title: Genetic modification of airway progenitors after lentiviral gene delivery to the amniotic fluid of murine fetuses.
abstract: Lentiviral vectors with the firefly luciferase or enhanced green fluorescent protein (EGFP) transgenes were delivered to the amniotic fluid of murine fetuses at Embryonic Day (E) 14.5 or E16.5. Whole-body imaging of luciferase recipients after birth demonstrated transgene expression in the peritoneal and thoracic regions. Organ imaging showed luciferase expression in lung, skin, stomach, and/or intestine. Histological immunofluorescence analysis of EGFP recipients demonstrated that small clusters (≤ three cells) of EGFP-positive epithelial cells were present in the large and small airways of recipients at up to 7 months (n = 11). There was no difference in the frequency of transgene expression in mice injected at E14.5 or E16.5 in respiratory or nonrespiratory organs. Analysis of the bronchoalveolar duct junctions on tissue sections of recipient mice identified multiple EGFP-positive epithelial cells. Cells coexpressing EGFP, Clara cell 10-kd protein, and surfactant protein C (SPC) were also found in lungs, consistent with the transduction of bronchoalveolar stem cells. Next, naphthalene lung injury in both luciferase and EGFP recipients was performed to determine whether transduced cells could contribute to tissue repair. In luciferase recipients, the whole-body luciferase signal increased 2- to 20-fold at 2 weeks after naphthalene treatment. Remarkably, immunohistological analysis of the lungs of EGFP recipients after lung injury repair demonstrated repopulation of airways with long stretches of EGFP-positive epithelial cells (n = 4). Collectively, these data demonstrate that lentiviral gene delivery to the amniotic fluid of murine fetuses genetically modifies long-lived epithelial progenitors capable of contributing to lung injury repair.
authors.affiliation: Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California, USA.
authors.forename: Suparna
authors.initials: S
authors.lastname: Mishra
authors.forename: Xingchao
authors.initials: X
authors.lastname: Wang
authors.forename: Nancy
authors.initials: N
authors.lastname: Smiley
authors.forename: Ping
authors.initials: P
authors.lastname: Xia
authors.forename: Chang Mu
authors.initials: CM
authors.lastname: Hong
authors.forename: Dinithi
authors.initials: D
authors.lastname: Senadheera
authors.forename: Kim Chi
authors.initials: KC
authors.lastname: Bui
authors.forename: Carolyn
authors.initials: C
authors.lastname: Lutzko
mesh_terms: D000653:Amniotic Fluid / Q000378:metabolism*; D000818:Animals; D016922:Cellular Senescence; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005333:Fetus / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000166:cytology* / Q000201:enzymology; D051379:Mice; D009928:Organ Specificity; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D011247:Pregnancy; D013234:Stem Cells / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D016019:Survival Analysis; D014132:Trachea / Q000166:cytology / Q000201:enzymology; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics; D051598:Whole Body Imaging; D014945:Wound Healing
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D010735:Phosphoglycerate Kinase