Development and validation of a qPCR based high-throughput assay to quanti-tatively assess the productive uptake of peptide conjugated antisense oligonu-cleotides (ASO) in a GLP1 receptor over-expressing HEK293 cell line
Ladda ner
Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2018
Författare
Dzenita, Bazdarevic
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Diabetes mellitus is the name of a collection of di˙erent disorders characterized by raised blood sugar levels due to either absolute or relative insulin insuÿciency to properly control the blood glucose levels in the body. The -cells in pancreas don’t have the ability to secrete insulin as much as required to meet the demand caused by obesity-driven insulin resistance. Diabetes is a growing public health issue, where type 2 diabetes is the most common form among the population. With the global increase of obesity and the emerging epidemic of type 2 diabetes there is a need for new medicines that cures type 2 diabetes. Researchers at AstraZeneca are working on developing new drugs that are specifi-cally targeting -cells using antisense oligonucleotides (ASOs). ASOs have a great potential to become a treatment because of the ability to bind and knockdown genes. There are still challenges that needs to be overcome regarding the delivery to specific cell types. One strategy to enhance the cellular uptake of ASOs is to use ASOs conjugated to a peptide hormone called Glucagon-like peptide 1, that can bind to a receptor found on the -cells surface. Currently successful uptake of ASOs is quantified by measuring the relative expression of gene inhibited with a manual and low throughput qPCR assay. To speed up the process and to be able to screen several ASOs, the aims of this study were to set up a two-step qPCR-based semi-automated assay on GLP1 receptor overexpressing HEK293 cell line and pri-mary mouse pancreatic islet cells to assess the delivery of a tool compound (ASO targeting MALAT1 ). The assay used in this study was based on another in-house assay, that involves cell lysis, cDNA synthesis of the accessible mRNA and qPCR amplification reaction. However, the assay needed modifications to generate robust and reproducible concentration-response curves showing the relative gene expression. This study have shown that setting up a high-throughput qPCR based assay is not straightforward and takes time to find the conditions for working assay in GLP1 receptor overexpressing HEK293 cells. Finding the right lysis bu˙er for the cell type, using a Poly-D-Lysine coated plate, 10% lysate in the cDNA synthesis and using hydrolysis (TaqMan) probes in the qPCR, solved the problem regarding the performance of amplification reaction and relative gene expression (concentration-response curves). The assay developed in this project can now be used for comparing and ranking of ASOs with di˙erent conjugated GLP1 peptides based on the eÿcacy and potency.
Beskrivning
Ämne/nyckelord
Cellbiologi , Cell- och molekylärbiologi , Medicinsk bioteknologi (med inriktning mot cellbiologi) , Livsvetenskaper , Cell Biology , Cell and Molecular Biology , Medical Biotechnology (with a focus on Cell Biology) , Life Science