Development of Cas9 protein based transcription regulators for the regulation of regulatory T-cells.

Avtoimunske bolezni, med katere spada tudi diabetes tipa 1, se pogosto razvijejo kot posledica upada mehanizmov imunske tolerance. Pomemben del slednje predstavljajo podskupina T limfocitov, imenovanih regulatorne T celice (Treg), ki s svojo imunosupresivno funkcijo preprečujejo avtoimunsko delovanje lastnih celic in katerih umanjkanje ali nedelovanje je lahko pogosto pomemben faktor v razvoju avtoimunskih bolezni. Za njihov razvoj iz timocitov je ključno stabilno izražanje transkripcijskega faktorja FOXP3, ki deluje kot glavni regulator diferenciacije Treg. Zadostna aktivacija izražanja FOXP3 v drugih T-limfocitih ali celo sorodstveno bolj oddaljenih celicah bi lahko bil zadosten signal za diferenciacijo v Treg in pridobitev imunosupresivnega fenotipa. V magistrskem delu smo poskušali ustvariti in preizkusiti transkripcijske dejavnike, vezane na Cas9 proteine, za specifično aktivacijo izražanja FOXP3. Za usmerjanje Cas9 proteinov na tarčno mesto smo ustvarili različne vodilne RNA (sgRNA) molekule, ki so prepoznale specifična mesta na ključnih regulatornih mestih gena FOXP3. S poskusi in vivo smo dosegli visoko povišanje ob uporabi trodelnega aktivatorja VPR, vezanega na Cas9 protein z izničeno nukleazno aktivnostjo (dCas9:VPR). Najvišjo aktivacijo izražanja FOXP3 smo dobili ob ciljanju promotorske regije gena FOXP3 ter regulatorne regije, katero smo poimenovali Cage1. Po optimizaciji aktivacije izražanja smo prav tako preverili spremembo izražanja ključnih tarčnih genov proteina FOXP3 v celicah, ki imajo FOXP3 utišan. Uvodni poskusi aktivacije izražanja FOXP3 v celični liniji HEK293T so pokazali določeno mero ujemanja izražanja tarčnih genov s Treg celicami. Z nalogo smo pokazali moč metode za tarčno aktivacijo izražanja ter potrdili pomembnost regije Cage1 za tarčno aktivacijo. Metoda bi lahko bila močno orodje pri poskusih diferenciacije celic v Treg, kar bi lahko bilo zlasti pomembno pri terapijah za razne avtoimunske bolezni.Autoimmune diseases, such as type 1 diabetes, often arrise as a result of a defect in one or mechanisms of immune tolerance. An important part of immune tolerance are a subpopulation of T cells, called regulatory T cells (Treg), which prevent the autoimmune activity of aggresive immune cells, and whose depletion or inactivity can lead to severe autoimmune reactions. Stable expression of the master regulator transcription factor FOXP3 is key for their development from thymocytes. Stable induction of FOXP3 expression could potentially drive the differentiation of other T cell population, as well as other cells, towards a Treg lineage and an acquisition of an immosupresive phenotype. In my masters thesis, we aimed at developing and testing transcription activators, bound to modified Cas9 proteins, for the specific induction of FOXP3 expression. We have designed novel short guide RNA (sgRNA), that direct the Cas9 to specific sites on key regulatory regions of the FOXP3 gene. We have achieved high in vivo expression induction of FOXP3 with the use of a tripartite transcription activator VPR, bound to a nuclease-null mutant of Cas9 protein (dCas9:VPR). The highest induction of expression was obtained when we used dCas9:VPR in combination with sgRNA which target the core promotor of FOXP3 and a regulatory region, which we termed Cage1. After the success in achieving transcription activation, we wanted to check the expression profile of key FOXP3 target genes in cells, which normally do not express FOXP3. Initial experiments in HEK293T cell line showed a certain degree of correlation of gene expression to that of Treg cells, after the activation of FOXP3 expression. With our work we have presented a powerfull tool for targeted gene expression, as well as the imporatance of the Cage1 region for the activation of FOXP3 expression. Our tool could be used in further experiments which aim at the generation of Treg cells, which could potentially be used to treat different autoimmune diseases

Development of a fast proteolysis-based response system for the secretion of proteins from the endoplasmic reticulum

Izločanje topnih ter premikanje membranskih proteinov na površino celic je pomemben celični proces, s katerim celice nadzorujejo množico različnih funkcij, kot so medcelična signalizacija, hormonska regulacija, prenos signala med sinapsami, homeostaza, obramba pred patogeni idr. Nadzorovanje izločanja proteinov s sintezno-biološkimi sistemi se ponuja kot obetavno orodje za obvladanje različnih bioloških procesov ter za uporabo v terapevtske namene. Večina do sedaj razvitih sistemov za nadzorovanje izločanja proteinov iz sesalskih celic temelji na indukciji izražanja izločenih proteinov, ki nato potujejo do zunanjosti celice po konvencionalni sekretorni poti. Vendar sta za takšen pristop potrebni predhodna transkripcija in translacija proteina, ki šele nato začne vstopati v sekretorno pot, zaradi česar se začne protein kopičiti v biološko relevantnih količinah šele več ur po indukciji izražanja proteina. Za nekatere, zlasti terapevtske aplikacije, pri katerih je potreben odziv v nekaj minutah, ne urah, je takšen odziv prepočasen. V tem doktorskem delu smo razvili dva sistema – lumER in membER – za izločanje topnih in membranskih proteinov, pri čemer smo izločanje nadzorovali s pomočjo inducibilnih proteaz. V obeh sistemih se izločeni proteini predhodno izrazijo ter se zadržijo na konvencionalni sekretorni poti v endoplazemskem retikulumu s pomočjo kratkih C-končnih zadrževalnih aminokislinskih zaporedij. Odstranitev zadrževalnih zaporedij je pod nadzorom ortogonalnih virusnih proteaz iz družine Potyviridae, katerih aktivnost lahko reguliramo z malimi molekulami. Ker nadzor izločanja poteka na ravni posttranslacijskih modifikacij, sistema povsem zaobideta potrebo po sintezi in akumulaciji proteina po indukciji, zaradi česar sta hitrejša od primerljivih sistemov, ki temeljijo na indukciji izražanja izločenega proteina. Pokazali smo, da sta oba sistema med sabo ortogonalna ter da lahko pri obeh sistemih nadzorujemo izločanje s pomočjo ortogonalnih in inducibilnih virusnih proteaz. Sistema omogočata večkratno zaporedno induciranje izločanja. Nadalje smo pokazali, da lahko izločanje s sistemoma membER in lumER nadzorujemo s konstrukti za procesiranje vhodnih signalov po načelu dvovhodnih logičnih operacij. Na koncu smo sistema uporabili za nadzorovanje izločanja terapevtsko relevantnih proteinov. Ker sta zadrževanje ter izločanje zagotovljena z modularnimi elementi, je mogoče sistema povezati tudi z drugimi sinteznimi in naravnimi sistemi, ki temeljijo na proteolizi, prav tako pa sta sistema primerna za prenos na številne druge topne in membranske proteine, zaradi česar se ponujata kot močno orodje za številne potencialne aplikacije.The secretion of soluble proteins and the movement of membrane proteins to the cell surface is an important process with which cells regulate a plethora of different functions, such as intercellular communication, hormonal regulation, signal transfer between synapses, homeostasis, host defence against pathogens etc. Controlling protein secretion with synthetic biological systems thus represents an attractive tool to regulate different biological processes and for use in therapeutic applications. Currently, most synthetic systems for regulated secretion from mammalian cells work by inducing the expression of the secreted protein, which then travel through the conventional secretory patway to the cells exterior. However, in this approach, the protein must first be transcribed and translated, before it can enter the secretory pathway and start to accumulate in the cells exterior, a process which can take several hours before the protein is secreted in biologically relevant amounts. For certain applications, especially therapeutic, where a response needs to occur within minutes, not hours, this delay in response makes secretions with such systems too slow for practical use. In the present doctoral dissertation we have developed two systems – lumER and membER – for regulated secretion of soluble and membrane proteins, in which secretion is controlled by inducible proteases. In both systems the secreted protein is expressed and retained in the conventional secretory pathway in the endoplasmic reticulum, with the help of appended C-terminal retention sequences. The removal of the retention sequence is under the control of orthogonal viral proteases from the Potyviridae family, whose activity is regulated by small molecules. Because secretion is regulated at the posttranslational level, both systems bypass the need for prior protein synthesis and are therefore faster than comparable systems based on regulating protein expression. We have shown that both systems are orthogonal to each other and are controllable with different orthogonal and inducible viral proteases. Both systems allow for repeated induction of secretion. Further, we have shown that secretion with the membER and lumER systems can be controlled by constructs used for two input logical operation processing. Finally, we have used the systems to control the secretion of therapeutic proteins. Because ER retention and secretion are achieved with the help of modular elements, the systems allow for their combination with other synthetic and natural systems, that rely on proteolysis, and can be extended to feature other soluble and membrane proteins, making both systems a powerfull tool for different applications

Robust saliva-based RNA extraction-free one-step nucleic acid amplification test for mass SARS-CoV-2 monitoring

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections