Lentiviral airway gene transfer in lungs of mice and sheep: successes and challenges

Abstract

These findings are the first to confirm the applicability of LPC pretreatment in the production of extensive lentiviral gene transfer in mouse lung airways. However, improved methodologies to increase transduction efficiency are required for adult sheep lung. The results suggest that continued in vivo development of LPC-enhanced lentiviral gene transfer is needed in the lungs of large animals to establish effective lentiviral gene transfer techniques suited to the treatment of airway disease.

Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways

“…Only the nasal epithelium in transgenic cystic fibrosis mice is suited to CF airway gene transfer modeling; 18 in our studies we utilize a 4 µl LPC pre-treatment followed by a 20 µl lentiviral vector dose. [1][2][3][4] When correctly delivered ( Figure 1, mice N1 and N2) the 4 µl volume remained on the treated side of the nasal cavity. In mouse N3 fluid can also be seen in the nasopharynx, but only along the treated side of the nasal airway.…”

“…It has the potential to improve the effectiveness and reduce the variability of gene therapy protocols where despite standardized procedures inconsistent levels and distributions of cell transduction are experienced in mouse nose and lung. 1,2,4 This new ability to directly visualize the delivery of a fluid in the airway at high temporal and spatial detail also suggests an immediate relevance and utility to other respiratory health and disease research modeling applications in small animals when pharmaceutical, infectious, allergenic and particulate fluid installations are utilized.…”

“…Our group uses the mouse nose and lung as in vivo model sites for developing a gene therapy treatment for cystic fibrosis (CF) airway disease. 1,2 Reporter genes and/or the therapeutic (CFTR) gene contained in a lentiviral gene vector are delivered by in vivo bolus instillation into the nose and trachea. To produce lasting gene expression after a single dose event we utilize a 4 µl 0.1 -1% (typically 0.3%) lysophosphatidylcholine (LPC) airway pre-treatment before delivering a 20 µl lentiviral gene vector dose (typically 10 7 -10 9 tu/ml) to the mouse nose.…”

“…To produce lasting gene expression after a single dose event we utilize a 4 µl 0.1 -1% (typically 0.3%) lysophosphatidylcholine (LPC) airway pre-treatment before delivering a 20 µl lentiviral gene vector dose (typically 10 7 -10 9 tu/ml) to the mouse nose. [1][2][3][4] Reliable dosing is essential for the success of these airway gene transfer protocols, but despite the use of standardized delivery techniques our group and others report large variability in reporter gene histological assessments and in electrophysiological gene transfer measurements. 2,5,6 One cause of this variability may be the heterogeneous dose distribution that may occur in the physically complex nose and trachea.…”

“…This method now enables the rational design of more effective delivery procedures in the airway gene transfer protocols used in normal and CF mouse models. 1,2,14,15 It should also permit improvements in reliability and repeatability to boost outcome effectiveness, reduce outcome variability, and decrease the volumes of costly gene vectors needed in mouse model studies.…”