In Vitro and In Vivo Transfer and Expression of Human Surfactant SP-A- and SP-B-Associated Protein cDNAs Mediated by Replication-Deficient, Recombinant Adenoviral Vectors

Robert J. Korst, Burkhard Bewig, Ronald Crystal

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Abstract

Congenital pulmonary alveolar proteinosis (CPAP) is a fatal disease of full-term infants that is unresponsive to current medical therapy. It is now recognized that at least some forms of this disorder are associated with a deficiency of SP-B, one of the surfactant-associated proteins, as well as probable aberrations in the surfactant-associated proteins SP-A and SP-C. Given these developments, it is logical to hypothesize that CPAP may be amenable to gene therapy, in which the human SP-B cDNA, and possibly the cDNAs of the other surfactant associated proteins, are transferred to the epithelium of the lower respiratory tract. We constructed replication-deficient, recombinant adenovirus vectors in which a constitutive viral promoter drives the expression of the DNAs for the surfactant-associated proteins, SP-B (AdCMV.SP-B) and SP-A (AdCMV.SP-A). Following infection of the human lung A549 epithelial cell line with these vectors in vitro, the appropriately sized mRNAs for these cDNAs were detected, whereas cells infected with a control virus or uninfected cells produced none. Western blots demonstrated expression of these proteins, including appropriate processing of the hydrophobic protein, SP-B. Following in vivo intratracheal infection of rats with these vectors, Northern analysis of the lungs revealed appropriately sized mRNAs for these cDNAs whereas rats infected with control virus or uninfected rats show no hybridization with the human surfactant-associated protein probes. In the AdCMV.SP-A-infected rats, Western blots confirmed the overproduction of the human SP-A protein in both the bronchoalveolar lavage and lung homogenates compared to controls. Thus, it is feasible to utilize adenovirus vectors to transfer and express the human surfactant associated protein cDNAs in vitro and in vivo, presenting a possible mode of therapy for CPAP, as well as other surfactant deficiency states such as the neonatal respiratory distress syndrome and possibly the adult respiratory distress syndrome.

Original languageEnglish
Pages (from-to)277-287
Number of pages11
JournalHuman Gene Therapy
Volume6
Issue number3
DOIs
Publication statusPublished - 1 Mar 1995
Externally publishedYes

Fingerprint

Pulmonary Surfactant-Associated Protein B
Surface-Active Agents
Complementary DNA
Pulmonary Surfactant-Associated Protein A
Proteins
Adenoviridae
Lung
Western Blotting
Viruses
Newborn Respiratory Distress Syndrome
Messenger RNA
Adult Respiratory Distress Syndrome
Bronchoalveolar Lavage
Infection
Genetic Therapy
Respiratory System
In Vitro Techniques
Epithelium
Epithelial Cells
DNA

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

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title = "In Vitro and In Vivo Transfer and Expression of Human Surfactant SP-A- and SP-B-Associated Protein cDNAs Mediated by Replication-Deficient, Recombinant Adenoviral Vectors",
abstract = "Congenital pulmonary alveolar proteinosis (CPAP) is a fatal disease of full-term infants that is unresponsive to current medical therapy. It is now recognized that at least some forms of this disorder are associated with a deficiency of SP-B, one of the surfactant-associated proteins, as well as probable aberrations in the surfactant-associated proteins SP-A and SP-C. Given these developments, it is logical to hypothesize that CPAP may be amenable to gene therapy, in which the human SP-B cDNA, and possibly the cDNAs of the other surfactant associated proteins, are transferred to the epithelium of the lower respiratory tract. We constructed replication-deficient, recombinant adenovirus vectors in which a constitutive viral promoter drives the expression of the DNAs for the surfactant-associated proteins, SP-B (AdCMV.SP-B) and SP-A (AdCMV.SP-A). Following infection of the human lung A549 epithelial cell line with these vectors in vitro, the appropriately sized mRNAs for these cDNAs were detected, whereas cells infected with a control virus or uninfected cells produced none. Western blots demonstrated expression of these proteins, including appropriate processing of the hydrophobic protein, SP-B. Following in vivo intratracheal infection of rats with these vectors, Northern analysis of the lungs revealed appropriately sized mRNAs for these cDNAs whereas rats infected with control virus or uninfected rats show no hybridization with the human surfactant-associated protein probes. In the AdCMV.SP-A-infected rats, Western blots confirmed the overproduction of the human SP-A protein in both the bronchoalveolar lavage and lung homogenates compared to controls. Thus, it is feasible to utilize adenovirus vectors to transfer and express the human surfactant associated protein cDNAs in vitro and in vivo, presenting a possible mode of therapy for CPAP, as well as other surfactant deficiency states such as the neonatal respiratory distress syndrome and possibly the adult respiratory distress syndrome.",
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