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.
The delivery of genes to the vasculature remains a promising and realistic approach for the treatment of diverse diseases that affect both the cardiac system and peripheral vasculature. A number of pre-clinical strategies have now progressed to clinical trial, largely using nonviral or adenoviral vectors.1 Recent clinical results for cardiovascular disease have generally been disappointing with only secondary endpoints being met.2, 3 This may, at least in part, be attributed to the mode of gene delivery and the vectors utilized. Most preclinical studies have utilized adenoviral vectors. At sufficiently high doses, adenoviral gene delivery to intact blood vessels,4 atherosclerotic lesions5 and vein grafts6 can be achieved. However, this is not without the complications of toxicity and immunogenicity and mediates a purely transient transgene expression when first-generation vectors are utilized.7
Adeno-associated viruses (AAV) have been suggested as potential alternative vectors for vascular gene therapy due to their longevity of transgene expression and relatively favourable safety profile. AAV serotype-2 (AAV-2)-based vectors have been used for cardiovascular gene delivery locally into blood vessels8, 9 and by direct intra-myocardial injection,10 the latter showing success therapeutically when delivered prior to surgical intervention, thereby allowing sufficient transgene to be expressed in the heart. However, poor transduction of the vascular endothelium by AAV-2 both in vitro and in vivo has been noted.9, 11, 12 This has been attributed to sequestration of AAV-2 in the extracellular matrix by heparan sulphate proteoglycans (HSPG), thus preventing cell entry12 and sensitivity to the proteasome,11 a mechanism previously established as an obstacle in the transduction of airway epithelial cells.13, 14 Implications for the proteasome-sensitivity in vascular cells is an important consideration for clinical applications since the ubiquitin-proteasome system remains active in diseased blood vessels.15, 16, 17 Alternate strategies to improve endothelial cell transduction by AAV have been actively sought.18 These include AAV-2 retargeting using vascular cell-selective peptides11, 19, 20 or the use of alternate serotypes.21 We previously reported that for vascular cells (including endothelial and smooth muscle cells) AAV-2 produced the highest levels of transduction compared to AAV-3, -4, -5 or -6, although these levels were lower than those achieved in the more permissive cell type HeLa.21 AAV-7 and -8, more recently isolated by Gao et al22 from primates, possess tropism that appears to make them favourable as in vivo gene therapy vectors compared to AAV-2. For example, Sarkar et al23 demonstrated complete correction of hemophilia A in mice using the AAV-8 vector as a liver directed gene therapy vector. AAV-7 has been shown to produce transgene levels equivalent to those achieved with AAV-1 in skeletal muscle, with AAV-1 previously showing the greatest transduction compared to the other serotypes to date.24 Since the cell binding, cell entry mechanisms and the sensitivity to proteasome degradation for AAV-7 and -8 is unknown, combined with the potential of AAV-7 and -8 as gene therapy vectors, we assessed vascular cell transduction in vitro and in intact blood vessels, and compared this to AAV-2.
As expected, AAV-2 transduction was low in primary human saphenous vein endothelial cells, competed by heparin and greatly enhanced in the presence of proteasome inhibition mediated by LLnL or MG132 (Figure 1). Transduction by AAV-7 and -8 was also disappointingly low under basal conditions and was also markedly enhanced by proteasome inhibition (Figure 1). Heparin failed to significantly modify basal transduction of AAV-7 or -8 consistent with alternate cell binding/entry pathways of both vectors compared to AAV-2.
We next assessed the transduction of murine endothelial cells to ascertain if sensitivity to the proteasome is a broad endothelial effect restricting gene delivery by AAV-7 and -8. Similar transduction profiles to primary human saphenous vein endothelial cells were observed for mouse endothelial cell lines (IP-1B and SVEC4-10) in the presence and absence of proteasome inhibitors, although the transgene levels achieved with proteasome inhibition for AAV-7 and -8 were lower than those achieved with AAV-2 (Figure 2). Previous studies have shown that AAV-2 possesses the ability to transduce vascular smooth muscle cells at levels substantially higher than endothelial cells.9 We therefore, assessed whether transduction of smooth muscle cells by AAV-7 and -8 was different to AAV-2 and secondly whether this was sensitive to proteasome inhibition. In both primary human smooth muscle cells (HSMC) and those derived from rat (RSMC) neither AAV-7 or -8 mediated higher levels of transduction compared to AAV-2 (Figure 3). In contrast to AAV-2, which showed sensitivity to proteasome degradation in human SMC and to a lesser extent in rat SMC, the transduction mediated by AAV-7 and -8 was not affected by inhibition of the proteasome (Figure 3). This suggests that alternate infection pathways may be used by AAV-2 compared to AAV-7 and -8 in smooth muscle cells although, due to heterogeneity between species and vascular beds it will be important to assess further smooth muscle cell types.
Previous studies have shown that inhibition of AAV degradation by the proteasome in airway epithelial cells leads to accumulation of AAV in the nucleus.13, 14 We therefore, assessed this in primary human saphenous vein endothelial cells. As expected LLnL coincubation markedly enhanced endothelial AAV-2 trafficking to the nucleus compared to incubation in the absence of LLnL (Figure 4). The profile in the absence of LLnL for AAV-7 and -8 was noticeably different. For each, an approximately equal proportion of virion DNA was located in the nuclear and cytosolic fractions (Figure 4) even though the transduction levels for AAV-7 and -8 in primary endothelial cells were approximately equal for AAV-2, -7 and -8 (Figure 1). However, for both AAV-7 and -8, nuclear localization was enhanced in the presence of LLnL (Figure 4). This again suggests that poor nuclear trafficking limits the level of cellular transduction for AAV-7 and -8 in a similar manner to AAV-2 in endothelial cells. Clearly, the pathways that mediate entry and translocation of AAV-7 and -8 will require further investigation once more knowledge of the cellular receptors for AAV-7 and -8 is elucidated.
Finally, we transduced intact rat aortae with each AAV in the presence or absence of proteasome inhibition (Table 1). It was clear that vessel wall infection by AAV-7 and -8 is poor since virion levels at 7 days postinfection in the absence of proteasome inhibition were substantially lower than AAV-2 (Table 1). In fact, levels of virion DNA were far lower than that achieved by AAV-2 (5947±1075 pM virion DNA/20 ng DNA for AAV-2 versus 2.78±1.36 for AAV-7 and 0.82±0.72 for AAV-8) in the absence of LLnL (Table 1). Additionally, in similarity to in vitro data, AAV-7 and -8 levels in the vessel wall were markedly enhanced in the presence of LLnL compared to the levels in the absence of proteasome inhibition (Table 1). We additionally assessed vessel homogenates for transgene activity using the CPRG Assay Kit that is effective on rat tissue.25 However, levels of transgene at 14 days were below the limits of detection of the assay for all treatments (data not shown) further enforcing the poor performance of AAV-2, -7 and -8 vectors in vascular tissue. This suggests that additional barriers to efficient transduction by AAV vectors are present in vascular tissue.
Unlike the substantial increases in transduction that AAV-7 and -8 achieve in defined tissues, example liver for AAV-8, compared to AAV-2,22 application of these vectors for the transduction of the vascular endothelium is limited by sensitivity to the proteasome. This, therefore, appears to be a common limitation for endothelial cell transduction by AAV. Like AAV-2,11 targeting strategies to improve uptake and/or trafficking may overcome these barriers. This study, therefore provides an important insight into the transduction of vascular cells by the recently isolated AAV-7 and -8 vectors.
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We thank Nicola Britton for technical assistance. This work was funded by the Medical Research Council (UK), National Kidney Research Fund (UK) and the Biotechnology and Biomedical Sciences Research Council.
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Denby, L., Nicklin, S. & Baker, A. Adeno-associated virus (AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation. Gene Ther 12, 1534–1538 (2005). https://doi.org/10.1038/sj.gt.3302564
- adeno-associated virus
- vascular gene therapy
- AAV serotypes
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