The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6—all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.

We use lentiviral-delivered RNA interference (RNAi) to inhibit the growth of a model of primary effusion lymphoma (PEL) in vitro and in vivo. RNAi is a phenomenon allowing the sequencespecific targeting and silencing of exogenous and endogenous gene expression and is being applied to inhibit viral replication both in vitro and in vivo. We show that silencing of genes believed to be essential for the Kaposi sarcomaassociated herpesvirus (KSHV) latent life cycle (the oncogenic cluster) has a varied effect in PEL cell lines cultured in vitro, however, concomitant silencing of the viral cyclin (vcyclin) and viral FLICE (Fasassociating protein with death domainlike interleukin-1␤-converting enzyme) inhibitory protein (vFLIP) caused efficient apoptosis in all PEL lines tested. We demonstrate that in a murine model of PEL, lentiviral-mediated RNA interference both inhibits development of ascites and can act as a treatment for established ascites. We also show that the administered lentiviral vectors are essentially limited to the peritoneal cavity, which has advantages for safety and dosage in a therapeutic setting. This shows the use of lentiviral-mediated RNA interference in vivo as a potential therapeutic against a virally driven human cancer. IntroductionRNA interference (RNAi) is being exploited to treat or prevent infection and as a therapeutic against cancer. 1,2 Attractive targets include foreign (eg, viral) and mutated, fused, or overexpressed genes (ie, cancer). [3][4][5] In vitro, RNAi has been shown to block infection and replication of various pathogens including human immunodeficiency virus (HIV-1), 6,7 influenza, 8 and viruses implicated in oncogenesis such as Epstein Barr virus (EBV), 9,10 hepatitis B, 11,12 hepatitis C, 13 and human papilloma virus (HPV). 14,15 In experimental in vivo models, RNAi has been shown to prevent chemical-and viral-induced hepatitis. [16][17][18] The efficient delivery of therapies that knock-down specific RNA remains one obstacle to translate RNAi into a realistic treatment option for human disease. 19 The direct delivery of antisense RNA to treat or prevent CMV retinitis is one of the few successful clinical applications thus far of RNA-targeted treatment. 20,21 Although antisense provides significantly less robust inhibition of gene expression compared to RNAi, the major problem with both therapies remains effective delivery to the site of disease. One use of RNAi is based around injecting large quantities of synthetic double-stranded RNA (dsRNA) or DNA encoding short hairpin RNA (shRNA) intravenously or using hydrodynamic transfection. 16,18 This approach is not realistic for treating human disease, except for diseases involving sites where delivery of synthetic dsRNA is more straightforward. The development of shRNA to knock-down gene expression 22,23 and the incorporation of shRNA into lentiviral- 24,25 or adenoviral-based 26,27 vectors offers the opportunity to use these vectors to target RNA efficiently in vivo. Vesicular stomatitis virus-g envelope (VSV-g)-pseudoty...

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Xenotransplantation of porcine cells, tissues, and organs may be associated with the transmission of porcine microorganisms to the human recipient. A previous, 2009, version of this consensus statement focused on strategies to prevent transmission of porcine endogenous retroviruses (PERVs). This version addresses potential transmission of all porcine microorganisms including monitoring of the recipient and provides suggested approaches to the monitoring and prevention of disease transmission. Prior analyses assumed that most microorganisms other than the endogenous retroviruses could be eliminated from donor animals under appropriate conditions which have been called "designated pathogen-free" (DPF) source animal production. PERVs integrated as proviruses in the genome of all pigs cannot be eliminated in that manner and represent a unique risk. Certain microorganisms are by nature difficult to eliminate even under DPF conditions; any such clinically relevant microorganisms should be included in pig screening programs. With the use of porcine islets in clinical trials, special consideration has to be given to the presence of microorganisms in the isolated islet tissue to be used and also to the potential use of encapsulation. It is proposed that microorganisms absent in the donor animals by sensitive microbiological examination do not need to be monitored in the transplant recipient; this will reduce costs and screening requirements. Valid detection assays for donor and manufacturing-derived microorganisms must be established. Special consideration is needed to preempt potential unknown pathogens which may pose a risk to the recipient. This statement summarizes the main achievements in the field since 2009 and focus on issues and solutions with microorganisms other than PERV.

Lentiviral vectors (LVs) are used in cell and gene therapies due to their ability to transduce both dividing and non-dividing cells while carrying a relatively large genetic payload and providing long-term gene expression via gene integration. Current cultivation methods produce titers of 105–107 transduction unit (TU)/mL; thus, it is necessary to concentrate LVs as well as remove process- and product-related impurities. In this work, we used a packaging cell line WinPac-RD-HV for LV production to simplify upstream processing. A direct capture method based on ion-exchange chromatography and cellulose nanofibers for LV concentration and purification was developed. This novel scalable stationary phase provides a high surface area that is accessible to LV and, therefore, has potential for high-capacity operation compared to traditional bead-based supports. We were able to concentrate LVs 100-fold while achieving a two-log removal of host cell protein and maintaining up to a 90% yield of functional vector.

Human stem cells could revolutionize the field of medicine by providing a diverse range of cell types for tissue replacement therapies and drug discovery. To achieve this goal, genetic tools need to be optimized and developed for controlling and manipulating stem cells ex vivo. Here we describe a lentiviral delivery system capable of high infection rates in human mesenchymal and embryonic stem cells. The lentiviral backbone was modified to express mono-and bi-cistronic transgenes and was also used to deliver short hairpin ribonucleic acid for specific silencing of gene expression in human stem cells. We show that lentiviral transduction can be used to alter gene expression without altering the genes' ability to differentiate in vitro. These vectors will enable rapid analysis of gene function in stem cells and permit the generation of knock-in / knock-out models of human disease in the rapidly developing field of gene therapy.

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