306 research outputs found
Scientific and medical basis of CRISPR/CAS9 and genome editing
No full textDie Verwendung von Stammzellen, iPS-Zellen sowie der Einsatz gentechnischer Methoden wie dem Genome Editing stellen für die Regenerative Medizin prinzipiell Möglichkeiten für die Entwicklung neuer Therapien dar. Die Umsetzung solcher Therapien wirft nicht nur naturwissenschaftlich-technische und medizinische Fragen auf, sondern auch ethische, rechtliche, soziale und ökonomische. Die erfolgreiche Realisierung regenerativmedizinischer Therapien ist daher auf eine disziplinübergreifende Herangehensweise angewiesen. Im Rahmen eines interdisziplinären und internationalen Symposiums sowie einer BMBF-Klausurwoche wurden die Aspekte der Verwendung von Stammzellen, iPS-Zellen sowie des Genome Editings diskutiert. Im Mittelpunkt standen u.a. die arzneimittelrechtliche Handhabung, verfassungsrechtliche und philosophische Fragen in Bezug auf die Kommerzialisierbarkeit menschlicher Körpersubstanzen sowie patentrechtliche Fragen im Umgang mit menschlichen Stammzellen und der Verfahren des Genome Editings.
Mit Beiträgen von
Insa S. Schröder; Susanne Müller und Timo Faltus; Inesa Chmurec; Tereza Hendl; Calvin Wai Loon Ho; Kalina Kamenova; Delphine Pichereau und Emmanuelle Rial-Sebbag; Hannah Schickl; Elena Buglo und Stephan Zuchner; Jochen Taupitz und Juliane Boscheinen; Winfried Kluth; Susanne Beck und Frederike Seitz; Timo Faltus; Rosario Isasi; Katrin Vohland, Julia Diekämper, Alexandra Moormann, Tobias Nettke und Wiebke Rössig; Hans Zillmann und Matthias Kaufmann; Ulrich Storz; Timo Faltus
Whole genome sequencing identifies causal variants in CMT
No full textFor the first time, medical sequencing has been successfully performed at the genome level to identify the causative gene in an individual with autosomal recessive Charcot-Marie-Tooth disease. The results of sequencing a proband with this condition highlight some of the opportunities and challenges of this seemingly ultimate approach to human genetics research and diagnostics
Untersuchungen zur Rolle von Neurozytokinen in Hirntumoren
No full textCiliary Neurotrophic Factor (CNTF) and Interleukin-6 (IL-6) influence the survival and the differentiation of neuronal and glial cells. CNTF induces reactive transformation of astrocytes in vivo and has been shown to promote proliferation of neuronal tumor cell lines in vitro. Thus, CNTF is a candidate growth and/or differentiation factor for neuronal and glial tumors. Effects of neurocytokines are mediated trough specific receptor components. In this study the expression of CNTF and IL-6 and its specific receptor components in vivo and in vitro were analyzed. In the majority of the examined astrocytomas and glioblastomas prominent CNTF-R alpha mRNA expression was detected. Likewise, two of four glioblastoma cell lines expressed CNTF-R alpha. Quantitative analysis of the performed northern blots revealed no significant elevated CNTF-R alpha levels of the gliomas compared to the controls. Nevertheless, huge amounts of CNTF-R alpha that are expressed by certain tumors support the thesis that CNTF is involved in the complex process of transformation of astrocytomas. In vitro studies with various cell lines showed differential expression patterns of CNTF, CNTF-R alpha, IL-6 and IL-6R alpha. Stimulation of different neuro-ectodermal and muscle cell lines with rCNTF and rIL-6 induced expression of the transcription factor c-fos. Interestingly, in most cell lines a co-expression of CNTF and IL-6 and its receptors was measured. Thus, it appears reasonable to speculate that CNTF and IL-6 influence growth and differentiation in vitro and in vivo via an autocrine loop
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The genetics of hereditary spastic paraplegia and implications for drug therapy
No full textHereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. A total of 30 chromosomal loci have been identified for autosomal dominant, recessive and X-linked HSP. The underlying genes for 15 of these loci have been described. The molecular dissection of the cellular functions of the related gene products has already greatly advanced our understanding of the most critical pathways involved in HSP. It is hoped that in the foreseeable future this knowledge will begin to translate into novel pharmacological approaches for this devastating disease
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The Genetic and Functional Analysis of the Obsessive-Compulsive Disorder Spectrum
No full textObsessive-compulsive disorder (OCD) and the spectrum of associated conditions, affect 2-4% of the population worldwide. Although heritability studies in OCD have shown a 3 - 12 times increased risk for first degree relatives, the identification of the underlying risk-conferring genetic variation using classic genetic association studies has proven to be difficult. The possibility of a larger contribution of rare genetic variants to the risk of psychiatric disorder has been suggested by several successful studies. We expect that a spectrum of risk allele frequencies exists, which includes not only common variation but also a substantial amount of rare genetic variants that contribute to OCD. This thesis is aimed at identifying and functionally characterizing rare genetic variation in the OCD spectrum. Identified statistically significant variants were scrutinized for changes related to synaptic function using high content screening and subsequent functional analyses. Identifying the genetic profile of rare variants found in the OCD spectrum cohort combined with the functional impact that these variants have has provided insight into the etiology of the OCD spectrum. With these approaches a foundation can be laid for the development of a predictive model of the OCD spectrum.</p
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Correcting a Pathogenic Mitochondrial DNA Mutation by Base Editing in Mice
No full textPrimary mitochondrial disorders are most often caused by deleterious mutations in the mtDNA. Here, I harnessed a mitochondrial base editor, DdCBE, to introduce a compensatory edit (m.5081G>A) in a mouse model that carries the pathological m.5024C>T mutation in the mitochondrial tRNAAla gene. For this, the DdCBE gene construct was packaged in recombinant AAV9 and systemically injected into mice. I found that total mt-tRNAAla levels, which are drastically reduced by the mutation, are restored by the m.5081G>A edit in a dose-dependent manner. However, an excessive expression of DdCBE also induces extensive mtDNA off-target editing, counteracting this positive outcome. To address this, I optimized the dosage to maximize the amount of compensatory edit generated with minimal off-target editing. These results show that mitochondrial base editors are promising candidates for gene therapy for mitochondrial disorders, but their expression needs to be carefully controlled.</p
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The Role of MTFMT in Mammalian Mitochondria
No full textThe Mitochondrial Methionyl-tRNA Formyltransferase (Mtfmt) is a nuclear gene encoding for a protein with exclusive function in mitochondria. During initiation of mitochondrial protein synthesis, the MTFMT enzyme N-formylates the first methionine that appears to be required for the synthesis of the 13 polypeptides encoded by the mitochondrial DNA. In humans, mutations in the MTFMT gene are associated with Leigh syndrome. Studies in fibroblasts from patients sharing the most frequent disease allele (c.626 C>T in exon 4), showed mitochondrial protein synthesis. Biochemical studies into the mutation suggest that protein synthesis depend upon residual levels of the mutated MTFMT protein. My dissertation work, aims to contribute to solve a fundamental but controversial question related to the absolute requirements of N-formylation in mammals and to gain insights on the disruption mechanism of MTFMT in Leigh syndrome. I generated conditional knockout (KO) mouse models in muscle and in the Central Nervous System. Unexpectedly, a novel and unique mutation in the 3’ loxP site of the conditional Mtfmt allele prevented the deletion of the gene in vivo. Ex vivo, I was able to delete the gene in dermal fibroblasts, producing a complete MTFMT KO cell model. I demonstrated that MTFMT activity is required to transfer the N-formyl group onto the mitochondrial Met-tRNAMet; N-formylation is not an absolute requirement for mitochondrial protein synthesis in mammals but has important roles in the efficiency of synthesis of certain mitochondrial protein subunits; and absence of N-formylation affects both the assembly of individual complexes I and IV and the respirasome.</p
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Impacts of Disrupting Early Motor Behaviors on Spatial and Temporal Patterning of Synapses in Zebrafish Spinal Cord
No full textTo generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitation and inhibition (E/I balance). Disruption of E/I balance is associated with many neurodevelopmental disorders, such as seizures, autism, startle disease and glycine encephalopathy. E/I balance is maintained at both the cellular and the systems levels, and is influenced by the relative distribution of excitatory and inhibitory synapses. While the spatial and temporal patterns of excitatory and inhibitory synapses strongly associate with E/I balance, it remains unclear how perturbations of E/I balance affect the spatial and temporal patterning of synapses in in vivo neural circuits. To answer this question, we investigated the spatial and temporal patterning of excitatory and inhibitory synapses in developing zebrafish spinal cord (Chapter 2). We hypothsized that excitatory and inhibitory synapses and neuronal processes follow a stable, systems-level spatial pattern on the medial-lateral axis in embryonic and larval spinal cord. Interestingly, this pattern is maintained in the zebrafish glycine transporter 1 mutant despite the presence of perturbation of E/I balance (Chapter 3). This mutant can naturally re-establish the spinal cord E/I balance with development. We found that though the general synapse pattern remains unchanged, subtle alterations of synapse spatial patterns take place at the beginning of the E/I balance re-establishment process. We also investigated how a perturbation of E/I balance impacts the synaptogenesis process. To understand this, we knocked down genes encoding particular subunits of glycine receptors (GlyRs) in zebrafish embryos, and analyzed how such knockdown affects glycinergic synaptogenesis and motor behaviors (Chapter 4). We found that disruption of different GlyR subunits impacts the formation of functional glycinergic synapses in different manners: knocking down the GlyR α1 subunit leads to a reduction of GlyRs while knocking down the βb subunit disrupts the clustering of GlyRs at the post-synapses. In addition, knockdown of either subunit alters the spatial pattern of glycinergic synaptogenesis. In conclusion, we found that E/I balance is associated with the spatial and temporal patterns of synapses at multiple levels. The systems-level pattern is stable and robust, while finer-scale patterns at cellular and dendritic levels are more flexible and likely to alter in response to perturbations of E/I balance.</p
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