John A. Dangerfield

Rafts, anchors and viruses — A role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors

Lipid rafts have been proposed as sites for the assembly of a number of viruses and are considered to play a major role in pseudotyping events. As a consequence, host glycosylphosphatidylinositol (GPI) anchored proteins commonly associated with lipid rafts can be found being incorporated into viral lipid envelopes with beneficial consequences for viral replication. In this review we will look at the link between lipid rafts, GPI-anchored proteins and retroviral particles and how these relationships can be exploited for the modification of enveloped viruses.

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.—Metzner, C., Mostegl, M. M., Günzburg, W. H., Salmons, B., Dangerfield, J. A. Association of glycosylphosphatidylinositol‐anchored protein with retroviral particles. FASEB J. 22, 2734–2739 (2008)

Magnetic field-controlled gene expression in encapsulated cells

Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches.

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.

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.

Postexit Surface Engineering of Retroviral/Lentiviral Vectors

Gene delivery vectors based on retroviral or lentiviral particles are considered powerful tools for biomedicine and biotechnology applications. Such vectors require modification at the genomic level in the form of rearrangements to allow introduction of desired genes and regulatory elements (genotypic modification) as well as engineering of the physical virus particle (phenotypic modification) in order to mediate efficient and safe delivery of the genetic information to the target cell nucleus. Phenotypic modifications are typically introduced at the genomic level through genetic manipulation of the virus producing cells. However, this paper focuses on methods which allow modification of viral particle surfaces after they have exited the cell, that is, directly on the viral particles in suspension. These methods fall into three categories: (i) direct covalent chemical modification, (ii) membrane-topic reagents, and (iii) adaptor systems. Current applications of such techniques will be introduced and their advantages and disadvantages will be discussed.

Surface Modification of Retroviral Vectors for Gene Therapy

If biomedicine is considered to be the use of biological or botanical agents in medicine then it is certainly the oldest kind of therapy. Since drugs, however, it is no longer the most prolifically used, at least in the developed world. Will this change, and if so will it be in the near or more distant future? It has been discussed amongst experts in the industry for some time that, despite ever-increasing funding and new high-tech methods of discovery and screening, amounts of new drugs and drug leads are declining. In part, this has opened the way for biologics. The frontrunner is of course antibody technologies, mainly monoclonals, the use of which has exploded over the last 5 years to the point where most of the major pharma players are involved or are getting involved in what has now become a multi-billion dollar industry. Antibodies have reached the point of being a well trusted and accepted form of medical product across the industry and communities from bench to bedside. Many believe the next similar success story will be for cell therapy, probably some differentiated form of stem cells. Although it has been happening for 20 years, it is only in the past 5 years that the amount of patients being treated with cells, as a therapy, has increased into the thousands per year. The vast majority of these are autologous cell treatments, undertaken by hospitals and private clinics on a patient to patient basis using the patients’ own cells to treat a wide variety of diseases and conditions. These are not officially approved medical products. However, there are several non-pharmaceutical giant companies now in clinical trials, notable amongst these are Geron in the USA, Mesoblast in Australia and ReNeuron in the UK, although there are some interesting endeavors in Southeast Asia too, e.g. Medipost in Korea. All of these are undergoing various stages and sizes of clinical trials for diverse indications, some of which are showing already very encouraging results. It is predicted that within the next few years one or more of these potential products will reach the market as an approved medical product. Having said this, other than showing ongoing safety and efficacy in the trials, some hurdles do remain in the industry as whole, such as up-scaling for mass production of the cells which is considered necessary for generating an off-the-shelf product. If this trend continues, it is only reasonable to assume that gene therapy will follow, most probably some years after cell therapy has been more widely accepted, but possibly in parallel to some extent, as there are also a number of human gene therapy trials already in progress. So, how significant will retroviral (RV) and lentiviral (LV) vectors be in this future gene therapy industry? One approach to answer this is to look at how popular they have

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.

Comment on Patel et al; “Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses” Nanomedicine, 2015. 11(5): p. 1097-107

We have read with great interest the article by Patel et al, entitled “Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses”. Having investigated the technique featured in this article, termed glycosylphosphatidylinositol (GPI)anchored protein transfer, or molecular painting (MP) for close to ten years, we would like to offer complementary information on the topic, mostly based on own work, which we believe would benefit authors and readers alike. Protein transfer is based on the property of purified GPI-anchored proteins to re-insert spontaneously into lipid bilayer membranes. GPI-anchoring is a type of post-translational modification and anchors consist of 2-3 lipid moieties linked to the protein via a carbohydrate core consisting mainly of mannose. The technology was pioneered by M.E. Medof in 1984 on erythrocytes and has since then been investigated for various biomedical applications in gene therapy, vaccine development and cancer therapy. We have adapted the method for different viral families and derived gene therapy vectors—retroviridae (i.e. HIV-1), herpesviridae (i.e. FHV-1) and orthomyxoviridae (i.e. Influenza)—and a range of GPI-anchored protein variants including monomeric green fluorescent protein, CD4, epidermal growth factor (EGF) and interleukin 2 (IL-2) in 2008. Additionally, we could demonstrate that the insertion is depending strictly on the presence of the lipid part of the anchor, that insertion increases with amount of available membranes and virus, that MP is not affecting infectivity and that more than one protein may be used for modification simultaneously. Additionally, calculations yielded an estimate of 5-150 molecules GPI-anchored proteins per virus particle. Main advantages of the technique are flexibility and speed of the approach (see Figure 1).Also, no geneticmanipulation of virus-producing cells is necessary. Disadvantages include the necessity of manipulations once the virus, viral vector or virus-like particle has left the cell (“post-exit”) which will reduce infectivity of viruses. Additionally, establishing and optimizing a protein purification strategy for a GPI-anchored protein is not a trivial task. Low abundance and strong hydrophobicity of the proteins may reduce yield and/or purity. Despite these hindrances, we strongly believe in the technology for the modification of nano-sized lipid

Singapore R&D and globetrotting

Worldwide, people are setting up or expanding into new careers, universities, institutes, and centers. This situation is particularly good for Singapore. From a biomedical perspective, three main movements may be observed: (1) pharmaceutical, biotechnological and supply companies establishing local-based subsidiaries, (2) renowned universities and colleges establishing biomedical and chemical biology institutes, and (3) researchers of international standing taking the lead to bring-in, train and sustain a first-class local workforce. By discussing the integrative nature of chemistry and biology, we shall attempt to address both local and international perspectives.We shall give an overview of the funding structure and collaborative opportunities in Singapore. As a theme, we have focused on drug discovery. For relevant complimentary information, please also refer to the Singapore special in BTJ’s November 2007 issue.