Optimizacija reakcije transkripcije in vitro s programom za načrtovanje eksperimentov

Valič, Tjaša (author), Bratkovič, Tomaž (mentor), Sekirnik, Rok (co-mentor)

Razvoj mRNA-cepiv med pandemijo SARS-Cov-2 je povzročil pravo revolucijo v medicini, pri čemer so se odprle neštete možnosti za različne terapevtske aplikacije molekule mRNA. Za učinkovito in poceni proizvodnjo čiste mRNA je ključnega pomena razumevanje procesnih parametrov transkripcije in vitro (IVT), reakcije, s katero pridobivamo terapevtsko mRNA. Med potekom reakcije poleg tarčne mRNA nastanejo tudi neželeni produkti, najbolj problematična je imunogena dvoverižna RNA (dsRNA). Nastanek le-te je odvisen od nukleotidnega zaporedja mRNA, vpliv pa imajo lahko tudi parametri reakcije IVT. S programom za načrtovanje eksperimentov MODDE smo za genski konstrukt eGFP (tj. gen za zeleni fluorescenčni protein) ustvarili model reakcije IVT. Spreminjali smo množinsko razmerje med Mg2+ in nukleotid trifosfati (NTP), koncentracijo NTP, matrične DNA in RNA-polimeraze T7 (RNAP). Koncentracijo nastale mRNA in porabo NTP smo spremljali s HPLC analitiko, vsebnost dsRNA v končnem produktu RNA pa z metodo točkovnega nanosa. Želeli smo raziskati, pri katerih pogojih bodo produkcija mRNA, izkoristek reakcije in stroškovna učinkovitost čim višji, vsebnost imunogene dsRNA pa čim nižja. Za ovrednotenje modela smo si pomagali s statistično analizo, ki nam jo omogoča program, in na podlagi rezultatov analize model izboljšali z dodatnimi eksperimenti. Rezultati so se izboljšanemu modelu dobro prilegali, poleg tega je imel model visoko napovedno moč, kar pomeni, da modelu lahko zaupamo in z njim napovemo nove rezultate. Pokazali smo, da za visoko produkcijo mRNA potrebujemo množinsko razmerje med Mg2+ in NTP okrog 1, visoke koncentracije plazmida in RNAP, tako prenizke kot previsoke koncentracije NTP pa produkcijo mRNA nižajo. Pokazali smo tudi, da višja produkcija mRNA ne pomeni nujno tudi dobre stroškovne učinkovitosti. Nadalje smo ugotovili, da višje množinsko razmerje med Mg2+ in NTP vodi v večjo vsebnost dsRNA v vzorcu, visoke koncentracije NTP pa jo znižajo. S tem smo pokazali, da se visoka produktivnost reakcije in nizka vsebnost dsRNA ne izključujeta. Optimalno šaržno reakcijo je model predvidel pri pogojih, ko množinsko razmerje med Mg2+ in NTP znaša 0,9, koncentracija NTP 10,4 mM, koncentracija plazmida 100 ng/μL in koncentracija RNAP 8,1 U/μL. V 90 min smo proizvedli 10 mg/mL mRNA z nizko vsebnostjo dsRNA in tako eksperimentalno pokazali, da se dobljeni rezultati ujemajo s predvidenimi. S pomočjo ugotovitev, ki smo jih pridobili na podlagi modela, smo nazadnje naredili načrt in izvedli reakcijo z dohranjevanjem ter tako še povečali produktivnost in stroškovno učinkovitost reakcije.

• Language: Slovenian
• Keywords: transkripcija in vitro, mRNA, RNA-polimeraza T7, dsRNA, načrtovanje eksperimentov
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-150105
• Publication date in RUL: 14.09.2023

Secondary language

• Language: English
• Title: Optimization of in vitro transcription reaction with design of experiments

Abstract

Development of mRNA vaccines during SARS-Cov-2 pandemics led to a revolution in medicine and options for therapeutic applications of mRNA molecules now seem limitless. The key to efficient and cost-effective mRNA production lies in a good understanding of both product- and process parameters involved in mRNA synthesis during in vitro transcription reaction (IVT). During IVT reaction not only target mRNA, but also by-products are formed, such as double-stranded RNA (dsRNA) which is a very potent immune system stimulant. dsRNA formation is dependent on mRNA sequence but can also be affected by IVT reaction parameters. In this study, we built a model of IVT reaction for enhanced green fluorescent protein (eGFP) gene construct with MODDE, a Design-of-Experiments software. Factors which were investigated comprised the ratio between concentration of Mg2+ and total nucleotide triphosphate (NTP) concentration, absolute concentrations of NTPs, DNA template and T7 RNA polymerase (RNAP). mRNA concentration and NTP consumption were measured by HPLC analysis and dsRNA content was estimated with the dot blot method. The aim of the study was to investigate which settings lead to the highest mRNA production, highest yields, most cost-effective mRNA production and lowest dsRNA content. We statistically evaluated the model and improved it with additional reactions. Results fit the model well and the model showed a high prediction power for further experiments. We showed that for high mRNA production, Mg2+ to total NTP molar ratio of approximately 1 and high concentrations of plasmid and RNAP are required, while both too low and too high concentrations of NTPs, lead to lower mRNA production. We observed that high mRNA production does not necessarily result in high cost-effectiveness. Furthermore, we showed that higher Mg2+ to total NTP molar ratio increases dsRNA content while higher NTP concentrations decrease it. This means that high mRNA production and low dsRNA content can be achieved during similar conditions of IVT reaction. Optimal batch reaction designed using the model was predicted at Mg2+ to total NTP molar ratio of 0,9, NTP concentration of 10,4 mM, plasmid concentration of 100 ng/μL and RNAP concentration of 8,1 U/μL. We produced 10 mg/mL of mRNA in 90 min and demonstrated that experimental results of the reaction match well with the results predicted by the model. Taking into account our findings, we performed fed-batch reaction with which we further improved mRNA production and cost-effectiveness of the process.

• Keywords: in vitro transcription, mRNA, T7 RNA polymerase, dsRNA, design of experiments