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

Motoyoshi Suzuki, Ravi N. Singh, Ronald Crystal

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 upregulated 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.

Original languageEnglish
Pages (from-to)1883-1893
Number of pages11
JournalHuman Gene Therapy
Volume7
Issue number15
Publication statusPublished - 1 Oct 1996
Externally publishedYes

Fingerprint

Cystic Fibrosis Transmembrane Conductance Regulator
5' Flanking Region
Response Elements
Regulator Genes
Genetic Therapy
Reporter Genes
Gene Expression
Up-Regulation
Epithelial Cells
Consensus Sequence
Intranasal Administration
Cell Line
Tandem Repeat Sequences
Phosphodiesterase Inhibitors
Luciferases
Human Activities
Genes
Plasmids
Epithelium
Escherichia coli

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

@article{3d0aa05efceb4b97832118b85452a3d6,
title = "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 upregulated 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.",
author = "Motoyoshi Suzuki and Singh, {Ravi N.} and Ronald Crystal",
year = "1996",
month = "10",
day = "1",
language = "English",
volume = "7",
pages = "1883--1893",
journal = "Human Gene Therapy",
issn = "1043-0342",
publisher = "Mary Ann Liebert Inc.",
number = "15",

}

TY - JOUR

T1 - Regulatable promoters for use in gene therapy applications

T2 - 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

AU - Suzuki, Motoyoshi

AU - Singh, Ravi N.

AU - Crystal, Ronald

PY - 1996/10/1

Y1 - 1996/10/1

N2 - 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 upregulated 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.

AB - 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 upregulated 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.

UR - http://www.scopus.com/inward/record.url?scp=0029858012&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029858012&partnerID=8YFLogxK

M3 - Article

C2 - 8894680

AN - SCOPUS:0029858012

VL - 7

SP - 1883

EP - 1893

JO - Human Gene Therapy

JF - Human Gene Therapy

SN - 1043-0342

IS - 15

ER -