Modular Transduction Targeting of Retroviral Vectors

Gene therapy vectors based on retroviruses (retroviral vectors, RV) are considered to hold great promise in the treatment of monogenic disorders like X-linked severe immunodeficiency (X-SCID) or chronic granulomatous disease (CGD) as well as cancer. A pre-requisite to the use or RVs in patients, especially after systemic administration, is targeting of transduction or infection to a specific subsets of cells in an organism. In retroviruses infection tropism is ensured by the interaction of the envelope glycoprotein (Env), a complex consisting of surface (SU) proteins, responsible for cell binding and transmembrane (TM) proteins, responsible for initiating cell entry by membrane fusion. Targeting of retroviral infection to specific cell types i.e. transduction targeting therefore mostly requires modification of the viral Env proteins. Several different approaches have been used for modification of Env proteins and usually the modified Env proteins are less efficient in initiating infection. To circumvent this effect, a relatively recent approach separates binding and fusion properties, by using binding- independent fusogenes with ablated binding activities, based on e.g. Sindbis or Influenza virus glycoproteins (SINmu and HAmu, respectively). In addition to the mentioned SINmu and HAmu systems, we also want to create a binding-independent fusogene based on the retroviral TM Env subunit, as this would provide a system more closely resembling retroviral infection. Binding activities will be provided preferentially by a process termed viral painting which allows for the post-budding modification of RVs with glycosylphosphatidylinositol (GPI)-anchored proteins, a type of post-translationally modified proteins carrying lipid residues. Combination of binding independent fusogenes and painting of RVs with binding properties should lead to a truly modular system for transduction targeting i.e, one and the same RV can be outfitted with different molecules and thus targeted to different subsets of cells. We will use a range of different binding molecules, which will be artificially anchored to GPI residues: epidermal growth factor (EGF) and vascular epithelial growth factor (VEGF) - for targeting to cells overexpressing the cognate receptors i.e. tumour cells; CD4 - for targeting HIV-1 infected cells and streptavidin, for providing a versatile link to biotinylated structures e.g., antibodies. Proof-of-principle will be tested by demonstrating strongly increased transduction rates in cells expressing the receptors compared to non- expressing cells. We believe that this project will contribute both to the basic understanding of the initial steps of retroviral infection as well as provide a novel approach to RV transduction targeting.

The project Modular Transduction Targeting of Retroviral Vectors was carried out during a period of 40 month. The main aim was to develop a strategy for the infection of a specific subset of cells by changing the binding properties of retroviral vectors. Mechanisms ensuring uptake of virus into cells would be provided by a second, independent system. The project focused on vectors based on HIV, due to their high infection efficiency and the ability to infect resting cells important factors for successful gene therapy. We have produced a range of binding factors that were equipped with a biological lipid anchoring structure the glycosylphosphatidylinositol or GPI anchor. Association of these factors to viral surfaces via the GPI anchors in a process termed Molecular Painting (MP) was investigated. Transfer was successful and protein remained active in most cases. Consistent with previous attempts at targeted infection, the initiation of virus uptake proved the most difficult part: we concentrated on using a modified fusion protein from Sindbisvirus unable to bind its natural receptors, which was shown to be effective in similar circumstances. Finally the changed attachment/infection spectrum was analyzed by using cell lines expressing receptors for the binding factors. First experiments demonstrated that changes in attachment can be determined by using fluorescent markers and are currently being repeated with the appropriate binding systems.