101. Development of Lentiviral Vectors with Respiratory Epithelial Specific Transgene Expression
Authors: Dinithi Senadheera, Benjamin Hendrickson, Denise Petersen, Karen Pepper, Carolyn Lutzko
Journal: Molecular Therapy (2005)

Long-term gene therapy for respiratory disorders requires stable gene transfer to progenitor or stem cells and efficient transgene expression in their differentiated progeny. Constitutive transgene expression may be suitable for some gene therapy applications; however, regulated transgene expression in specific lineages will be required in others. In this study we developed and evaluated a series of lentivectors for respiratory epithelial lineage specific transgene expression. Specifically, we incorporated promoters from the surfactant protein C (SPC), Clara cell 10kd protein (CC10) or Jaagskiete sheep retrovirus (JSRV) promoters for expression in alveolar epithelial II cells (AECII; SPC), Clara cells (CC10) or both AECII and Clara cells (JSRV) into SIN lentiviral vectors. Vectors were evaluated by the expression of an eGFP reporter gene by flow cytometry. The lentivectors were tested in alveolar epithelial type II (AECII; MLE-12 and MLE-15), Clara cell (MTCC1-2, H441), kidney, muscle, blood and brain cell lines. In the first experiment, each cell line was transduced with a serial dilution of positive control lentivector supernatant (with the constitutive MND promoter), to determine the optimal virus concentration for single proviral integrants. This virus concentration producing 5-10% positive cells was used in subsequent experiments to minimize the proportion of cells containing multiple integrants. Four experiments were performed with each of the respiratory epitehlial promoters. In each experiment, cells were transduced by a single addition of viral supernatant and expression was evaluated by FACS for 8-weeks for the level of eGFP expression. The SPC promoted vector expressed eGFP with high specificity, in MLE-12 and -15 and H441 cells (mean fluorescent intensity [MFI]: 56-100), but not other cell types (<30) (p<0.05). The CC10 lentivector expressed the highest level of eGFP in MTCC1-2 and H441 (MFI: 113-148), at lower levels in MLE-12 and -15 (MFI: 48-56), and at background levels (<30) in all other cell types and the no promoter control (p<0.05). Surprisingly, expression from the JSRV promoter was observed in all epithelial and hematopoietic cell lines tested (MFI: 40-101), indicative of disregulated expression from this promoter in a lentiviral backbone. To confirm that all samples contained provirus, all transduced cell lines were subject to semi-quantitative eGFP PCR analysis. This analysis showed similar levels of gene transfer in all transduced cells samples, indicating that differences in transgene expression were due to differences is levels of expression from each promoter and not due to differences in the level of gene transfer. In on-going experiments we are evaluating the lineage and tissue expression profile from these vectors following intra-venous administration of vector supernatant to neonatal mice. The development of lentiviral vectors with respiratory epithelial specific expression patterns will provide a useful tool for the study of the biology of the respiratory epithelium and the development of gene therapy for respiratory disease.