Adeno-associated virus (AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation

Abstract

Transduction of the vascular endothelium by adeno-associated virus (AAV) vectors would have broad appeal for gene therapy. However, levels of transduction by AAV serotype-2 are low, an observation linked to deficiencies in endothelial cell binding, sequestration of virions in the extracellular matrix and/or virion degradation by the proteasome. Strategies to improve transduction of endothelial cells include AAV-2 capsid targeting using small peptides isolated by phage display or the use of alternate serotypes. Previously, we have shown that AAV serotypes-3 through -6 transduce endothelial cells with poor efficiency. Recently, AAV serotypes-7 and -8 have been shown to mediate efficient transduction of the skeletal muscle and liver, respectively, although their infectivity profile for vascular cells has not been addressed. Here, we show that AAV-7 and -8 also transduce endothelial cells with poor efficiency and the levels of transgene expression are markedly enhanced by inhibition of the proteasome. In both cases proteasome blockade enhances the nuclear translocation of virions. We further show that this is vascular cell-type selective since transduction of smooth muscle cells is not sensitive to proteasome inhibition. Analysis in intact blood vessels corroborated these findings and suggests that proteasome degradation is a common limiting factor for endothelial cell transduction by AAV vectors. Gene Therapy (2005) 12, 1534-1538.

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

“…3 However, transduction efficiency of endothelial cells was found to be very low in vivo 3,4 and in vitro. 3,5,6 Transduction efficiency of endothelial cells could be increased by introduction of an endothelial-targeting peptide identified by phage display within the AAV capsids or selection of random AAV display peptide libraries on endothelial cells. 7--13 Such retargeted vectors resulted in an increase in transduction rates by about 40-fold.…”

Celastrol enhances AAV1-mediated gene expression in mice adipose tissues

“…20 Many studies suggested that the proteasome inhibitors primarily acted on the viral intracellular trafficking and resulted in an increase of AAV genome copies inside the nucleolus instead of preventing the degradation of cellular internalized AAV particles. 17,18,21,22 Celastrol (Figure 1b) is one of the potent bioactive monomers isolated from the root bark of an herb in traditional Chinese medicine, Tripterygium wilfordii or Thunder God vine (Figure 1a). Clinically, the celastrol-containing plant extract has been used for treating sepsis, autoimmune diseases, various cancers and neurodegenerative diseases.…”

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

“…Proteasome inhibitors (PIs) were first described to enhance recombinant AAV (rAAV) polarized airway cell transduction (6), and since then PIs, including N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) (6,(14)(15)(16)(17)(18)(19), MG132 (5,6,8,11,14,17,(20)(21)(22)(23)(24), bortezomib (11,25), and celastrol (26), have been observed to enhance transduction in many cell types both in vitro and in vivo. Nevertheless, questions remain regarding the mechanism of this enhancement.…”