Distinct Classes of Proteasome-Modulating Agents Cooperatively Augment Recombinant Adeno-Associated Virus Type 2 and Type 5-Mediated Transduction from the Apical Surfaces of Human Airway Epithelia

Abstract

Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (rAAV-2) and rAAV-5 serotypes. In the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines, polarized human airway epithelia, and mouse lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines. However, these two cell types demonstrated cell specificity in the ability of N-acetyl-L-leucyl-L-leucyl-L-norleucine (LLnL) or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2,000-fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggests that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of proteasome function can be effectively used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

Intracellular trafficking of adeno-associated viral vectors

“…32,66 Rather, inhibition of the proteasome enhances nuclear uptake of viruses in a number of cell systems. 32,35 These findings suggest that modulation of the ubiquitinproteasome system might alter AAV intracellular processing at the level of endosomal trafficking, endosomal escape, nuclear transport, and/or uncoating of the virus. The enhanced transduction of rAAVs in the presence of proteasome inhibitors does not appear to be a direct effect of modulating second-strand genome synthesis, but rather a result of increasing the concentration of viral genomes in the nucleus.…”

“…[32][33][34][35]66 The mechanism of enhanced transduction in the presence of proteasome inhibitors does not appear to be due to blocking AAV genome degradation. 32,66 Rather, inhibition of the proteasome enhances nuclear uptake of viruses in a number of cell systems.…”

“…The enhanced transduction of rAAVs in the presence of proteasome inhibitors does not appear to be a direct effect of modulating second-strand genome synthesis, but rather a result of increasing the concentration of viral genomes in the nucleus. 34,35 Furthermore, there appears to be some level of cell-type specificity in the action of proteasome inhibitors since they do not enhance rAAV transduction in all cell types and tissues. 32,65 Although the mechanism of proteasome inhibitor action remains unclear, the finding that both rAAV2 and rAAV5 capsids are a substrate for ubiquitination in vivo and in vitro 32,66 suggests that the extent of capsid ubiquitination may be important in directing intracellular movement of the virion.…”

“…Several agents have been identified that promote nuclear accumulation of AAV and rAAV transduction. These include proteasome inhibitors, 32,33,35 hydroxyurea, 38 and adenovirus. 36 It is currently thought that these agents enhance endosomal and/or cytoplasmic processing of the virus rather than directly facilitating nuclear translocation.…”

“…Increasing evidence has suggested that intracellular trafficking of rAAV may be a universal rate-limiting step in AAV transduction. [32][33][34][35][36][37][38] However, entry pathways of AAVs likely determine the paths by which these viruses move through the cell to the nucleus. As such, discussions of intracellular trafficking must be placed in the context of viral receptors and coreceptors that facilitate endocytosis and movement of AAVs through the endosomal compartment.…”