Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus
Dongsheng Duan, Yongping Yue, Ziying Yan, Jusan Yang, John F. Engelhardt
Dongsheng Duan, Yongping Yue, Ziying Yan, Jusan Yang, John F. Engelhardt
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Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus

Abstract

The restriction of viral receptors and coreceptors to the basolateral surface of airway epithelial cells has been blamed for the inefficient transfer of viral vectors to the apical surface of this tissue. We now report, however, that differentiated human airway epithelia internalize rAAV type-2 virus efficiently from their apical surfaces, despite the absence of known adeno-associated virus–2 (AAV-2) receptors or coreceptors at these sites. The dramatically lower transduction efficiency of rAAV infection from the apical surface of airway cells appears to result instead from differences in endosomal processing and nuclear trafficking of apically or basolaterally internalized virions. AAV capsid proteins are ubiquitinated after endocytosis, and gene transfer can be significantly enhanced by proteasome or ubiquitin ligase inhibitors. Tripeptide proteasome inhibitors increased persistent rAAV gene delivery from the apical surface >200-fold, to a level nearly equivalent to that achieved with basolateral infection. In vivo application of proteasome inhibitor in mouse lung augmented rAAV gene transfer from undetectable levels to a mean of 10.4 ± 1.6% of the epithelial cells in large bronchioles. Proteasome inhibitors also increased rAAV-2–mediated gene transfer to the liver tenfold, but they did not affect transduction of skeletal or cardiac muscle. These findings suggest that tissue-specific ubiquitination of viral capsid proteins interferes with rAAV-2 transduction and provides new approaches to circumvent this barrier for gene therapy of diseases such as cystic fibrosis.

Authors

Dongsheng Duan, Yongping Yue, Ziying Yan, Jusan Yang, John F. Engelhardt

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Optimization of LLnL-enhanced rAAV transduction in polarized bronchial e...
Optimization of LLnL-enhanced rAAV transduction in polarized bronchial epithelia. Differentiated airway epithelia were infected with AV.GFP3ori (10,000 particles per cell) from either the basolateral (a) or the apical (b) surface with the indicated treatments involving LLnL and/or EGTA. All infections were carried out for 24 hours, and GFP transgene expression was monitored by indirect fluorescent microscopy at the indicated times. Data represent the mean (± SEM; n = 6) for each experimental condition. Experiments were performed in triplicate on transwells derived from samples obtained from two different patients. The following conditions were evaluated for basolateral infection in a: single infection with AV.GFP3ori alone (solid line); single infection with AV.GFP3ori in the presence of 40 μM LLnL (filled circles); single infection with AV.GFP3ori in the presence of 40 μM LLnL followed by repeated 5-hour exposure to 40 μM LLnL in the basal compartment culture medium every third day thereafter (open circles); single infection with AV.GFP3ori in the presence of 40 μM LLnL followed by the continued exposure to 40 μM LLnL in the basal medium after rAAV was removed (filled triangles); and repeated infection with AV.GFP3ori on days 0 and 15 in the presence of 40 μM LLnL for 24 hours at the time of infection (open triangles). The following conditions were evaluated for apical infection in b: single infection with AV.GFP3ori alone (filled circles); single infection with AV.GFP3ori after pretreatment with 3 mM hypotonic EGTA before the viral infection (open circles); single infection with AV.GFP3ori in the presence of 40 μM LLnL (filled triangles); and single infection with AV.GFP3ori in the presence of 40 μM LLnL after pretreatment with 3 mM hypotonic EGTA before the viral infection (open triangles).