siRNA-BASED THERAPEUTICS: DELIVERY AND TARGETING TO PEL TUMOR BY USING CATIONIC LIPOSOMES

Aims of this research was to develop a “nanomedicine” approach based on siRNA delivery for the treatment of primary effusion lymphoma (PEL). The therapeutic use of antitumoral siRNA requires the development of specifically designed functional vectors, allowing improve¬ment of siRNA stability after systemic admin¬istration, and enabling targeted delivery directly into the neoplastic cells. In this context, liposomes, and particularly cationic liposomes, appears particu¬larly suitable to generate complexes with highly degradable siRNAs, as well as to specifically deliver siRNAs directly into the cytoplasm of the target tumor cells, where RNA interference processes take place. Generally, the electrostatic interaction between the positively charged lipids and the negatively charged nucleic acids leads to the formation of stable lipoplexes, protecting the cargo against nuclease attack and improving the cellular uptake and activity [1. In this context, we are investigating innovative target strategies to improve the treatment of human herpesvirus 8 (HHV8)-associated primary effusion lymphoma (PEL). Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin’s lymphoma, affecting the serous cavities (such as the pleu¬ral, pericardial and abdominal cavities) and preferentially arising in immunocompromised or elderly patients, typically affected by several comorbidities and organ function impairments. PEL therapy has been revealed to be unsuccessful in the vast majority of patients, who are invariably characterized by a poor prognoses. Recently, small interfering RNAs (siRNAs), able to knock-down viral oncogenic proteins, were shown to induce efficient PEL cell apoptosis in vitro and PEL regressions in mice treated with intracavitary injection of lentiviral vectors expressing siRNA precursors[2. Moving from our promising preliminary results in the field of nanotechnologies[3-4, we are developing different lipid-based nanocarriers (cationic and stealth-cationic liposomes), to deliver specific siRNAs to knock-down novel molecular targets (HHV8-encoded microRNAs, viral oncogenic proteins, or host transcription factors) with relevant functions in PEL pathogenesis [5. We are presently testing the delivery efficiency of these nanocarriers and the antineoplastic activity in vitro and in vivo using different PEL-derived cell lines and a previously established PEL mouse model[6. We performed several preliminary technological experiments aimed at optimizing the operative condition to obtain the efficient liposomes/siRNAs complexes. Chemic-physical properties of both liposomes and lipoplexes were evaluated by exploiting microscopic, spectroscopic and gel electrophoresis techniques. In vitro experiments demonstrated a high transfection efficiency of some of our carriers, which stably protected and efficaciously delivered siRNAs into PEL cells. Preliminary experiments using a mixture of siRNAs targeting a specific cellular gene showed a remarkable dose-dependent apoptosis, measured by annexin-V staining, in lipoplexes-transfected PEL cells. Moreover, the in vivo delivery of these therapeutic siRNAs significantly increased the survival time of treated mice compared with control treatment (log-rank test, lipoplexes vs empty liposomes, p=0.002), indicating that our lipoplexes exerted a significant antineoplastic activity. The empty carriers were not toxic in control mice and did not delay PEL development respect untraeted mice. Our data indicate that our lipoplexes may therefore be considered as the basis for the development of useful short interfering RNA delivery vectors to treat PEL tumor. Moreover, we identified a target gene whose suppression exerts a relevant tumoricidal activity on PEL cells in vitro and in vivo, opening new perspectives for PEL treatment.