1084. Converting Non-Infectious Virus-Like Particles into Hybrid Gene Therapy Vectors Using Polyethylenimine

Abstract

Current approaches for delivering genetic material to a target tissue predominately rely on viral or non-viral vectors. Each vector possesses a unique set of advantages and disadvantages. Viral vectors are highly efficient and offer the potential for stable gene expression while nonviral vectors are generally more robust, provide for the possibility of tailored cell targeting, and are less pathogenic and immunogenic. We have explored combining advantages traditionally associated with these two vector types, and possibly reducing disadvantages, through the development of a hybrid viral/nonviral vector based on murine leukemia virus (MLV) virus-like particles (VLP) and high molecular weight branched polyethylenimine (PEI). Non-infectious VLP are produced from cells expressing the MLV gag-pol genes. VLP lack virus envelope protein and thus any means of traversing the cell lipid bilayer. The chimeric hybrid vector that has been developed is an electrostatic complex formed between cationic PEI and negatively charged VLP. PEI functions in place of the virus envelope protein to aid in the endocytic uptake of the complexes and escape from the endolysosomal network. Once in the cell cytoplasm the virus directs intracellular transport to the cell nucleus and integration into the cell genome. Our results show gene expression levels using the hybrid vector range from 20% to 40% those observed using DNA/PEI. The effective diameter of hybrid vector complexes is slightly larger than DNA/PEI complexes (917 nm versus 786 nm), and like DNA/PEI complexes hybrid vector complexes have positive zeta-potentials. Although the transfection efficiency of the hybrid vector is currently less than DNA/PEI, we believe the potential advantages such as tailored cell targeting and stable gene expression warrant the continued study of such hybrid vectors. We will be reporting transfection results for PEI/VLP complexes and characterization of the vector including sizing using dynamic light scattering, surface charge characteristics based zeta- potential measurements, and vector morphology observed in TEM micrographs. Current approaches for delivering genetic material to a target tissue predominately rely on viral or non-viral vectors. Each vector possesses a unique set of advantages and disadvantages. Viral vectors are highly efficient and offer the potential for stable gene expression while nonviral vectors are generally more robust, provide for the possibility of tailored cell targeting, and are less pathogenic and immunogenic. We have explored combining advantages traditionally associated with these two vector types, and possibly reducing disadvantages, through the development of a hybrid viral/nonviral vector based on murine leukemia virus (MLV) virus-like particles (VLP) and high molecular weight branched polyethylenimine (PEI). Non-infectious VLP are produced from cells expressing the MLV gag-pol genes. VLP lack virus envelope protein and thus any means of traversing the cell lipid bilayer. The chimeric hybrid vector that has been developed is an electrostatic complex formed between cationic PEI and negatively charged VLP. PEI functions in place of the virus envelope protein to aid in the endocytic uptake of the complexes and escape from the endolysosomal network. Once in the cell cytoplasm the virus directs intracellular transport to the cell nucleus and integration into the cell genome. Our results show gene expression levels using the hybrid vector range from 20% to 40% those observed using DNA/PEI. The effective diameter of hybrid vector complexes is slightly larger than DNA/PEI complexes (917 nm versus 786 nm), and like DNA/PEI complexes hybrid vector complexes have positive zeta-potentials. Although the transfection efficiency of the hybrid vector is currently less than DNA/PEI, we believe the potential advantages such as tailored cell targeting and stable gene expression warrant the continued study of such hybrid vectors. We will be reporting transfection results for PEI/VLP complexes and characterization of the vector including sizing using dynamic light scattering, surface charge characteristics based zeta- potential measurements, and vector morphology observed in TEM micrographs.