Research & Clinical Collaboration

Research & Clinical Collaboration

Aims and techniques

The ultimate goal is to help patients and families to better understand their disease and find effective treatment options.

We aim to understand the pathogenic mechanism underlying human genetic diseases and to develop therapies to treat the patient´s condition. We make use of patient material (e.g. blood samples and skin biopsies).

A multitude of technologies is applied to help families to understand the molecular basis of their diseases. Among others, these technologies include high-throughput sequence analysis (Next Generation Sequencing) or Sanger Sequencing, and different technologies originating from molecular biology, cell culture and therapy development. A detailed characterization of the pathogenic processes associated with genetic alterations is performed by detailed transcript analyses, in cell culture assays, or in in vivo disease models. The results of these analyses build the basis to develop therapeutic approaches.

Figure legend: This scheme summarizes our research aims and clinical collaborations, as well as frequently used lab techniques. A patient’s sample is required for these analyses. Frequently, high throughput sequencing / Next Generation Sequencing is performed to identify disease-associated genes and mutations. We verify our genetic findings in cell culture and make use of patient-derived cell lines (e.g. fibroblasts from a skin biopsy as small as a pinhead). This assists in establishing the clinical diagnosis and helps in genetic counseling of patients and families. A deeper understanding of pathological processes leading to genetic disease and the development of approaches are major aims of our work.

Human Genetics

We are broadly interested in the genetic and molecular basis of human diseases. Our research focus includes ciliopathies, retinal dystrophies, hearing deficits and brain malformations, all of which are among the most heterogeneous genetic diseases known so far. We analyze family pedigrees and search for genetic causes of inherited disorders.

Genetic heterogeneity and clinical variability are obstacles to the identification of disease-associated genes. Next Generation Sequencing is the method of choice to overcome these obstacles. It enables the analysis of all known genes (whole exome or genome sequencing) in parallel.

Figure legend: The complexity of retinal diseases illustrating the challenges to identify genetic causes in heterogeneous disorders. Retinal degenerations are associated with mutations in over 250 different genes. Several clinical diagnoses can be distinguished. Clear genotype-phenotype correlation rarely exist and the same mutation(s) may cause different phenotypes in patients. Only about 60-70% of the cases can be explained by the known disease-associated genes, implicating that additional genetic causes are to be discovered. Modified after "The molecular basis of human retinal and vitreoretinal diseases". Berger W, Kloeckener-Gruissem B, Neidhardt J. 2010, Prog Retin Eye Res., p335-375.

Pathomechanism

We aim to understand the pathogenic processes underlying genetic diseases. After identifying pathogenic sequence variants (mutations) in disease-associated or novel candidate genes, the biological function of the disease genes is characterized. We culture patient-derived cell lines or make use of in vivo disease models to identify disease-relevant mechanisms. Additional examples of techniques applied to study the consequence of mutations include DNA/RNA/protein analyses, different cell culture techniques, and immunohistochemical stainings.

Frequently, mutations in disease associated genes belong to the group of ciliopathies. RPGR mutations cause ciliopathies (see figure) and are associated with retinal degenerations in patients.

Figure legend: Immunofluorescence detection of RPGR in control and patient-derived fibroblast cells illustrating the sensitivity of techniques applied to understand pathogenic processes induced by mutations. The analysis shows that RPGR mutations may lead to a mis-localization of the RPGR protein along the primary cilium. The cilium is a protrusion from the cell surface that functions as a signaling antenna and regulates biological pathways. The photoreceptors of the retina contain a specialized cilium, the so-called connecting cilium. Indeed, retinal degenerations are frequently caused by mutations in genes that affect the function of the cilium. Mutations in ciliopathy associated genes can cause non-syndromic or syndromic forms of diseases with a wide range of phenotypes.

Therapy Development

Research labs around the world develop therapeutic approaches to treat genetic diseases. Replacing the defective gene by a functional copy (replacement therapy) is one of the more common therapeutic strategies. Furthermore, we successfully applied therapeutic strategies to treat the underlying genetic defects and corrected the ciliopathy phenotype in patient-derived cell lines and in vivo models.

We develop gene therapeutic approaches in the lab. One example is described below and uses techniques to correct defective transcript processing:

Figure legend: Mutations occurring in the genomic DNA of patients can result in splice defects. The vast majority of human genes are composed of coding (exons) and non-coding regions (introns). Gene transcription results in the pre-messenger RNA (pre-mRNA), which is subsequently modified by removal of the intronic regions, a complex process that is known as the splice process. Splicing produces the mature mRNAs transcripts which can be translated into proteins.

On average, about 20% of the patients show disease-causing mutations that lead to defects in the splicing of genes. This observation is largely independent from the affected disease gene. Thus, the pathogenic effect of many mutations in monogenetic diseases primarily alters the gene transcript (and only subsequently affects the protein composition). We developed gene therapies to treat diseases that are caused by mutations affecting the splice process. We used splice factors (e.g. U-snRNAs) and designed them to correct the patient-specific splice defects. Splicing assays (using antisense-oligonucleotides (AONs), minigenes, or patient-derived cell lines) were utilized to optimize the gene therapeutic approaches. We demonstrated that these therapeutic strategies are functional in patient-derived cell lines and in vivo models of diseases.

Figure legend: We design splice factors to recognize the mutated splice site as if it was a normal splice site. This therapeutic approach leads to correction of the mutation-induced splice defects. Alternatively, antisense oligonucleotides (AONs) may silence the effect of the mutation. Replication-deficient lenti or adeno-associated viruses (AAV) are used to transfer the gene therapeutics. The therapeutic approaches result in correctly spliced transcripts and normal protein products.

Research labs around the world develop therapeutic approaches to treat genetic diseases. Replacing the defective gene by a functional copy (replacement therapy) is one of the more common therapeutic strategies. Furthermore, we successfully applied therapeutic strategies to treat the underlying genetic defects and corrected the ciliopathy phenotype in patient-derived cell lines and in vivo models.

We develop gene therapeutic approaches in the lab. One example is described below and uses techniques to correct defective transcript processing:

Figure legend: Mutations occurring in the genomic DNA of patients can result in splice defects. The vast majority of human genes are composed of coding (exons) and non-coding regions (introns). Gene transcription results in the pre-messenger RNA (pre-mRNA), which is subsequently modified by removal of the intronic regions, a complex process that is known as the splice process. Splicing produces the mature mRNAs transcripts which can be translated into proteins.

On average, about 20% of the patients show disease-causing mutations that lead to defects in the splicing of genes. This observation is largely independent from the affected disease gene. Thus, the pathogenic effect of many mutations in monogenetic diseases primarily alters the gene transcript (and only subsequently affects the protein composition). We developed gene therapies to treat diseases that are caused by mutations affecting the splice process. We used splice factors (e.g. U-snRNAs) and designed them to correct the patient-specific splice defects. Splicing assays (using antisense-oligonucleotides (AONs), minigenes, or patient-derived cell lines) were utilized to optimize the gene therapeutic approaches. We demonstrated that these therapeutic strategies are functional in patient-derived cell lines and in vivo models of diseases.

Figure legend: We design splice factors to recognize the mutated splice site as if it was a normal splice site. This therapeutic approach leads to correction of the mutation-induced splice defects. Alternatively, antisense oligonucleotides (AONs) may silence the effect of the mutation. Replication-deficient lenti or adeno-associated viruses (AAV) are used to transfer the gene therapeutics. The therapeutic approaches result in correctly spliced transcripts and normal protein products.
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