Less is More: an overview on the use of RNAi as a tool to achieve Substrate Reduction in Mucopolysaccharidoses

Lysosomal storage diseases (LSDs) are a group of genetic disorders caused by dysfunction in enzymes responsible for the intralysosomal degradation of particular compounds. Given their complex nature and the limitations of available therapies, the shift towards the development of combination treatments to counteract more effectively the pathological burden of these disorders is in the agenda of current research viewing to improve the clinical outcome of LSD patients. We consider that treatment strategies relying on RNA interference (RNAi), as well as in other RNA-based methodologies, may be feasible and particularly promising if designed in the context of a synergistic combinatorial therapeutic approach. We are currently evaluating an RNAi-dependent strategy based upon the selective downregulation of genes involved in the biosynthesis of glycosaminoglycans (GAGs), the major substrates that accumulate in patients suffering from a subset of LSDs called mucopolysaccharidoses (MPSs). Although enzyme replacement therapy is already available for some MPSs, it has some serious drawbacks, justifying the challenge of developing additional therapies targeting this group of disorders. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPSs’ symptoms. It should be noticed that, even though some substrate reduction therapy (SRT) drugs have already been approved (miglustat for GD) or are undergoing clinical trial (genistein and/or rhodamine B for MPSs), clinical evaluation of those same drugs has unveiled a few side effects, the most well-known being those observed for miglustat, which included osmotic diarrhea and weight loss. Nevertheless, chemical drugs aren’t the only way to achieve substrate reduction. Ours is fully molecular, drug-free approach, whose major focus relies on the biosynthetic pathways giving origin to each one of the GAGs whose degradation is impaired in MPS. Taking advantage of the RNAi technology potential, we have designed and assayed specific siRNAs targeting genes on those biosynthetic cascades to decrease the levels of production of each one of the four substrates. Their efficiency is currently being evaluated in vitro. Here we present an overview of the preliminary results of this project and unveil its next steps towards a full characterization/evaluation of its potential therapeutic effect.This work was partially supported by Fundação Millennium bcp (bcp/LIM/DGH/2014). Coutinho MF is grantee from the FCT (SFRH/BPD/101965/2014).N/

Coutinho et al. Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders. Int. J. Mol. Sci. 2016, 17, 1065

Erratum for Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders. [Int J Mol Sci. 2016]n/a.info:eu-repo/semantics/publishedVersio

Genetically modulated Substrate Reduction Therapy for Mucopolysaccharidoses – in vitro studies

Mucopolysaccharidoses (MPSs) are caused by dysfunction in enzymes responsible for the intralysosomal degradation of glycosaminoglycans (GAGs). We have designed an RNA-based strategy based upon the selective downregulation of genes involved in the biosynthesis of GAGs, which is currently under evaluation. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPSs’ symptoms. Taking advantage of the RNA interference (RNAi) technology potential, we have designed and assayed specific siRNAs targeting genes on those biosynthetic cascades to decrease the levels of production of each one of the four substrates: dermatan sulphate (DS), heparan sulphate (HS), keratan sulphate (KS), and chondroitin sulphate (CS). MPSs were divided into two major sub-groups: (1) those that accumulate DS/CS and (2) those that accumulate HS. ‘Group 1’ included MPS types I, II, VI and VII, while ‘group 2’ includes the Sanfilippo syndrome, or MPS III, which subdivides into four different diseases: IIIA, IIIB, IIIC and IIID. Proof of principle on the effect of siRNAs targeting CHSY1 and XYLT1 was achieved for two independent control cell lines, with 8-12 fold decreases on the target mRNA levels, after 24h of incubation with concentrations of each siRNA as low as 20nM. Subsequent analysis on the effect of those same siRNAs on patients’ cell lines resulted in significant CHSY1 expression decrease in MPS I/MPS VI cell lines (‘group 1’), as well as that of XYLT1 in MPS IIIA and IIID fibroblasts (‘group 2’). Initial studies evaluated mRNA levels after 24-48h incubation with each siRNA. Even though relevant decreases were observed for all tested cell lines, it became evident that the treatment efficacy may depend on the features of each specific MPS cell line, with some lines requiring higher siRNA concentrations to promote similar inhibition levels. In order to assess the effect of that treatment on substrate reduction, we have used both the routine Alcian blue and a modified, more sensitive 1,9-dimethylmethylene blue assay on the culture media collected after seeding and incubation, at different time points. Nevertheless, the low confluency levels required for siRNA transfection did not allow detection of GAGs excreted to the culture media. Similar problems have been noted by other authors, particularly in small samples, like the ones we used. Thus, we are currently quantifying GAGs’ storage by direct measurement of tissue samples after papain extraction. By doing so, we can access the intralysosomal levels of GAGs instead of their excretion. Here we present an overview of the preliminary results of this project and unveil its next steps towards a full characterization/evaluation of its potential therapeutic effect.This work was partially supported by Fundação Millennium bcp (bcp/LIM/DGH/2014). Coutinho MF and Santos JI are grantees from the FCT (SFRH/BPD/101965/2014 and SFRH/BD/124372/2016, respectively).N/

Genetically Modulated Substrate Reduction Therapy for Sanfilippo Syndrome: Proof of Principle

About proof of principle in genetically modulated substrate reduction therapy for Sanfilippo syndrome.FCT SFRH/BPD/101965/2014; SFRH/BD/124372/2016N/

Development of Engineered-U1 snRNA Therapies: Current Status

Review(This article belongs to the Special Issue Future Challenges and Trends of Nucleic Acids)Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.Funding: This work was financially supported with funding from FCT/MCTES (UIDB/00211/2020) through national funds, and also partially supported by the MPS Society project (2019DGH1642) and the LA/P/0059/2020—Laboratório Associado para a Ciência Animal e Veterinária/Associate Laboratory for Animal and Veterinary Sciences (AL4animalS).info:eu-repo/semantics/publishedVersio

Lysosomal storage diseases in Portugal: 10 years of experience in molecular studies at National Health Institute (2006-2016)

As Doenças Lisososomais de Sobrecarga (DLS) são um grupo de mais de 50 doenças hereditárias do metabolismo, sendo a maioria causada por defeitos em enzimas lisossomais específicas. A característica distintiva das DLS é a acumulação lisossomal do(s) substrato(s) não degradado(s), bem como a acumulação de outro material secundariamente à disfunção lisossomal. A apresentação clínica destas patologias é bastante heterogénea, variando desde formas pré-natais, até apresentações infantis ou na idade adulta, sendo frequente a presença de atraso psicomotor e neurodegeneração progressiva. Neste artigo são apresentados os resultados de vários estudos de caracterização molecular efetuados ao longo da última década (2006-2016) em doentes portugueses com as seguintes DLS: Mucopolissacaridose II, Mucopolissacaridose IIIA, Mucopolissacaridose IIIB, Mucopolissacaridose IIIC, Sialidose, Galactosialidose, Gangliosidose GM1, Mucolipidose II alfa/beta, Mucolipidose III alfa/beta, Mucolipidose III gama e Doença de Unverricht-Lundborg. De um modo geral, estes trabalhos permitiram conhecer as variações genéticas associadas a estas DLS, analisar a sua distribuição na população portuguesa e compreender o seu papel na forma de apresentação clínica destas patologias.Lysosomal Storage Diseases (LSDs) are a group of more than 50 inherited metabolic diseases which in the majority of cases, result from a defective function of specific lysosomal enzymes. The classical hallmark of LSDs is the lysosomal accumulation of unhydrolyzed substrate(s) as well as storage of other material secondary to lysosomal dysfunction. LSD clinical presentation has considerable heterogeneity, ranging from prenatal forms to later onset infantile or adult presentations, often with psycho-motor delay and progressive neurodegeneration. In this paper we present the results of several molecular studies performed over the last decade (2006-2016) in Portuguese patients with the following LSDs: Mucopolysaccharidosis II, Mucopolysaccharidosis IIIA, Mucopolysaccharidosis IIIB, Mucopolysaccharidosis IIIC, Sialidosis, Galactosialidosis, GM1 Gangliosidosis, Mucolipidosis II alpha/beta, Mucolipidosis III alpha/beta, Mucolipidosis III gamma and Unverricht-Lundborg disease. Globally these studies have provided valuable data regarding the underlying genetic causes of these LSDs and the frequency of their causative mutations the Portuguese population, further contributing to a better understanding of their role in the clinical presentation of the symptoms.Estudos parcialmente financiados pela Comissão de Fomento da Investigação em Cuidados de Saúde, Ministério da Saúde (P.I. nº 99/2007 e nº 100/2007) e pela Fundação para a Ciência e a Tecnologia (PIC/IC/83252/2007 e PIC/IC/82822/2007)

Usability of functional electrical stimulation in upper limb rehabilitation in post-stroke patients: A narrative review

Stroke leads to significant impairment in upper limb (UL) function. The goal of rehabilitation is the reestablishment of pre-stroke motor stroke skills by stimulating neuroplasticity. Among several rehabilitation approaches, functional electrical stimulation (FES) is highlighted in stroke rehabilitation guidelines as a supplementary therapy alongside the standard care modalities. The aim of this study is to present a comprehensive review regarding the usability of FES in post-stroke UL rehabilitation. Specifically, the factors related to UL rehabilitation that should be considered in FES usability, as well a critical review of the outcomes used to assess FES usability, are presented. This review reinforces the FES as a promising tool to induce neuroplastic modifications in post-stroke rehabilitation by enabling the possibility of delivering intensive periods of treatment with comparatively less demand on human resources. However, the lack of studies evaluating FES usability through motor control outcomes, specifically movement quality indicators, combined with user satisfaction limits the definition of FES optimal therapeutical window for different UL functional tasks. FES systems capable of integrating postural control muscles involving other anatomic regions, such as the trunk, during reaching tasks are required to improve UL function in post-stroke patients.info:eu-repo/semantics/publishedVersio

Postural control during turn on the light task assisted by functional electrical stimulation in post stroke subjects

Postural control mechanisms have a determinant role in reaching tasks and are typically impaired in post-stroke patients. Functional electrical stimulation (FES) has been demonstrated to be a promising therapy for improving upper limb (UL) function. However, according to our knowledge, no study has evaluated FES infuence on postural control. This study aims to evaluate the infuence of FES UL assistance, during turning on the light task, in the related postural control mechanisms. An observational study involving ten post-stroke subjects with UL dysfunction was performed. Early and anticipatory postural adjustments (EPAs and APAs, respectively), the weight shift, the center of pressure and the center of mass (CoM) displacement were analyzed during the turning on the light task with and without the FES assistance. FES parameters were adjusted to improve UL function according to a consensus between physiotherapists’ and patients’ perspectives. The ANOVA repeated measures, Paired sample t and McNemar tests were used to compare postural control between the assisted and non-assisted conditions. When the task was assisted by FES, the number of participants that presented APAs increased (p= 0.031). UL FES assistance during turning on the light task can improve postural control in neurological patients with UL impairments.info:eu-repo/semantics/publishedVersio

Neutral Sterols of Cephalic Glands of Stingless Bees and Their Correlation with Sterols from Pollen

Sterols are essential molecules in the membrane lipid composition and precursors of important sterol hormones that regulate many developmental processes. Insects are unable to synthesize sterols de novo and, thus, all phytophagous insects depend on an exogenous source of sterols for growth, development, and reproduction. The sterol requirements of social bees are not fully known due to the fact that there is no well-defined diet available throughout the year with regard to floral resources. Our study aimed to characterize the sterols present in pollen stored in Melipona marginata and Melipona scutellaris colonies, as well as evaluating their presence in the mandibular, hypopharyngeal, and cephalic salivary gland secretions. We analyzed the chemical composition of pollen stored in the colonies and the composition of the cephalic glands of workers in three adult functional phases (newly emerged, nurses, and foragers) by gas chromatography and mass spectrometry. The results showed that the pollen analyzed contained campesterol, stigmasterol, sitosterol, isofucosterol, lanosterol, and small amounts of cholesterol. The glands showed the same compounds found in the pollen analyzed, except lanosterol that was not found in M. scutellaris glands. Surprisingly, cholesterol was found in some glands with relative ratios greater than those found in pollen