Genic Therapy for CMT2A: researchers from the Dino Ferrari Center involved in the research thanks to the support of the Mitofusin 2 Project Association.
Dr. Federica Rizzo, Dr. Monica Nizzardo, Prof. Stefania Corti, Prof. Giacomo P. Comi, Prof Nereo Bresolin
Dino Ferrari Center, University of Milan, I.R.C.C.S. Foundation Ca ’Granda Ospedale Maggiore Policlinico.
Our research project aims to develop a possible therapeutic approach for CMT2A, a sensorimotor polyneuropathy, characterized by the death of motor and sensory neurons. The pathology is caused by mutations in the Mitofusin 2 (MFN2) gene, that codes for the MFN2 protein, located in the membrane of a cell organelle, the mitochondria, which represents the power station of cells and performs essential biological functions. As yet, unfortunately, no resolutive therapy is available and very few research groups are involved in studying this disease. Gene therapy represents a promising strategy, since it is designed to correct the genetic cause underlying the disease itself. This type of strategy is giving excellent results in clinical trials with adeno-associated type 9 vectors (AAV9) for the common form of Spinal Muscular Atrophy (SMA), associated with mutations in the SMN gene (www.clinicaltrials.gov). The clinical trial is currently underway also at the Policlinico of Milan and is followed by our researchers. Therefore, taking advantage of the skills we have acquired in this sector, we propose to use the same type of approach also for the CMT2A. Nevertheless, the causes of this pathology are both the lack of the "healthy" gene, and the presence of the "sick" MFN2 protein are the cause of the pathology. Thus, this aspect of the disease requires, in anticipation of a therapy for the patients, to introduce the "healthy" gene -as it can be done for SMA or for other diseases only caused by the lack of a protein- but also to turn off the sick gene. Furthermore, the MFN2 protein, due to its important role in mitochondria function, must be present "in appropriate quantity", neither too low nor too high (another aspect that for other proteins isn’t so essential, in relation to their function).
For all these reasons, the development of a gene therapy for CMT2A requires additional efforts in terms of number of experiments and time.
On the bases of these premises, our therapeutic strategy for CMT2A requires to:
administer two therapeutic vectors: the first one enables to turn off the "sick" gene, the second one allows to express the "healthy" gene. A possible alternative may be the production of a single, larger vector, able to perform both functions.
obtain optimal MFN2 levels (neither too low nor too high) in order to avoid the appearance of adverse effects.
We have already generated created the necessary constructs, as well as demonstrated the silencing of the “sick” MFN2 gene and the correct expression of the “healthy” MFN2 protein, through the use of the vectors described in point 1, both in cellular and in animal models of this disease. In particular, in the cellular model we observed a marked improvement of the pathological phenotype (Figure 1).
We propose to complete these experiments and publish them in an indexed scientific journal, contributing to laying the first bases for an effective gene therapy for patients. Before proposing a clinical trial to the FDA/EMA, however, a more detailed analysis of this strategy with further studies in pre-clinical models is required. In this regard, the development of pre-clinical in vivo models of disease -that can more faithfully reproduce the clinical and neuropathological aspects of human CMT2A- will certainly be useful to assess the effectiveness of gene therapy.
The progress that has been made so far in this project will be presented in a session at the 71st Congress of the American Academy of Neurology, the largest professional organization of neurologists in North America, to be held on May 4-10, 2019 in Philadelphia. The presentation, entitled "RNAi/gene therapy combined approach and therapeutic strategy for Charcot-Marie-Tooth 2A" (Rizzo F, Bono S, Salani S, Bordoni A, Melzi V, Ruepp M, Pagliarani S, Barbullushi K, Abati E, Cordiglieri C, Bresolin N, Comi G, Nizzardo M, Corti S), will serve help to share the data and to focus the attention of a wide audience of researchers and clinicians on this disease. The commitment of our research group in recalling the scientific interest in this disease is also demonstrated by the recent publication in which we conducted a literature review concerning available in vitro and in vivo models of CMT2A and possible therapeutic approaches (Barbullushi K, Abati E, Federica Rizzo F, Bresolin N, Comi GP, Corti S. Disease modeling and therapeutic strategies in CMT2A: state of the art. in press Molecular Neurobiology).
Milan, March 4, 2019
FUNCTIONAL ANALYSIS AND GENOME-WIDE RNA-SEQ OF HUMAN MOTOR NEURONS IMPLICATE SELECTIVE MITOCHONDRIAL DEPLETION, RESISTANCE TO APOPTOSIS AND INCREASED MITOPHAGY IN CHARCOT-MARIE-TOOTH 2A
Rizzo F, Ramirez A., Ronchi R., Salani S, Nizzardo M, Fortunato F, Bordoni A, Bresolin N, Comi GP, Corti S.
Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation(DEPT), Univerity of Milan, Neurology Unit, IRCCS Fondation Cà Granda Ospedale Maggiore Policlinico, Milan, Italy
The disease type 2A Charcot-Marie-Tooth (CMT2A) is a sensorimotor polyneuropathy, characterized by the death of motor neurons and sensory, which results in progressive limb weakness, muscle atrophy and loss of sensory. The disease is due to mutations in the gene Mitofusina2 (MFN2), which encodes a protein localized at the mitochondria, organelles very important for the survival of the cells and their proper functioning. To date, unfortunately, there are no conclusive therapies for this condition.
The reprogramming of these mature cells in induced pluripotent stem cells (iPSC) offers the possibility of obtaining patient-specific cells, such as human motor neurons and sensory neurons, typically affected in the disease, but impossible to obtain directly from patients with other strategies. In the study published online August 9, 2016, on Human Molecular Genetics, the research team of the Centro Dino Ferrari, University of Milan, Fondazione IRCCS Ca 'Granda Ospedale Maggiore Policlinico , differentiated motor neurons (MN) from iPSCs derived from biopsy skin of patients with CMT2A. In this way, we have generated an in vitro model of the disease, currently not available. The cells thus obtained showed some typical aspects of the disease, such as reducing the amount of mitochondria and changes in their location, with no significant differences in survival. These defects are most apparent in neuronal cells results compared to skin fibroblasts, in agreement with the neuronal specificity of the disease. Overall, these data suggest that the reduction of mitochondria in motor neurons that express the mutated form of MFN2 , is not the result of a reduced production of mitochondria, but more likely the result of the death of these organelles. These mechanisms represent possible new molecular therapeutic targets for the development of an effective treatment for this disease.
Last updated on Oct 27, 2016
Dr. Federica Rizzo, Dr. Stefania Corti, Prof. Giacomo P. Comi
Centro Dino Ferrari Università degli Studi di Milano
Charcot-Marie-Tooth disease type 2A (CMT2A) is a sensory-motor polyneuropathy, characterized by the involvement of motor and sensitive neurons, resulting in progressive weakness, limb muscle atrophy and loss of sensitivity. Mitofusin2 (MFN2) gene has been identified as causative of the disease. The MFN2 protein,
located in the outer mitochondrial membrane, is involved in the mitochondrial functions. A treatment for CMT2A is not currently available. In our project, we aim to develop an effective therapy, based on the understanding of the disease molecular mechanisms, helpful not only to identify new therapeutic targets, but also to define specific disease hallmarks.
We first generated fibroblasts from CMT2A patients with different MFN2 mutations. The reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) provides the derivation of disease-specific cell types, such as motor and sensitive neurons, affected in the disease. Based on this method, we successfully generated human iPSCs from a CMT2A patient and demonstrated their differentiation into motor neurons. In particular, we observed an alteration in mitochondria localization, a reduction in the amount of mitochondrial DNA and a dysfunction of the mitochondrial respiratory chain, identifying specific hallmarks of the disease phenotype. These defects are more evident in neuronal cells compared to fibroblasts, in agreement with neuronal specificity of the disease. In addition in vitro models, the analysis of these aspects has been conducted in the only currently available mouse model of CMT2A (MitoCharc 1) to extend its characterization, searching for biomarkers of disease phenotype.
We aimed to develop a therapeutic approach for this disorder. We silenced endogenous MFN2 gene by short harpin RNA (shRNA) in CMT2A fibroblasts. At the same time, in order to restore correct MFN2 protein levels, we transfected a MFN2 c-DNA modified to be resistant to shRNA-mediated silencing. The results of this strategy were very promising in CMT2A fibroblasts, and preliminary data were also obtained in the CMT2A mouse model.
This study contributed to deepen the knowledge about disease molecular mechanisms, generating an in vitro model of CMT2A by patient-specific iPSCs, and to identify a possible therapeutic strategy for CMT2A.
The future developments of our research project will be:
- to increase the number of iPSC lines obtained from CMT2A patients and to differentiate them into neurons
-to test our RNAi therapeutic strategy in CMT2A neurons
-to apply our strategy in CMT2A mouse model, using adeno-viral type 9, capable of transferring our constructs into neuronal cells.
Recently, we decide to test CRISPR-CAS9 genome editing system to correct the disease MFN2 gene mutation directly at the DNA level. We will establish gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, guiding Cas9 in its cutting. At the same time, we will also deliver a DNA template strand. Cells repair the damage produced by Cas9 copying from the template and thus introducing new genetic material into the genome, which results in the correction of MFN2 mutation. The CRISPR system is considered a gold standard strategy for genome editing, such as demonstrated by positive results obtained in other pathologies.
Updated in Jan 2015
Corti S., Rizzo F., Ronchi D., Del Bo R., Nizzardo M., Comi G.P.
Charcot-Marie-Tooth disease type 2A (CMT2A), the most common type of CMT2, is caused by mutations in the mitofusin 2 gene (MFN2). For CMT2A treatment, no effective therapy is available. In this project, we propose two different approaches as a possible therapeutic strategy: one based on cell therapy and second on Morpholino antisense oligomer. To test these strategies, we will use MitoCharc1 mouse model, characterized by R94Q (Arginine to Glutamine) amino acid substitution to mimic the most common mutation found in CMT2A (Cartoni R et al.; 2010).
Regarding cell therapy strategy, a possible approach is to perform transplantation of stem cells to deliver growth factors which could have a therapeutic role on CMT2A neurons. In fact we have previously demonstrated that neural stem cell (NSC) transplantation can improve the phenotype in motor neuron disease (MND) mouse models (Corti S. et al.; 2006-2010). Growth factors such as Glia-derived neurotrophic factor (GDNF) are known to have neuroprotective effects in vitro and in vivo models of neurodegenerative diseases (Henderson CE et al.; 1994; Suzuki M et al.; 2007; 2008). Based on these data, we will evaluate the therapeutic potential of human NSCs and Myogenic Stem Cells (MySCs) obtained by patient specific Induced Pluripotent Stem Cells (iPSCs), alone or combined with growth factors delivery. Our goal is to isolate and characterize NSCs and MySCs from iPSCs, to genetically manipulate them to overexpress GDNF and to define an adequate transplantation protocol for their delivery into MitoCharc1 mice (intrathecal of NSCs and intramuscular of MySCs). Once determined survival and differentiative fate of NSCs/MySCs following transplantation, we will investigate the potential of wild-type and genetically modified NSCs/MySCs in protecting neurons and neuromuscular junctions improving neuromuscular functions.
Concerning Morpholino antisense oligomer strategy, we would like to use phosphorodiamidate morpholino (MO), third-generation backbone-modified antisense oligonucleotides (ASO), is designed to reduce human MNF-2 protein level. The use of MO can offer great promise for treatment of human disease, based on data recently described about motor neuron disease mouse models (Porensky PN et al.; 2011). In this project, we want to transfer this approach into CMT2A field. In particular we will test MO sequences, capable to silence MNF2 protein, in human or mouse cell lines to evaluate in vitro protein level reduction. Then we define the best MO administration protocols into MitoCharc1 mice, determining the dose and modalities of injection, considering the local (intracerebroventricular and/or intratechal) and/or systemic (intravenous) approaches. We will study the biodistribution of MO and their capacity to decrease mRNA and protein MNF-2 level in the mice by gene expression and protein analysis. We will also evaluate therapeutic efficacy of MO in improving neuromuscular phenotype, delaying disease progression and providing a survival benefit in treated MitoCharc1 mice. Our intention is to define a pre-clinical protocol with morpholino to high reduction of MNF2 level with minimum toxic consequences, offering a therapeutic clinical realistic approach.
Updated in Nov 2013