(324e) Recovery of Retroviral Infectivity Via Non-Covalent Attachment of Chitosan to Murine Leukemia Virus-Like Particles

Gene therapy offers the promise of revolutionizing healthcare for millions of people. However, it has yet to become a common treatment for the variety of diseases that could benefit from the delivery of therapeutic genes. Limited progress is primarily due to the lack of a safe and efficient means of delivering genetic material. Viral vectors, for instance, are extremely efficient but potentially pathogenic and immunogenic. They also typically possess a tropism towards specific cellular receptors which is difficult to modify without significant loss in efficiency. Non-viral vectors are typically non-pathogenic and non-immunogenic, yet lack the efficiency necessary for gene therapy and are also typically toxic at clinically useful concentrations. It is clear that the current implementation of gene therapy must be preceded by the investigation of vectors with improved characteristics. We believe that the development of a hybrid vector combining viral and non-viral entities would eliminate disadvantages of either vector design and provide a safer and efficient vector design. The research proposed here focuses on producing improved gene therapy vectors through association of a biodegradable polysaccharide, chitosan (χ) with retrovirus-like particles (RVLPs).

RVLPs are essentially intact viruses lacking the envelope protein most necessary for transfection, thus making them inactive. The envelope protein is the source of many disadvantages associated with using retroviruses as a clinical gene therapy vector. We have reported previously, that synthetic polycations (PC) such as 750 kDa Polyethylenimine (PEI) and 150-300 kDa Poly-L-Lysine (PLL) can restore infectivity to RVLPs without the need of the envelope protein. The PCs electrostatically combine with the RVLPs to reintroduce activity to the RVLPs leading to successful transfection. In essence, PC acts as a synthetic envelope protein-substitute. In this study, we provide proof-of-principle of granting infective capabilities to RVLPs via complexing with Chitosan as a polymer based-synthetic envelope and expand upon the new class of hybrid viral/non-viral gene delivery vector that we have shown in our past studies. We show that chitosan is a non-toxic alternative to PEI and PLL and provides higher transfection efficiency in HEK293 cells. The density of viral particles used to form hybrid vectors significantly affects the optimal stoichiometry required for efficient gene transfection, varying from 6-9 µg/billion RVLPs to 40 µg/billion RVLPs. The size of these vectors is between 400-1000 nm which is an important determinant in cellular uptake. We have also visualized the morphology of these hybrid vectors that prove that they are multi-particle entities rather than singular gene delivery vehicles. We see this combination of a biocompatible synthetic agent with an inactive virus to form a highly efficient hybrid vector as a significant extension to the development of novel gene delivery platforms.