Regulatable Promoters for Use in Gene Therapy Applications: Modification of the 5′-Flanking Region of the CFTR Gene with Multiple cAMP Response Elements to Support Basal, Low-Level Gene Expression That Can Be Upregulated by Exogenous Agents That Raise Intracellular Levels of cAMP

Abstract

This study focuses on the design, construction, and evaluation of a chimeric promoter for gene therapy applications where it is desirable to have low-level basal expression of the newly transferred gene, which can be induced to higher levels of expression by the administration of pharmacologic agents that can be safely used locally and/or systemically in humans. To achieve this, a chimeric promoter was constructed using fragments of the 5′-flanking region of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, and multiple tandem repeats of the consensus sequence and flanking elements of the cAMP response element (CRE), promoter sequences that support increased transcription in response to elevations in intracellular cAMP levels. Preliminary studies using plasmid vectors demonstrated that: (i) the 5′-flanking sequences from the CFTR gene have low promoter activity in the human airway epithelial cell lines; (ii) chimeras using –718 bp fragment from the 5′-flanking sequence of CFTR gene as the base, with the addition of 4–10 units of a 25-bp sequence containing the CRE consensus sequence, were all inducible by a rise in intracellular cAMP, with the chimera having eight CRE repeats the most responsive; and (iii) a CF126(CRE8) chimera, consisting of the –126 bp fragment from the 5′-flanking region of CFTR gene together with eight CRE repeats, yielded low-level basal activity but maximal upregulation by cAMP, resulting in expression of the reporter gene that was 51–58% of an RSV-LTR control. On the basis of these observations, replication-deficient adenoviral vectors containing the CF126(CRE8) chimera and the luciferase reporter gene [AdCF126(CRE8).Luc] or the Escherichia coli lacZ (β-Gal) reporter gene [AdCF126(CRE8).βgal] were constructed. In several human airway epithelial cell lines, the AdCF126 (CRE).Luc vector provided low basal activity, but was significantly up-regulated by agents that increase cAMP levels. Intranasal administration of the β-Gal-expressing AdCF126(CRE8)βgal vector into C57B1/6 mice demonstrated cAMP-induced upregulation of the reporter gene in airway epithelial cells. Quantification of the inducibility of the chimeric promoter activity in the airway epithelium using the AdCF126(CRE8).Luc vector demonstrated an 11-fold upregulation of the basal promoter activity in the lung with the administration of a phosphodiesterase inhibitor and a cAMP analog. These observations demonstrate the feasibility of using a chimeric promoter comprised of a minimal fragment of the CFTR 5′-flanking region, together with added multiple CRE, to control genes delivered in vivo. Importantly, because there are many drugs used in humans that raise cAMP, the concept of using a cAMP-regulatable promoter may also be a useful approach to enhance the safety, efficacy, and feasibility of a variety of human gene therapy strategies. This study focuses on the design and evaluation of chimeric promoters for gene therapy that provide a low basal level of expression, but could be upregulated by pharmacologic agents that may be safely administered to the host. This was accomplished by using a minimal fragment from 5′-flanking sequence of the CFTR gene and adding tandem repeats of consensus sequences of cAMP response elements (CRE) to confer the ability to upregulate gene expression by increasing intracellular cAMP levels. The ability of a CRE/CFTR chimeric promoter to respond to agents that elevate cAMP was demonstrated in replication-deficient adenovirus vectors in vitro in human airway epithelial cells and in vivo in the lung following intranasal administration. This chimeric promotor is potentially applicable to a variety of gene therapy strategies requiring intermittent upregulation of expression of the newly transferred gene.