To date, variants have been described in more than 500 human genes that can lead to inborn errors of immunity (IEI). In addition to deficiencies of the immune system, both autoimmunity and auto-inflammation can be part of the clinical manifestations of patients. The diagnosis of these diseases includes genetic analysis, in which the variants in the genomic DNA of the patients are detected, as well as the elucidation of the pathomechanistic processes that lead to the various diseases in the presence of the individual gene variants.

These pathophysiological analyses can sometimes be very complex and can lead to the use of cell models, such as human inducible stem cells, into which the individual variants can be introduced for subsequent experimental analyses using the CRISPR/Cas9 system. Time and again, new genetic defects can be identified in patients and insights into the complex functioning of an intact human immune system can be gained from the identified disorders of immunobiological processes.

Early detection of bacteraemia, identification of the pathogen and possible accompanying pathogenicity factors and antimicrobial resistance are of great clinical importance. In the context of sepsis, for example, the survival rate drops by around 8% per hour if treatment is delayed. In the context of transfusion medicine, pathogen detection plays a role, for example, in the context of stem cell transplants or in the manufacture of blood products. Contamination of blood products, especially platelet concentrates, with bacteria, viruses and other pathogens is a serious problem that affects both the quality of the transfusion product and the safety of the recipient.

In the context of sepsis, the dilemma of delayed diagnosis of infectious diseases and sepsis in particular is reflected in the current diagnostic criteria for sepsis, which require a suspected or confirmed infection in combination with an increase of two points in the Sequential Organ Failure Assessment (SOFA) score. As the name suggests, this score increases when organ failure is already recognisable. The department is developing new methods for this problem.

It is estimated that there are around 10,000 monogenetic human diseases. These include X-linked severe combined immunodeficiency (X-SCID), which accounts for around 50 % of all SCID cases. It is caused by mutations in the IL2RG gene. The availability of the CRISPR/Cas system has now given a significant boost to research into non-viral gene correction approaches for such diseases. Prime editing (PE) is a promising platform, not least due to the use of a Cas9 variant that introduces a single-strand break ("nick") instead of a DNA double-strand break. Further improvements to the methodology are continually being introduced. These concern both the design of the PE components and the manipulation of the target cell metabolism, for example to eliminate undesirable effects of the intrinsically necessary "mismatch" repair mechanism.

The desired goal would be to correct mutations in the IL2RG gene in human ^CD34+ haematopoietic stem cells ex vivo with efficiencies > 50 %. After treatment, the cells should a) retain their stem cell properties and b) their capacity for long-term repopulation. Basically, we want to define a protocol that requires as few molecular tools as possible. The first task should therefore be to improve efficiency within the given experimental framework.

Third-generation sequencing (TGS) refers to a novel sequencing technology that can sequence DNA or RNA fragments in their entire length. While TGS has disadvantages in terms of processing time and throughput compared to conventional second-generation sequencing technology, TGS offers advantages in terms of sequencing complex genetic questions, as it is both a scalable technology and enables the analysis of long DNA or RNA fragments.

Long reads and other advantages enable a number of applications related to transfusion medicine. Current areas of application include the sequencing of microbial pathogens, including virulence and resistance determination. The technique is also used to resolve some genes such as the NCF1 gene, which is difficult to analyse using conventional short-read sequencing due to the problem of two pseudogenes. This and the resolution of haplotypes is also a field of application in blood group diagnostics, for example as shown in the Rhesus blood group system.

The flow cytometry method is mainly used in the department to analyse leukocytes, thrombocytes and stem cells. One specific application example is the elucidation of congenital defects of the immune system in order to quantitatively analyse haematopoietic cells using surface markers in a multi-parametric manner and to purify target cells by sorting. Four flow cytometers are available for this purpose: Two pure analysers with two lasers and five evaluation channels or with five lasers and 30 evaluation channels as well as two cell sorters with two lasers and eight evaluation channels or with three lasers and 15 evaluation channels.

Our department uses a broad repertoire of molecular genetic and molecular biological methods as part of its research and diagnostic work. A central group of molecular genetic and molecular biological methods involves working with nucleic acids and analysing them. This includes the polymerase chain reaction (PCR), which enables the selective amplification of certain segments of DNA. Numerous advanced variants of PCR, such as reverse transcriptase quantitative PCR (RT-qPCR), which enables the detection and quantification of specific RNA sequences, have been established in the department.

There is also a microbiological laboratory in which, for example, the recombinant expression of proteins from genetically modified E. coli bacteria is carried out. Other methods enable the expression products to be analysed: Western blotting, for example, allows the identification of the target protein in a protein mixture based on its size and specific fluorescence-coupled antibodies against the protein. These methods are frequently used in the department for the expression of recombinant Cas9 proteins. In combination with a target-specific guide molecule, the guide RNA (gRNA), targeted cuts can be made in the genome. With further modifications of the technique, even specific modifications of certain bases or the capture of targeted DNA segments from the entire genome are possible.

The department is also equipped for the development and realisation of cell-based assays. Several cell culture banks and incubators enable sterile cultivation of human cells. Various microscopes, including a laser scanning microscope, are available for follow-up analyses.

The availability of this wide range of established methods and equipment enables the department to work on a wide variety of issues, both in routine diagnostics projects and in the field of research.

The human immune system is a complex network of cells and organs that plays a crucial role in the defence against diseases. One of the central organs of the immune system is the thymus, which plays a key role in the formation and maturation of T cells. In recent years, research has made significant progress in the development of "artificial thymus organoids" (ATO). This innovative technology allows us to focus in detail on the disruption of T cell development in various genetic defects.

The thymus organoids are composed of a genetically optimised murine stromal cell line and human haematopoietic stem cells. These CD34-positive stem cells can either be derived from T-cell-deficient patients and control subjects or we can generate them from human induced pluripotent stem cells (hIPSC) into which genetic variants of interest are introduced using CRISPR/Cas technology.

The ATOs allow us to precisely monitor T cell differentiation across a series of defined progenitor stages. The detection and classification of these different progenitor populations is carried out in our laboratory using flow cytometry. In this way, we can very precisely assign blockages in development in the presence of the various genetic defects to individual T cell differentiation stages.

We can then isolate conspicuous cell populations using flow cytometry-based cell sorting and then comprehensively characterise them genetically (e.g. gene expression analyses using RNASeq) as well as cell and molecular biologically (e.g. determination of surface markers, activation assays, signal transduction analyses) in order to comprehensively analyse and clarify the pathomechanisms underlying the individual congenital disorders of the T-cellular immune system.