Demirdöğen R. E., Emen F. M., Dükel M., Pavlopoulou A., Ayar Kayali H., Meuwissen R. L. J.
TÜBİTAK Projesi, 2022 - 2025
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Proje Türü:
TÜBİTAK Projesi
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Başlama Tarihi:
Ocak 2022
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Bitiş Tarihi:
Ocak 2025
Proje Özeti
Triple Negative Breast Cancer accounts for 20% of all BC cases, and survival rates of patients are very low in late stages. Majority of TNBC patients are resistant to chemotherapy drugs and thus overcoming drug resistance (DR) is a critical step in the cancer treatment process.
Crispr/Cas9 genome wide sequencing is a common method to understand the drug resistance (DR) process. A library consisting of thousands of sgRNA is used and ~ 20000 genes are mutated to understand the effect of loss of function of these genes on DR. Crispr/Cas9 carrier systems are promising for treatment of cancer via gene modification both in vivo and in vitro. However, since their therapeutic effect depends on the carrier sytem, new effective carrier systems should be developed.
In this Project, after investigating resistance against Afatinib in TNBC cells via Crispr/Cas9 genome wide sequencing, the sgRNAs related with DR and Afatinib will be carried into the cells by an artificial viral carrier system that will be developed in this study both in vitro and in vivo. DR against Afatinib shall be overcome by knocking-out the related gene and hence the therapeutic effect of Afatinib will be availed. To the best of our knowledge, there is no study in the literature on use of Crispr/Cas 9 genome wide sequencing for overcoming Afatinib resistance in TNBC cells.
This project consists of two parts: developing new artificial virüs carrier system and the genetic studies. In this study, a multifunctional nucleus targeted “core-shell” structured artificial virüs will be developed for to carry Crispr/Cas9, sgRNA and Afatinib. The amine and fluoride conjugated hyperbranched amphiphilic polymer that can luminesce in the NIR region (BHX-HFBE-NH2-F774) will be synthesized for the first time will allow monitoring. The RRPHFC artificial virüs will consist of Crispr/Cas9-sgRNA-Affatinib loaded BHX-HFBE-NH2-F774 core and a multifunctional RGD-R8-PEG-HA-FA (RRPH) shell.
In the genetic studies, the Crispr/Cas9 activity and Afatinib DR will be tested on HCC1143 and MDA-MB-157 cells. Then, virüs will be produced for the sgRNA in the Crispr Library and the HCC1143 or MDA-MB-157 cells shall be infected with thus produced virüs. The cells infected with the Crispr/Cas9 library will be subjected to Afatinib and genomic DNA isolation will be made. Then, a 2 step PCR and Crispr/Cas9 genome sequencing shall be made for the samples. After new generation sequencing (NGS), via bioinformatic analysis at least 3 target genes and the sgRNAs that would inactivate these genes will be determined. The effect of these target genes on Afatinib resistance will be validated. The drug and Crispr/Cas9 loading capacity of the developed artificial virüs carrier system in the cell culture shall be determined. Then, how Afatinib and sgRNA via Crispr/Cas 9 carrier system delivery affects tumor growth and DR shall be investigated in vivo.
This Project will be carried under the supervision of Çankırı Karatekin University by experts working in the fields of chemistry, molecular biology and bioinformatics from Burdur Mehmet Akif Ersoy, Dokuz Eylül İBG and Ege Univeristy. If our Project is supported, data that would elucidate Afatinib resistance will be obtained and via the in vivo ve in vitro activity that the Crispr/Cas9-sgRNA-Afatinib carrier artificial virüs system that would be developed in this study would provide direct targeting and thus would contribute to TNBC treatment and lower mortality due to TNBC and improve welfare of patients. Turkey will be able to have a voice in the international arena by developing systems that will make CRISPR technology effective with its own resources. The project will contribute to the scientific and technological research power of Turkey, the training of scientists and the production of new projects.