Monash University

Restricted Access

Reason: Access restricted by the author. A copy can be requested for private research and study by contacting your institution's library service. This copy cannot be republished

Investigation of the potential of adenoviral proteins for construction of a novel and safer gene delivery system

posted on 2017-02-27, 00:15 authored by Sharma, Gaurav
The experimental technique that delivers genes for the treatment of pathological conditions is known as gene therapy or gene delivery. Gene-based therapeutics involves modulating the target gene expression either using viral or non-viral vectors. Viral gene delivery, although often quite effective has potential safety concerns. In contrast, non-viral approaches such as polyplexes and lipoplexes are less effective but are generally assumed to be safer. Therefore, a system that has the valuable attributes of both the viral and non-viral approaches would be ideal, and is the subject of the current research. The adenovirus is one of the most studied virus that express transiently in hosts cells. It can travel through the cell cytoplasm by interaction with microtubular dynein motors and can deliver its DNA to the nucleus via nuclear pore core complex. Though it has been the most popular vector in clinical studies, the adenovirus is now considered a poor clinical candidate for gene delivery due to immunogenicity and liver toxicity. To design and produce a novel and effective non-viral gene delivery system that mimics the adenovirus gene delivery system but retains the safety profile of non-viral approaches, is an objective that would constitute a major advance in the field. One step towards the design of a biologically active gene delivery system is to explore the use of the adenoviral proteins that are responsible for DNA condensation and nuclear delivery. This study aimed to characterize and assess the DNA condensation and gene delivery potential of adenoviral core protein V and VII. Furthermore, the project aimed to consider the inclusion of a fragment that could potentially facilitate intracellular trafficking of the gene delivery system. The capsid protein hexon is known to shuttle the adenovirus from cytoplasm to the nucleus using cellular motor, dynein. It can be assumed that hexon could not be used in gene delivery due to it being immunogenic. The epitopes that elicit immunogenic responses against adenovirus are located in the hypervariable regions of hexon protein. A fragment of hexon, referred to in this thesis as fragment 6.1 does not lie in the immunogenic region of hexon and has been previously hypothesized to mimic hexon’s function of microtubular trafficking. In the current study, the localisation of a 6.1-βGal-GFP fusion protein displayed similar behaviour to hexon, and also showed some unusual cellular distribution patterns (for example, GFP distribution at the nuclear membrane) that may indicate an important role for fragment 6.1 in designing an effective gene delivery system. Using expression studies of GFP fusion proteins, the cellular distribution patterns of individual adenoviral proteins II (hexon), V and VII were investigated using confocal fluorescence microscopy and widefield fluorescence microscopy combined with differential interference contrast (DIC) microscopy. To quantify the localisation of GFP fusion proteins in the cytoplasm, nucleus and nucleolus in multiple cells, fluorescence and DIC images were analysed in approximately 50 cells for each fusion protein. Protein II-GFP was distributed evenly through the cell and did not accumulate in the nucleus. Proteins V and VII were taken into the nucleus and protein V was specifically delivered to nucleoli. Interestingly, protein II restricted the distribution of the core proteins significantly in the cytoplasm when transfected with II-V-GFP and II-VII-GFP fusion constructs. In addition, fusion proteins were constructed with fragment 6.1, on the basis that a non-viral gene delivery system could be assembled using fragment 6.1 as the microtubular trafficking component and V or VII as the DNA condensing and potential gene delivery component. Fragment 6.1-βGal-GFP was observed to translocate passively into the nucleus of the majority of the cells, however, a nuclear membrane distribution in some cells was also noticed. 6.1-V-GFP sometimes showed both the nuclear membrane and nucleolar localization, suggesting that 6.1, being part of hexon, may localize intermittently at the nuclear membrane, finally arriving at the nucleolus of the transfected cells. Having studied their localization patterns, the next aim was to obtain recombinant proteins by production in E.coli and purification of individual core proteins using chromatographic techniques. The expression and purification of the adenoviral core proteins from bacteria has been reported to be challenging in several published reports. In this study, for the first time, methods were developed for purification of full-length proteins VII and V from E.coli with up to 98% and 90% purity respectively. These proteins were functional with respect to condensation of DNA and had comparable efficiency to poly-L-lysine and protamine. In addition protein VII was studied as a potential transfection agent using a luciferase reporter gene expression system. Lipid/VII/DNA displayed 2.8 times and 1.1 times greater transfection efficiency than lipid/PEI/DNA and lipid/protamine/DNA respectively, conferring its potential in designing a novel gene delivery system. However, due to instability of proteins V and VII in solution and lack of time, studying the in vitro gene expression efficiency of the fusion proteins was beyond the scope of this study. As a result of this study it is now possible to express and purify core proteins, thus expression and purification of 6.1-V and 6.1-VII constructs in E.coli are the most important objectives for work in the near future.


Campus location


Principal supervisor

Colin W. Pouton

Additional supervisor 1

Paul White

Year of Award


Department, School or Centre

Drug Discovery Biology

Additional Institution or Organisation

Monash Institute of Pharmaceutical Sciences


Doctor of Philosophy

Degree Type



Faculty of Pharmacy and Pharmaceutical Sciences