Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection.

RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) lung infection in vivo. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The study presented here demonstrates an attractive alternate delivery strategy for the prophylactic treatment of both future and emerging respiratory viral diseases.

Aurora kinases are a novel therapeutic target for HPV-positive head and neck cancers.

OBJECTIVES: Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells. MATERIALS AND METHODS: To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment. RESULTS: RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells. CONCLUSION: Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.

Prevalence and types of high-risk human papillomaviruses in head and neck cancers from Bangladesh.

BACKGROUND: There is a dramatic rise in the incidence of Human papillomavirus (HPV) - associated head and neck squamous cell carcinoma (HNSCC) in the world, with considerable variation by geography, gender and ethnicity. Little is known about the situation in Bangladesh, where tobacco- and areca nut-related head and neck cancers (HNCs) are the most common cancers in men. We aimed to determine the prevalence of HPV in HNSCC in Bangladesh and to explore the possible value of cell cycle markers in clinical diagnostic settings. METHODS: One hundred and ninety six archival HNSCC tissue samples were analysed for the presence of HPV DNA. The DNA quality was assured, and then amplified using a nested PCR approach. The typing of HPV was performed by automated DNA sequencing. Cellular markers p53, Cyclin D1 and pRb were tested on all samples by immunohistochemistry (IHC), as well as p16 as a putative surrogate for the detection of HPV. RESULTS: HPV DNA was detected in 36/174 (~21%) samples: 36% of cancers from the oropharynx; 31% of oral cancers, and 22% from the larynx. HPV-16 was most common, being present in 33 samples, followed by HPV-33 (2 samples) and HPV-31 (1 sample). Twenty-eight out of 174 samples were positive for p16, predominantly in HPV-positive tissues (p < 0.001). No statistically significant association was observed between the cellular markers and HPV DNA positive cases. However, p16 positivity had excellent predictive value for the presence of HPV by PCR. CONCLUSION: There is a significant burden of HPV-associated HNSCC in Bangladesh, particularly in the oropharynx but also in oral and laryngeal cancers. Whilst a combination of PCR-based DNA detection and p16 IHC is useful, the latter has excellent specificity, acceptable sensitivity and good predictive value for carriage of HPV in this population and should be used for prognostic evaluation and treatment planning of all HNSCC patients in South Asia, as in the Western world.

Can gene editing and silencing technologies play a role in the treatment of head and neck cancer?

Conventional treatment strategies have done little to improve the prognosis or disease-free survival in head and neck cancer (HNC) patients. Recent progress in our understanding of molecular aspects of head and neck squamous cell carcinoma (HNSCC) has provided insights into the potential use of molecular targeted therapies in combination with current treatment strategies. Here we review the current understanding of treatment modalities for both HPV-positive and HPV-negative HNSCCs with the potential to use gene editing and silencing technologies therapeutically. The development of sequence-specific RNA interference (RNAi) with its strong gene-specific silencing ability, high target specificity, greater potency and reduced side effects, has shown it to be a promising therapeutic candidate for treating cancers. CRISPR/Cas gene editing is the newest technology with the ability to delete, mutate or replace genes of interest and has great potential for treating HNSCCs. We also discuss the major challenge in using these approaches in HNSCC; that being the choice of target and the ability to deliver the payload. Finally, we highlight the potential combination of RNAi or CRIPSR/Cas with current treatment strategies and outline the possible path to the clinic.

Targeted drug delivery to the virus-infected airway; complications and remedies.

Drug delivery to the airway and lower respiratory tract by aerosol inhalation has become a successful, non-invasive method of preventing and treating local disease of the lung. Consequently, it has been a promising route for clinical trials using highly specific and novel therapies to overcome viral pulmonary infection such as RNA interference, neutralising monoclonal antibodies and microparticle treatments. Yet despite this great potential, this form of delivery has proven somewhat ineffective due to airway remodeling, inflammation and mucus hypersecretion that results from viral symptoms in the respiratory tract. Here we review the research into the delivery technologies available as well as the types of therapeutics used for respiratory virus disease and examine how virus infection-induced airway inflammation modulates its success. We discuss the future of aerosol administration and present potential alternative methods for efficient drug delivery so as to improve postinfection virus control therapies.

A novel method for screening viral interferon-resistance genes.

Many viruses have evolved mechanisms to antagonize the interferon (IFN) system, targeting all the major components involved in receptor binding and signaling. Although a number of these vital proteins are homologous to cellular proteins involved in IFN downregulation (e.g., viral IFN regulatory factors [vIRFs]), many share little resemblance to known proteins. To determine the IFN-blocking properties of these proteins, functional assays are required. Here, we present a new and rapid functional screening method, based on the 2fTGH cell line, which is able to determine viral gene products that inhibit the IFN-alpha/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-alpha and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-alpha. We also demonstrate that selection occurs if members of the Jak-Stat signaling pathway are lost. To show the utility of our system, we have used a known suppressor of IFN signaling, the human papillomavirus (HPV) E7 gene. Expression of E7 causes the loss of ability of 2fTGH cells to respond to IFN-alpha treatment because of a functional disruption of the signaling pathway. This approach offers a new strategy for identifying novel viral genes or new functions of already described viral genes that have a role in IFN-alpha signaling inhibition.