Gene therapy vectors based on adeno-associated virus-2 (AAV2) offer considerable promise for human gene therapy. Applications for AAV vectors are limited to tissues efficiently transduced by the vector due to its natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. Tropism modification to elevate efficiency and/or selectivity to individual cell types would enhance the scope of AAV for disease therapies. The vascular endothelium is implicitly important in cardiovascular diseases and cancer, but is relatively poorly transduced by AAV vectors. We therefore genetically incorporated the peptide SIGYPLP, which targets endothelial cells (EC), into position I-587 of AAV capsids. SIGYPLP-modified AAV (AAVsig) showed enhanced transduction of human EC compared with AAV with a wild-type capsid (AAVwt), a phenotype independent of heparan sulphate proteoglycan (HSPG) binding. In contrast, AAVsig did not enhance transduction of primary human vascular smooth muscle cells or human hepatocytes, principal targets for AAV vectors in local or systemic gene delivery applications, respectively. Furthermore, infection of EC in the presence of bafilomycin A(2) indicated that intracellular trafficking of AAV particles was altered by targeting AAV by means of SIGYPLP. AAV vectors with enhanced tropism for EC will be useful for diverse gene therapeutics targeted at the vasculature.
The efficiency of rAAV-mediated gene transfer into breast cancer cells is significantly higher than previously reported and can be further enhanced by co-administration of chemotherapeutic agents. We also confirmed that breast cancer cells can activate human dendritic cells after infection with a CD40L-encoding rAAV.
The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.
PurposeRecombinant adeno‐associated viruses 2 (rAAV2) represent a nonpathogenic and safe alternative to other viral delivery systems. However, their transduction efficiency in corneal endothelial cells (CEC) is limited. As the level of transgene expression is dependent on the conversion of single‐stranded (ss)‐ into double‐stranded (ds)‐DNA, self‐complementary (sc)‐AAV vectors have been developed to circumvent this problem. The aim of this study was to evaluate the use of scAAV2 in terms of transduction efficiency in CEC. Additionally, the impact of transduction on cell viability was investigated.MethodsA human corneal endothelial cell line (HCEC‐12) as well as organ‐cultured human donor corneas were transduced with different titers of ss‐ or sc‐AAV2. Transduction efficiencies were compared by means of GFP‐transgene expression. GFP‐expression in HCEC‐12 cells was evaluated by flow cytometry over a period of 28 days. GFP‐expression in human donor corneas was analyzed by confocal microscopy on day 6. 7‐AAD staining and flow cytometry as well as MTT‐assay were performed to determine cell viability after transduction.ResultsGFP‐expression was significantly higher in cells transduced with scAAV2 than in cells transduced with ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. The highest transgene expression rate using scAAV2 was 86.9% compared to 80.5 % using ssAAV2. In human donor corneas GFP‐expression was observed in 72.2% (scAAV) and 44.1% (ssAAV) of CEC respectively. There was no significant difference between viability of transduced and untreated cells.ConclusionsScAAV2 vectors are an effective tool to enhance transduction efficiency in CEC. Allowing higher transduction rates with lower vector titers, this could improve AAV2‐mediated gene therapy to protect CEC in corneal allografts.
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