Virus AND gene edit

Treating human genetic conditions in vivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with in vivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduces drug development flexibility and manufacturing efficiency...

Synaptic function in neurons is modulated by local translation of mRNAs that are transported to distal portions of axons and dendrites. The metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) is broadly expressed across cell types, almost exclusively as a nuclear long noncoding RNA. We found that in differentiating neurons, a portion of Malat1 RNA redistributes to the cytoplasm. Depletion of Malat1 using antisense oligonucleotides (ASOs) stimulates the expression of particular pre- and postsynaptic proteins, implicating Malat1 in their regulation...

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers...

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma...

BACKGROUND: The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus...

A hallmark of the last decades is an extensive development of genome editing systems and technologies propelling genetic engineering to the next level. Specific and efficient delivery of genome editing tools to target cells is one of the key elements of such technologies. Conventional vectors are not always suitable for this purpose due to a limited cargo volume, risks related to cancer and immune reactions, toxicity, a need for high-purity viral material and quality control, as well as a possibility of integration of the virus into the host genome leading to overexpression of the vector components and safety problems...

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-colour genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F...

In the world's first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy...

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors...

BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship...

Soybean (Glycine max ) is an important oilseed, protein and biodiesel crop. It faces significant threats from bacterial, fungal and viral pathogens, which cause economic losses and jeopardises global food security. In this article, we explore the relationship between soybeans and these pathogens, focusing on the molecular responses that are crucial for soybeans defence mechanisms. Molecular responses involve small RNAs and specific genes, including resistance (R) genes that are pivotal in triggering immune responses...

Infection with the hepatitis B virus (HBV) is highly prevalent globally. Over 250 million people suffer from chronic hepatitis B, and more than 800,000 patients die each year due to hepatitis B complications, including liver cancer. Although protective HBV vaccines are recommended for all newborns, global coverage is suboptimal. In adults, sexual transmission is by far the most frequent route of contagion. The WHO estimates that 1.5 million new HBV infections occur annually. Oral nucleos(t)ide analogues entecavir and tenofovir are the most frequent antivirals prescribed as HBV therapy...

Although often located at a distance from their target gene promoters, enhancers are the primary genomic determinants of temporal and spatial transcriptional specificity in metazoans. Since the discovery of the first enhancer element in simian virus 40, there has been substantial interest in unraveling the mechanism(s) by which enhancers communicate with their partner promoters to ensure proper gene expression. These research efforts have benefited considerably from the application of increasingly sophisticated sequencing- and imaging-based approaches in conjunction with innovative (epi)genome-editing technologies; however, despite various proposed models, the principles of enhancer-promoter interaction have still not been fully elucidated...

PURPOSE: Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues...

Glaucoma and uveitis are non-vascular ocular diseases which are among the leading causes of blindness and visual loss. These conditions have distinct characteristics and mechanisms but share a multifactorial and complex nature, making their management challenging and burdensome for patients and clinicians. Furthermore, the lack of symptoms in the early stages of glaucoma and the diverse aetiology of uveitis hinder timely and accurate diagnoses, which are a cause of poor visual outcomes under both conditions...

UNLABELLED: Paramyxo- and filovirus genomes are equipped with bipartite promoters at their 3 ' ends to initiate RNA synthesis. The two elements, the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2), are separated by a spacer region that must be precisely a multiple of 6 nucleotides (nts), indicating these viruses adhere to the "rule of six." However, our knowledge of PE2 has been limited to a narrow spectrum of virus species. In this study, a comparative analysis of 1,647 paramyxoviral genomes from a public database revealed that the paramyxovirus PE2 can be clearly categorized into two distinct subcategories: one marked by C repeats at every six bases (exclusive to the subfamily Orthoparamyxovirinae ) and another characterized by CG repeats every 6 nts (observed in the subfamilies Avulavirinae and Rubulavirinae )...

The role of CD8+ T-cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T-cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T-cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss...

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells...

INTRODUCTION: Signal peptide peptidase ( SPP ) is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although SPP is essential for many viral infections, its role in silkworms remains unclear...

Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance...