Research intents

RNAs play a key role in all biological processes, and therapeutics based on modified RNA molecules or molecules that regulate RNA transcription, splicing or processing, or translation can be used in the treatment of many diseases, including those for which there are no effective therapies to date.

The aim of the project is research, divided into three research intents (RI), which should lead to new types of RNA therapeutics:

• Transcription and its regulation (RI1)
• RNA modifications and mechanisms of post-transcriptional regulation (RI2)
• Control of translation and protein synthesis by rational design (RI3)

The study of transcription and the design and study of DNA modifications to regulate the transcription of certain genes. The results of the regulation will be used to design potential therapeutics that prevent or enhance protein expression. The goal may be to design next-generation antibiotics or more efficient gene repair for gene therapy.

Traditionally, most RNA-based drugs have targeted cytoplasmatic molecular mechanisms, for example various antisense mechanisms or transient translation of mRNA-based drug in vivo for vaccination. To expand the portfolio of RNA-based drugs, the RI attempts to therapeutically harness nucleic acid analogues to mimic or antagonize endogenous RNA processes associated with transcription. As the proposed research direction is at the limit of current technology, the RI1 aims at:

• development of new approaches to the chemical and enzymatic synthesis of various nucleobase- and/or sugar-modified RNA molecules;
• development of novel robust transcriptional systems that can synthetize RNA analogues (including XNA) that carry the chemical modifications for the use in RNA therapeutics and biomedical applications;
• designing and studying novel RNA drug candidates to target helicases and nucleases which control transcriptional processes;
• studying transcriptional regulators in bacteria, especially those involved in virulence and essential ones that might represent targets for the development of new antibiotics;
• studying transcriptional arrest or modulation when encountering specific DNA modifications;
• the modified RNA and XNA developed in this RI will also be used in RI2 and RI3 to regulate processing of RNA or translation.

Study of mRNA splicing and processing and study of RNA interference and regulatory functions of RNA. The results may lead to the design of RNA therapeutics for the treatment of diseases arising from poor editing or reading frame shift, viral infections, genetic diseases or cancer.

Newly synthesized RNA needs to be properly processed and modified to fulfil its biological role and maintain control of cellular homeostasis. Work package 2 (RI2) of RNA4T project explores the enormous space of post-transcriptional regulations of RNA and seeks to develop new applications in this area. The immense diversity of this field is mirrored by diversity of research areas covered by participants and by their model systems and methodologies.

The RI2 has been divided into two main objectives, which represent major molecular themes with application and/or therapeutic potential: I. Exploring and exploiting chemical modifications of RNA and II. Dissecting and targeting medically important ribonucleoprotein (RNP) complexes. The broad and diverse RI2 program includes research on human pathologies associated with defects in RNA metabolism and on specific adaptations in RNA metabolism of human pathogens. Activities coupled with main objectives will aim at high relevance for medicine and practical applications, namely:

• identification of the role of NAD capping of human snRNA and its relevance to metabolism of free NAD in various pathologies;
• identification of the role of 5’ NAD capping of RNAs in Bordetella pertussis and its medical relevance;
• development of RNA aptamers recognizing specific 5′ caps, which could serve for selective detection and targeting of specifically capped mRNAs;
• analysis of internal and terminal modifications of mammalian RNAs, their function in gene expression, and potential for mRNA-based therapy;
• development of synthetic antisense oligonucleotides interfering with 3' RNA binding factors in trypanosomatids and assessment of their ability to serve as RNA-based inhibitors of the parasites;
• deciphering the nexus between mutations in spliceosome RNPs and development of retinitis pigmentosa and investigating ability of modified oligonucleotides to relieve impact of the mutations;
• solving structures of RNA editing RNPs in trypanosomatids & identifying their druggable components;
• developing and testing new chemically modified siRNA and miRNA therapeutics;
• determining the spectrum of defects caused by AGO & Dicer mutations in human pathologies;
• engineering the smallest effective variant of mammalian Dicer and test its antiviral role and ability to remedy Dicer mutations in cancer cells and Dicer deficiency in the retinal pigment epithelium.

Taken together, RI2 will help to understand molecular causes of pathologies linked to RNA metabolism. It will aim at uncovering new therapeutic paths and development of strategies targeting or modifying RNAs and specific aspects of their metabolism with application potential in biotechnology and medicine.

The study of translation and regulation and the study of more stable and efficient mRNA derivatives and analogues will enable the design and development of effective mRNA therapeutics for virtually any disease caused by abnormal expression of a protein, RNA vaccines or regulation of protein expression

Protein synthesis/translation captures the flow of genetic information from DNA into proteins and determines its egulá output (generated proteome) under defined conditions. This work package (RI) aims to exploit egulátory mechanisms of translation towards production of stable and efficient custom-tailored RNA therapeutics with tunable properties to treat various diseases. The ultimate goal of this RI is to modulate efficiency of translation in a spatiotemporal manner and thus either mitigate or boost expression of a protein-of-interest in relevant pathogenic cells/tissues. Obtained results will also be utilized to develop new tools to study deregulated molecular mechanisms underlying more complex illnesses such as cancer.

Specifically, RI3 aims:

• to develop modified nucleic acids with minimal immunogenicity as tools to purposefully modulate efficiency of prokaryotic and eukaryotic translation in cell free systems, as well as in living cells, including the pathogenic ones. There is only very limited information on how modified or alternative nucleotides affect the mRNA’s stability and translatability. Our goal is to explore the applicability of the modified mRNAs developed here as RNA therapeutics;
• to design therapeutics, such as short modified RNA or XNA regulators, various cap analogues, as well as small molecule inhibitors to directly target factors facilitating individual steps of protein synthesis;
• to study cellular activation, proliferation and differentiation of wild-type naïve T-cells and transformative translatome changes manifested in various blood cancer cells, as well as the ability of these cells to translate modified mRNAs developed throughout this project as prospective therapeutics;
• to develop novel chemo-enzymatic approaches for synthesis of modified tRNAs to study the role of tRNA levels, trafficking and modifications in codon biased translation, a possibility of using the tRNA molecule as a specific therapeutic for the treatment of a wide range of genetic diseases, and the applicability of modified tRNAs as antiparasitic drugs.