RNA technologies - Fraunhofer ITEM

RNA for targeted therapies

Um die molekularen Signalwege in gesunden und kranken Herzen besser zu verstehen, analysieren Forschende nicht-codierende Mikro-RNAs mithilfe der Real-time-PCR. Mikro-RNAs, die im Zusammenhang mit der Krankheit stehen, wie etwa die Mikro-RNA-132, können somit identifiziert werden. — © Fraunhofer ITEM, Ralf Mohr
In order to better understand the molecular signaling pathways in healthy and diseased hearts, researchers analyze non-coding microRNA molecules by real-time PCR.

© Fraunhofer ITEM, Ralf Mohr
Process development and GMP production of mRNA therapeutics for early-phase clinical trials can take place directly at Fraunhofer ITEM.

RNA-based therapeutics are a drug class with huge potential for medicine — and scientists have barely scratched the surface of their possible use. Fraunhofer ITEM researchers develop novel drugs and methods for RNA-based therapeutic concepts.

The targeted use of RNA compounds based on coding or non-coding RNA sequences enables a tailored response of the respective target cells under certain pathological conditions. Fraunhofer researchers use a wide range of RNA-based compounds, such as small interfering RNA, nucleoside-modified messenger RNA or RNA blockers — from target discovery through to preclinical development programs. Bioinformatic models play a key role when it comes to selecting disease-associated RNAs, studying their interaction with other genes, and optimizing drug design.

Special viral and non-viral administration technologies, both at the molecular and equipment level, are being developed for the targeted use of RNA compounds. Local delivery via the airways is a promising solution in particular for RNA therapeutics used to combat lung diseases.

Safety and efficacy testing of RNA compounds

For safety and efficacy testing of therapeutics, researchers use proven in-vitro and in-vivo animal models and are continuously developing new model systems that are based on human tissues as well as mini organs and cells derived from stem cells.

Process development and GMP production of RNA-based drugs

Researchers at Fraunhofer ITEM are developing bioprocessing methods and production technologies for the modular and automated manufacture of RNA molecules and RNA nanocarriers that can be scaled right up to industry level. Rapid, safe and reliable production technologies for the manufacture of RNA-based vaccines and drugs are a fundamental requirement for successful translation into marketable products. To support this, process development and GMP production of mRNA therapeutics for early-phase clinical trials take place at Fraunhofer ITEM, and the early-phase clinical trials can also be conducted in-house.

RNA molecules as biomarkers

RNA molecules are attracting increasing interest for diagnostic purposes as well. The expression profiles of RNA molecules are altered in many pathologies, such as lung diseases and cancer. RNA can be obtained from various liquid biopsies, such as blood, urine or cerebrospinal fluid. Taking a liquid biopsy is much less invasive for patients than a tissue biopsy. Next generation sequencing technologies allow the RNA composition of a sample to be determined. RNA expression profiles can thus be used as biomarkers to characterize diseases, predict the response to a specific therapy, and monitor treatment success. Fraunhofer ITEM researchers have developed technologies that enable the analysis of even very small quantities of RNA as well as determination of the expression profiles of single cells or cell-free, circulating RNA.

Our technologies and methods

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Target Discovery

Identification of target structures

Computer-based models can provide clues to molecular RNA targets in different human organ pathologies.

Based on these structures, RNA drug candidates can be synthesized and subsequently validated in translational model systems.

LEAD OPTIMIZATION

Lead optimization

Analytical methods are essential for the optimization of RNA-modulating agents. Oligonucleotide-based binding assays are used to validate the structure of the active ingredient in advance and adjust it accordingly.

The ensuing efficacy testing and pharmacokinetics studies are also performed with the help of bioanalytical methods. Microscale thermophoresis (MST) and HPLC platforms are available as well.

EFFICACY TESTING AND RNA-BASED ANALYSES

Efficacy testing in human model systems and RNA-based analytical methods (PK/PD studies)

Human and animal tissue culture models

Heart

Cell cultures
Cardiac tissue slices
3D organoid models

Lung

Cell cultures
Isolated perfused lung
Precision-cut lung slices (PCLS)
3D organoid models

Liver

Cell cultures
Precision-cut liver slices(PCLiS)

Toxicology

Toxicology (in vitro/in vivo)

Preclinical testing in vivo
Preclinical tests using different in-vitro and ex-vivo lung and heart models
Safety pharmacology
Toxicological risk assessment
- Identification and use of predictive biomarkers for toxicological endpoints
- Generation of mechanistic data to support next generation risk assessment
- Assessment of genotoxic impurities according to ICH M7, including review of literature, QSAR analysis, and PDE derivation

MANUFACTURE

mRNA manufacture

Plasmid production and purification up to 400 L fermentation scale; Fraunhofer ITEM holds an EU GMP manufacturing authorization
Enzymatic in-vitro transcription, functionalization, and purification of mRNA as active pharmaceutical ingredient (up to gram scale)
Microfluidic formulation of mRNA LNPs as formulated bulk
Automated or manual aseptic filling of mRNA LNPs as medicinal products in up to 7000 10R vials (grade A (EU) / class 100 (US) in grade-B clean-room environment, validated media fill). Other filling formats are available on request.
QC analyses for IPC and release testing of medicinal products in-house or, when necessary, by qualified external service providers
In-house QP available, mRNA therapeutics can be released and supplied as IMP for clinical use

RNA targeted delivery

Different technologies and cardiopulmonary 2D and 3D model systems are used to develop the targeted, organ-specific delivery of RNA therapeutics.

Bioanalytics for therapeutic RNA interaction with the target sequence
Manufacturing and validation of nanoparticles as carriers of RNA therapeutics
Biological validation of RNA-nanoparticle complexes
Development of cell-specific nanoparticles
Nebulization of RNA-nanoparticle complexes

Clinical trials phases I/II

Proband wird von Studienarzt untersucht und informiert — © Fraunhofer ITEM, Ralf Mohr

Phase-I unit and infrastructure for clinical research
Broad range of diagnostic methods for safety and efficacy assessment
- MRI to visualize lung ventilation and blood flow
- ECG, echocardiography, and telemetry
Controlled aerosol inhalation
Broad range of challenge models for early proof of concept

RNA research: recent projects and highlights

Circular RNA: an innovative RNA therapy agent?

Project RNAuto

Fraunhofer develops automated production technologies for mRNA-based drugs.

RNAi therapy to treat viral respiratory diseases

In the iGUARD project, a team from Fraunhofer ITEM and MHH is working on the rapid development of customized RNA-based antiviral therapies.

Platform for RNA-based therapeutics

The Fraunhofer Cluster of Excellence Immune-Mediated Diseases CIMD has been funding the development of novel RNA-based therapeutic concepts.

Project ZET-O-MAP

Researchers from Fraunhofer ITEM have succeeded in developing an analysis pipeline to identify biomarkers for developmental toxicity.

RNA drug against cardiac fibrosis

The EU is funding this research project with around 2.5 million euros.

Project archive

Here you can find more projects sorted by our research and development competences.

Types of RNA

There are many different types of RNA. The most widely known is messenger RNA (mRNA). It carries the genetic information needed to produce proteins, such as for the SARS-CoV-2 spike protein in coronavirus vaccines. But in addition to mRNA, a large number of other RNA molecules are present in the organism. These are referred to as non-coding RNAs, including, for example, microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA), and they play essential regulatory roles inside cells. Their dysregulation can result in a broad range of diseases, which is why they are crucial therapeutic targets.

Messenger RNA

Messenger-RNA (mRNA) is a single-stranded ribonucleic acid that carries and transmits genetic information for building a certain protein inside a cell.

mRNA-based drugs introduce the blueprint for a particular protein into cells and the cells will then synthesize this protein. In the case of mRNA vaccines, the synthesized protein serves as antigen, triggering an immune response to build protection.

MicroRNA

MicroRNA is a class of highly conserved, short, non-coding RNAs that play a crucial role in the complex network of gene regulation.

The various miRNA molecules regulate gene expression with a high degree of specificity at the post-transcriptional level and can thus serve as therapeutic targets.

Small interfering RNA

The term small interfering RNA (siRNA) refers to synthetically produced RNA molecules that are used in research, but also as therapeutic agents. siRNA molecules with any desired sequence can be produced by chemical synthesis.

To introduce siRNA into cells, a method known as liposomal transfection or lipofection is mainly used. It couples RNA with liposomes that fuse with the target cell membrane, thereby delivering the siRNA into the cell.

Circular RNA

Circular RNA (circRNA) is a type of single-stranded ribonucleic acid which, unlike linear RNA, forms a covalently closed continuous loop. Because circular RNA does not have 5' or 3' ends, it is resistant to exonuclease-mediated degradation and is presumably more stable than most linear RNA in cells.

circRNA is involved in gene regulation and has been linked to some diseases such as cancer. The biological function of most circRNA molecules is as yet unclear.

IncRNA

Long non-coding RNAs (lncRNA) are generally defined as transcripts with more than 200 nucleotides that are not translated into protein.

They are involved in gene regulation at the transcriptional, post-transcriptional and epigenetic levels and have an impact on DNA replication timing and chromosome stability.

modRNA

Nucleoside-modified messenger RNA (modRNA) is a synthetic, chemically modified mRNA, in which some nucleosides have been replaced by other naturally modified nucleosides or by synthetic nucleoside analogues.

Such RNA molecules are used in experimental or therapeutic contexts to induce the production of a desired protein in certain cells.

Publications

Ahangari, F., Price, N. L., Malik, S., Chioccioli, M., Barnthaler, T., Adams, T. S., Kim, J., Pradeep, S. P., Ding, S., Cosmos, C., Jr., Rose, K. S., McDonough, J. E., Aurelien, N. R., Ibarra, G., Omote, N., Schupp, J. C., DeIuliis, G., Villalba Nunez, J. A., Sharma, L., Ryu, C., Dela Cruz, C. S., Liu, X., Prasse, A., Rosas, I., Bahal, R., Fernandez-Hernando, C., Kaminski, N. (2023). microRNA-33 deficiency in macrophages enhances autophagy, improves mitochondrial homeostasis, and protects against lung fibrosis. JCI Insight 8(4). doi: 10.1172/jci.insight.158100 https://insight.jci.org/articles/view/158100 - Open Access
Biss, S., Teschler, M., Heimer, M., Thum, T., Bar, C., Mooren, F. C., Schmitz, B. (2023). A single session of EMS training induces long-lasting changes in circulating muscle but not cardiovascular miRNA levels - A randomized crossover study. J Appl Physiol. [Epub ahead of print]. doi: 10.1152/japplphysiol.00557.2022 https://journals.physiology.org/doi/abs/10.1152/japplphysiol.00557.2022
Kandil, R., Baldassi, D., Bohlen, S., Muller, J. T., Jurgens, D. C., Bargmann, T., Dehmel, S., Xie, Y., Mehta, A., Sewald, K., Merkel, O. M. (2023). Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma. Journal of Controlled Release 354: 305-315. doi: 10.1016/j.jconrel.2023.01.014 https://www.sciencedirect.com/science/article/abs/pii/S0168365923000159?via%3Dihub
Lu, D., Cushman, S., Thum, T., Bär, C. (2023). Gene Therapy and Cardiovascular Diseases. In: Genome Editing in Cardiovascular and Metabolic Diseases. Advances in Experimental Medicine and Biology. Xiao, J. (ed.) Springer, Singapore 1396: 235-254.
Neufeldt, D., Cushman, S., Bär, C., Thum, T. (2023). Circular RNAs at the intersection of cancer and heart disease: potential therapeutic targets in cardio-oncology. Cardiovascular Research [Epub ahead of print]. doi: 10.1093/cvr/cvad013 https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvad013/6991260?redirectedFrom=fulltext
Thum, T., Lam, C. S. P. (2023). Accelerating developments in heart failure. Cardiovascular Research 118(18): 3401-3402. doi: 10.1093/cvr/cvac185 https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac185/6965791 - Open Access