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Obtaining human glioblastoma cells LAMP2A knockout via CRISPR/Cas9

Grant number: 22/12651-5
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): November 01, 2022
Effective date (End): October 31, 2023
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Clarissa Ribeiro Reily Rocha
Grantee:Beatriz Silva de Oliveira
Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil


Glioblastoma is an extremely aggressive type of cancer and patients with this type of tumor have a median survival between 12 and 15 months. Almost all patients succumb to the disease due to chemotherapy resistance. Standard chemotherapy treatment is performed through the administration of temozolomide (TMZ) which is an alkylating agent that induces cell death through the formation of various DNA damage. Among the different types of lesions caused by TMZ, O6-methylguanine is the most toxic. This lesion is repaired by the enzyme O6-methylguanine methyltransferase (MGMT) and if not repaired, this lesion can lead to cell death via the mismatch repair (MMR) pathway activity. As a consequence, MGMT expression and MMR activity are strongly associated with TMZ resistance. However, due to the high recurrence rate after TMZ treatment even in patients who do not have alterations in these pathways, other cellular processes must be involved in TMZ resistance. The main objective of this scientific initiation project is to establish, validate and characterize the LAMP2A knockout human glioblastoma lineage. For that, we will use T98G human glioblastoma cells and establish knockout lines for the LAMP2A gene using the CRISPR/Cas9 gene editing tool. After establishing the cell line, we will evaluate the induction and processing of TMZ-induced DNA lesions, as well as evaluate cell cycle arrest and proliferation rate compared to the control line. We will also evaluate induction of cell death after treatment with TMZ in the control strain and in the knockout LAMP2A strain. We will then evaluate the cellular responses in these strains against TMZ treatment. With this, we intend to evaluate the role of these two resistance mechanisms and, based on these results, establish a protocol for a therapeutic combination between TMZ and inhibitors of these resistance pathways.

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