Abstract
Vectors based on adeno-associated virus (AAV) are promising tools for gene therapy. The production of strongly toxic vectors, for example for cancer-directed gene transfer, is often unfeasible due to uncontrolled expression of toxic genes in vector-producing cells. Using an approach based on transcriptional repression, we have created novel AAV vectors carrying the genes coding for diphtheria toxin A (DTA) and the pro-apoptotic PUMA protein. The DTA vector had a significant toxic effect on a panel of tumor cell lines, and abrogation of protein synthesis could be shown. The PUMA vector had a toxic effect on HeLa and RPMI 8226 cells, and sensitized transduced cells to doxorubicin. To permit targeted gene transfer, we incorporated the DTA gene into a genetically modified AAV-2 capsid previously developed by our group that mediates enhanced transduction of murine breast cancer cells in vitro. This vector had a stronger cytotoxic effect on breast cancer cells than DTA vectors with wildtype AAV capsid or vectors with a random capsid modification. The vector production and application system presented here allows for easy exchange of promotors, transgenes and capsid specificity for certain target cells. It will therefore be of great possible value in a broad range of applications in cytotoxic gene therapy and significantly broadens the spectrum of available tools for AAV-based gene therapy.
Highlights
Vectors derived from adeno-associated virus (AAV) hold promise as safe and efficient tools for gene therapy
This construct, designated pAAVTetO2, provides the inverted terminal repeat sequences required for packaging of transgenes into vector capsids, as well as tet operator sequences to allow for transcriptional inhibition of transgenes by the
In several proof of principle experiments, we showed that the vectors produced with our method and carrying the genes coding for the diphtheria toxin A (DTA) protein and the PUMA protein under the control of the cytomegalovirus immediate early promoter (CMV) promoter are functional, and both in a targeted and non-targeted vector capsid context
Summary
Vectors derived from adeno-associated virus (AAV) hold promise as safe and efficient tools for gene therapy. Their desirable safety profile has been confirmed in a number of clinical trials [1]. The spectrum of strategies in the former field is wide [5,6] It includes the exploitation of the natural diversity of AAV serotypes [7], the insertion of targeted peptides [8,9,10], the use of bispecific conjugates [11,12,13], and the insertion of random peptide libraries exposed at the capsid surface, which enables the identification of suitable capsid variants by biopanning [14,15,16,17,18]
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