Gene transfer to the alveolar epithelium is an attractive therapeutic approach for a number of acute and chronic acquired lung diseases, including pulmonary inflammation, pulmonary edema, acute lung injury (ALI), the acute respiratory distress syndrome (ARDS), and pulmonary fibrosis (1, 5, 15, 43). In addition, due to its large surface area and proximity to the vascular endothelium, the alveolar epithelium represents a desirable target for delivery of therapeutic genes encoding secreted proteins. However, in contrast to the large number of studies that have utilized a variety of vectors to achieve gene transfer to tracheal and bronchial epithelium in the upper airways, particularly in the context of gene therapy for cystic fibrosis (54), relatively few studies have examined gene transfer into the alveolar epithelium of the distal respiratory tract.
Non-viral strategies for delivery of exogenous DNA to the lung have been limited by low efficiency of transduction (56). Of the various vectors that have been evaluated for gene therapy thus far, each exhibits characteristic disadvantages, and none has proven effective in achieving efficient, long-term expression in the distal respiratory tract. Due to the relative quiescence of the cells that constitute the alveolar epithelium, viral vectors must be able to efficiently transduce cells that are not actively dividing. In this regard, adenoviral vectors have been shown to effectively transduce the alveolar epithelium (15). However, use of these vectors in vivo has been limited by immune responses, which can be especially problematic in the lung alveoli due to the potential for inducing serious pulmonary inflammation. In any event, use of this non-integrating vector system would also require repeated viral administration to achieve long-term gene expression (12). Adeno-associated virus (AAV) vectors show episomal expression and eventual integration following cell division and have been used for gene delivery to the distal respiratory tract (16, 17); however, their use has been limited by low packaging capacity and difficulty obtaining high titer preparations (20). Repeated administration of AAV vectors has been limited by the development of neutralizing antibodies (8). Thus, investigation of alternative vectors, such as retroviruses, for gene delivery to the alveolar epithelium is warranted.
Two types of retrovirus vectors, murine leukemia virus-(MLV) and lentivirus-based, have been tested for their efficiency in transducing alveolar epithelial cells (“AEC”). Under normal in vivo conditions, the cells that constitute the alveolar epithelium undergo very low rates of proliferation (2, 4, 46, 47). The efficiency of standard MLV-based retroviruses for gene transfer to the adult alveolar epithelium is therefore predictably inefficient (14, 50). This limitation has been partially overcome by inducing cell proliferation with growth factors, but overall transduction efficiency is still quite low. Newer lentivirus vectors, however, have recently been shown to transduce several non-dividing cell types including neuronal cells, myocytes, liver cells and tracheal epithelial cells, with long term persistence of transgene expression (21, 23, 34, 40).
Pseudotyping of the lentivirus with different envelope proteins has expanded the range of host cells that can be transduced by lentivirus vectors, and has also allowed the virus to be easily concentrated to high titers, especially when pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) (38). Lentivirus vectors would therefore appear to be ideally suited for gene transfer to the relatively quiescent cells of the alveolar epithelium. However, the lack of a well-characterized virus receptor and the polarized nature of its distribution makes transfection efficiency in different cell types unpredictable.
Studies to date have focused on lentivirus-based gene transfer to the tracheal or bronchial epithelium of the proximal airways. In polarized, well-differentiated airway epithelia, only minimal transduction by VSV-G pseudotyped vectors introduced from the apical surface (the only directly accessible surface in vivo) has been observed. The use of lentivirus vectors for transduction of adult alveolar epithelial cells in the distal respiratory tract has not been evaluated to date. In particular, whether the polarized cells that constitute the alveolar epithelium present a similar barrier to apical transduction by lentivirus has not been explored.
Based on the 1984 paper by Fuller et al., it was widely believed in the art that receptors for VSV-G were exclusively located on the basolateral cell surface (41). Because only the apical surface is accessible in AEC in vivo, it was believed that it would be futile to use VSV-G pseudotyped lentivirus to transduce these cells.
The present invention overcomes these hurdles and provides a composition and method for highly efficient transduction of AEC from the apical surface using the lentivirus vector.