Browsing Theses and Dissertations by Author "Pokiniewski, Katie Ann"
Understanding the cellular mechanism of Adeno-Associated Virus genome stabilizationXiao, Weidong; Tsygankov, Alexander Y.; Gallucci, Stefania; Skorski, Tomasz; Yang, Xiao-Feng (Temple University. Libraries, 2016)The Adeno-Associated Virus(AAV) is a small, single stranded DNA virus that has been developed as a gene transfer vector. Early clinical trials using recombinant AAV vectors (rAAV) have identified the following concerns that need to be addressed in order to increase efficiency of these vectors. It has since been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. The purpose of this study is to elucidate mechanisms that help stabilize the AAV genome in order to make it a more efficient vector for gene therapy. Previously, there have been studies using fluorescent labeling to track the movement of AAV into the nucleus. An integral part of AAV genomic stability may be the obstacles it encounters in the cytoplasm prior to entering the nucleus. Previous studies on improving AAV transduction have focused primarily on the nucleus. The study will hopefully shed light on the hurdles AAV encounters as it moves through the cytoplasm. Thus, this project has designed and utilized a new system for specifically tracking the status of AAV genomes in the cytoplasm as well as in the nucleus. This project utilizes a novel dual luciferase reporter system to track the movement of AAV particles from the cytoplasm into the nucleus to elucidate mechanisms that could contribute to the stabilization of the AAV genome. The novel dual reporter system is comprised of a single-stranded vector containing two different types of secreted luciferases: Cypridina Luciferase and Gaussia Luciferase. Cypridina Luciferase is placed under the control of a nuclear promoter and therefore it is expressed only in the nucleus. The second, Gaussia Luciferase, is under the control of a cytoplasmic promoter that will only be expressed in the cytoplasm upon the presence of T7 RNA polymerase. Using this dual reporter luciferase system along with RT qPCR quantification in a Hek293 cell line expressing the T7 RNA polymerase, demonstrated that genomes are present in the cytoplasm at 18 and 24 hours post infection. The second part of this study is using the dual reporter system to understand the trafficking patterns of rAAV and looking at ways to enhance transduction. One method could be administering rAAV vectors in conjunction with a drug; this approach may help overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Cidofovir (CDV) is a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase. In vitro, CDV actively inhibits a number of DNA viruses including herpes viruses, adenovirus, polyomavirus, papillomavirus, and poxviruses. Cidofovir has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, has not been documented. Results have demonstrated that CDV is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 5 fold, depending on the cell type and concentration, in vitro, using both rAAV2 and rAAV8 luciferase reporter vectors. These results have been able to replicated using other reporter vectors: rAAV2-LacZ reporter, rAAV2-GFP, rAAV2-hAAT, and a rAAV8-GFP in vitro. Results have shown a dose-dependent increase in rAAV genomes with CDV pretreatment in HelaS3 cells via Southern Blot. Also southern blot analysis of cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hours post infection, suggesting CDV does not enhance viral entry but that CDV may enhance at steps downstream of viral entry. Using RNA sequencing and Ingenuity software analysis of HelaS3 cells pretreated with CDV, an increase in several genes of interest including those involved in the mechanism of viral exit were observed. These genes include Actin and vacuolar proteins, these molecules are involved in and associated with endosomal sorting complexes as well as required for transport inside the cell. This finding along with southern blot data supports the theory that CDV may enhance rAAV trafficking since we observed that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection. Also utilizing the dual luciferase reporter system an increase in transgene expression present with CDV pretreatment compared to PBS in both the cytoplasm and nucleus was observed suggesting that CDV may enhance with rAAV trafficking. These results taken together demonstrate that this dual reporter system is a powerful tool for understanding and improving rAAV trafficking. Also drugs like CDV can greatly contribute to the understanding of rAAV trafficking, and eventually lead to the development of novel strategies to increase overall efficiency of AAV transduction.