Drug Repurposing for Mitochondrial Disorders Using iPSCs Derived Neural Cells
- Conditions
- Leigh Syndrome (Maternally Inherited, MILS)Leigh Syndrome (AR, AD, XR)
- Interventions
- Procedure: skin biopsyOther: generation of iPSCsDiagnostic Test: blood drawingDrug: off-label compassionate drug use
- Registration Number
- NCT06967831
- Lead Sponsor
- Charite University, Berlin, Germany
- Brief Summary
In this project, the investigators are using iPSC lines derived from patients with Leigh syndrome that carry mutations in the mitochondrial (mtDNA) and in the nuclear DNA (nDNA) to reprogram them into neural progenitor cells and into dopaminergic neurons. The researchers are using this experimental system to screen FDA (Food and Drug Administration, USA) and EMA (European Medicines Agency) approved drugs for a positive effect on Leigh patient-derived neuronal cells (drug repurposing) using various biochemical, optic, and morphological outcome measures. Confirmed positive hits may be used for compassionate off-label use in Leigh patients when no standard treatment is available.
- Detailed Description
Leigh syndrome is a rare severe hereditary neurological disease that typically manifests during early childhood and is characterized by the progressive loss of motor and intellectual skills. A hallmark of the disease is the degeneration of neuronal cells in the brainstem and in the basal ganglia, particularly the dopaminergic neurons therein. The genetic underpinnings of this condition are multifaceted, encompassing mutations in both nuclear genes and in those contained within the mitochondrial DNA (mtDNA). The investigators hypothesize that these mutations share a common principle in their capacity to induce dysfunction of mitochondrial processes and of bioenergetic metabolism. The precise mechanism underlying neuronal death remains to be elucidated as researchers presently lack suitable disease models. Notably, the generation of a mouse model for mtDNA mutations has not been achieved, necessitating the exclusive reliance on patient derived material for research into the pathogenesis of these diseases. Moreover, there are currently no pathogenesis-based treatment approaches that have been demonstrated to improve patients' symptoms. Here, the investigators aim to utilize reprogramming technologies to engineer innovative human-derived disease models for research into Leigh syndrome. To this end, the investigators plan generating induced pluripotent stem cells (iPSCs) from fibroblasts of different Leigh syndrome patients who carry both nuclear (e.g. in SURF1) and mtDNA mutations (e.g. in MT-ATP6). Pluripotent progenitor cells offer a novel approach to better understand the pathogenesis of genetic diseases. In the case of Leigh syndrome, accessible cells, such as skin or blood cells, are almost never clinically affected. However, the nerve cells of the basal ganglia, which cannot be obtained via biopsies, are predominantly affected. The underlying mechanisms by which these dopaminergic neurons are particularly vulnerable to mitochondrial dysfunction and subsequent death remain to be elucidated. The objective of this study is to differentiate these induced pluripotent stem cells (iPSCs) into neural precursor cells (NPCs) and then into a neuronal cell population that is predominant inside the basal ganglia, such as dopaminergic neurons. Subsequently, a detailed analysis of these neurons will be conducted to ascertain mitochondrial and metabolic parameters, with the objective of elucidating neuronal changes associated with mitochondrial disease. Consequently, based on the identified dysfunction, imaging test procedures will be developed that are aimed at high sample throughput. This should enable high-throughput screening of molecule libraries on patient-specific iPSC-based neuronal cells for drug repurposing. The initial phase of the study has identified four potential metrics to be used for the screening of EMA and FDA approved drugs (repurposing): \[1\] measurement of the mitochondrial membrane potential using fluorophores, \[2\] measurement of calcium transients using fluorophores and reporter constructs, \[3\] measurement of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using a Seahorse flux analyzer, and \[4\] investigation of the axonal outgrowth and branching patterns of the iPSC-derived neuronal cells by high-content screening. Compounds for which the investigators are able to confirm a positive effect by the above mentioned read-out methods will subsequently be provided to a selected number of patients for off-label compassionate use in cases where no standard treatment is available.
Recruitment & Eligibility
- Status
- RECRUITING
- Sex
- All
- Target Recruitment
- 80
- Patient has a disease causing mutation in one of the genes causing Leigh syndrome if mutated, 2. Patient has the characteristic cranial MRI abnormalities of Leigh syndrome
- bleeding disorder that precludes a skin biopsy, 2. retraction of consent
Study & Design
- Study Type
- OBSERVATIONAL
- Study Design
- Not specified
- Arm && Interventions
Group Intervention Description Leigh Syndrome (AR, AD, XR) generation of iPSCs Patients with mitochondrial diseases that are caused by mutations in the nuclear DNA, which may be inherited in an autosomal recessive (AR), autosomal dominant (AD), or X-chromosomal recessive (XR) mode of inheritance. Leigh Syndrome (Maternally Inherited, MILS) off-label compassionate drug use Patients with Leigh syndrome carrying disease-causing mutations in their mtDNA. This subtype of Leigh syndrome is called Maternally Inherited Leigh Syndrome (MILS) Leigh Syndrome (AR, AD, XR) skin biopsy Patients with mitochondrial diseases that are caused by mutations in the nuclear DNA, which may be inherited in an autosomal recessive (AR), autosomal dominant (AD), or X-chromosomal recessive (XR) mode of inheritance. Leigh Syndrome (AR, AD, XR) blood drawing Patients with mitochondrial diseases that are caused by mutations in the nuclear DNA, which may be inherited in an autosomal recessive (AR), autosomal dominant (AD), or X-chromosomal recessive (XR) mode of inheritance. Leigh Syndrome (AR, AD, XR) off-label compassionate drug use Patients with mitochondrial diseases that are caused by mutations in the nuclear DNA, which may be inherited in an autosomal recessive (AR), autosomal dominant (AD), or X-chromosomal recessive (XR) mode of inheritance. Leigh Syndrome (Maternally Inherited, MILS) blood drawing Patients with Leigh syndrome carrying disease-causing mutations in their mtDNA. This subtype of Leigh syndrome is called Maternally Inherited Leigh Syndrome (MILS) Leigh Syndrome (Maternally Inherited, MILS) generation of iPSCs Patients with Leigh syndrome carrying disease-causing mutations in their mtDNA. This subtype of Leigh syndrome is called Maternally Inherited Leigh Syndrome (MILS) Leigh Syndrome (Maternally Inherited, MILS) skin biopsy Patients with Leigh syndrome carrying disease-causing mutations in their mtDNA. This subtype of Leigh syndrome is called Maternally Inherited Leigh Syndrome (MILS)
- Primary Outcome Measures
Name Time Method Reduction of the increased mitochondrial membrane potential 1 year The investigators will measure the mitochondrial membrane potential in Leigh patient iPSC-derived neural cells. If the membrane potential can be normalized, a drug will be considered a positive hit. The membrane potential will be determined indirectly by the fluorescence intensity measurement of a membrane potential sensitive dye (TMRM). The patients' fluorescence readings will be compared to the fluorescence readings of neural cells that have been generated from healthy control iPSCs.
- Secondary Outcome Measures
Name Time Method Reconstitution of the neural outgrowth pattern 1 year The invesitgators will measure the neural axonal outgrowth and branching pattern in Leigh patient iPSC-derived neural cells using high content screening. If a normal outgrowth and branching pattern can be reconstituted in the patient iPSC-derived neural cells, a drug will be considered a positive hit. Axonal outgrowth and branching will be determined by high throughput parallel microscopy. This will determine the axonal growth velocity and the number of branch points per axon. Patients' data will be compared to healthy control iPSC-derived neural cells.
Measuring calcium transients in iPSC derived neural cells 1 year The investigators will measure calcium transients in Leigh patient iPSC-derived neural cells after electrical stimuation or after chmical stimulation using histamin. This essay will be used for high content screening. If a normal calcium signalling pattern can be reconstituted, a drug will be considered a positive hit. The mitochondrial calcium content will be determined using a fluorescent reporter construct (CEPIA) that is targeted into the mitochondrial matrix. No calibrated absolute measurements are possible. The calcium transients of the patient iPSC-derived neural cells will be compared to those of healthy control iPSC-derived neural cells.
Trial Locations
- Locations (2)
Universitätsklinikum Düsseldorf
🇩🇪Düsseldorf, Nordrhein-Westfalen, Germany
Charite - Universtaetsmedizin Berlin
🇩🇪Berlin, Germany