VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.

Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.

Non-viral engineering of NK cells.

The last decade has witnessed great progress in the field of adoptive cell therapies, with the authorization of Kymriah (tisagenlecleucel) in 2017 by the Food and Drug Administration (FDA) as a crucial stepstone. Since then, five more CAR-T therapies have been approved for the treatment of hematological malignancies. While this is a great step forward to treating several types of blood cancers, CAR-T cell therapies are still associated with severe side-effects such as Graft-versus-Host Disease (GvHD), cytokine release syndrome (CRS) and neurotoxicity. Because of this, there has been continued interest in Natural Killer cells which avoid these side-effects while offering the possibility to generate allogeneic cell therapies. Similar to T-cells, NK cells can be genetically modified to improve their therapeutic efficacy in a variety of ways. In contrast to T cells, viral transduction of NK cells remains inefficient and induces cytotoxic effects. Viral vectors also require a lengthy and expensive product development process and are accompanied by certain risks such as insertional mutagenesis. Therefore, non-viral transfection technologies are avidly being developed aimed at addressing these shortcomings of viral vectors. In this review we will present an overview of the potential of NK cells in cancer immunotherapies and the non-viral transfection technologies that have been explored to engineer them.

Cellular and Natural Viral Engineering in Cognition-Based Evolution.

Neo-Darwinism conceptualizes evolution as the continuous succession of predominately random genetic variations disciplined by natural selection. In that frame, the primary interaction between cells and the virome is relegated to host-parasite dynamics governed by selective influences. Cognition-Based Evolution regards biological and evolutionary development as a reciprocating cognition-based informational interactome for the protection of self-referential cells. To sustain cellular homeorhesis, cognitive cells collaborate to assess the validity of ambiguous biological information. That collective interaction involves coordinate measurement, communication, and active deployment of resources as Natural Cellular Engineering. These coordinated activities drive multicellularity, biological development, and evolutionary change. The virome participates as the vital intercessory among the cellular domains to ensure their shared permanent perpetuation. The interactions between the virome and the cellular domains represent active virocellular cross-communications for the continual exchange of resources. Modular genetic transfers between viruses and cells carry bioactive potentials. Those exchanges are deployed as nonrandom flexible tools among the domains in their continuous confrontation with environmental stresses. This alternative framework fundamentally shifts our perspective on viral-cellular interactions, strengthening established principles of viral symbiogenesis. Pathogenesis can now be properly appraised as one expression of a range of outcomes between cells and viruses within a larger conceptual framework of Natural Viral Engineering as a co-engineering participant with cells. It is proposed that Natural Viral Engineering should be viewed as a co-existent facet of Natural Cellular Engineering within Cognition-Based Evolution.

Generation of Mature DENVs via Genetic Modification and Directed Evolution.

Maturation of dengue viruses (DENVs) alters the structure, immunity, and infectivity of the virion and highly mature particles represent the dominant form in vivo. The production of highly mature virions principally relies on the structure and function of the viral premature membrane protein (prM) and its cleavage by the host protease furin. We redeveloped a reliable clonal cell line (VF1) which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs in all serotypes independent of cell or host, usually with minimal impact on viral yield. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic partially mature forms. Given the clinical importance of mature DENVs in immunity, our study provides reliable strategies and reagents for the production of stable, high-titer mature DENVs for DENV antibody neutralization and vaccination immunity studies. Biologically, our data from directed evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, verifying the substrate preference between mammalian and insect furin, while hinting at an evolutionary equilibrium of DENV prM cleavage site between its host and vector in nature. IMPORTANCE Mature DENVs represent the dominant form in vivo and are the target for vaccine development. Here, we used multiple strategies, including protein engineering and natural and directed evolution to generate DENV1, -2, -3, and -4 variants that are highly mature without compromising replication efficiency compared to the parental strains. Given the clinical importance of mature DENVs in immunity, this work provides a roadmap for engineering highly mature DENV that could apply to future vaccine development. Our directed-evolution data also shed light on the divergent evolutionary relationship of DENVs between its host and vector.

Immunogenic cell death: The cornerstone of oncolytic viro-immunotherapy.

According to the World Health Organization, cancer is one of the leading global health concerns, causing nearly 10 million deaths in 2020. While classical chemotherapeutics produce strong cytotoxicity on cancer cells, they carry limitations of drug resistance and off-target effects and sometimes fail to elicit adequate antitumor protection against tumor relapse. Additionally, most cancer cells have developed various ways to escape immune surveillance. Nevertheless, novel anticancer strategies such as oncolytic viro-immunotherapy can trigger immunogenic cell death (ICD), which can quickly grasp the attention of the host defense machinery, resulting in an ensuing antitumor immune response. Specifically, oncolytic viruses (OVs) can infect and destroy targeted cancer cells and stimulate the immune system by exposing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to promote inflammatory reactions, and concomitantly prime and induce antitumor immunity by the release of neoantigens from the damaged cancer cells. Thus, OVs can serve as a novel system to sensitize tumor cells for promising immunotherapies. This review discusses the concept of ICD in cancer, centralizing ICD-associated danger signals and their consequence in antitumor responses and ICD induced by OVs. We also shed light on the potential strategies to enhance the immunogenicity of OVs, including the use of genetically modified OVs and their combination with ICD-enhancing agents, which are helpful as forthcoming anticancer regimens.

RNA Viral Vectors for Accelerating Plant Synthetic Biology.

The tools of synthetic biology have enormous potential to help us uncover the fundamental mechanisms controlling development and metabolism in plants. However, their effective utilization typically requires transgenesis, which is plagued by long timescales and high costs. In this review we explore how transgenesis can be minimized by delivering foreign genetic material to plants with systemically mobile and persistent vectors based on RNA viruses. We examine the progress that has been made thus far and highlight the hurdles that need to be overcome and some potential strategies to do so. We conclude with a discussion of biocontainment mechanisms to ensure these vectors can be used safely as well as how these vectors might expand the accessibility of plant synthetic biology techniques. RNA vectors stand poised to revolutionize plant synthetic biology by making genetic manipulation of plants cheaper and easier to deploy, as well as by accelerating experimental timescales from years to weeks.

Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering.

As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

Engineered AAV vectors for improved central nervous system gene delivery.

Adeno-associated viruses (AAV) are non-pathogenic members of the Parvoviridae family that are being harnessed as delivery vehicles for both basic research and increasingly successful clinical gene therapy. To address a number of delivery shortcomings with natural AAV variants, we have developed and implemented directed evolution-a high-throughput molecular engineering approach to generate novel biomolecules with enhanced function-to create novel AAV vectors that are designed to preferentially transduce specific cell types in the central nervous system (CNS), including astrocytes, neural stem cells, and cells within the retina. These novel AAV vectors-which have enhanced infectivity in vitro and enhanced infectivity and selectivity in vivo-can enable more efficient studies to further our understanding of neurogenesis, development, aging, and disease. Furthermore, such engineered vectors may aid gene or cell replacement therapies to treat neurodegenerative disease or injury.

Sorting Out Pandora's Box: Discerning the Dynamic Roles of Liver Microenvironment in Oncolytic Virus Therapy for Hepatocellular Carcinoma.

Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete anti-viral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, natural killer cells are greatly enriched in the liver, contributing to the innate defense against viruses. The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus (OV) therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, OVs can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of OV therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing. This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome.