Viruses are considered highly efficient gene delivery vectors. Adenovirus, in particular, is a relatively safe virus with neither severe pathogenicity nor oncogenecity. Adenovirus has been extensively used as a gene delivery vector. Usefulness of the viral vector, however, is limited by drawbacks such as immunogenicity, promiscuous tropism, and inefficient infectivity of certain types of cells. Many efforts have been made to develop non-viral gene delivery vectors that are safer, less immunogenic, and targeted, but these vectors typically suffer from poor efficiency.
Our goal was to develop a hybrid vector that provides advantages over both viral and non-viral vectors. For this purpose, we developed a vector formed of a fiberless adenovirus (Ad-FL), polyethylene glycol (PEG), poly-L-lysine (PLL), and an RNA aptamer that served as a targeting ligand for prostate cancer cells expressing prostate-specific membrane antigen (PSMA). The virus fiber, which is known to play a role in both virus tropism and initiating an inflammatory response and virus clearance, was replaced with the PEG-PLL grafted copolymer. This approach reduced the drawbacks associated with the fiber and provided advantages often associated with PEG, such as reduced immunogenicity and improved biodistribution. In addition, incorporating the PSMA targeting aptamer allowed for targeting prostate cancer cells expressing PSMA.
The PEG-PLL/Ad-FL hybrid vector was prepared by conjugating different molecular weights (1, 2, 5 and 10 kDa) of heterobifunctional PEG with a thiol-reactive maleimide (MAL) group and an amine-reactive N-hydroxyl succinimidyl ester (NHS) group (MAL-PEG-NHS), with different molecular weights of PLL (15-30 and 30-70 kDa) at 10% and 25% degrees of PEGylation. The MAL-PEG-PLL grafted copolymer was then complexed with Ad-FL before finally conjugating a thio-modified RNA targeting aptamer. The targeting efficiency and overall performance of the hybrid vector was evaluated by targeting the PSMA-positive LNCaP prostate cancer cell line and the PSMA-negative PC3 prostate cancer cell line that served as a negative control. The effects of PEG and PLL molecular weights and degree of PEGylation on hybrid vector performance were evaluated and compared to the native adenovirus. The nanoparticle size and surface charge were characterized using dynamic light scattering, and the transduction efficiency was determined using a beta-galactosidase reporter protein measured using a chemiluminescence-based assay.
The results showed that the PEG-PLL/Ad-FL hybrid gene delivery vector was able to overcome some of the significant drawbacks of adenoviral vectors and simultaneously retain high transduction efficiency and impart flexible targeting.