In VitroandIn VivoTransfer 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. Replication deficient, recombinant adenovirus vectors were constructed that contained the cDNAs for the human surfactant associated proteins SP-A and SP-B (AdCMV.SP-A and AdCMV.SP-B). Following infection with these vectors, the human A549 lung epithelial cell line showed appropriate sized mRNA transcripts by Northern analysis. Western blots confirmed the production of SP-A in both the supernatant and cell lysate of these cells. Human SP-A mRNA and protein were observed in the lungs of Sprague-Dawley rats infected with AdCMV.SP-A. Western blots of A549 cell lysate following infection with AdCMV.SP-B revealed both the primary translation product as well as fully processed SP-B, and appropriately sized mRNAs for human SP-B were demonstrable in lung homogenates of rats infected with this vector. These vectors may be useful in vitro for the production of recombinant human surfactant proteins, and in vivo as a possible therapeutic modality for surfactant deficiency states.