Neurological disease modeling for Sanfilippo: a key step towards understanding and treating a rare genetic disorder

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The disease modelling value of baby teeth: A new way to unlock knowledge about a special group of genetic disorders

Publication . Carvalho, Sofia; Santos, Juliana Inês; Moreira, Luciana; Gaspar, Paulo; Gonçalves, Mariana; Matos, Liliana; Encarnação, Marisa; Ribeiro, Diogo; Duarte, Ana Joana; Prata, Maria João; Coutinho, Maria Francisca; Alves, Sandra

Mucopolysaccharidoses (MPS), are a group of genetic, metabolic, and rare diseases investigated since the early years of the 20th century. One of the first steps to collect information about the underlying mechanisms of those disorders is the development and analysis of in vitro models. Furthermore, those models provide an appropriate platform for the evaluation of future therapeutics. Among all the possible disease cell models, patient-derived ones are those which allow us to get better disease insights. However, finding the best cell type that recapitulates diseaserelevant features is not always easy: two systems largely involved in MPS pathology are the brain and the musculoskeletal ones, which reflects an issue once both are hard to access. Here, our main goal is to establish an innovative non-invasive method to generate disease-relevant cell models from stem cells from deciduous (baby) teeth (SHED), which may then be differentiated into our MPS-target cell lines. So far, we have already implemented and optimized the protocol for collection, isolation, establishment and cryopreservation of those stem cells. Then, our rationale is simple: for those obtained from MPS patients suffering from multisystemic disease with marked musculoskeletal alterations, we are using a chondrogenesis differentiation protocol. For those derived from patients with neurological pathology, we will establish mixed neuronal/glial cultures. As soon as we can get the SHED-derived differentiated cells, various cellular and molecular processes from our target disorders may be unveiled and used as a target for possible future therapeutics. Acknowledgements This work is partially supported by the Portuguese Society for Metabolic Disorders (SPDM - Bolsa SPDM de apoio à investigação Dr. Aguinaldo Cabral 2018;2019DGH1629/SPDM2018I&D), Sanfilippo Children's Foundation (2019DGH1656/SCF2019I&D) and FCT (EXPL/BTM-SAL/0659/2021).

2022-11-08Conference object

Open access

Splicing Modulation as a Promising Therapeutic Strategy for Lysosomal Storage Disorders: The Mucopolysaccharidoses Example

Publication . Santos, Juliana Inês; Gonçalves, Mariana; Matos, Liliana; Moreira, Luciana; Carvalho, Sofia; Prata, Maria João; Coutinho, Maria Francisca; Alves, Sandra

Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.

2022-04-19Journal article

Open access

The 2020s Tooth Fairy: from loose tooth to neuronal cell cultures, an innovative method for in vitro genetic disease modeling of a rare neurological disorder

Publication . Carvalho, Sofia; Santos, Juliana Inês; Ribeiro, Diogo; Moreira, Luciana; Duarte, Ana Joana; Encarnação, Marisa; Gaspar, Paulo; Gonçalves, Mariana; Matos, Liliana; Prata, Maria João; Luísa, Pereira de Almeida; Coutinho, Maria Francisca; Alves, Sandra

The development of adequate in vitro disease models is a major issue in Biomedical Genetics. Those models allow for the initial screening of novel therapeutics and help us get an insight on the cellular mechanisms that underly pathology in each case. In fact, one of the best ways to get those insights is the analysis of patient-derived cells. Yet, not every cell holds potential to recapitulate relevant disease features. For neurodegenerative diseases in particular, it is challenging to grow neuronal cultures that accurately represent them because of the obvious inability to access live neurons. This scenario changed significantly when induced pluripotent stem cells (iPSC) were first described. From then on several differentiation protocols to generate neurons from iPSC were developed. Still, iPSC generation is a laborious/expensive protocol with significant limitations in terms of production and subsequent uses. Here we present an alternative to establish patient-derived neuronal cells in a much more expedite way. We are taking advantage of the existence of a population of multipotent stem cells (SC) in human dental pulp, the dental pulp stem cells (DPSC), to establish mixed neuronal and glial cultures for a rare neurological genetic disorder: the Sanfilippo syndrome. Sanfilippo-derived DPSC have never been used for differentiation into specific cell types even though they represent a natural source of SC that may be used to investigate human disease especially for the infantile forms. This is a total innovation in the field and we believe it holds potential to set a new trend for investigating the cellular/gene expression changes that occur in Sanfilippo and other related diseases as it relies on a non-invasive, cost-effective approach that can be set as a routine in any lab with standard cell culture conditions. Ultimately, the same method may be applied for virtually any monogenic disorder with neurological presentation.

2022-06Conference object

Restricted access

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step towards Understanding and Treating Mucopolysaccharidoses

Publication . Carvalho, Sofia; Santos, Juliana Inês; Moreira, Luciana; Gonçalves, Mariana; David, Hugo; Matos, Liliana; Encarnação, Marisa; Alves, Sandra; Coutinho, Maria Francisca

Despite extensive research, the links between the accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of mucopolysaccharidoses (MPSs) have yet to be further elucidated. This is particularly true for the neuropathology of these disorders; the neurological symptoms are currently incurable, even in the cases where a disease-specific therapeutic approach does exist. One of the best ways to get insights on the molecular mechanisms driving that pathogenesis is the analysis of patient-derived cells. Yet, not every patient-derived cell recapitulates relevant disease features. For the neuronopathic forms of MPSs, for example, this is particularly evident because of the obvious inability to access live neurons. This scenario changed significantly with the advent of induced pluripotent stem cell (iPSC) technologies. From then on, a series of differentiation protocols to generate neurons from iPSC was developed and extensively used for disease modeling. Currently, human iPSC and iPSC-derived cell models have been generated for several MPSs and numerous lessons were learnt from their analysis. Here we review most of those studies, not only listing the currently available MPS iPSC lines and their derived models, but also summarizing how they were generated and the major information different groups have gathered from their analyses. Finally, and taking into account that iPSC generation is a laborious/expensive protocol that holds significant limitations, we also hypothesize on a tempting alternative to establish MPS patient-derived neuronal cells in a much more expedite way, by taking advantage of the existence of a population of multipotent stem cells in human dental pulp to establish mixed neuronal and glial cultures.

2023-04-21Journal article

Open access

The disease modelling value of a folklore FAIRYtale: SHEDing light over a special group of genetic disorders

Publication . Carvalho, S.; Santos, J.I.; Moreira, L.; Gaspar, P.; Gonçalves, M.; Encarnação, M.; Ribeiro, D.; Duarte, A.; Prata, M.J.; Coutinho, M.F.; Alves, Sandra

The problem we are addressing: Despite extensive research, the links between accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of Mucopolysaccharidoses (MPSs) have yet to be further elucidated. These Lysosomal Storage Diseases (LSDs) present symptoms, which may (or may not) include critical musculoskeletal and cardiovascular alterations, respiratory problems, and serious neurological dysfunctions. The skeletal and brain systems are the hardest ones to access and, consequently, those in greatest need of additional knowledge and novel therapeutic solutions.

2023-05Conference object

Restricted access