Title High-throughput microRNA screening identifies potential CHO cells engimiRs for enhancement of recombinant protein production Title (croatian) Visoko-protočno pretraživanje za identifikaciju microRNA molekula u cilju poboljšanja CHO proizvodnje rekombinantnih proteina Author Ivan Labik Mentor Mirela Sedić (mentor)Committee member Mirela Sedić (predsjednik povjerenstva)Committee member Ivana Ratkaj (član povjerenstva)Committee member Sandra Kraljević Pavelić (član povjerenstva)Granter University of Rijeka Defense date and country 2016-09-20, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Global demand for biopharmaceuticals is increasing and requires constant improvement in manufacturing processes. Chinese hamster ovary (CHO) cells are dominant host for production of recombinant therapeutic proteins with human-like post-translational modifications. In the past, engineering of mammalian cell systems was focused on the manipulation of single target genes or proteins. Although cell performance is enhanced, numerous limitations and bottlenecks remain. Therefore, novel technologies
... More and improvements in biopharmaceutical manufacturing processes are necessary to address this issue. Several unique characteristics make microRNAs (miRNAs) attractive alternative for CHO cell engineering. MicroRNAs are small non-coding RNAs that regulate gene expression at post-transcriptional level. Mature miRNA can simultaneously repress or degrade multiple target mRNAs involved in many cellular processes such as proliferation, apoptosis, cell cycle, metabolism, and protein synthesis. Importantly, mature miRNAs are highly conserved between species and due to low-target-specificity, they are capable of regulating hundreds of genes. Hence, several reports showed successful application of miRNA (engimiR) in CHO optimizations. However, due to insufficient improvement and cell line or recombinant protein dependency, none of them have made their way into industrial setting. The goal of this thesis was to explore phenotypic change after transient transfection with miRNA and identify engimiRs that improve CHO cell-specific productivity and viable cell concentration (VCC). Following protocol set-up, successful transfection of CHO cell line expressing recombinant erythropoietin (rEPO) with five pre-selected miRNAs confirmed proof-of-principle. Cell counting and ELISA assay provided insight into the effect of miRNA transfection on the VCC and rEPO concentration. In addition, functional high-throughput miRNA screening of non-viral delivery of human miRNA mimics library to CHO-rEPO was employed. Using this approach, 26 from 2042 miRNAs were identified to improve rEPO concentration at the cellular level by 3.5 to 6.0-fold without decreasing VCC. Highest effect, 6-fold increase in cell-specific productivity was detected after transient transfection with miR-3153. Finally, bioinformatics tools miRBase and miRWalk were utilized to predict conservation of human miRNA in CHO cells and to identify potential targets. Further studies are necessary in order to validate results and to select cell line and protein independent engimiRs in large-scale production. Less Abstract (croatian) Globalna potražnja za biološkim lijekovima je u porastu i zahtjeva stalno unapređivanje proizvodnih procesa. Stanice ovarija kineskog hrčka (CHO) su najčešće korišteni domaćini u proizvodnji rekombinantnih proteina zbog post-translacijskih modifikacija sličnih ljudskima. U prošlosti, inženjerstvo staničnih sustava je bilo usmjereno prema manipulaciji pojedinačnih gena ili proteina. Iako je stanična učinkovitost time poboljšana, brojna ograničenja su preostala. Stoga, nužna je primjena novih
... More tehnologija i unapređivanje proizvodne bioloških lijekova. Nekoliko jedinstvenih osobina čine mikro RNA (miRNA) primamljivom alternativom za inženjerstvo CHO stanica. Mikro RNA su male, nekodirajuće RNA molekule koje reguliraju gensku ekspresiju na post-transkripcijskoj razini. Zrela miRNA može istovremeno potisnuti ili razgraditi nekoliko mRNA meta uključenih u različite stanične procese, poput proliferacije, apoptoze, staničnog ciklusa, metabolizma i sinteze proteina. Nadalje, miRNA su visoko-konzervirane među vrstama i radi niske specifičnosti, sposobne su regulirati stotine gena. Nekoliko istraživačkih grupa je pokazalo uspješnu primjenu miRNA u optimizaciji CHO stanica. Međutim, nedovoljna efikasnost i ovisnost o korištenoj staničnoj liniji i rekombinantnom proteinu su razlog izostanka industrijske primjene. Cilj ovog rada je bio istražiti fenotipske promjene uslijed prolazne transfekcije s miRNA i identificirati one koje će poboljšati produktivnost i rast CHO stanica. Učinkovitost uspostavljenog protokola je pokazana uspješnom transfekcijom pet odabranih miRNA u CHO stanice koje izražavaju rekombinantni eritropoetin (rEPO). Brojanje stanica i ELISA esej su omogućili uvid u utjecaj miRNA transfekcije na koncentraciju živih stanica i eritropoetina. U nastavku, funkcionalni visoko-protočni skrining knjižnice ljudskih miRNA je primijenjen na CHO-rEPO stanicama. Takvim pristupom identificirano je 26 od 2042 miRNA koje su povećale razinu rEPO na staničnoj razini za 3.5 do 6 puta uz izostanak negativnog utjecaja na broj stanica. Najizraženiji utjecaj, povećanje za 6 puta je zabilježen nakon transfekcije s miR-3153. Konačno, bioinformatička analiza pomoću alata miRBase i miRWalk je korištena u svrhu predikcije konzerviranosti ljudskih miRNA u CHO stanicama te za identifikaciju potencijalnih meta. Dodatna ispitivanja su nužna kako bi se potvrdili dobiveni rezultati i kako bi se pronašla miRNA neovisna o staničnoj liniji i rekombinantnom proteinu te učinkovita u masovnoj proizvodnji. Less Keywords
CHO cells
MicroRNA
High-throughput screening
Cell-specific productivity
Viable cell density
EngimiR
Keywords (croatian)
CHO stanice
MikroRNA
Visoko-protočno pretraživanje
Stanično-specifična produktivnost
Koncentracija živih stanica
Language english URN:NBN urn:nbn:hr:193:168706 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Closed access Terms of use Created on 2016-10-31 14:38:44
