Dissertations, Theses, and Capstone Projects

Date of Degree

9-2018

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Benjamin Ortiz

Committee Members

Laurel Eckhardt

Patricia Rockwell

Barbara Studamire

Derek Sant'Angelo

Subject Categories

Biology

Keywords

Translational science, gene regulation, biotechnology

Abstract

Despite the promise of emerging stem cell transplant gene therapy strategies, there remains a gap in the ability to predictably generate robust, temporally controlled long-lasting, therapeutic gene expression in specific target cell lineages following stem cell differentiation. There exists a locus control region (LCR) in the mouse T-cell receptor (TCR)-α locus that confers an ab TCR-like spatiotemporal expression pattern upon a linked transgene, regardless of its site of integration in the genome. These properties are well suited to direction of high-level, physiological expression of therapeutic antigen receptor genes to the T cell progeny of vector-transduced stem cells. The endogenous LCR spans over 12.5-kb of DNA. To accommodate LCR activity within the space constraints of gene delivery vectors, we aim to reduce the size of the LCR, while retaining its characteristics. Mini-LCRs have been constructed with varying combinations of previously characterized sub-elements of the TCRα LCR, ranging in size from 1.3- to 4.0-kb. These mini LCRs are tested in an in vitro mouse embryonic stem (ES) cell differentiation system for their ability to recapitulate hallmark LCR properties. In T cells derived from ES cells, reporter gene expression driven by TαLCR4.0 approximates that observed in the presence of the full length LCR, in that TCRα LCR-linked reporter gene expression levels are robust, mostly cell type-restricted and are upregulated during the expected stage transition of T cell development. Reporter gene expression driven by a smaller mini LCR, TαLCR1.3, while robust in all clones, displays early activation kinetics suitable for directing therapeutic gene expression to correct inherited immunodeficiency. TαLCR2.9 and TαLCR3.0 drive tighter spatiotemporal expression by restoring deleted elements from TαLCR1.3, and two other mini LCR cassettes have been designed to further narrow down the sub-elements conferring function. This structure/function information contributes to characterization of the elements of TCRα LCR, and can enable a better understanding of the basic scientific mechanisms underlying the gene regulation in the TCRα locus. Furthermore, incorporating TCRα-derived mini LCRs into gene delivery vectors may provide the controlled and predictable therapeutic gene expression required to overcome some of the current hurdles to the use of genetically engineered stem cells as vehicles for delivering T cell gene therapy against cancer and a variety of other diseases.

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