Since we and others discovered the involvement of the LMNA gene encoding the nuclear proteins Lamins A/C, in Hutchinson-Gilford Progeria Syndrome (OMIM #176670), one of the best known premature aging syndromes (De Sandre-Giovannoli et al., Science 2003), our team focuses on translational research on premature aging (progeroid) syndromes (PS), nuclear protein-linked lipodystrophies, as well as other premature aging diseases, with or without an identified molecular basis. Our research aims to identify novel genes involved in these syndromes, increase our understanding of their pathophysiological mechanisms and to develop new therapeutic approaches using patients’ cell lines or mouse models as our main preclinical tools.
PS are a group of rare genetic disorders characterized by many clinical features that mimic those associated to physiological aging. Many PS share the dysfunction of a prenylated protein. This is the case in Hutchinson-Gilford Progeria Syndrome (HGPS) and related PS, where prenylated progerin or prelamin A aberrantly accumulate in cells’ nuclei, exerting multiple toxic effects and ultimately leading to the systemic severe phenotypes observed in patients. Besides, several other PS have been discovered as being caused by activating mutations in genes encoding permanently prenylated proteins of the RAS‐MAPK pathways. Among these “RASopathies”, research on Costello syndrome, due to H-RAS mutations, will be part of this project. Finally, several lines of evidence suggest that progerin production may also be involved in natural aging pathomechanisms.
Our research has focused until now on the following axes:
a. Extend the clinical spectrum of syndromes associated with prelamin A deficient processing.
We identified several novel or related syndromes mainly characterized by premature aging 7-25 or lipodystrophy / metabolic syndrome 9, 26 linked to mutations of LMNA or functionally related genes; as well as previously undiscovered genetic associations 10.
b. Produce in vitro and in vivo models for studying rare progeroid syndromes
A large panel of data and primary fibroblast cell lines from children affected with typical Progeria or atypical PS, and progeroid RASopathies have been collected through national and international collaborations and are stored in the Biological Resources Center (CRB-TAC), at La Timone hospital, Marseille. Human iPS cell lines (hiPSC) have also been derived and used to study the pathophysiology of the syndrome 27 or compare ongoing or proposed treatments 28. Primary endothelial cells and progenitors issued from human umbilical vein or cord blood were also used as in vitro models to study HGPS pathophysiology 29. Importantly, in collaboration with Pr Lopez-Otin in Oviedo, we generated the mouse model of progeria which most closely mimics the human disease (Knock-In LmnaG609G/G609G) by reproducing the same aberrant splicing mechanism leading to progerin production 30. Additionally, we are involved in a consortium with the mouse clinical institute and the medical genetics team in Bordeaux, to explore a KI model of CS carrying the most common H-RAS p.G12S mutation, based on a project funded by the ANR (Cf. Appendix 7).
c. Identify disease mechanisms in progeroid syndromes
- We demonstrated a common toxic pathway involving prenylated prelamin A accumulation in many PS ranging from restrictive dermopathy and MAD-B to HGPS-like syndromes 8, 12, 24, 25, 31-36.
- We showed that the microRNA miR‐9 negatively controls lamin A and progerin expression in HGPShiPSC derived neural cells 27, 28, having a protective effect on the patients’ central nervous system, which is clinically spared.
- A miRNome analysis by qRT-PCR on dermal fibroblasts of 5 HGPS patients and 5 healthy individuals at early and late passages, allowed the identification of 29 deregulated miRNAs (15 overexpressed, 14 underexpressed; Frankel et al., publication in preparation). Based on these in vitro results, the potential benefit of miRNA-based therapeutic approaches (antagomiRs or mimics) in HGPS is under evaluation.
- We contributed to scientific reviews on progeroid laminopathies and related syndromes 7, 8, 37
- We reported HRAS mutations in CS patients, confirming the involvement of the activated prenylated RAS proteins 38 in RASopathies, shown to trigger senescence in vitro 39.
d. Establish preclinical proofs of principle for therapeutic approaches and launch clinical trials
- We demonstrated the synergistic effect of pravastatin (statin) and zoledronate (N-BisPhosphonate) (ZoPra) and its effectiveness in reducing prenylation and rescuing HGPS cells defects and progeroid phenotypes in Zmpste24-/- mice 40.
- this preclinical work allowed our clinical team to design, and conduct a phase II therapeutic trial (ClinicalTrials.gov, NCT00731016) using zoledronate and pravastatin to evaluate its safety and efficacy in 12 HGPS patients included for 3.5 years each, with partially positive results on primary and secondary endpoints and without severe adverse effects (De Sandre‐Giovannoli, Sigaudy et al, submitted). An Orphan drug designation for this drug combination, “ZoPra”, was also obtained from EMA for progeria, under patent with an exclusive licence to ProGeLife, a spin-off of our academic team, cf. Appendix 6.
- We showed an efficient reduction of progerin production in vitro at the mRNA and protein levels and phenotypic rescue in the LmnaG609G/G609G mice, using systemic antisense oligonucleotide (AON) treatment 41, providing the first preclinical proof of principle in vivo of the efficacy of this novel and promising approach.
- We further provided a preclinical proof of principle for the use of a similar, but personalized antisense treatment in patients affected with HGPS-like and type B Mandibuloacral Dysplasia syndromes 42, who may therefore also be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients.
- We collaborated in preclinical studies using high-throughput screening of already marketed compounds with HGPS-derived iPS-MSC, leading to the identification of potential novel drugs 43, 44 or comparisons of the efficacy of treatments being tested in clinical trials 28.
Importantly, we recently identified that a specific class of proteasome inhibitors, including MG132, are able to reduce progerin levels by inducing both its degradation through autophagy and inhibition of its production by mRNA splicing modulation45. These findings pave the way for the development of a novel promising class of drugs for the treatment of patients affected with Hutchinson-Gilford Progeria and related syndromes. They may as well find larger applications in diseases involving the accumulation of prenylated proteins or natural aging. This discovery was patented and is co-exploited by ProGeLife (co-owner) and SATT-Sud Est (SATT-SE).
Our future research will be developed on similar strategies:
- Pursue the data and biological samples’ collection from patients affected with PS and RASopathies stored in the labelled Biological Resource Center (CRB TAC), Department of Medical Genetics, Timone Hospital of Marseille; by establishing iPSC lines which are undergoing differentiation in cell lines of interest, depending on the patient’s phenotypes (collaboration with X. Nissan, iSTEM and F. Magdinier, U910 iPSC platform), by developing future CRISPR mutated iPS cell lines with mutations of interest. Our center also participates to the National registry “OPALE” (https://epidemiologie-france.aviesan.fr/epidemiologie-france/fiches/observatoire-des-patients-atteints-de-laminopathies-et-emerinopathies), collecting longitudinal informations on patients affected with laminopathies and carrying mutations of the LMNA gene.
- Identify further biomarkers and pathophysiological pathways of premature aging: To dissect the mechanisms of aging caused by dysfunctions of prenylated proteins, we will combine OMICs data (genome, transcriptome/miRNome, proteome, and microbiome), with bioinformatics and systems’ biology interpretation tools. We aim to identify disease biomarkers, pro-and anti-aging factors, and druggable targets using primary fibroblasts from HGPS, Atypical Progeroid Syndromes (APS) due to LMNA mutations, CS patients and age and sex-matched controls; fibroblast-derived hiPSCs cells; 2) iPS-derived differentiated cells (coll. F. Magdinier) from selected APS patients and compared to similar age-matched HGPS and control cells; 3) mouse tissues or tail-tip fibroblast cultures of HGPS (LmnaG609G/G609G) and CS (HrasG12S/G12S) models and controls (wild type mice from the same crosses).
- Identify and validate Proof-of-Concepts for molecules exhibiting a therapeutic potential in cellular and animal models: we propose a multi-step, sequential translational approach from patients’ cells explorations to the preclinical validation of compounds in order to advance with solid bases towards novel clinical trials. Both pharmacologic and gene therapy approaches are under study in the team. Concerning the first approach, we recently identified that MG132 and other aldehyde compounds of the same class of proteasome inhibitors are able to dramatically reduce progerin intranuclear levels and ameliorate several disease parameters in HGPS patients’ cell lines. These molecules act by preventing progerin synthesis and increasing progerin degradation both in vitro (HGPS fibroblasts, iPS-MSC and iPS-VSMC from HGPS iPSC) and in vivo (muscles of LmnaG609G/G609G mice)45. Preliminary in vivo pilot studies indicate that the systemic administration of MG132 will require the formulation of a novel galenic form to increase its half-life: this work will be developed in collaboration with industrial and academic partners.In this research axis, the different compounds that are found efficient alone will be tested in combination. Their ability to improve classical disease biomarkers in vitro will be evaluated, with dose-response studies. This study will be completed in vivo, exploring: the drug pharmacokinetics, safety/toxicity, the effects on progerin levels and OMICS studies to validate drug activity and to search for side-effects.
Interestingly, since progerin has been shown to accumulate in cells’ nuclei during natural aging 3, the proposed research on HGPS and related syndromes may have spinoffs in the field of natural aging.
- Identification of novel genes involved in premature aging syndromes: Efforts will be continued towards: 1) identifying novel causal genes in patients affected with sporadic PS or lipodystrophy syndromes of unknown origin (ex. WiedemannRautenstrauch, Hallermann-Streiff syndromes, atypical progeroid syndromes…), and genomic modulators that account for phenotypic variability in PS with extreme phenotypes
- Functional characterization of pathogenic variants in Lamin A and LINC complex proteins in nuclear protein-linked lipodystrophies: Recent studies have revealed the important role played by the nuclear envelope (NE) in the cell response to various external environmental constraints. In particular, A-type Lamins and proteins of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex are key actors of nucleo-cytoskeletal coupling, playing a role in nuclear signal mechanotransduction 46. We’ll use patients’ cells to investigate the relationship between NE and LINC complex proteins and pathologies.