(324g) Reinstatement of Retroviral Infectivity Via Non-Covalent Attachment of DOTAP, DOPE & Cholesterol 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 biocompatible lipids 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) and 190-310 kDa chitosan 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 explore the design of associating RVLPs with a synthetic mixture of a) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), b) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and c) cholesterol as a lipid 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 the composition of these 3 lipids is the greatest determinant of efficient transfection onto HEK293 cells which has also been observed in non-viral vectors with the most optimal composition being DOTAP:DOPE:Cholesterol=3:2:5. Compositions which had DOTAP < 50% were more efficient than those which had > 50%. The transfection efficiency of these hybrid vectors is also on the same order of magnitude as their biological cousins, the enveloped viruses. Additionally, we have observed that the cellular uptake of these lipid-hybrid complexes was not correlated with efficient transfection. Pure cationic lipid complexes showed higher uptake but very low transfection efficiency whereas mixed composition lipids showed reduced uptake but superior transfection efficiency. The size of these vectors is much higher than regular RVLPs which restrict their future clinical application under the current design strategy. We also visualized these hybrid vectors under TEM which proves that these lipids envelope multiple RVLP particles to form an aggregated mixture of complexes. We see this combination of a synthetic lipid mixture with an inactive virus to form a highly efficient hybrid vector as an ideal replacement for the biological retrovirus in gene therapy applications.