| The ultimate goal of gene therapy is to cure both inherited and acquired diseases by replacing, adding or correcting genes. Vectors designed for gene delivery can be divided into two general categories: viral and non-viral. Viral vectors use replication deficient viruses to transfer genetic material into cells. Non-viral vectors, such as polyethylenimine (PEI) and liposome-based vectors, are synthetic carriers that utilize physico-chemical methods for gene delivery. Currently, the main limitations of non-viral vectors for gene therapy are their relatively inefficient gene delivery, lack of tissue specificity, and toxicity. This dissertation outlines the development and evaluation of novel PEI and liposome-based non-viral transfection vectors designed to improve gene transfer efficiency and vector stability while reducing toxicity. Initially, using amine reactive reducible cross-linking reagents, relatively high molecular weight (HMW) PEI polymers were synthesized from low molecular weight (LMW) PEI monomers. The goal was to take advantage of the high transfection efficiency observed using transfection vectors prepared with HMW PEI, along with the decreased toxicity obtained using vectors prepared with LMW PEI. Using this strategy, efficient gene transfer was observed in vitro, and transfection related toxicity was reduced (Chapter 2). Next, in an effort to optimize transfection vectors comprised of polycation condensed DNA (polyplex) complexed with anionic liposomes, termed LPDII, a novel anionic liposome formulation containing diolein was developed. Diolein/cholesteryl hemisuccinate liposomes released increasing amounts of encapsulated calcein and displayed a time-dependent size increase in response to decreasing pH. Furthermore, diolein-based LPDII vectors mediated improved gene expression compared with PEI-based transfection, and overcame the serum sensitivity of previous LPDII formulations (Chapter 3). Finally, diolein-based LPDII vectors were further modified by incorporating reversible covalent cross-linking, to improve vector stability, and lipid-anchored ligands, such as folate, to promote receptor-mediated internalization of transfection complexes. Cross-linking improved the stability of polyplexes used for LPDII formation, but had a negative impact on gene transfer efficiency. Inclusion of folate in the anionic liposomes used to prepare LPDII vectors led to an increase in gene expression (Chapter 4). The applications of incorporating folate into liposome formulations utilized for gene and drug delivery are also described in detail (Chapter 1). |
