Development of an autoregulatory gene expression system for in vivo gene therapy application in a Huntington's disease model

Abstract

Regulation of expression of genes with therapeutic potential remains one of the key issues limiting widespread clinical application of gene therapy. Ideally, therapeutic gene expression should be coupled to the physiological need of the cell, such that expression is quiescent under basal conditions but active when required. To address the unmet need for a regulatory gene expression system for use in gene therapy, a novel autoregulatory gene expression cassette was developed. Within this cassette, transgene expression is driven by a regulatory factor (RF) via its response element (RE) in response to cell stress-induced activation of cell proteases, caspase-3 or calpain, and thus is physiologically regulated. Cistron 1 of the cassette encodes a constitutively expressed RF which is fused to a dominant nuclear export signal (NES) via a linker containing the protease cleavage site. Under basal conditions, the NES restricts the RF to the cytosol, and thus the RF is unable to bind to its response element (RE) in the Cistron 2, controlling expression of the therapeutic gene. However, following cell stress-induced activation of the proteases, the NES-RF fusion protein is cleaved allowing the RF to translocate to the nucleus and bind to the RE, thus driving transcription of the downstream therapeutic gene. The transcription of the therapeutic gene remains active only for as long as the cell remains stressed and the proteases active. Hence, this is a fully self-regulating and homeostatic system in which transgene expression is tightly coupled to cell stress This thesis has focused on the development and following in vitro analysis of different candidate RF/RE systems in the context of the autoregulatory cassette with the aim to create a regulated gene expression system for the in vivo gene therapy in HD model. As a result, two regulatory systems, the ARF5/AuxRE and Cre/lox, were developed and determined to be a promising tool for application in gene therapy of neurodegenerative diseases. In addition, protease sensor vectors employing the regulatory principle similar to that of the expression cassettes were developed as a novel tool for detecting protease activity at the level of individual cells.