Methods and compositions for the treatment of lung diseases and disorders

Abstract

A method of treating injured or diseased alveolar epithelial tissue in the lung of a mammal comprising transplanting into the lung a population of differentiated transgenic stem cells, or progeny thereof, which have an alveolar type II phenotype, effective to repair at least a portion of the injured or diseased alveolar epithelial tissue; an expression vector for transgenically modifying stem cells, comprising a DNA sequence encoding human surfactant protein C promoter operably linked to a DNA sequence encoding at least one drug-resistance gene; transgenic stem cells comprising such expression vector; and a method of preparing such transgenic stem cells and progeny thereof, a high percentage of which have an alveolar type II phenotype.

1 . A construct comprising a DNA sequence encoding human surfactant protein C promoter operably linked to a DNA sequence encoding at least one drug -resistance gene. 
     
     
         2 . The construct of  claim 1 , wherein said drug-resistance gene is a neomycin-resistance gene. 
     
     
         3 . A linear expression vector comprising the construct of  claim 2  operably linked downstream of a DNA sequence encoding a 3′-hprt vector, wherein said 3′-hprt vector comprises a puromycin-resistance gene. 
     
     
         4 . A transgenic stem cell comprising the expression vector of  claim 3 . 
     
     
         5 . A method of preparing a population of in vitro cultured cells of alveolar epithelial type II (ATII) cell lineage derived from at least one transgenic stem cell, comprising:
 (a) culturing transgenic stem cells in vitro in a medium comprising laminin, collagen and growth factors that promote differentiation and proliferation of said stem cells, to produce differentiated cells without formation of an embryonic body, wherein at least some of said differentiated cells are of ATII cell phenotype, at least one said differentiated cell containing an expressible transgene comprising the DNA construct of  claim 1 ;   (b) selecting said differentiated cells for drug resistance, to isolate the differentiated cells having ATII cell phenotype from those not expressing the drug resistance; and   (c) cloning the isolated cells to produce a population of cells at least 95% of which having ATII cell phenotype.   
     
     
         6 . The method of  claim 5  wherein at least 99% of the produced population of cells have an ATII cell phenotype. 
     
     
         7 . The method of  claim 5  wherein the transgenic stem cells are transgenic embryonic stem cells. 
     
     
         8 . The method of  claim 5  wherein said isolated cells express at least one biomarker selected from the group consisting of surfactant protein C, cystic fibrosis transmembrane conductance receptor, α-1 -antitrypsin, complement protein C3 and complement protein C5. 
     
     
         9 . The method of  claim 5  wherein said transgenic stem cells comprise a therapeutic transgene operably linked to a cell-specific promoter. 
     
     
         10 . The method of  claim 5  wherein in (c), cloning the isolated cells to produce a population of cells having ATII cell phenotype comprises producing a population of more than 10 6  cells within 15 days of differentiation, wherein at least 99% of said population have ATII phenotype. 
     
     
         11 . A method of preparing a population of in vitro cultured cells of alveolar epithelial type II (ATII) cell lineage derived from at least one transgenic induced pluripotent stem cell, comprising:
 (a) culturing transgenic induced pluripotent stem cells in vitro in a medium comprising laminin, collagen and growth factors that promote differentiation and proliferation of said stem cells, to produce differentiated cells without formation of an embryonic body, wherein at least some of said differentiated cells are of ATII cell phenotype, at least one said differentiated cell containing an expressible transgene comprising the DNA construct of  claim 1 ;   (b) selecting said differentiated cells for drug resistance, to isolate the differentiated cells having ATII cell phenotype from those not expressing said drug-resistance; and   (c) cloning the isolated cells to produce a population of cells at least 95% of which having ATII cell phenotype.   
     
     
         12 . The method of  claim 11 , wherein said at least one drug resistance gene comprises a puromycin resistance gene and a neomycin resistance gene. 
     
     
         13 . An in vivo method of repairing injured or diseased alveolar epithelial tissue in the lung of a mammal, comprising transplanting into said lung containing injured or diseased alveolar epithelial tissue, a population of differentiated stem cells, or progeny thereof, at least 95% of which have ATII phenotype, wherein said population of cells is prepared in accordance with the method of  claim 1 , and is effective to repair at least a portion of said injured or diseased alveolar epithelial tissue. 
     
     
         14 . The method of  claim 13  wherein at least 99% of said population of cells have a ATII cell phenotype. 
     
     
         15 . The method of  claim 13  wherein said stem cells comprise embryonic stem cells. 
     
     
         16 . The method of  claim 13  wherein said at least one said differentiated stem cell, or progeny thereof, comprises a therapeutic transgene operably linked to a cell-specific promoter, wherein said transgene encodes a therapeutic gene product. 
     
     
         17 . The method of  claim 16 , wherein said mammal suffers from a genetic disease affecting alveolar epithelial tissue in the lung, and said therapeutic transgene encodes a gene product for ameliorating the detrimental effects of said genetic disease in said alveolar epithelial tissue. 
     
     
         18 . The method of  claim 13 , wherein said transplanting comprises administering said population of cells directly to injured or diseased alveolar epithelial tissue in said lung. 
     
     
         19 . The method of  claim 13 , wherein said transplanting comprises administering said population of cells into said lung endotracheally via oropharynx intubation. 
     
     
         20 . The method of  claim 13  wherein said isolated cells isolated cells express at least one biomarker selected from the group consisting of surfactant protein C, cystic fibrosis transmembrane conductance receptor, α-1-antitrypsin, complement protein C3 and complement protein C5.