Modulacija ekspresije genov z uporabo protismernih oligonukleotidov

Zabret, Nika (avtor), Jerala, Roman (mentor)

Na uravnavanje izražanja genov lahko vplivamo na nivoju transkripcije, zorenja primarnega transkripta pre-mRNA in translacije. V tem magistrskem delu predstavimo pristop uravnavanja izražanja genov z uporabo protismernih oligonukleotidov (ASO) na nivoju translacije. Načrtovali smo ASO, ki se tarčno vežejo na zaporedja v 5' regiji UTR, ki imajo zaradi oblikovanja sekundarnih struktur potencialno inhibitorne učinke na iniciacijo translacije. Vezava ASO lahko poruši elemente sekundarne strukture in olajša iniciacijo translacije in posledično poveča izražanje gena. Ta pristop smo preizkusili na genu CTNNB1, ki zapisuje protein β-katenin, ki ima v celici pomembno strukturno in signalizacijsko vlogo. Prisotnost mutacije v enem izmed alelov gena CTNNB1 zaradi zmanjšanega izražanja ali nefunkcionalnosti β-katenina vodi v razvoj sindroma CTNNB1, zato ima uporaba ASO za povišanje izražanja β-katenina potencial za terapevtske namene. Z uporabo TopFlash/FopFlash reporterskega sistema smo z merjenjem luminiscence pokazali, da s transfekcijo ASO v celice HEK293T dosežemo povečano aktivacijo signalne poti Wnt, v katero je vključen β-katenin. Po transfekciji ASO v celične linije HEK293T, SH-SY5Y in iPSC pa smo z imunodetekcijo proteinov na membrani pokazali, da pride ob dodatku tarčnih ASO do povišanja količine β-katenina v celicah vseh testiranih celičnih linij. Ker smo pokazali, da z uporabo načrtovanih ASO povečamo izražanje in obenem dosežemo povečano aktivnost β-katenina, bi bil tak pristop v kombinaciji z ustrezno dostavno metodo obetaven za klinično uporabo.

• Jezik: Slovenski jezik
• Ključne besede: 5' regija UTR, β-katenin, protismerni oligonukleotidi, translacija
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Leto izida: 2023
• PID: 20.500.12556/RUL-151121
• COBISS.SI-ID: 166588419
• Datum objave v RUL: 30.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Modulation of gene expression using antisense oligonucleotides

Izvleček

The regulation of gene expression can be influenced at the level of transcription, maturation of the primary transcript pre-mRNA and translation. In this master's thesis, we present the approach of regulating gene expression at the level of translation using antisense oligonucleotides (ASO). We designed ASO that bind specifically to sequences in the 5' UTR region, which have potentially inhibitory effects on translation initiation due to the formation of secondary structures. ASO binding can disrupt secondary structures and facilitate translation initiation and consequently increase gene expression. We tested this approach on the CTNNB1 gene, which encodes the protein β-catenin, that has an important structural and signaling role in the cell. The presence of a mutation in one of the alleles of the CTNNB1 gene, due to the reduced expression of β-catenin, causes CTNNB1 syndrome, therefore the application of ASO to increase the expression of β-catenin has the potential for therapeutic use. Using the TopFlash/FopFlash reporter, we showed by measuring luminescence that transfection of ASO into HEK293T cells increases activation of the Wnt signaling pathway, in which β-catenin is involved. After transfection of ASO into HEK293T, SH-SY5Y and iPSC cell lines, we showed by immunodetection of proteins on the membrane that the addition of target ASO leads to an increase in the amount of β-catenin in the cells of all tested cell lines. As we have shown that the use of designed ASO increases the expression and at the same time achieves increased activity of β-catenin, this approach combined with an appropriate delivery method would be promising for clinical use.

• Ključne besede: 5' UTR, β-catenin, antisense oligonucleotides, translation