Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates.
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 beta-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-DeltaCFTR 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 (1.005x10(6) copies/mug DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24x10(5) copies/microg) and mRNA expression. Immunoprecipitation and (32)P 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.
Fischer
Anne C
AC
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Smith
Carolina I
CI
Cebotaru
Liudmila
L
Zhang
Xuemei
X
Askin
Frederic B
FB
Wright
Jerry
J
Guggino
Sandra E
SE
Adams
Robert J
RJ
Flotte
Terence
T
Guggino
William B
WB
D000818:Animals; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008253:Macaca mulatta; D010446:Peptide Fragments / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins