Tarčna integracija heterolognih genov monoklonskega protitelesa v genom celic CHO

Vivod, Petra (author), Gunčar, Gregor (mentor)

Celice CHO sože vrsto let vodilni ekspresijski sistem za proizvodnjo bioloških zdravil. Trenutni razvoj celičnih linij temelji na naključni integraciji ekspresijskega vektorja v genom celic CHO, s čimer pridobimo klone, ki se med sabo zelo razlikujejo tako v stopnji kot tudi stabilnosti produkcije proteinov. Za razvoj visoko producirajoče celične linije, je tako potrebno generirati in presejati ogromno število klonov. Predpostavili smo, da bi s tarčno integracijo mAb ekspresijskega vektorja v regije genoma celic CHO, ki se nahajajo blizu super-ojačevalcev določenih z metodo ATAC-seq, lažje in hitreje razvili visoko producirajočo celično linijo. V okviru magistrske naloge smo želeli vzpostaviti enostaven in učinkovit način tarčne integracije. Za vstavitev lineariziranega mAb ekspresijskega vektorja v tarčno mesto smo izkoristili popravljalni mehanizem spajanja nehomolognih koncev, ki sodeluje pri popravilu dvojnega preloma DNA. Prelom dvojne vijačnice smo v celicah tarčno sprožili z uporabo orodja za urejanje genoma. Z razvito metodo smo dosegli učinkovitost tarčne integracije med 3 in 11 %, odvisno od učinkovitosti delovanja sistema za urejanje genoma in tarčne regije. V drugem delu magistrske naloge smo pri klonih s tarčno integriranim ekspresijskim vektorjem ovrednotili vpliv super-ojačevalcev na izražanje heterolognih genov ter na produktivnost posameznih klonov. Pokazali smo, da s tarčno integracijo dosežemo 1,7-krat višjo stopnjo izražanja heterolognih genov ter 1,5-krat višjo specifično produktivnost. Z analizo,v kateri smo ugotavljali stopnjo izražanja heterolognega gena na posamezno kopijo ekspresijskega vektorja pa smo ugotovili, da med kloni s tarčno in naključno integracijo ni statistično pomembne razlike. Poleg tega smo pokazali, da z metodo tarčne integracije na osnovi popravljalnega mehanizma spajanja nehomolognih koncev v genom celic CHO integriramo večje število kopij heterolognega gena kot z naključno integracijo. V okviru magistrske naloge smo torej uspeli razviti način za pripravo celičnih linij z izboljšano produktivnostjo.

• Language: Slovenian
• Keywords: tarčna integracija, NHEJ, super-ojačevalec, celice CHO
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-113722
• COBISS.SI-ID: 1538524611
• Publication date in RUL: 29.01.2020

Secondary language

• Language: English
• Title: Targeted integration of heterologous monoclonal antibody genes into the genome of CHO cells

Abstract

For many years, CHO cells have been the leading expression system for the production of biological drugs. The current cell line development process is based on the random integration of the expression vector into the genome of CHO cells, which leads to the generation of clones that differ significantly in both the rate and stability of protein production. In order to develop a highly producing cell line, it is necessary to generate and screen a huge number of clones. We hypothesized that targeted integration of the mAb expression vector into regions of the genome located close to the super-enhancers, determined by the ATAC-seq method, would allow easier and more rapid development of a high-producing cell line. As part of the master's thesis, we wanted to establish a simple and effective targeted integration approach. For the insertion of a linearized mAb expression vector into the target site, we utilized a DNA double-strand break caused by genome editing tool, which is repaired by the non-homologous end joining(NHEJ). With the developed method, we achieved between 3 and 11 % efficiency of targeted integration, depending on the activity of the genome editing tool and the target region. In the second part of the thesis, we evaluated the influence of super-enhancers on the expression of heterologous genes and the productivity of individual clones with a target-integrated mAb expression vector. Targeted integration has been shown to increasethe expression of heterologous genes and specific productivity of generated clones by 1,7-foldand 1,5-fold, respectively, when compared to random integration. However, when analyzing the mRNA expression levels per copy of the heterologous gene, no statistically significant difference between clones with targeted and random integration was observed. Furthermore, we have shown, for the first time,that when using NHEJ based targeted integration of expression vector into the CHO cell genome, more copies of heterologous genes are introduced into the genome compared to random integration, leading to higher specific productivity. In summary, we developed a targeted integration approach for generating production cell lines with improved productivity.

• Keywords: targeted integration, NHEJ, super-enhancers, CHO cells