Drug Repurposing for Mitochondrial Disorders Using iPSCs Derived Neural Cells

Официальное название

Using iPSC Derived Human Basal Ganglia Neurons From Patients With Leigh Syndrome for Metabolic Studies, Assay Development, and Drug Repurposing.

Обзор

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.

Подробное описание

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.

Вмешательства

• Процедура skin biopsy
  Taking a punch skin biopsy of 3 mm diameter under local anesthesia and culturing skin fibroblasts from them.
• Другое generation of iPSCs
  Using cultured skin fibroblasts of the patients, iPSCs will be generated according to standard procedures.
• Диагностический тест blood drawing
  Drawing blood from a peripheral vein for DNA and RNA isolation. The degree of heteroplasmy (mutation load) for the mtDNA mutation will be determined in the blood DNA.
• Препарат off-label compassionate drug use
  In case the investigators identify a positive hit during drug repurposing with FDA and EMA approved substances, they will offer it as off-label compassionate use to patients for whom no standard treatment is available.

Первичные конечные точки

• Reduction of the increased mitochondrial membrane potential [Срок оценки: 1 year]

Вторичные конечные точки (2)

• Reconstitution of the neural outgrowth pattern [Срок оценки: 1 year]
• Measuring calcium transients in iPSC derived neural cells [Срок оценки: 1 year]

Критерии участия

Inclusion Criteria: 1. 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

Exclusion Criteria: 1. bleeding disorder that precludes a skin biopsy, 2. retraction of consent

Критерии приведены из реестра в оригинале (на английском). Окончательную оценку соответствия проводит исследовательский центр.

Здоровые добровольцы: Нет

Дизайн исследования

Модель наблюдения

Только случаи

Центры проведения

Германия · 2 центра

• Universitätsklinikum Düsseldorf — Düsseldorf
• Charite - Universtaetsmedizin Berlin — Berlin

Публикации

• Lorenz C, Lesimple P, Bukowiecki R, Zink A, Inak G, Mlody B, Singh M, Semtner M, Mah N, Aure K, Leong M, Zabiegalov O, Lyras EM, Pfiffer V, Fauler B, Eichhorst J, Wiesner B, Huebner N, Priller J, Mielke T, Meierhofer D, Izsvak Z, Meier JC, Bouillaud F, Adjaye J, Schuelke M, Wanker EE, Lombes A, Prigione A. Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological PMID 28132834
• Diodato D, Schiff M, Cohen BH, Bertini E, Rahman S; Workshop participants. 258th ENMC international workshop Leigh syndrome spectrum: genetic causes, natural history and preparing for clinical trials 25-27 March 2022, Hoofddorp, Amsterdam, The Netherlands. Neuromuscul Disord. 2023 Aug;33(8):700-709. doi: 10.1016/j.nmd.2023.06.002. Epub 2023 Jun 15. No abstract available. PMID 37541860
• Henke MT, Zink A, Diecke S, Prigione A, Schuelke M. Generation of two mother-child pairs of iPSCs from maternally inherited Leigh syndrome patients with m.8993 T > G and m.9176 T > G MT-ATP6 mutations. Stem Cell Res. 2023 Mar;67:103030. doi: 10.1016/j.scr.2023.103030. Epub 2023 Jan 17. PMID 36669241
• Steiner T, Zink A, Henke MT, Cecchetto G, Buenning M, Rossi A, Schuelke M, Prigione A. RNA-based generation of iPSCs from a boy carrying the mutation m.9185 T>C in the mitochondrial gene MT-ATP6 and from his healthy mother. Stem Cell Res. 2022 Oct;64:102920. doi: 10.1016/j.scr.2022.102920. Epub 2022 Sep 15. PMID 36137325
• Carli S, Levarlet A, Diodato D, Bertini ES, Martinelli D, Malandrini A, Lopergolo D, Gallus GN, Ganetzky RD, La Morgia C, Carelli V, Primiano G, Dominguez-Gonzalez C, Serrano-Lorenzo P, Martin MA, Ardissone A, Lamperti C, Nicoletta V, Klopstock T, Distelmaier F, Zeng L, Buchner B, Mancuso M, Schuelke M, Prigione A, Garone C. Natural History of Patients With Mitochondrial ATPase Deficiency Due to P PMID 40112238
• Inak G, Rybak-Wolf A, Lisowski P, Pentimalli TM, Juttner R, Glazar P, Uppal K, Bottani E, Brunetti D, Secker C, Zink A, Meierhofer D, Henke MT, Dey M, Ciptasari U, Mlody B, Hahn T, Berruezo-Llacuna M, Karaiskos N, Di Virgilio M, Mayr JA, Wortmann SB, Priller J, Gotthardt M, Jones DP, Mayatepek E, Stenzel W, Diecke S, Kuhn R, Wanker EE, Rajewsky N, Schuelke M, Prigione A. Defective metabolic progra PMID 33771987

Идентификаторы

NCT: NCT06967831 · EA2_131_13

Первоисточники (государственные реестры)

• ClinicalTrials.gov (NIH, США)

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