Recombinant AAV serotype and capsid mutant comparison for pulmonary gene transfer of alpha-1-antitrypsin using invasive and noninvasive delivery

Abstract

Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing alpha-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 x 10(10) vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT ("long serpin") indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency-related emphysema.

Figure 1

Comparison of serum human α-1-antitrypsin (hAAT) levels among the different recombinant adeno-associated viruses (rAAVs) and delivery routes. The blood of C57B16 mice receiving various AAV serotypes or AAV2 capsid mutants intratrachealy or intranasally were collected every 4 weeks. The serum hAAT levels were assayed by enzyme-linked immunosorbent assay. (a) rAAVs delivered intratrachealy, (b) rAAVs delivered intranasally. Refer to Table 2 for statistical analysis. Data are presented as group averages ± SEM (n = 5).

Figure 2

Human α-1-antitrypsin (hAAT) expression in broncho-alveolar lavage fluid (BALF). Four months after being injected with the rAAVs, animals were killed and their lungs were lavaged to measure hAAT levels present in the lung with an enzyme-linked immunosorbent assay. Data are presented as group averages ± SEM (n = 5).

Figure 3

Recombinant AAV genome copies in lung tissue. Four months after being injected with AAVs, animals were killed and lungs were collected and genomic DNA was extracted. Purified DNA was assayed by Taqman quantitative PCR for AAV copies. Data are presented as group averages ± SEM (n = 5).

Figure 4

Immunostaining for α-1-antitrypsin in the Lung. Representative lung histology sections images (×10 and ×20) of mice receiving 1 × 10¹⁰ vg of various rAAV serotypes via both routes of administration. (a) rAAV2-intranasal, (b) rAAV5-intranasal, (c) rAAV8-intranasal, (d) rAAV9-intranasal, (e) rAAV2-intratracheal, (f) rAAV5-intratracheal, (g) rAAV8-intratracheal, (h) rAAV9-intratracheal.

Figure 5

Inflammatory changes after rAAV administration. (a) Pathological score of inflammation, 4 months after being injected with virus or PBS control, mice lungs were harvested and pathology scoring was done to evaluate the possible inflammation. (b) Hyperplasia in bronchial epithelial cells in mice receiving AAV mutant E mouse 4 months after intranasal delivery, the hyperplasia was found in the bronchial epithelial (×10). Macrophages are also observed in the region (arrows). (c) Vesicles in bronchial epithelial in AAV mutant E: mouse 4 months after intranasal delivery, the vesicles were found in the bronchial epithelial (×20), along with an occasional macrophage (arrows). (d) Macrophage infiltration and staining in rAAV8 transduced lung 4 months after intratracheal delivery. Data are presented as group averages (×40) (n = 5).