Development and functional testing of a novel in vitro delayed scratch closure assay.

As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro. Chronic wounds such as those suffered by later stage diabetic patients are characterised by degrees of slowness to heal caused by a combination of continued localised physical trauma and pro-inflammatory signalling at the wound. To recreate this in a cell-based assay, a defined physical scratch was created and stimulated by combinations of pro-inflammatory factors, namely interferon, the phorbol ester PMA, and lipopolysaccharide, to delay scratch closure. The concentrations of these factors were characterised for commonly used human keratinocyte (HaCaT) and dermal fibroblast (HDF) cell lines. These models were then tested for scratch closure responsiveness to a proprietary healing secretome derived from human Wharton's jelly mesenchymal stem cells (MSCs) previously validated and shown to be highly effective on closure of acute wound models both in vitro and in vivo. The chronically open scratches from HaCaT cells showed closure after exposure to the MSC secretome product. We propose this delayed scratch closure model for academic and industrial researchers studying chronic wounds looking for responsiveness to drugs or biological treatments prior to testing on explanted patient material or in vivo.

Biomedical applications of glycosylphosphatidylinositol-anchored proteins.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) use a unique posttranslational modification to link proteins to lipid bilayer membranes. The anchoring structure consists of both a lipid and carbohydrate portion and is highly conserved in eukaryotic organisms regarding its basic characteristics, yet highly variable in its molecular details. The strong membrane targeting property has made the anchors an interesting tool for biotechnological modification of lipid membrane-covered entities from cells through extracellular vesicles to enveloped virus particles. In this review, we will take a closer look at the mechanisms and fields of application for GPI-APs in lipid bilayer membrane engineering and discuss their advantages and disadvantages for biomedicine.

Extracellular Vesicles and Their Membranes: Exosomes vs. Virus-Related Particles.

Cells produce nanosized lipid membrane-enclosed vesicles which play important roles in intercellular communication. Interestingly, a certain type of extracellular vesicle, termed exosomes, share physical, chemical, and biological properties with enveloped virus particles. To date, most similarities have been discovered with lentiviral particles, however, other virus species also frequently interact with exosomes. In this review, we will take a closer look at the similarities and differences between exosomes and enveloped viral particles, with a focus on events taking place at the vesicle or virus membrane. Since these structures present an area with an opportunity for interaction with target cells, this is relevant for basic biology as well as any potential research or medical applications.

A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells.

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

Association of glycosylphosphatidylinositol-anchored protein with retroviral particles.

We describe for the first time the association of glycosylphosphatidylinositol (GPI) -anchored proteins with retroviral and lentiviral particles, similar to a process well established for cells, termed "painting." The aim of the study was to assess the feasibility of modification of retroviral vectors by exogenous addition of recombinant protein, removing the need for genetic engineering of virus producer cell lines. The recombinant GPI protein CD59his was purified via fast protein liquid chromatography and associated with concentrated virus stock in a controlled incubation procedure. Reaction mixtures were purified in order to remove nonassociated GPI protein and endogenous protein. Analysis of samples by immunoblotting revealed that CD59his was only detectable in the presence of viral particles. From this, we conclude that CD59his could be stably associated with retroviral particles. In addition, we demonstrated by flow cytometry that virus particles remain infectious after these procedures. As well as suggesting a novel possibility for interaction between enveloped virus and host, we believe that the stable association of recombinant GPI proteins to retroviral particles can be developed into an important tool for both research and clinical applications, especially in the fields of gene therapy and vaccine development.

Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications.

HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression.

We present evidence that the HIV-1 Rev protein can heterologously regulate expression of the simple beta retrovirus mouse mammary tumour virus (MMTV). Up to 10-fold upregulation was seen in a functional assay system when specific MMTV sequences were substituted for the HIV-1 Rev responsive element (RRE). RNA gel shift analysis showed that purified recombinant Rev could specifically bind to MMTV unique region 3 prime (U3) RNA and that these sequences could compete for wild-type Rev-RRE binding approximately 20-fold more efficiently than a non-specific competitor RNA. Using a combination of in silico and deletion mutation analyses, it was not possible to define any single specific secondary structure responsive to Rev, suggesting that a structure or combination of structures that only form in the context of the complete U3 transcript is/are required to interact with Rev. Taken together, these results suggest that HIV-1 Rev can directly bind to MMTV RNA as well as mediate upregulation of MMTV gene expression.

Fluorescence molecular painting of enveloped viruses.

In this study, we describe a versatile, flexible, and quick method to label different families of enveloped viruses with glycosylphosphatidylinositol-modified green fluorescent protein, termed fluorescence molecular painting (FMP). As an example for a potential application, we investigated virus attachment by means of flow cytometry to determine if viral binding behavior may be analyzed after FMP of enveloped viruses. Virus attachment was inhibited by using either dextran sulfate or by blocking attachment sites with virus pre-treatment. Results from the FMP-flow cytometry approach were verified by immunoblotting and enzyme-linked immunosorbent assay. Since the modification strategy is applicable to a broad range of proteins and viruses, variations of this method may be useful in a range of research and applied applications from bio-distribution studies to vaccine development and targeted infection for gene delivery.