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Glioblastoma (GBM) is the most frequent and aggressive primary brain tumour in adults. Despite advances in surgical and medical neuro oncology, prognosis for GBM patients remains dismal, with a median survival of about 15 months. It has been demonstrated that the modest benefit of conventional therapies depends on a small population of cancer stem cells within the tumor, named Glioma Stem Cells (GSCs) that cause tumor relapse and chemoresistance and therefore could play a key role in GBM recurrence. Thus, the identification of new specific ligands for GSCs could be a fundamental challenge for the development of effective glioma therapies. Here, we developed an in vitro evolution based approach, named differential whole cellSELEX; it is used to generate nucleic acid ligands, named aptamers, with high affinity and specificity for GSCs. Aptamers, were obtained through the iterative evolution of a random pool of sequences using human primary GSCs as target. Among different potential candidates we focused on one sequence, named 40L. The 40L aptamer and its truncated form, 40S, were selective for human GSCs distinguishing them from tumor differentiated cells, obtained from the stem cells induced to differentiate. 40L revealed to be functionally active on target cells and able to inhibit stemness, cell growth and migration. 40s preserves binding ability of 40L sequence and it has further proven to strongly reduce tumor proliferation in in vivo experiment. Moreover, both 40L and 40s were able to rapidly internalize upon target binding and therefore may serve as selective vehicle for therapeutics.In conclusion, our results indicate that 40L and its short form 40s can selectively target GSCs both in vitro and in vivo. Given the crucial role of these cells in GBM recurrence and therapy resistance, 40L and 40s represent innovative drug candidates with a great potential in the GBM treatment. Citation Format: Alessandra Affinito, Cristina Quintavalle, Maurizio Albero, Claudia Vilardo, Francesco Palma, Giuseppina Roscigno, Lucia Ricci Vitiani, Roberto Pallini, Carla Lucia Esposito, Vittorio de Franciscis, Gerolama Condorelli. Identification of RNA aptamers selectively recognizing and affecting glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 169.

Gliomas are the most common primary central nervous system tumors with a dismal prognosis. Despite recent advances in surgery, radiotherapy, and chemotherapy, current treatment regimens have a modest survival benefit. Due to a combination of its complex phenotype and organ-specific clinical manifestations, efforts to refine glioma treatment with targeted therapies have largely been frustrated. Hence, finding specific ligands capable of detecting and measuring the altered pattern of gene expression and to discriminate between different tumor phenotypes, is a strategic and plausible objective for the diagnosis and therapy of glioma. To date, antibody-based approaches have been developed for in vivo applications but, in most cases, they show toxicity in vivo and do not reach adequate sensitivity. Thanks to their unique characteristics (low size, good affinity for the target, no immunogenicity, chemical structures that can be easily modified to improve their in vivo applications), single-stranded nucleic acid ligand molecules, named aptamers, represent a valid alternative to antibodies for in vivo targeted recognition as therapeutics or delivery agents for nanoparticles, small interfering RNAs, chemotherapeutic cargos and molecular imaging probes. By applying cell-SELEX on cancer cells we have generated and characterized different nuclease resistant 2′fluoro-pyrimidines RNA aptamers as high affinity ligands and inhibitors of human receptor tyrosine kinases (RTKs) with a crucial role in glioma, including EGFR, EGFRVIII, EphB3, Axl and PDGFRβ. All the aptamers are able to efficiently and specifically inhibit tumor growth in cell based assays and in animal models of human glioma. Further, some of these aptamers strongly cooperates in inducing inhibition of tumor growth thus providing the basis for further development of antitumor combination therapies. Citation Format: Simona Camorani, Carla L. Esposito, Silvia Catuogno, Paola Amero, Anna Rienzo, Gennaro De Vita, Gerolama Condorelli, Vittorio de Franciscis, Laura Cerchia. An RNA aptamer-based approach for human glioma treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4196. doi:10.1158/1538-7445.AM2014-4196

The most recent advances in the field of cancer therapy have shown the great potential of using RNA-based molecules for human cancer treatment. RNA-based oligonucleotides, such as siRNA, miRNA mimic or miRNA inhibitors are being developed to modulate the expression of genes important in cancer, thus blocking tumourigenesis, inhibiting tumour growth or preventing metastasis. However a major limitation in the translation to clinic of RNA oligonucleotides is represented by the need of technologies that mediate their selective delivery improving their efficacy and safety. Conjugation with nucleic acid aptamers represents an innovative and promising approach to overcome this obstacle. Aptamers are single-stranded oligonucleotides that have been shown as high-affinity ligands and potential antagonists of cancer-associated proteins. They discriminate between closely related targets and are characterized by high specificity and low toxicity thus revealing as compounds of choice for in vivo cell recognition as delivery agents for various secondary reagents such as nanoparticles, siRNA bioconjugates, chemotherapeutic cargoes and molecular imaging probes. We have recently generated and characterized two 2’fluoro-pyrimidines containing RNA aptamers, named GL21.T and Gint4, that bind at high affinity and high specificity to human Axl tyrosine kinase receptor and PDGFRβ, respectively. These aptamers not only bind to their proper RTK but are also rapidly internalized into target cell, getting about 30% of cell internalization following 15 min-incubation and reached about 60% following 2h of aptamer treatment. By using different approaches, we have generated several completely RNA-based chimeric molecules containing internalizing aptamers coupled to therapeutic miRNAs that are down-regulated in human tumors and whose expression results in selective tumour growth inhibition. We have shown that when applied to cells expressing the specific aptamer target, the chimeric molecules are internalized and processed by Dicer, thus increasing miRNA cellular level and inhibiting miRNA target protein. As a consequence, the depletion of the targeted protein leads to miRNA herapeutic effect. Remarkably, no binding or functionality of the chimeras has been detected in cells that do not express aptamer target receptor. In addition, since miRNAs are frequently up-regulated in different alignancies, acting as oncogenes, we have developed different pproaches to conjugate internalizing aptamers to therapeutic miRNA inhibitors. The ability of these molecules to bind to and internalize in aptamer target-positive cells has been verified. The characterization in vitro and in vivo of the different chimeras is ongoing in our laboratory revealing these hybrid compounds as potential tools for innovative anti-cancer targeted therapeutics. Citation Format: Carla L. Esposito, Silvia Catuogno, Simona Camorani, Antonella di Costanzo, Margherita Iaboni, Gerolama Condorelli, Laura Cerchia, Vittorio de Franciscis. Aptamer-mediated cancer cell-specific delivery of therapeutic microRNAs and miRNA inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2013-3053

Stem cells are a group of cells, which have two important fundamental properties: self-renewal and multipotency. Stem cells have been identified also in many human cancers on the basis of being both morphologically and functionally distinct from other cells within the heterogeneous tumor mass. According to “cancer stem cell hypothesis,” the cancer stem cells (CSC) would remain unaffected by conventional therapies, and capable of repopulating the tumor and giving rise to cancer recurrence. The possibility to identify highly selective biomarkers on CSC would greatly improve cancer diagnosis and treatment. Recently, nucleic acid-based aptamers have proven useful as reagents for identifying cell surface proteins and for cell typing. Further, their high specificity and low toxicity render them a valid alternative to antibodies for in vivo cell recognition. We have developed the SELEX technology on intact glioblastoma cancer stem cells to generate aptamers as biologically active high affinity ligands for CSC-specific cell surface proteins. The approach has been applied by utilizing glioblastoma differentiated tumor cells as a negative selection, and cancer stem cells obtained from two different patients, as positive selection. Upon 16 cycles of SELEX, we generated a set of 2′-fluoro-pyrimidines containing RNA aptamers that, based on sequence analysis, have been grouped into 5 main families. Best aptamers have been then selected by binding experiments for their ability to distinguish glioblastoma CSC from differentiated tumor cells. We thus determined the binding affinity of each molecule to the cell surface specific targets. Functional experiments will be as well presented to further insight the biological role of the most promising aptamer molecules in the acquisition and maintenance of stem cells properties of glioma cancer cells. Results demonstrate the possibility to generate RNA-based aptamers as potential innovative tools for the selective targeting of cancer stem cells. A proteomic approach will define the specific membrane targets of the selected aptamers Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 883. doi:1538-7445.AM2012-883

Aptamers are single-stranded oligonucleotides able to bind with high affinity to specific protein or non-protein targets by folding into complex tertiary structures. They have proven useful as reagents for identifying cell surface proteins and for cell typing. Further, their high specificity and low toxicity render them a valid alternative to antibodies for in vivo targeted recognition as therapeutics or delivery agents for nanoparticles, small interfering RNAs, chemotherapeutics and molecular imaging probes. We have generated aptamers as biologically active ligands for cancer cell surface proteins. The approach has been applied to different tumor systems with the generation of different 2′fluoro-pyrimidines containing RNA aptamers that specifically bind to the ectodomain of human receptor tyrosine kinases (RTKs). Three of these aptamers (named GL21, CL4 and M4) bind at high affinity and high specificity to Axl, EGFR and PDGFR, respectively. Following binding, the aptamers hamper ligand-dependent receptor dimerization and autophosphorylation. All the selected aptamers bind to the correspondent protein target with Kd values in the order of 10 nM and show excellent correlation between cell surface binding and the expression of the target on the cell surface. The anti-Axl GL21 aptamer strongly inhibits cell migration and invasion in vitro, reduces the number of colonies in soft agar colony formation assay and interferes with spheroid formation by malignant glioma U87MG cells. Furthermore, it strongly inhibits tumor growth in mouse xenograft models of human cancer. The anti-EGFR CL4 aptamer strongly induces apoptosis of EGFR-positive cells and at low doses it works even on cells that are resistant to the most frequently used EGFR-inhibitors, as gefitinib and cetuximab. In addition, the combined treatment of cetuximab with the aptamer shows clear synergy in vitro and in vivo. Moreover, we demonstrated that GL21 and M4 aptamer not only bind to Axl and PDGFR, respectively, but also internalize within the target cell thus they can be used as cargoes for tissue specific internalization. The generation of RNA-chimeras (aptamer/miRNA) is on going in our laboratory. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1100. doi:1538-7445.AM2012-1100

A great promise in developing specific molecular probes for disease biomarker discovery and for diagnostic and therapeutic applications is recently represented by the intact cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment) strategy that allow to select nucleic acid aptamers against living cells. Indeed, using cell-SELEX to target proteins expressed on the cell surface permits to develop aptamers that are able to discriminate between even closely related target cells. This strategy has the advantage of a direct selection of ligands without prior knowledge of the target molecules and enables the identification of aptamers that bind large cell surface-specific markers, in their native conformation. Recently, by coupling a differential whole-cell SELEX protocol to human glioma and NSCLC cell lines we addressed the possibility to isolate aptamer molecules that may target cancer specific cell surface bound proteins. We isolated a set of 2’fluoro-pyrimidines modified RNA aptamers that are specific for different targets that are highly expressed on the tumor cell type used and underepresented on other cancer cell types tested. Remarkably, two of these aptamers bind at high affinity and high specificity to the extracellular domain of two human receptor tyrosine kinases (RTKs), EGFR and Axl respectively. Following binding, both aptamers hamper ligand-dependent receptor dimerization and autophosphorylation. Further, depending on the targeted RTK, the selected aptamers cause inhibition of cell proliferation and selective cell death in vitro and in vivo. Since an obvious advantage of RNA-based aptamers that bind cell surface receptors is the possibility to be conjugated to a siRNA and used as cargoes for tissue specific internalization, we thus developed a cell SELEX based protocol to enrich for aptamers that following binding to cell surface are rapidly internalized in a receptor-mediated manner. The whole-cell SELEX strategy holds a great promise in developing specific molecular probes for cancer biomarker discovery and for cancer diagnostic and therapeutic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2011-626