LAUSR

Simple Summary Gene therapy for the treatment of malignancies is an emerging and promising area of particular interest. Therefore, it is imperative to develop effective delivery systems for the specific transfer of nucleic acids into tumors. In comparison to viral vectors, non-viral delivery systems tend to be simple to manufacture, show lower immunogenicity, and are associated with fewer regulatory issues when translated into clinical settings. The aim of our study was to develop single-chain antibody conjugated cyclodextrin-modified poly(propylene imine) (PPI) nanocarriers, comprising β-cyclodextrin-modified PPI, mono-biotinylated, maltose-modified PPI, neutravidin and mono-biotinylated prostate stem cell antigen (PSCA)-specific single-chain antibodies for the targeted transposition of minicircle DNA into PSCA-positive tumor cells. Remarkably, we achieved long-term expression of a therapeutic p53 gene in PSCA-positive tumor cells by combining our tumor-specific hybrid polyplexes with the Sleeping Beauty transposon system in minicircle format. Abstract Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53−/−/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.