Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages
Andrew A. Wilson, … , Bela Suki, Darrell N. Kotton
Andrew A. Wilson, … , Bela Suki, Darrell N. Kotton
Published December 21, 2009
Citation Information: J Clin Invest. 2010;120(1):379-389. https://doi.org/10.1172/JCI36666.
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Technical Advance Genetics

Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages

Abstract

Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human α1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.

Authors

Andrew A. Wilson, George J. Murphy, Hiroshi Hamakawa, Letty W. Kwok, Sreedevi Srinivasan, Avi-Hai Hovav, Richard C. Mulligan, Salomon Amar, Bela Suki, Darrell N. Kotton

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Figure 4

Quiescent transduced macrophages persist in lung tissue despite a transient acute pulmonary inflammatory response.

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Quiescent transduced macrophages persist in lung tissue despite a transi...
(A) BAL, bone marrow, and blood cells from the same mouse analyzed for GFP expression 2 weeks after infection with CMV-GFP lentivirus. Note the transduction of BAL, but not bone marrow or circulating blood cells. (B) Bone marrow and AMs from mice infected with CMV-GFP and then exposed to BrdU for 2 weeks: 86% of marrow cells were BrdU labeled, compared with less than 2% of AMs, regardless of whether they were transduced (GFP+) or untransduced (GFP–). (C) BAL cell counts were increased above control 1 week after lentiviral infection, indicating a transient inflammatory response (n = 4; *P < 0.05 by ANOVA). Data are presented as means ± SEM. (D) Forward/side scatter analyses of BAL samples showed that more than 85% of BAL cells in normal mice were AMs. However, 1 week after intratracheal lentiviral instillation, there was an influx of neutrophils (26%) as well as monocytes and lymphocytes (23%; also quantified by cytospin differentials in Supplemental Figure 4). By week 6, BAL forward/side scatter profiles and cell counts were indistinguishable from baseline (n = 4 per time point). (E) A representative mouse treated with PKH26, infected the next day with CMV-GFP lentivirus, and analyzed 1 week later revealed that all transduced (GFP+) cells were resident in the lung at the time of infection (GFP+/PKH26+), whereas recruited inflammatory cells (68% of BAL cells after infection) were PKH26– and GFP–. Numbers within the plots denote the percentage of cells in the gated portion or quadrant of the plot.