(257d) Improving Hybrid VLP/Polymer Transduction Efficiency through VLP Purification

Viral gene therapy vectors have been used successfully in clinical trials to treat challenging diseases such as severe combined immunodeficiency disease, cancer, and blinding retinal disease. Serious setbacks have occurred, however, due to drawbacks associated with viral vectors such as oncogenicity and immunogenicity. While some of these drawbacks can be overcome by using nonviral vectors, poor efficiency has hindered the use of synthetic vectors. These current limitations highlight the need for continued development of safe, yet efficient gene delivery vectors.

We have previously proposed and demonstrated that some advantages can be obtained through the use of hybrid viral/nonviral vectors composed of noninfectious virus-like particles (VLP) and cationic polymer. Our hybrid vector system combines the advantages associated with viral and nonviral vectors while avoiding some of the drawbacks. The major limitation of our hybrid vector system was poor efficiency, relative to native retrovirus, which we hypothesized was due to interference of VLP/polymer complex formation by serum and cellular proteins present in the VLP supernatant. With the ultimate objective of improving hybrid vector efficiency, we developed a method for purifying retrovirus VLPs and studied VLP/polymer complex formation and gene delivery efficiency using purified VLPs. The retrovirus VLPs were purified using ultra-filtration and size exclusion chromatography, and the extent of purification was determined by SDS-PAGE. Purified VLPs were used subsequently to form hybrid vector complexes. Particle size and zeta-potential were measured using dynamic light scattering, and particle morphology was determined from transmission electron microscopy. The ultimate effectiveness of the vector was determined by gene delivery efficiency of a luciferase reporter gene to HEK-293 target cells. We will present our results demonstrating that purified VLPs allow for more precise control over VLP/polymer particle formation and ultimately improve gene delivery efficiency.