Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.

Abstract

Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.

CFTR gene transduction in neonatal rabbits using anadeno-associated virus (AAV) vector

“…These antisera have been previously characterized in our laboratory. 3,11 Antitrates with scattered neutrophils, eosinophils and mononuclear cells. Figure 5b and c are representative of n = 4 serum 169 will specifically immunoprecipitate human CFTR from transfected heterologous cells (Cheng SE, control animals, and Figure 5d is representative of n = 8 vector-treated animals (dose range 10 9 -10 10 particles).…”

“…The lung disease is characterized virus is nonpathogenic in humans, and single doses of by chronic, progressive respiratory obstruction and infec-AAV vectors have induced long-term (up to 6 months) tion. Children with CF have structurally normal lungs at human CFTR expression in both adult rabbit 3 and pribirth, but evidence of inflammation can be found very mate models, 4 as well as in cultured CF bronchial epiearly in life. 1 It is therefore likely that the optimal time thelial cells.…”

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus

“…[18][19][20][21] In treated muscle of immunocompetent mice, this expression was shown to persist for 1.5 years. 19 Potentially therapeutic proteins that have been shown to be delivered and expressed by AAV vectors include erythropoietin, 20 cystic fibrosis transmembrane conductance regulator, 22 blood coagulation factor IX, 23,24 and GUS. 21 In none of these cases, however, were experiments designed to show that the AAV-mediated gene therapy actually affected the long-term course of a disease.…”

Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus

“…In vivo studies using an AAV vector system have documented transgene expression in well-differentiated postmitotic adult rat striatal neurons and rabbit lung epi- thelial cells. 5,6 In addition, these studies have demonstrated prolonged expression of the transgene, up to 6 months, without any adverse host response.…”

“…5,6 The mechanism(s) involved in longterm AAV-mediated transgene expression is complex and is only now being understood. Persistence of AAV within tissues may be due to either its integration within the genome of the transduced cell or its retention as an unintegrated episome.…”