Evaluation of the respiratory epithelium of normals and individuals with cystic fibrosis for the presence of adenovirus E1a sequences relevant to the use of E1a- adenovirus vectors for gene therapy for the respiratory manifestations of cystic fibrosis.
Lung disease associated with disorders such as cystic fibrosis (CF) may be amenable to somatic gene therapy in which there is delivery of the normal gene directly to the respiratory epithelium using E1a- adenovirus (Ad) type 2- or 5-based vectors. For safety reasons, the Ad vectors are rendered replication deficient by deletion of the E1a region. Because there is the theoretical possibility of an E1a- replication-deficient vector replicating as a result of recombination or complementation with Ad 2/5 E1a sequences present in the target cell, this study is directed toward evaluating respiratory epithelium of normals and individuals with CF for the presence of E1a sequences. Using Ad 2/5 E1a-specific primers and the polymerase chain reaction to evaluate DNA recovered from freshly isolated nasal and bronchial epithelium recovered by brushing, E1a sequences were detected in respiratory epithelium of 19 of 91 normals (21%). In the E1a-positive samples, the average of E1a copy number was 55 +/- 18/10(3) recovered cells. In CF individuals, 7 of 52 (13%) had detectable E1a sequences in the respiratory epithelium, with E1a copy number in the positive samples of 80 +/- 21/10(3) recovered cells. These results demonstrate that there are detectable Ad 2/5 E1a sequences in the respiratory epithelium of a small percentage of normals and individuals with CF. Because of the theoretical potential of such sequences supporting replication of E1a- Ad vectors, human gene therapy protocols for CF utilizing such vectors should consider evaluating study individuals for the presence of Ad 2/5 E1a sequences in the respiratory epithelium.
Pulmonary Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
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Chu
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D000257:Adenoviridae Infections / Q000150:complications / Q000821:virology*; D017500:Adenovirus E1A Proteins / Q000235:genetics; D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D000328:Adult; D000914:Antibodies, Viral / Q000097:blood; D001483:Base Sequence; D001980:Bronchi / Q000473:pathology / Q000821:virology*; D001992:Bronchoalveolar Lavage Fluid / Q000821:virology; D003550:Cystic Fibrosis / Q000150:complications / Q000821:virology*; D004279:DNA, Viral / Q000302:isolation & purification*; D003673:Defective Viruses / Q000235:genetics; D004848:Epithelium / Q000821:virology; D005260:Female; D015316:Genetic Therapy; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007962:Leukocytes / Q000821:virology; D008297:Male; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000821:virology*; D016133:Polymerase Chain Reaction; D012449:Safety; D014766:Viremia / Q000821:virology; D014779:Virus Replication
D017500:Adenovirus E1A Proteins; D000914:Antibodies, Viral; D004279:DNA, Viral