| Adenovirus is currently the most widely used vector in gene therapy trials as it can infect both dividing and quiescent cells for a wide range of tissues. The majority of adenoviral vectors currently employed are only minimally deleted in their viral genomes. The low level of viral gene expression leads to immunogenicity from, along with silencing of most currently used viral promoters, and the lack of any maintenance mechanism, results in transient expression of the transgene.;The goal of this dissertation was to generate hybrid vectors based on a fully deleted Helper Dependent adenovirus (HDAd) delivering an Epstein-Barr virus (EBV) episome, capable of long term maintenance. The work builds upon previous vectors designed in the lab using minimally deleted (E1 deleted) adenovirus to deliver a small EBV episome using a dual (binary) vector set with co-infection. We present two separate vector systems, the first iteration uses the HDAd system in a similar binary strategy, requiring co-infection of target cells and driving expression from the strong cytomegalovirus immediate-early promoter/enhancer. The second system consolidates the two vectors into a single hybrid vector system, and uses a liver specific promoter derived from the alpha 1-antirypsin promoter.;The fully deleted Helper Dependent adenoviral vector, lacks all coding regions of the virus, retaining only the extreme ends with the viral origin of replication, inverted terminal repeats, and the Psi packaging element. Within this vector, we place the elements to produce an EBV episome. These episomes are able to replicate once per S phase, and with the action of the Epstein-Barr virus Nuclear Antigen 1 (EBNA-1), successfully segregate during mitosis, to ensure the episomes are retained in daughter cell nuclei.;We demonstrate successful EBV episome delivery and long term persistence with both the Binary Hybrid Vector system and Single Hybrid Vector system in immune compromised mice. We detect a marked difference in expression profiles between the two vector systems, with steadier and longer term expression in the Single Hybrid Vector system. This is likely due to the use of the liver specific promoter.;We have also demonstrated preliminary data comparing the two vector systems in immune competent mice. A stark difference in expression profiles exist between the two vector systems, with complete loss of detection with the Binary Hybrid Vector and very stable expression with the Single Hybrid Vector system. Once more this may be ascribed to the liver specific promoter.;Further studies will be required to better characterize these differences between the Hybrid Vector systems, and continue development of the system to possible disease models. |
