Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis.
A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.
Rich
D P
DP
Genzyme Corporation, Framingham, MA 01701.
Couture
L A
LA
Cardoza
L M
LM
Guiggio
V M
VM
Armentano
D
D
Espino
P C
PC
Hehir
K
K
Welsh
M J
MJ
Smith
A E
AE
Gregory
R J
RJ
D000260:Adenoviruses, Human / Q000235:genetics* / Q000254:growth & development; D000328:Adult; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015316:Genetic Therapy; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001616:beta-Galactosidase