Priprava plazmida z zapisom za citolizin A za zdravljenje raka s cepljenjem in situ

Samac, Zvezdana (author), Starčič Erjavec, Marjanca (mentor), Kamenšek, Urška (co-mentor)

Cilj imunoterapije proti raku je sprožiti trajen protitumorski imunski odziv, medtem ko normalne gostiteljske celice ostanejo nepoškodovane. Eden od imunoloških pristopov k zdravljenju raka je in situ cepljenje. Pri tem pristopu zvišamo količino in vidljivost tumorskih antigenov (TA) z uporabo terapij, za katere je znano, da sprožijo imunogeno celično smrt. Ta pa lahko privede do nastanka specifičnega imunskega odziva proti sproščenim TA. Za in situ cepljenje so toksini, ki tvorijo pore in povzročajo lizo celic, še posebej privlačni, saj lahko sprostijo TA. V to skupino toksinov uvrščamo tudi citolizin A (ClyA). Da bi raziskali uporabo ClyA za zdravljenje raka z in situ cepljenjem, smo pripravili dva plazmida z zapisom za clyA, pORF-ClyAhsp in pORF-ClyAhsp-Xmark. Slednjega smo uporabili za pripravo plazmida pORF-ClyAhsp-ORT, ki nima gena za odpornost proti antibiotiku in tako zagotovili varnost v skladu s standardi regulatornih agencij. Preverili in potrdili smo ohranjanje pripravljenih plazmidov v bakterijskih sevih Escherichia coli JM109 in DH1-ORT. Zanimalo nas je tudi, ali je pripravljen pORF-ClyAhsp-ORT funkcionalen in se ClyA po transfekciji izraža v evkariontskih celicah. Dobljene rezultate smo primerjali s transfekcijo celične linije B16-F10 s plazmidom pEGFP-N1. Glede na dobljene rezultate bi bilo smiselno v prihodnje opraviti še več raziskav, da bi lahko z gotovostjo trdili, da je ClyA, ki nastane na podlagi kloniranega gena v uporabljenih plazmidih, citotoksičen in tako uporaben za zdravljenje raka z in situ cepljenjem.

• Language: Slovenian
• Keywords: plazmidi, citolizin A, zdravljenje raka z in situ cepljenjem, tehnologija ORT, citotoksičnost
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [Z. Samac ]
• Year: 2022
• PID: 20.500.12556/RUL-142062
• UDC: 616-006
• COBISS.SI-ID: 127251971
• Publication date in RUL: 19.10.2022

Secondary language

• Language: English
• Title: Construction of cytolysin A encoding plasmid for in situ cancer vaccination

Abstract

The aim of cancer immunotherapy is to induce a sustained immune response against the tumor while leaving the normal host cells intact. One of the immunological approaches to cancer treatment is in situ vaccination. In this approach, the amount and visibility of tumor antigens (TA) is increased by therapies known to induce immunogenic cell death. This in turn can lead to the development of a specific immune response against released TAs. For in situ vaccination, pore-forming toxins that cause cell lysis are particularly attractive because they can release TA. Cytolysin A (ClyA) also belongs to this group of toxins. To investigate the use of ClyA for cancer treatment by in situ vaccination, two clyA-encoding plasmids were constructed, pORF-ClyAhsp and pORF-ClyAhsp-Xmark. The latter was used to produce the plasmid pORF-ClyAhsp-ORT without the antibiotic resistance gene to ensure safety according to regulatory agency standards. Stable maintenance of the constructed plasmids in Escherichia coli bacterial strains JM109 and DH1-ORT was confirmed. In addition, we were interested in whether the constructed pORF-ClyAhsp-ORT is functional and ClyA is expressed in eukaryotic cells after transfection, and we compared the obtained results with the transfection of the B16-F10 cell line with the pEGFP-N1 plasmid. Based on the results obtained, it would be reasonable to carry out further studies in the future to assay whether the ClyA produced based on the cloned gene in the plasmids is cytotoxic and thus useful for the treatment of cancer by in situ vaccination.

• Keywords: plasmids, cytolysin A, in situ cancer vaccination, ORT technology, cytotoxicity