• Bioengineering Artificial Chromosomes For The Production Of Human Mirna

      Altherr, Delon
      Glioblastoma is a devastating disease with dismal patient outcomes. With current treatment options such as chemotherapy, radiation, and resection providing little hope to patients diagnosed with glioblastoma, research has turned to mesenchymal stem cells (MSCs) as a potential cellular delivery vehicle. The ability of MSCs to cross the blood brain barrier has increased the range of prospective treatment options available to patients with diseases like glioblastoma. Gene silencing through miRNA targeted therapy is one of the treatment options available through the utilization of MSCs. MSCs naturally package and secrete exosomes with miRNAs which have been shown to inhibit glioblastoma growth through gene silencing. Up until this point, miRNA targeted therapy has been limited to current gene therapy vectors and miRNA mimics. This project proposes an alternative to these approaches through the utilization of artificial chromosomes. The central hypothesis of this work is that artificial chromosomes can be bioengineered to produce multiple miRNAs for potential therapeutic applications. In order to test this hypothesis, a murine-derived artificial chromosome (platform ACE) was bioengineered to upregulate the expression of human miRNAs let-7b and miR124-1. The overall design of the project involved two major components. First was to identify a stable expression system for production of miRNA from the platform ACE. The second component consisted of subsequent analysis of miRNA production from the platform ACE. Findings from this project suggest that inducible vectors are more stable in the production of miRNA from artificial chromosomes compared to constituently activated vectors. Additionally, this project reports the successful transcription of the engineered miRNA constructs from the platform ACE in the Chinese Hamster Ovary (CHO) engineering cell line. Although this study was successful in producing primary transcripts from the platform ACE, this project further reports the disruption of miRNA processing beyond the initial transcript to a mature miRNA product. This study is in support of previous reports that CHO cells are ineffective in the production of mature miRNA from non-endogenous sources. Furthermore, suggesting that production of mature miRNA products must occur in species specific cell lines or with species specific flanking segments. Although more research is needed in the eventual utilization of artificial chromosomes in the production of miRNA, this project proposes an alternative to the use of current gene therapy vectors and miRNA mimics. In contrast to current approaches in miRNA targeted therapy, artificial chromosomes are stably maintained and not limited in their carrying capacity. Thereby opening the door for the potential delivery of multiple biological anti-cancer therapeutics in one cell mediated therapy vehicle. The use of artificial chromosomes has a long history for the delivery of large genetic payloads as well as multiple anticancer therapeutics. Thus, the potential of this project will allow for autonomous cell mediated therapy and targeting of multiple aberrant cell processes common to the etiology of glioblastoma.