RNA Interference Approaches and Assessment of New Delivery Systems to Target Heparanase in Soft-tissue Sarcomas

Arrighetti, Noemi (2019). RNA Interference Approaches and Assessment of New Delivery Systems to Target Heparanase in Soft-tissue Sarcomas. PhD thesis The Open University.

Abstract

Soft tissue sarcomas are rare tumours of mesenchymal origin characterised by high biomolecular complexity and heterogeneity. The clinical management of advanced diseases remains a major challenge which needs new therapeutic strategies.

Mounting evidence supports that the heparanase/heparan sulfate proteoglycan system plays a key role in sustaining tumour progression and dissemination and preclinical studies suggest its potential relevance as a therapeutic target.

To better understand the specific role of heparanase, we performed RNA interference in rhabdoid tumour and synovial sarcoma models, and assessed the functional effects of heparanase silencing. We tested both miRNA and siRNA targeting heparanase, but only siRNAs were able to silence mRNA and down-regulate the protein following transient transfection. The reduced expression and enzymatic activity of secreted heparanase observed upon siRNA transfection were consistent with a strong inhibition of cell migration and invasion capability. The expression of pro-angiogenic factors was also reduced both in rhabdoid tumour and synovial sarcoma cell lines. In contrast, we observed that heparanase localised in specific subcellular compartments was resistant to the siRNA effect.

The clinical application of siRNA-based therapeutic strategies faces significant challenges, mainly due to siRNA in vivo poor stability and inefficient delivery. In this work, we evaluated the use of different nanodelivery systems aimed at overcoming these limitations.

The first approach consisted of hybrid nanoparticles with a silica core and a pH-responsive hydrogel shell. These nanoparticles showed promising features, such as remarkable loading and efficient release of siRNA, endo-lysosomal escape and in vivo accumulation at the tumour site. Nevertheless, siRNA delivered through this approach did not inhibit heparanase expression. The second nanodelivery system was leukosome, a biomimetic nanoparticle with leukocyte proteins included in a phospholipidic bilayer. Leukosomes efficiently delivered siRNA into the cells avoiding the endo-lysosomal entrapment. Moreover, these biomimetic nanoparticles mainly accumulated at the tumour primary and metastatic sites. Despite these promising features, also leukosomes were unable to deliver an active siRNA.

Overall, these findings demonstrate that specific inhibition of heparanase by gene silencing is a potential therapeutic approach for soft tissue sarcoma treatment. However, the siRNA delivery systems tested in this work, although promising in terms of tumour and metastasis targeting, still need further improvement to be suitable for nucleic acid delivery.

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