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Identification of a drug-delivery aptamer against human glioblastoma stem-like cells

Grant number: 16/03686-9
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): April 20, 2017
Effective date (End): April 19, 2018
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Alexander Henning Ulrich
Grantee:Erika de Simone Molina
Supervisor abroad: Guenter Mayer
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Local de pesquisa : Universität Bonn, Germany  
Associated to the scholarship:14/00462-7 - Identification of DNA aptamers to human glioblastoma stem-like cells, BP.DR

Abstract

Glioblastoma (GBM) stem-like cells (GSCs) represent a subpopulation of intratumoral undifferentiated cells, which would be able to reinitiate tumorigenesis after treatment and recapitulate the heterogeneity of the original tumor. Similar to physiological neural stem cells, GSCs are postulated to self-renewal and differentiate into progenitors cells, which in turn gradually differentiate into more committed cells. Although both cell populations share several common molecular stemness markers, only GSC would perform tumorigenic activity. Besides, only GSC would evade chemotherapeutic treatment, being able to further reinitiate GBM propagation. The objective of this proposal is to answer whether it is possible to use an aptamer for designing a drug delivery system against GSCs. To achieve this, the present study proposes to identify and modify an aptamer to internalize and release a cytotoxic drug specifically into GSCs in collaboration with Prof. Dr. Günter Mayer from the Aptamer Center of The Life & Medical Sciences (LIMES) Institute, University of Bonn. Previously, the library of ssDNA molecules (pre aptamers candidates) have been exponentially enriched through Cell-Selex technique, in which positive cycles of selection were performed against resistant viable GBM cells after treatment with chemotherapeutic agent, which were alternated with negative cycles of selection against non-resistant GBM cells during the ongoing Fapesp Fellowship Process Number 2014/00462-7. In the following, the pool of molecules presenting higher affinity for the GSCs will be first used for prediction of three-dimensional structures and selection of aptamers candidates. For this, sequence analysis will be performed aiming to identify and modify strategic G-quadruplex motifs aiming to obtain internalizing aptamers. The potential of candidates for internalizing into GSCs will be evaluated by internalization kinetics assays and confocal microscopy. Next, the prospective aptamers will be conjugated with a cytotoxic agent and the aptamer-drug delivery system will be assessed regarding GSC uptake followed by GBM cellular death. By the demonstration of an aptamer internalizing and cargo releasing specifically into GSCs, the proposed study attempts to provide a specific drug delivery system targeting chemoresistant GSCs avoiding misidentification of NSC. Perspectives of this study include further identification of aptamer-bound target molecules for specific GSC recognition and internalization, also contributing for the elucidation of therapeutic targets in tumor biology and validating aptamers as potent drug-delivery tools.