenoviral treatment methods are available, the development of alternative anti- adenovirus treatment strategies to decrease adenovirus-mediated mortality among immunocompromised patients is of paramount importance. In our project we want to investigate if RNA interference (RNAi) has the capacity to inhibit or impair the multiplication of adenoviruses in a cell culture system in vitro. We aim to knock down the gene expression of key players of the adenoviral life cycle: (i) genes encoding proteins essential for DNA replication and (ii) genes encoding structural proteins which are part of the mature virion. In the initial phase of the project we will employ small interfering RNAs (siRNAs) to knock down gene expression. Subsequently, the most effective siRNAs will constitute the basis for the generation of an RNAi vector system for the expression of the siRNAs in the form of microRNAs (miRNAs). In a third step we plan to insert the miRNA cassette into a replication-deficient adenoviral vector and express it from an adenoviral promoter. The final vector is expected to only allow efficient miRNA expression in cells infected with wild-type virus since only the wild-type virus is able to provide the gene products necessary for vector replication and for the full activation of the adenoviral promoter driving the expression of the miRNA cassette. Such a system may constitute the basis for the development of a therapeutic for the treatment of life-threatening adenovirus infections.

Adenoviruses can be life-threatening or even lethal to immunocompromised patients, and due to the lack of sufficiently effective anti-adenoviral drugs, alternative treatment strategies are heavily needed. In this project we investigated the potential of RNA interference (RNAi) as a tool to knock down adenoviral gene expression, and consequently, to inhibit adenoviral multiplication. We could demonstrate that synthetic, small interfering RNAs (siRNAs) are capable of efficiently decreasing the multiplication of adenoviruses in infected cells in vitro. Upon introduction into infected cells, these siRNAs exert their function by specifically binding to viral RNA, thereby preventing this viral RNA from becoming active. Our experiments indicated that blocking the function of a viral RNA that is required for the multiplication of the viral genome represents a crucial step in inhibiting adenoviruses. Further investigations demonstrated that virus inhibition can not only be achieved with siRNAs but also artificial microRNAs (amiRNAs). These amiRNAs can be introduced into cells in the form of DNA fragments, which are subsequently converted into small pieces of RNA that can block gene expression in a way analogous to siRNAs. In the case of our project, the output of infectious virus particles from such cells was heavily decreased in both cases. Our study also revealed that amiRNAs can be utilized to, at least in vitro, enhance the performance of drugs commonly used to treat adenovirus infections. In an envisioned clinical application, siRNAs or amiRNAs may be able to decrease the symptoms of severe and potentially life-threatening adenovirus infections in immunocompromised patients, or to support the function of conventional anti-adenoviral drugs.