Species-specific differences in mouse and human airway epithelial biology of recombinant adeno-associated virus transduction

Abstract

Differences in airway epithelial biology between mice and humans have presented challenges to evaluating gene therapies for cystic fibrosis (CF) using murine models. In this context, recombinant adeno-associated virus (rAAV) type 2 and rAAV5 vectors have very different transduction efficiencies in human air-liquid interface (ALI) airway epithelia (rAAV2 approximately = rAAV5) as compared with mouse lung (rAAV5 >> rAAV2). It is unclear if these differences are due to species-specific airway biology or limitations of ALI cultures to reproduce in vivo airway biology. To this end, we compared rAAV2 and rAAV5 transduction biology in mouse and human ALI cultures, and investigated the utility of murine deltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) ALI epithelia to study CFTR complementation. Our results demonstrate that mouse ALI epithelia retain in vivo preferences for rAAV serotype transduction from the apical membrane (rAAV5 >> rAAV2) not seen in human epithelia (rAAV2 approximately = rAAV5). Viral binding of rAAV2 and rAAV5 to the apical surface of mouse ALI airway epithelia was not significantly different, and proteasome-modulating agents significantly enhanced rAAV2 transduction to a level equivalent to that of rAAV5 in the presence of these agents, suggesting that the ubiquitin/proteasome pathway represents a more significant intracellular block for rAAV2 transduction of mouse airway epithelia. Interestingly, cAMP-inducible chloride currents were enhanced in deltaF508CFTR mouse ALI cultures, making this model incompatible with CFTR complementation studies. These studies emphasize species-specific differences in airway biology between mice and humans that significantly influence the use of mice as surrogate models for rAAV transduction and gene therapy for CF.

Figure 1.

Differentiation and polarization of cultured murine tracheal epithelia. Murine tracheal epithelia grown at an air–liquid interface (ALI) were evaluated by (A, B) scanning electron microscopy and (C) transmission electron microscopy at 2–3 wk of culture. Cilia are marked by arrows. (D–I) Keratin 14 and tubulin IV immunostaining of native mouse tracheal section (D, E) are compared with the staining pattern in section of murine ALI airway epithelia (MAE) (G, H) and whole-mount staining of MAE for ZO-1 and aquaporin 4 (F, I). C, ciliated cell; NCC, noncilated cell.

Figure 2.

Short-circuit current (Isc) profiles of cystic fibrosis (CF) and non-CF primary cultures of human and murine proximal airway epithelium. Isc tracings from human and mouse airway epithelial cultures were assessed under secretory conditions as described in Materials and Methods after sequential addition of amiloride, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS), cAMP agonists (3-isobutyl-l-methylxanthine [IBMX]/ forskolin), and bumetanide. (A and B) Representative recordings of Isc from human ALI cultures of non-CF (A) and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) (B) airway epithelia. (D and E) Representative recordings of Isc from mouse ALI cultures of non-CF and ΔF508CFTR airway epithelia. (C and F) Delta responses inIsc (ΔIsc) after the addition of amiloride, DIDS, cAMP agonists, and bumetanide to human (C) and mouse (F) airway epithelial cultures. ΔIsc was calculated as the peak current after stimulation minus the current immediately before chemical addition. Values are the mean ± SEM for n number of independent epithelia measured.

Figure 3.

Species-specific differences in the polarity of recombinant adeno-associated virus (rAAV) 5 and rAAV2 transduction in murine (MAE) and human (HAE) airway epithelial cultures. Mouse and human airway epithelial ALI cultures were infected with 2.0 × 10³ particles/cell of rAAV luciferase virus (serotype 2 or 5) from the apical (A and C) or basolateral (B and D) surface. The relative luciferase activity was measured on Days 3 and 15 after infection. Data represent the mean (± SEM) relative luciferase activity (per well) from three independent experiments (n = 10 transwells for each experimental point). (E and F) In vivo rAAV transduction in mouse trachea (E) and lung (F) was performed as described in Materials and Methods, using 2 × 10¹⁰ particles of rAAV vector per mouse. The relative luciferase activity was measured at 2 wk and 3 mo after the infection, and data represent the mean (± SEM) of the relative luciferase activity (per mg protein) from three independent experiments (n = 4 animals for each experimental point).

Figure 3.

Species-specific differences in the polarity of recombinant adeno-associated virus (rAAV) 5 and rAAV2 transduction in murine (MAE) and human (HAE) airway epithelial cultures. Mouse and human airway epithelial ALI cultures were infected with 2.0 × 10³ particles/cell of rAAV luciferase virus (serotype 2 or 5) from the apical (A and C) or basolateral (B and D) surface. The relative luciferase activity was measured on Days 3 and 15 after infection. Data represent the mean (± SEM) relative luciferase activity (per well) from three independent experiments (n = 10 transwells for each experimental point). (E and F) In vivo rAAV transduction in mouse trachea (E) and lung (F) was performed as described in Materials and Methods, using 2 × 10¹⁰ particles of rAAV vector per mouse. The relative luciferase activity was measured at 2 wk and 3 mo after the infection, and data represent the mean (± SEM) of the relative luciferase activity (per mg protein) from three independent experiments (n = 4 animals for each experimental point).

Figure 4.

Proteasome-modulating agents augment rAAV-mediated transduction in mouse and human airway epithelial cultures. The primary mouse (MAE) and human (HAE) airway epithelial cultures were infected with 2.0 × 10³ particles/cell of rAAV luciferase (serotype 2 or 5) viral vectors from the apical surface in the presence and absence of proteasome inhibitors (PI). The relative luciferase activity was measured on Day 3 after infection. Data represent the mean (± SEM) relative luciferase activity (per well) from three independent experiments (n = 10 transwells for each experimental point).

Figure 5.

Immunostaining for AAV binding receptors and Southern blot analysis of proviral DNA after infection of murine tracheal ALI culture. (A) Heparan sulfate proteoglycan (HSPG) and Maackia amurensis (MAA) lectin immunofluorescent staining on sections of mouse trachea and murine ALI cultures was conducted as previously reported (10, 41). Panels were processed as marked with 4′,6-diamidino-2-phenylindole staining of nuclei (blue) and FITC staining of the specific antigen (green). Arrowheads mark the basal membrane of the epithelia. Note: The dense staining of the transwell filter (TF) with MAA is specific, and did not occur when empty collagen-coated transwell filters were stained (data not shown). (B and C) Hirt DNA from rAAV luciferase (serotype 2 or 5)–infected or mock-infected murine ALI cultures were extracted for Southern blotting against a [P³²]-labeled luciferase probe. Hirt Southern blots after apical and basolateral infection for (B) 1 h at 4°C and (C) 1 h at 37°C. Note: different exposure times were used in (B) and (C).