Expression of a truncated cystic Fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates

Anne C. Fischer, Carolina I. Smith, Liudmila Cebotaru, Xuemei Zhang, Frederic B Askin, Jerry Wright, Sandra E. Guggino, Robert John Adams, Terence Flotte, William B Guggino

Research output: Contribution to journalArticle

Abstract

Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken β-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-δCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005×106copies/μg DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24×105copies/μg) and mRNA expression. Immunoprecipitation and 32P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.

Original languageEnglish (US)
Pages (from-to)756-763
Number of pages8
JournalMolecular Therapy
Volume15
Issue number4
DOIs
StatePublished - Apr 2007

Fingerprint

Cystic Fibrosis Transmembrane Conductance Regulator
Primates
Genes
Dependovirus
DNA
Green Fluorescent Proteins
Cytomegalovirus
Immunoprecipitation
Cystic Fibrosis
Genetic Therapy
Actins
Microscopy
Chickens
Proteins
Epithelium
Safety
Gene Expression
Lung
Messenger RNA
Therapeutics

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Expression of a truncated cystic Fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates. / Fischer, Anne C.; Smith, Carolina I.; Cebotaru, Liudmila; Zhang, Xuemei; Askin, Frederic B; Wright, Jerry; Guggino, Sandra E.; Adams, Robert John; Flotte, Terence; Guggino, William B.

In: Molecular Therapy, Vol. 15, No. 4, 04.2007, p. 756-763.

Research output: Contribution to journalArticle

Fischer, Anne C. ; Smith, Carolina I. ; Cebotaru, Liudmila ; Zhang, Xuemei ; Askin, Frederic B ; Wright, Jerry ; Guggino, Sandra E. ; Adams, Robert John ; Flotte, Terence ; Guggino, William B. / Expression of a truncated cystic Fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates. In: Molecular Therapy. 2007 ; Vol. 15, No. 4. pp. 756-763.
@article{39da8729b5724870aead115ff1632c87,
title = "Expression of a truncated cystic Fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates",
abstract = "Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken β-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-δCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005×106copies/μg DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24×105copies/μg) and mRNA expression. Immunoprecipitation and 32P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.",
author = "Fischer, {Anne C.} and Smith, {Carolina I.} and Liudmila Cebotaru and Xuemei Zhang and Askin, {Frederic B} and Jerry Wright and Guggino, {Sandra E.} and Adams, {Robert John} and Terence Flotte and Guggino, {William B}",
year = "2007",
month = "4",
doi = "10.1038/sj.mt.6300059",
language = "English (US)",
volume = "15",
pages = "756--763",
journal = "Molecular Therapy",
issn = "1525-0016",
publisher = "Nature Publishing Group",
number = "4",

}

TY - JOUR

T1 - Expression of a truncated cystic Fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates

AU - Fischer, Anne C.

AU - Smith, Carolina I.

AU - Cebotaru, Liudmila

AU - Zhang, Xuemei

AU - Askin, Frederic B

AU - Wright, Jerry

AU - Guggino, Sandra E.

AU - Adams, Robert John

AU - Flotte, Terence

AU - Guggino, William B

PY - 2007/4

Y1 - 2007/4

N2 - Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken β-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-δCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005×106copies/μg DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24×105copies/μg) and mRNA expression. Immunoprecipitation and 32P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.

AB - Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken β-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-δCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005×106copies/μg DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24×105copies/μg) and mRNA expression. Immunoprecipitation and 32P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.

UR - http://www.scopus.com/inward/record.url?scp=33947255488&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33947255488&partnerID=8YFLogxK

U2 - 10.1038/sj.mt.6300059

DO - 10.1038/sj.mt.6300059

M3 - Article

VL - 15

SP - 756

EP - 763

JO - Molecular Therapy

JF - Molecular Therapy

SN - 1525-0016

IS - 4

ER -