Research projects finished

The use of iPS cells for the development of therapeutic strategies

The use of iPS cells as a model for the study of mitochondrial encephalopathies caused by defects in mitochondrial translation: an experimental approach for the development of therapeutic strategies.

INSTITUTIONS:
1. Instituto de Investigación Sanitaria Hospital 12 de Octubre, i+12
Avenida de Córdoba s/n, 28041. Madrid

2. Faculty of Medicine (Universidad Autónoma de Madrid)
Avenida Arzobispo Morcillo nº4, 28019. Madrid

BACKGROUND INFORMATION

Mitochondrial diseases (MDs) constitute a large group of genetic disorders which are linked by a dysfunction in the Oxidative Phosphorylation System (OXPHOS) (1). Generally speaking, they are multi-system disorders that tend to affect preferentially tissues with a high demand for energy like the brain, skeletal muscle and heart. However, any organ can be affected and mitochondrial defects can also cause unique clinical manifestations such as blindness, sensorineural hearing loss or kidney dysfunction (1). Although MDs are rare individually, as a whole they group together a wide variety of genetic disorders and we are currently beginning to foresee that in a not so distant future the development of new methods of diagnosis will demonstrate to us the mitochondrion’s involvement, at a primary or secondary level, in an enormous number of human diseases. Since the biogenesis of the OXPHOS system depends on two different genomes, mitochondrial (mtDNA) and nuclear (nDNA), mitochondrial diseases can be caused by mutations in mtDNA or in genes encoded in nDNA (2). MtDNA is a circular double-stranded molecule that codes for 22 tRNAs, 2 rRNAs and 13 proteins that code for at least one structural subunit of each OXPHOS complex (complexes I, III, IV and V) except complex II, which is encoded entirely in the nucleus. The over 70 remaining OXPHOS subunits as well as the proteins responsible for the maintenance of the mtDNA, mitochondrial transcription, translation and assembly of the OXPHOS complexes are encoded by the nuclear genome (3).

The estimated incidence of MDs at birth is 1: 5000. Current therapies for this type of pathology are based fundamentally on trying to alleviate the symptoms, more often than not unsuccessfully. Consequently, there is an urgent need to develop new therapeutic approaches (4). In this respect, the development of new technologies that enable the reprogramming of different types of somatic cells to induced pluripotent stem cells (iPSCs) by the ectopic expression of four transcription factors, OCT4, SOX2, KLF4 and c-MYC, has opened up enormous possibilities for the generation of new disease models, particularly, in order to study the effect of pathogenic mutations in their target tissues (5). Following Yamanaka’s discovery of iPSCs, up to now success has been achieved in generating iPSCs from patients with different mitochondrial disorders. They include iPSCs from a patient with Pearson syndrome that manifested a deletion in mtDNA of 2501 bp (6) and from two patients with diabetes, who were carriers of the m.3243>G mutation in the MT-TL1 gene (7). In addition, successful results have been achieved in reprogramming fibroblasts obtained from patients with MELAS Syndrome, who were carriers of the mutations, m.1513G>A located in the MT-ND5 gene of complex I of the CR and m.3243A>G located in the MT-TL1 gene (8; 9). Our group recently generated iPSCs from patients with Leigh syndrome, caused by heteroplasmic mutations in the MT-ND5 gene (m. 13513G>A; p.Asp393GIn) and in the MT-ATP6 gene (c.8993T>G; p.Leu156Arg) (10; 11), from a patient with a defect in intergenomic communication caused by a homozygotic mutation in the POLG gene (c.2243>C; p.Trp748Ser) (12) and from a patient with a phenotype of optic atrophy plus syndrome caused by a heterozygotic mutation in the OPA1 gene (C.1861C>T; P.GIn621Ter) (13). These results are promising since they are going to enable us to discover the effect that mitochondrial alterations have on their target tissues, which will constitute a clear step forward in our knowledge of the physiopathogenic mechanisms of this group of disorders.

The possibility of reprogramming patients’ fibroblasts to iPSCs and of studying the molecular phenotype that they cause in the differentiated target cells of the disorder proves to be of enormous benefit not only for the study of the disorder in their target tissue, but also for their use as a platform for the identification of new drugs and, potentially, for their use in cell therapy. Moreover, it is important to point out that the transfer into clinical practice of the knowledge gained by using iPSCs has increased significantly and that successful therapies based on the use of these cells already exist for animal models of human disorders. Likewise, iPSCs have already been successfully generated from patients’ cells and their defect corrected, differentiating the cells from functional cell lines (14; 15). In Japan the first clinical trial was even started on a patient with macular degeneration to whom differentiated cells obtained through iPSCs were transplanted. Although this trial has been suspended for different reasons, which have arisen as a result of changes in Japanese legislation, so far the patient does not exhibit any alterations that might indicate a failure in the trial (16). At present, although regenerative medicine is already starting to be implemented for certain pathologies, it still has not been possible to apply it in the field of MDs. The great challenge for regenerative medicine is to repair or replace damaged tissue cells and organ cells by transplanting healthy cells or tissue. Ideally, this would be done by using stem cells or embryonic stem cells (ESCs) generated in the laboratory from the patient’s own cells or from a compatible donor’s. However, the study of human stem cells entails several drawbacks. Firstly, the production of human ESCs raises ethical and, in some countries, legal issues. Moreover, the possibility of obtaining stem cells from adult tissues raises several problems: (a) for most of them there are no specific genetic markers to allow their identification and/or isolation, (b) with rare exceptions, their ex vivo expansion is not technically possible for prolonged periods of time, (c) it is not yet possible to obtain adult stem cells from all tissues and (d) their potential is rather limited as they are only able to generate the cell types of the tissue that they come from. To overcome these difficulties the possibility of being able to use iPSCs constitutes a clear step forward in the field of regenerative medicine and therapy (17). In fact, one of the great advantages of using iPSCs lies in the fact that they enable us to generate each patient’s own specific cell types, thus reducing the risk of immunological rejection in a transplant (autologous transplant) (18).

A considerable group of MDs is associated with mutations in genes involved in mitochondrial translation. Mitochondrial translation is carried out in the mitochondrial matrix with a protein synthesis machinery which is mostly independent of the mechanism responsible for the translation of the genes contained in the nuclear genome, which takes place in the cytosol (19). Besides the tRNAs and rRNAs encoded in the mtDNA, the translation requires other components including approximately 50 ribosomal proteins; several tRNA maturation enzymes; the aminoacyl tRNA synthetases; the initiation, elongation and termination translation factors; and probably a large number of as yet unidentified factors, including ribosome assembly factors (20). Anomalies in these genes, whether mitochondrial or nuclear, can compromise mitochondrial translation and consequently create biochemical defects that give rise to failures in the OXPHOS system and disease. Although most of the components of the mitochondrial translation system are encoded by the nucleus, most of the mutations associated with defects in the synthesis of mitochondrial proteins have been identified in the mtDNA. These include temporary mutations in tRNAs and the 16S rRNA, as well as large deletions which eliminate various tRNAs. These defects are associated with a wide spectrum of clinical phenotypes which predominately affect the nervous system, skeletal muscle and heart. Among the mutations identified in components of the mitochondrial translation system encoded in the nucleus the following have been described: 1) mutations in tRNA-modifying enzymes (PUS1, TRMU, MTO1), 2) aminoacyl tRNA synthetases (RARS2, DARS2, YARS2, SARS2, HARS2, AARS2, MARS2, EARS2, FARS2), 3) ribosomal proteins MRPS16, MRPS22, MRPL3, MRPL12), 4) a termination factor (C12orf65), 5) translational activators (LRPPRC, TACO1), 6)C12orf62 which couples COX1 synthesis with the assembly of cytochrome c oxidase and elongation factors (EFTs, EFTu, GFM1) (19). One particularly interesting type of defect in the synthesis of mitochondrial proteins is that caused by mutations in elongation factor G1 (GFM1) which codes for protein EFG1 (21). As yet, we do not know which molecular mechanisms it is that lead to the development of these pathologies or whether an effective treatment exists for them, partly due to the lack of suitable experimental models. In our laboratory, we recently generated iPSCs from fibroblasts obtained from the patient that this Foundation is named after (22). The patient exhibits mutations in GFM1 and she was five years old when the biopsy was carried out. She suffers from mitochondrial encephalopathy, due to a genetic disorder in mitochondrial elongation factor GFM1 (compound heterozygote with mutations in both copies of the gene: a deletion that causes a phase change (c.1401delA) and a transition c.2011C>T which causes amino acid change Arg671Cys (23). It should be pointed out that unlike other published cases, this patient shows a stable clinical course that has enabled her to survive beyond early childhood. The generation and characterization of this model of iPSCs will not only enable us to reach a better understanding of the physiopathological mechanisms of this type of disorder, but will also open up new paths for identifying pharmacological and cell therapy treatments for this type of pathology.

REFERENCES

1. DiMauro, S and Schon, E.A. (2003) “Mitochondrial respiratory chain diseases” N. Engl.
J. Med. 348: 2656-2668.
2. Garesse, R and Vallejo, CG (2001) “Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes” Gene 263: 1-16.
3. Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N. (2009) “Importing mitochondrial proteins: machineries and mechanisms” Cell. 138(4):628-44.
4. Galera T, Zurita-Díaz F, Garesse R, Gallardo ME. iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial Disorders: Pros and Cons. (2016) J Cell Physiol (in press).
5. Takahashi K, Yamanaka S. (2006) “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”. Cell 126:663-676
6. Cherry AB, Gagne KE, McLoughlin EM, Baccei A, Gorman B, Hartung O, Miller JD, Zhang J, Zon RL, Ince TA, Neufeld EJ, Lerou PH, Fleming MD, Daley GQ, Agarwal S. “Induced Pluripotent Stem Cells with a Pathological Mitochondrial DNA Deletion”. (2013) Stem Cells 31(7):1287-97.
7. Fujikura J, Nakao K, Sone M, Noguchi M, Mori E, Naito M, Taura D, Harada-Shiba M, Kishimoto I, Watanabe A, Asaka I, Hosoda K, Nakao K. “Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation”. (2012) Diabetologia 55: 1689-1698.
8. Folmes CD, Martinez-Fernandez A, Perales-Clemente E, Li X, McDonald A, Oglesbee D, Hrstka SC, Perez-Terzic C, Terzic A, Nelson TJ. “Disease-causing Mitochondrial Heteroplasmy Segregated within Induced Pluripotent Stem Cell Clones Derived from a MELAS Patient”. (2013) Stem Cells 31(7):1298-308.
9. Hämäläinen RH, Manninen T, Koivumäki H, Kislin M, Otonkoski T, Suomalainen A.
Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model. (2013) Proc Natl Acad Sci U S A. 110(38):E3622-30.
10. Teresa Galera; Francisco Zurita; Cristina Gónzalez-Páramos; Ana Moreno-Izquierdo; Mario F. Fraga; Rafael Garesse; M. Esther Gallardo. Generation of a human iPSC line from a patient with Leigh syndrome. (2016) Stem Cell Research 16(1):63-66.
11. Teresa Galera-Monge; Francisco Zurita-Díaz; Cristina González-Páramos; Ana Moreno-Izquierdo Cristina; Mario F. Fraga; Agustin F. Fernández; M. Esther Gallardo.
Generation of a human iPSC line from a patient with Leigh Syndrome caused by a mutation in the MT-ATP6 gene. (2016) Stem Cell Research (en prensa).
12. Francisco Zurita; Teresa Galera; Cristina González-Páramos; Ana Moreno-Izquierdo; Peter Schneiderat; Mario F. Fraga; Agustin F. Fernández; Rafael Garesse; M. Esther Gallardo. Generation of a human iPSC line from a patient with a defect of intergenomic communication. (2016) Stem Cell Research. 16(1):120-123.
13. Teresa Galera-Monge; Francisco Zurita-Díaz; Ana Moreno-Izquierdo; Mario F. Fraga; Agustin F. Fernández; Carmen Ayuso; Rafael Garesse; M. Esther Gallardo. Generation of a human iPSC line from a patient with an optic atrophy ‘plus’ phenotype due to a mutation in the OPA1 gene. (2016) Stem Cell Research 16(3): 673-676.
14. Huang X, Wang Y, Yan W, Smith C, Ye Z, Wang J, Gao Y, Mendelsohn L, Cheng L. Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation. (2015) Stem Cells. 33(5):1470-1479
15. Song B, Fan Y, He W, Zhu D, Niu X, Wang D, Ou Z, Luo M, Sun X. Improved
hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. (2015) Stem Cells Dev 24(9):1053-1065.
16. Garber, K. RIKEN suspends first clinical trial involving induced pluripotent stem cells. (2015) Nat. Biotechnol. 33: 890–891.
17. Pareja-Galeano H, Sanchis-Gomar F, Pérez LM, Emanuele E, Lucia A, Gálvez BG, Gallardo ME. iPSCs-based anti-aging therapies: Recent discoveries and future challenges. (2016) Ageing Res Rev 27:37-41.
18. Pareja-Galeano H, Sanchis-Gomar F, Emanuele E, Gallardo ME, Lucia A. IPSCs, a Promising Tool to Restore Muscle Atrophy. (2016) J Cell Physiol 231(2):259-260.
19. Ott M, Amunts A, Brown A. Organization and Regulation of Mitochondrial Protein Synthesis. (2016) Annu Rev Biochem.
20. Valente L, Tiranti V, Marsano RM, Malfatti E, Fernandez-Vizarra E, Donnini C,
Mereghetti P, De Gioia L, Burlina A, Castellan C, Comi GP, Savasta S, Ferrero I, Zeviani M. Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. (2007) Am J Hum Genet. 80(1):44-58.
21. Coenen MJ, Antonicka H, Ugalde C, Sasarman F, Rossi R, Heister JG, Newbold RF, Trijbels FJ, van den Heuvel LP, Shoubridge EA, Smeitink JA. Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. (2004). N Engl J Med. 351(20):2080-6.
22. Francisco Zurita-Díaz; Teresa Galera-Monge; Moreno-Izquierdo Ana; Mario F. Fraga; Carmen Ayuso; Agustin F. Fernández; Rafael Garesse; M. Esther Gallardo. Generation of a human iPSC line from a patient with a mitochondrial encephalopathy due to mutations in the GFM1 gene. (2016) Stem Cell Research. 16(1):124-127.
23. Brito S, Thompson K, Campistol J, Colomer J, Hardy SA, He L, Fernández-Marmiesse A, Palacios L, Jou C, Jiménez-Mallebrera C, Armstrong J, Montero R, Artuch R, Tischner C, Wenz T, McFarland R, Taylor RW. Long-term survival in a child with severe
encephalopathy, multiple respiratory chain deficiency and GFM1 mutations. (2015) Front Genet. 6:102.

OBJECTIVES:

The specific objectives of the project are:

1) To study the physiopathogenic mechanisms of mitochondrial encephalopathies caused by mutations in the GFM1 gene using as a model system iPS cells generated from the patient that the Foundation is named after.
2) A pilot study to identify pharmacological treatments for patients with defective mitochondrial translation.

METHODOLOGY:

1) To study the physiopathogenic mechanisms of mitochondrial encephalopathies caused by mutations in the GFM1 gene using as a model system iPS cells generated from the patient that the Foundation is named after.

By this stage we have already developed the protocol for differentiating iPS cells to neural stem cells, neurons and astrocytes and the next step is to carry out an electrophysiological characterization to enable us to confirm the functionality of these cell types as a preliminary study to any analysis of mitochondrial function. This characterization will consist of: 1) Identifying and quantifying the different subpopulations (glial cells and different neuronal cell types) obtained by differentiating neural stem cells to neurons. 2) An analysis of neuronal functionality through the study of calcium intermediaries by means of confocal microscopy. 3) “Time Lapse” to analyze mitochondrial transport in neurons and their quantification with ImageJ. 4) Electrophysiological characterization of neuronal functionality using patch clamp technique (membrane potential, action potential properties, sodium and potassium currents …) and 5) Analysis of neurons’ capacity to form synapses by analyzing pre- and post-synaptic antibodies.

Once neuron and astrocyte functionality has been confirmed electrophysiologically, studies will be carried out on mitochondrial function. To do so, we will analyze the impact that mutations in GFM1 have: 1) on the original fibroblasts (from which the iPS cells were generated), 2) on the iPS cells themselves and 3) on neurons and astrocytes. For this purpose, laboratory techniques will be used, including: a) Mitochondrial translation trials; b) Lactate production; c) High resolution respirometry testing; d) Measurement of enzyme activity in the respiratory chain complexes with the use of spectrophotometry; e) Analysis of mitochondrial membrane potential; f) ROS production and g) Apoptosis measurement.

2) A pilot study for the identification of pharmacological treatment for patients with defective mitochondrial translation.

Once the neurons and/or astrocytes have been characterized and obtained from the patient, and an/some altered phenotype/s has/have been identified through functional studies, a platform for therapeutic testing will be developed to identify possible drugs to rescue the alterations observed. As part of this objective we also aim to carry out a primary screening using the Prestwick Chemical Library, which we already have in the laboratory. This chemical compound library is presented in trays of 96 well plates that contain a collection of over 1200 molecules comprising off-patent drugs approved by the FDA. 85% of them have been approved and marketed as drugs suitable for human use to treat a wide variety of pathologies, including cardiovascular, infectious and neurodegenerative diseases and cancer. These drugs are selected for their high structural diversity, wide range of functions and mechanisms of action and well-characterized pharmacological, pharmacokinetic and toxicological properties. Once the primary screening is complete we will identify any positive hit(s) and carry out a second screening which will include dose-response curves. Due to the fact that the compounds tested are drugs that already exist and are not toxic for humans, those drugs that give positive hits in the second screening could much sooner be made available for the treatment of patients since they are already on the market.

WORK PLAN

The different activities outlined in the project will be undertaken by the members of the research team: the PI, Dr María Esther Gallardo (MEG); the co PI, Rafael Garesse Alarcón (RGA) and the pre-doctoral investigators: Francisco Zurita Díaz (FZD) and Teresa Galera Monge (TGM), for whom we request 9 months’ funding. Throughout the duration of this project the PI (MEG) will be responsible for supervising all the activities planned. The discussion of the results generated by the rest of the research team will be carried out by MEG and RGA. The distribution of tasks listed by objectives is as follows:

1. To study the physiopathogenic mechanisms of mitochondrial encephalopathies caused by mutations in the GFM1 gene using as a model system iPS cells generated from the patient that the Foundation is named after.

FZD will be responsible for fulfilling this objective by means of iPS cell differentiation experiments on neurons and astrocytes. The electrophysiological characterization of these cells will be carried out by graduate TGM, who has undergone training for this work in the Stem Cells, Aging and Neurodegeneration Laboratory of the Stem Cell Center, University of Lund, Sweden. We request nine months’ project funding for TGM (from April to December 2017). The studies of mitochondrial function characterization will be carried out by FZD and TGM (both have experience in these techniques as they have been using them routinely in the laboratory for over four years).

2. A pilot study for the identification of pharmacological treatments for patients with defective mitochondrial translation.

FZD and TGM will be in charge of drug screening .

BUDGET DETAILS:

1. Personnel Expenses:
Graduate Research Assistant’s salary for 9 months (April – December 2017) — 22.125€
Subtotal of Personnel Expenses: 22.125€

2. Running Costs:
A large part of the budget will be used for consumables required for the experiments proposed with iPSCs and central nervous system cultures. These include iPSC culture media, neuronal differentiation media, KO serum replacement, HyClone defined serum, growth factors, essential amino acids, supplements, antibiotics, antibodies, radioactive isotopes, enzymes for enzymatic method of analysis, biochemistry, cellular and molecular biology reagents for all the functional analyses programmed, culture plasticware and capillary tubes for mechanical alteration of iPSCs.

Subtotal of consumables (first year): 30,000€
Subtotal of consumables (second year): 30,000€

The rest of the expenses are to cover the cost of the different services required to run the project: storage of specimens in liquid nitrogen, genomics service, microscopy service, flow cytometry analysis etc.

Subtotal of service costs (first year): 10,000€
Subtotal of service costs (second year): 10,000€

Subtotal of Running Costs (first year): 40,000€
Subtotal of Running Costs (second year): 40,000€

Total Running Costs: 80,000€
Total Personnel Expenses: 22,125€

TOTAL PROJECT BUDGET: 102.125€

Execution: 100%