Brent Larsen

Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering

This study illustrates the application of asymmetrical flow field-flow fractionation (AF4) and light scattering analysis during the development of a gene delivery vehicle based on virus-like particles (VLPs) derived from the human polyoma JC virus. The analytical system was created by connecting an AF4 apparatus to the following detectors: diode array, fluorescence, multiangle light scattering, dynamic light scattering, and refractometer. From a single analysis, the molar mass, root mean square and hydrodynamic radii, composition, and purity of the sample could be determined. The VLPs were purified from baculovirus-infected Sf158 insect cells overexpressing the recombinant VP1 protein using weak anion exchange chromatography. The VLPs were dissociated to VP1 pentamers, and the contaminating DNA and proteins were removed using strong anion exchange chromatography. The gene delivery vehicle was created by reassembling the VP1 pentamers in the presence of the desired DNA. The newly formed VLPs encapsulated the DNA and were shown to be capable of delivering the gene of interest to target cells where it was translated into protein. This paper describes the scalable process that was derived to produce the VLPs and demonstrates how the AF4-based analytical characterization was indispensable during the development process.

ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution

The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials.

170. In Vitro Transduction of Cells To Determine Tropism Using Viral-Like Particles Derived from JC Virus VP1

Top of pageAbstract Gene therapy for treatment of neurological diseases is a rapidly emerging new field. Virus-Like Particles (VLP) originating from the human polyoma virus JCV are being evaluated as a delivery vector for the central nervous system (CNS) because JCV preferentially infects both oligodendrocytes and astrocytes. In developing a gene delivery system with JCV VLP, we have optimized both the production of recombinant VP1 from JCV in insect cells and the packaging conditions for purified VP1 and plasmid DNA. Using VP1-VLP containing plasmid DNA expressing EGFP, we have performed transduction assays in a panel of human and rodent brain-derived and non-brain derived cells in order to characterize the in vitro tropism and species specificity of VP1-VLP. From the survey of human brain-derived cell lines, TC620 (oligodendroglioma) had the highest transduction efficiency with VP1-VLP. Up to 80% of the cells were expressing EGFP after transduction in vitro. In these studies, we added 0.3 ng/cell of VP1 protein containing 0.8 |[mu]|g of plasmid DNA to 5x104 cells. SK-N-SH, a human neuroblastoma cell line, and a population of primary normal human astrocytes also showed high transduction efficiencies following treatment with VP1-VLP. The rodent cell line NG108-15, a hybrid between a rat glioma and mouse neuroblastoma, and rat brain primary cells expressed EGFP after treatment with human VP1-VLP. These results demonstrate that VP1-VLP can transduce both human and rodent brain cells, at least in vitro. With the exception of one human prostate cell line (PC-3), significant VP1-VLP transduction was not observed in any non-brain-derived human or rodent cells studied including cell lines from kidney, liver, lung, skeletal muscle, and vascular endothelium. Thus, the VP1-VLP system from JCV may provide an efficient and selective delivery system for therapeutic genes to target specific cells in the brain.