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Machado–Joseph disease (MJD) or spinocerebellar ataxia 3 (SCA3) is a rare, inherited, monogenic, neurodegenerative disease, and the most common SCA worldwide. MJD/SCA3 causative mutation is an abnormal expansion of the triplet CAG at exon 10 within the ATXN3 gene. The gene encodes for ataxin-3, which is a deubiquitinating protein that is also involved in transcriptional regulation. In normal conditions, the ataxin-3 protein polyglutamine stretch has between 13 and 49 glutamines. However, in MJD/SCA3 patients, the size of the stretch increases from 55 to 87, contributing to abnormal protein conformation, insolubility, and aggregation. The formation of aggregates, which is a hallmark of MJD/SCA3, compromises different cell pathways, leading to an impairment of cell clearance mechanisms, such as autophagy. MJD/SCA3 patients display several signals and symptoms in which the most prominent is ataxia. Neuropathologically, the regions most affected are the cerebellum and the pons. Currently, there are no disease-modifying therapies, and patients rely only on supportive and symptomatic treatments. Due to these facts, there is a huge research effort to develop therapeutic strategies for this incurable disease. This review aims to bring together current state-of-the-art strategies regarding the autophagy pathway in MJD/SCA3, focusing on evidence for its impairment in the disease context and, importantly, its targeting for the development of pharmacological and gene-based therapies. CureCSB project info:eu-repo/semantics/publishedVersion

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in phagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ders. national funds through FCT (Foundation for Science and Technology) -Brain Health2020 projects CENTRO-01-0145-FEDER-000008, UID/NEU/04539/2020, CENTRO-01-0145-FEDER-022095, PTDC/NEU-NMC/0084/2014|POCI-01-0145-FEDER-016719, POCI-01-0145-FEDER-029716, POCI-01-0145-FEDER-016807, POCI-01-0145-FEDER-016390, POCI-01-0145-FEDER-032309, info:eu-repo/semantics/publishedVersion

ATXN2 gene, encoding for ataxin-2, is located in a trait locus for obesity. Atxn2 knockout (KO) mice are obese and insulin resistant; however, the cause for this phenotype is still unknown. Moreover, several findings suggest ataxin-2 as a metabolic regulator, but the role of this protein in the hypothalamus was never studied before. The aim of this work was to understand if ataxin-2 modulation in the hypothalamus could play a role in metabolic regulation. Ataxin-2 was overexpressed/re-established in the hypothalamus of C57Bl6/Atxn2 KO mice fed either a chow or a high-fat diet (HFD). This delivery was achieved through stereotaxic injection of lentiviral vectors encoding for ataxin-2. We show, for the first time, that HFD decreases ataxin-2 levels in mouse hypothalamus and liver. Specific hypothalamic ataxin-2 overexpression prevents HFD-induced obesity and insulin resistance. Ataxin-2 re-establishment in Atxn2 KO mice improved metabolic dysfunction without changing body weight. Furthermore, we observed altered clock gene expression in Atxn2 KO that might be causative of metabolic dysfunction. Interestingly, ataxin-2 hypothalamic re-establishment rescued these circadian alterations. Thus, ataxin-2 in the hypothalamus is a determinant for weight, insulin sensitivity and clock gene expression. Ataxin-2’s potential role in the circadian clock, through the regulation of clock genes, might be a relevant mechanism to regulate metabolism. Overall, this work shows hypothalamic ataxin-2 as a new player in metabolism regulation, which might contribute to the development of new strategies for metabolic disorders.

Machado-Joseph disease (MJD), also named spinocerebellar ataxia type 3 (SCA3), is a dominantly inherited neurodegenerative disease caused by abnormal CAG expansions in MJD1 gene, which translate to an overexpanded tract of glutamines in the ataxin-3 (ATXN3) protein. Since the identification of the causative gene, a huge effort was made toward the development of animal models for MJD/SCA3, to increase the understanding of the molecular mechanisms underpinning disease pathogenesis, and to develop therapeutic strategies for the disease. Nevertheless, until now there are no therapies available capable of stopping or delaying the disease progression, which culminates with the death of the patients. Therefore, there is an urgent unmet need for therapeutic solutions, for which gene therapy stands out. The RNA interference (RNAi) mechanism discovery allowed the identification of small RNA molecules with the ability to regulate gene expression. For gene therapy, RNAi provided a way to silence mutant genes, which are particularly useful in dominantly inherited diseases. In the last years, several studies have focused on using RNAi molecules to target mutant ATXN3. The results showed that this could be an efficient and safe strategy for modifying MJD/SCA3 progression. Now, an additional effort must be done to translate these results into clinical trials.

Polylgutamine (polyQ) diseases are a group of neurodegenerative disorders caused by abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cell roles, the pathogenic mutant proteins bearing an expanded polyQ sequence share a tendency to self-assemble, aggregate, and engage in abnormal molecular interactions. Understanding the shared paths that link polyQ protein expansion to the nervous system dysfunction and degeneration that takes place in these disorders is instrumental for identifying targets for therapeutic intervention. Among polyQ diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve the functional compromise of the cerebellum, resulting in the hallmark ataxic signs. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, spinocerebellar ataxias type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. PolyQ disease pathophysiology has been described to involve an inability to properly respond to cell stress. Taking into consideration, we evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and in stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyQ protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from SCA2 and SCA3 patients, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in a SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioral deficits. Overall, our results suggest that a decrease in G3BP1 levels may be a component of SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyQ disorders.

Spinocerebellar ataxia type 2 (SCA2) is a rare autosomal, dominantly inherited disease, in which the affected individuals have a disease onset around their third life decade. The molecular mechanisms underlying SCA2 are not yet completely understood, for which we hypothesize that aging plays a role in SCA2 molecular pathogenesis. In this study, we performed a striatal injection of mutant ataxin-2 mediated by lentiviral vectors, in young and aged animals. Twelve weeks post-injection, we analyzed the striatum for SCA2 neuropathological features and specific aging hallmarks. Our results show that aged animals had a higher number of mutant ataxin-2 aggregates and more neuronal marker loss, compared to young animals. Apoptosis markers, cleaved caspase-3, and cresyl violet staining also indicated increased neuronal death in the aged animal group. Additionally, mRNA levels of microtubule-associated protein 1 light-chain 3B (LC3) and sequestosome-1 (SQSTM1/p62) were altered in the aged animal group, suggesting autophagic pathway dysfunction. This work provides evidence that aged animals injected with expanded ataxin-2 had aggravated SCA2 disease phenotype, suggesting that aging plays an important role in SCA2 disease onset and disease progression. info:eu-repo/semantics/publishedVersion

Koppenol et al. show that overexpression of G3BP1 in cell models of SCA2 and SCA3 leads to a reduction in ataxin-2 and ataxin-3 aggregation. G3BP1 lentiviral delivery reduces motor deficits and neuropathology in preclinical models, suggesting that G3BP1 may be a potential therapeutic target for polyQ disorders. Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders. PPBI-POCI-01-0145-FEDER-022122 info:eu-repo/semantics/publishedVersion

Polyglutamine (polyQ) diseases are a group of 9 rare neurodegenerative disorders caused by an abnormal expansion of the CAG trinucleotide in the codifying regions of the respective disease-associated gene. The trinucleotide abnormal expansion leads to the translation of a protein containing an overexpanded tract of glutamines. PolyQ mutant proteins undergo a gain of toxic function disrupting normal cellular pathways leading to neuronal death and, consequently, leading to selective neurodegeneration of specific brain regions. Spinocerebellar ataxia (SCA) 2 and SCA3 (also known as Machado-Joseph disease) are two different polyQ diseases in which the ataxin-2 and ataxin-3 proteins, respectively, bear abnormally long polyQ tracts. Until now, there is no treatment for these fatal diseases or therapies that could delay the normal pathologic progression. Stress granules (SGs) are important structures formed in response to cellular stress, having an important role in mRNA triage. A core component of SGs is the RNA-binding protein (RBP) Ras GTPase-activating protein-binding protein 1 (G3BP1), which is also implicated in the SGs assembly. Furthermore, G3BP1 is known to have endoribonuclease activity and an important role in modulating RNA metabolism. In this study, we showed that G3BP1 is decreased in context of SCA2 and SCA3 disease. For that, we assessed whether restoring the expression levels of G3BP1 might positively impact the SCA2 and SCA3 pathology. We showed that gene delivery of G3BP1 in two distinct lentiviral mouse model of SCA2 and SCA3 was able to i) reduce the number of aggregates and ii) reduce the loss of neuronal marker associated with the mutant toxic proteins. Importantly, in a polyQ transgenic mouse model, lentiviral delivery of G3BP1 in the cerebellum was able to i) preserve the number of Purkinje cells, ii) reduce the number of HA-ataxin-3 and, importantly iii) improve the motor performance, balance and coordination. Additionally, we identify the nuclear transport nuclear transport factor 2-like (NTF2-like) domain and the ser149 phosphorylation site of G3BP1 as a key players in the reduction of mutant ataxin-2 and ataxin-3 levels and aggregation. Altogether these results showed that gene delivery of G3BP1 is able of mitigating the disease-associated phenotype in SCA2 and SCA3 disease, in three different disease mouse models. Therefore, this study suggests G3BP1 as a novel therapeutic target for SCA2 and SCA3 diseases.

Spinocerebellar ataxia type 2 (SCA2) is an incurable and genetic neurodegenerative disorder. The disease is characterized by progressive degeneration of several brain regions, resulting in severe motor and non-motor clinical manifestations. The mutation causing SCA2 disease is an abnormal expansion of CAG trinucleotide repeats in the ATXN2 gene, leading to a toxic expanded polyglutamine segment in the translated ataxin-2 protein. While the genetic cause is well established, the exact mechanisms behind neuronal death induced by mutant ataxin-2 are not yet completely understood. Thus, the goal of this study is to investigate the role of autophagy in SCA2 pathogenesis and investigate its suitability as a target for therapeutic intervention. For that, we developed and characterized a new striatal lentiviral mouse model that resembled several neuropathological hallmarks observed in SCA2 disease, including formation of aggregates, neuronal marker loss, cell death and neuroinflammation. In this new model, we analyzed autophagic markers, which were also analyzed in a SCA2 cellular model and in human post-mortem brain samples. Our results showed altered levels of SQSTM1 and LC3B in cells and tissues expressing mutant ataxin-2. Moreover, an abnormal accumulation of these markers was detected in SCA2 patients’ striatum and cerebellum. Importantly, the molecular activation of autophagy, using the compound cordycepin, mitigated the phenotypic alterations observed in disease models. Overall, our study suggests an important role for autophagy in the context of SCA2 pathology, proposing that targeting this pathway could be a potential target to treat SCA2 patients.

Stress granules (SGs) are membraneless cell compartments formed in response to different stress stimuli, wherein translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins coalesce together. SGs assembly is crucial for cell survival, since SGs are implicated in the regulation of translation, mRNA storage and stabilization and cell signalling, during stress. One defining feature of SGs is their dynamism, as they are quickly assembled upon stress and then rapidly dispersed after the stress source is no longer present. Recently, SGs dynamics, their components and their functions have begun to be studied in the context of human diseases. Interestingly, the regulated protein self-assembly that mediates SG formation contrasts with the pathological protein aggregation that is a feature of several neurodegenerative diseases. In particular, aberrant protein coalescence is a key feature of polyglutamine (PolyQ) diseases, a group of nine disorders that are caused by an abnormal expansion of PolyQ tract-bearing proteins, which increases the propensity of those proteins to aggregate. Available data concerning the abnormal properties of the mutant PolyQ disease-causing proteins and their involvement in stress response dysregulation strongly suggests an important role for SGs in the pathogenesis of PolyQ disorders. This review aims at discussing the evidence supporting the existence of a link between SGs functionality and PolyQ disorders, by focusing on the biology of SGs and on the way it can be altered in a PolyQ disease context. ALG-01-0145-FEDER-29480, SFRH/BD/133192/2017, SFRH/BD/133192/2017, SFRH/BD/148533/2019 info:eu-repo/semantics/publishedVersion

Aims Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is the most common autosomal dominantly-inherited ataxia worldwide and is characterised by the accumulation of mutant ataxin-3 (mutATXN3) in different brain regions, leading to neurodegeneration. Currently, there are no available treatments able to block disease progression. In this study, we investigated whether carbamazepine (CBZ) would activate autophagy and mitigate MJD pathology. Methods The autophagy-enhancing activity of CBZ and its effects on clearance of mutATXN3 were evaluated using in vitro and in vivo models of MJD. To investigate the optimal treatment regimen, a daily or intermittent CBZ administration was applied to MJD transgenic mice expressing a truncated human ATXN3 with 69 glutamine repeats. Motor behaviour tests and immunohistology was performed to access the alleviation of MJD-associated motor deficits and neuropathology. A retrospective study was conducted to evaluate the CBZ effect in MJD patients. Results We found that CBZ promoted the activation of autophagy and the degradation of mutATXN3 in MJD models upon short or intermittent, but not daily prolonged, treatment regimens. CBZ up-regulated autophagy through activation of AMPK, which was dependent on the myo-inositol levels. In addition, intermittent CBZ treatment improved motor performance, as well as prevented neuropathology in MJD transgenic mice. However, in patients, no evident differences in SARA scale were found, which was not unexpected given the small number of patients included in the study. Conclusions Our data support the autophagy-enhancing activity of CBZ in the brain and suggest this pharmacological approach as a promising therapy for MJD and other polyglutamine disorders. Fundação para Ciência e Tecnologia; Richard Chin and Lily Lock Machado-Joseph Disease Research Fund; American Portuguese Biomedical Research Fund (APBRF); National Ataxia Foundation (USA); European Union H2020 program, GA No.643417; EU Joint Program - Neurodegenerative Disease Research (JPND); Competitiveness Factors Operational Program (COMPETE 2020); ERDF through the Regional Operational Program Center 2020 info:eu-repo/semantics/publishedVersion

Polyglutamine spinocerebellar ataxias (PolyQ SCAs) are a group of 6 rare autosomal dominant diseases, which arise from an abnormal CAG repeat expansion in the coding region of their causative gene. These neurodegenerative ataxic disorders are characterized by progressive cerebellar degeneration, which translates into progressive ataxia, the main clinical feature, often accompanied by oculomotor deficits and dysarthria. Currently, PolyQ SCAs treatment is limited only to symptomatic mitigation, and no therapy is available to stop or delay the disease progression, which culminates with death. Over the last years, many promising gene therapy approaches were investigated in preclinical studies and could lead to a future treatment to stop or delay the disease development. Here, we summed up the most promising of these therapies, categorizing them in gene augmentation therapy, gene silencing strategies, and gene edition approaches. While several of the reviewed strategies are promising, there is still a gap from the preclinical results obtained and their translation to clinical studies. However, there is an increase in the number of approved gene therapies, as well as a constant development in their safety and efficacy profiles. Thus, it is expected that in a near future some of the promising strategies reviewed here could be tested in a clinical setting and if successful provide hope for SCAs patients. Foundation (FCT) project (ALG-01-0145-FEDER-29480) info:eu-repo/semantics/publishedVersion

PurposeThis study aimed to endow the cell-penetrating peptide (CPP) S4(13)-PV with adequate features towards a safe and effective application in cancer gene therapy.MethodsPeptide/siRNA complexes were prepared with two new derivatives of the CPP S4(13)-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S4(13)-PV peptide, being named C12-H-5-S4(13)-PV and H-5-S4(13)-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum.ResultsPeptide/siRNA complexes prepared with the C12-H-5-S4(13)-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H-5-S4(13)-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes.ConclusionsThese encouraging results pave the way for a potential application of the C12-H-5-S4(13)-PV peptide as a promising tool in cancer gene therapy. European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme [CENTRO-010145-FEDER-000008] European Regional Development Fund (ERDF), through the COMPETE 2020 -Operational Programme for Competitiveness and Internationalisation Portuguese national funds via FCT - Fundacao para a Ciencia e a Tecnologia [POCI-01-0145-FEDER-016390: CANCEL STEM, POCI-01-0145-FEDER-007440, UIDB/04539/2020] Fundacao para a Ciencia e Tecnologia (FCT, Portugal)Portuguese Foundation for Science and Technology [UID/QUI/50006/2013, UID/MULTI/04378/2013, IF/00092/2014, SFRH/BD/79077/2011, PD/BD/106035/2015] info:eu-repo/semantics/publishedVersion

This study aimed to endow the cell-penetrating peptide (CPP) S4Peptide/siRNA complexes were prepared with two new derivatives of the CPP S4Peptide/siRNA complexes prepared with the C12-HThese encouraging results pave the way for a potential application of the C12-H

Objective Compromised brain cholesterol turnover and altered regulation of brain cholesterol metabolism have been allied with some neurodegenerative diseases, including Huntington’s disease (HD). Following our previous studies in HD, in this study we aim to investigate in vitro in a neuroblastoma cellular model of HD, the effect of CYP46A1 overexpression, an essential enzyme in cholesterol metabolism, on huntingtin aggregation and levels. Results We found that CYP46A1 reduces the quantity and size of mutant huntingtin aggregates in cells, as well as the levels of mutant huntingtin protein. Additionally, our results suggest that the observed beneficial effects of CYP46A1 in HD cells are linked to the activation of autophagy. Taken together, our results further demonstrate that CYP46A1 is a pertinent target to counteract HD progression. This work was supported by Brainvectis and E.rare: E-Rare Joint Transnational Call for Proposals 2017 “Transnational Research Projects for Innovative Therapeutic Approaches for Rare Diseases”. CN laboratory is supported by the French Muscular Dystrophy Association (AFM-Téléthon), the Ataxia UK, and the Fundação para a Ciência e Tecnologia (project ALG-01-0145-FEDER-29480 “SeGrPolyQ”). AM is supported by a Ph.D. fellowship from FCT (SFRH/BD/133192/2017) info:eu-repo/semantics/publishedVersion

As it was already mentioned in the first chapter of this book, but will also be made clear throughout the remaining chapters, the term gene therapy encapsulates different approaches and applications based on the use and delivery of different nucleic acids. The most straightforward and perhaps most simple strategy for a gene-based therapy consists in adding a new protein-coding gene, which is an approach called gene augmentation therapy (Fig. 6.1). For monogenic recessive disorders in which the causative mutated is nonfunctional, the therapeutic gene to be delivered will be the normal wild-type form of the gene. The delivery of a correct copy of the gene is expected to restore the production of the defective or missing protein and thus revert the disease phenotype. This type of gene augmentation therapy is often also referred to as gene replacement therapy. As already discussed, this rationale was behind the first gene therapy clinical trial performed and is also at the basis of the majority of the gene therapy clinical trials performed so far.

The development of therapeutics to treat human disease has always been a major goal for biomedical research and innovation. Nevertheless, the complexity of the majority of human diseases poses an important and difficult obstacle to overcome. Moreover, the genetic contribution to these conditions complicates the targeting of endogenous and normal processes of cellular functioning, such as transcription and translation. In the past century, the understanding of DNA structure and the development of techniques to manipulate and recombine this molecule allowed the conception of new strategies that could use DNA as a therapeutic agent. In the 1970s, it was proposed for the first time that some human genetic conditions could be treated by the administration of exogenous DNA. The enormous technological advance in the field of biomedicine, with the human genome sequencing or the development of high-throughput techniques, for example, contributed to an effective application of gene therapy in the human context. Recently, the approval of several gene therapy medicines in Europe and the USA definitively established a new paradigm in human disease treatment and opened a new era for gene therapy.

Presently, more than 3400 genes have been associated with diseases [1], some of these pathologies are debilitating, mortal, and without any effective therapeutic options, and this number is expected to increase in the next decade as genomic studies advance. Gene therapy is a therapeutic strategy that seeks to target the genes behind the disorders in order to cure patients. Thus, this approach holds the promise of establishing therapies that treat the causes, rather than the symptoms, by manipulating deficient genes, removing or silencing pathologic genes, or adding missing genes. However, these ambitious strategies have been hampered by several problems, such as the lack of safety and efficiency of some of the developed approaches, which can be explained by the very nature of most gene-manipulating tools. Naked DNA plasmids, for example, are rapidly degraded in biological fluids, they are unable to efficiently cross the cellular membranes and therefore reach the target cells and they activate the immune system, which is programmed to identify and eliminate vehicles containing foreign genetic information [2]. Thus, although a large number of preclinical and clinical studies have been performed using naked nucleic acids, the use of delivery vectors yields better results, namely because they protect the nucleic acids from nuclease degradation and increase intracellular delivery. Still, despite the big efforts made in the last decades with gene therapy strategies, very few gene therapy-based drugs or therapeutic products have reached the market, revealing that much still has to be done in the field of vectors for gene therapy.

The selection of the delivery system is one of the most critical points for the success (and safety) of gene therapy. First, the chosen vector must ensure that the gene is delivered to the correct target cells, or at least predominantly to those cells. Second, the introduced transgene has to be activated, that is, it must go to the nucleus, be transcribed, and then translated. In a one-time cure setting, ideally the transgene should be integrated into the host genome or endure episomally, ensuring a long-term persistent expression. The ideal delivery system should meet several criteria,including (i) a good safety profile; (ii) easy production; (iii) good stability in target cells, and (iv) a high transgene capacity. As mentioned, the delivery systems can be roughly divided into two main categories: non-viral and viral systems. The non-viral methods were described in the previous chapter, whereas in this chapter the viral vectors used in gene therapy will be explained.

The development of a therapy for human use entails a complex and long process of studies and validations, first in cells and animal models and later in human subjects. The first step is commonly referred to as preclinical research or development, being important to access the safety and also the efficacy of the therapy proposed. In a gene therapy context, the in vivo preclinical studies, using relevant animal models, are particularly important, as most of the therapies are being studied for the first time. If preclinical studies yield relevant results, therapy can continue its process of development, being then evaluated in clinical trials, which refer to research studies performed in human subjects. In their simplest design, clinical trials aim to evaluate the outcomes of a particular therapy in human subjects under an experimental condition, being also referred to as interventional clinical studies.

It was already mentioned throughout this book that gene therapy was primarily developed as a strategy targeting recessive monogenic disorders caused by non-functional mutant genes, where in theory the simple addition of a functional (non-mutated) copy of the causative gene would be enough to counteract the disease phenotype and cure the disease. Nevertheless, for more complex diseases, the addition of a normal gene is not enough to revert the disease phenotype. For example, in dominantly inherited disorders, the presence of a single abnormal allele is sufficient to lead to disease manifestation, and thus a gene therapy should instead be able to shut down (or silence) the expression of that abnormal gene. Although this strategy is not so linear and easy, the possibility of silencing the expression of mutant genes using gene therapy approaches became possible with the development of antisense oligonucleotides (ASOs) and with the discovery of the RNA interference (RNAi) pathway. Despite several differences that will be explored further in this chapter, gene therapy strategies based on these two tools have been demonstrated to be efficient in treating dominant genetic diseases. More recently, gene editing tools have also been used to perform gene silencing, including at the DNA level, for example by introducing a premature stop codon or by removing the defective gene; however, this and other gene editing approaches will be discussed in Chap. 8.

Background Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 aggregation and neuronal degeneration. There is no treatment available to block or delay disease progression. In this work we investigated whether trehalose, a natural occurring disaccharide widely used in food and cosmetic industry, would rescue biochemical, behavioral and neuropathological features of an in vitro and of a severe MJD transgenic mouse model. Methods Two MJD animal models, a lentiviral based and a transgenic model, were orally treated with 2% trehalose solution for a period of 4 and 30 weeks, respectively. Motor behavior (rotarod, grip strength and footprint patterns) was evaluated at different time points and neuropathological features were evaluated upon in-life phase termination. Results Trehalose-treated MJD mice equilibrated for a longer time in the rotarod apparatus and exhibited an improvement of ataxic gait in footprint analysis. Trehalose-mediated improvements in motor behaviour were associated with a reduction of the MJD-associated neuropathology, as MJD transgenic mice treated with trehalose presented preservation of cerebellar layers thickness and a decrease in the size of ataxin-3 aggregates in Purkinje cells. In agreement, an improvement of neuropathological features was also observed in the full length lentiviral-based mouse model of MJD submitted to 2% trehalose treatment. Conclusions The present study suggests trehalose as a safety pharmacological strategy to counteract MJD-associated behavioural and neuropathological impairments.

As mentioned before, the choice of a particular vector for gene delivery is a critical point in the success of gene therapy. From the details presented in the previous chapters concerning the different possible vectors, important conclusions emerge. First, an ideal vector should combine several features, ensuring both safety and efficacy. Second, there are advantages and disadvantages for each of the described vectors, and the choice between them is very much dependent on the target disease/condition. All in all, a correct selection is a crucial point for the success of a particular gene therapy.

Stem cells are non-differentiated and unspecialized cells characterized by their ability of self-renewing by proliferating through symmetric division, which maintains the pool of stem cells. On the other hand, these cells can also undergo asymmetric division, resulting in two different cells, one stem cell and another cell that becomes compromised into one cell lineage and gives rise to mature specialized cells. Stem cells are present in all stages of human development, from the zygote to adulthood. During embryogenesis, they are responsible for tissue and organ formation. Although in adults they are present in much smaller numbers, they are still present in specific regions, germinative niches, with the important role of tissue maintenance and regeneration. However, in adults the tissue regeneration ability of these cells in some organs like the brain is very limited.

Machado-Joseph disease (MJD) is a neurodegenerative disorder caused by an abnormal expansion of citosine-adenine-guanine trinucleotide repeats in the disease-causing gene. This mutation leads to an abnormal polyglutamine tract in the protein ataxin-3 (Atx3), resulting in formation of mutant Atx3 aggregates. Despite several attempts to develop a therapeutic option for MJD, currently there are no available therapies capable of delaying or stopping disease progression. Recently, our group reported that reducing the expression levels of mutant Atx3 lead to a mitigation of several MJD-related behavior and neuropathological abnormalities. Aiming a more rapid translation to the human clinics, in this study we investigate a pharmacological inhibitor of translation-cordycepin-in several preclinical models. We found that cordycepin treatment significantly reduced (i) the levels of mutant Atx3, (ii) the neuropathological abnormalities in a lentiviral mouse model, (iii) the motor and neuropathological deficits in a transgenic mouse model and (iv) the number of ubiquitin aggregates in a human neural model. We hypothesize that the effect of cordycepin is mediated by the increase of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) levels, which is accompanied by a reduction in the global translation levels and by a significant activation of the autophagy pathway. Overall, this study suggests that cordycepin might constitute an effective and safe therapeutic approach for MJD, and probably for the other polyglutamine diseases. European Union through the European social fund, funds Fundo Europeu de Desenvolvimento Regional through the Competitive Factors Operational Program-COMPETE, POPH and QREN French Muscular Dystrophy Association (AFM-Telethon) [18776] Ataxia UK Fundacao para a Ciencia e TecnologiaPortuguese Foundation for Science and Technology [SFRH/BD/133192/2017] FCT - Fundacao para a Ciencia e a Tecnologia, I.P. [UID/BIM/04773/2013 CBMR]

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3, the most common dominant spinocerebellar ataxia (SCA) worldwide, is caused by over-repetition of a CAG repeat in the ATXN3/MJD1 gene, which translates into a polyglutamine tract within the ataxin-3 protein. There is no treatment for this fatal disorder. Despite evidence of the safety and efficacy of mesenchymal stromal cells (MSCs) in delaying SCA disease progression in exploratory clinical trials, unanticipated regression of patients to the status prior to treatment makes the investigation of causes and solutions urgent and imperative. In the present study, we compared the efficacy of a single intracranial injection with repeated systemic MSC administration in alleviating the MJD phenotype of two strongly severe genetic rodent models. We found that a single MSC transplantation only produces transient effects, whereas periodic administration promotes sustained motor behavior and neuropathology alleviation, suggesting that MSC therapies should be re-designed to get sustained beneficial results in clinical practice. Furthermore, MSC promoted neuroprotection, increased the levels of GABA and glutamate, and decreased the levels of Myo-inositol, which correlated with motor improvements, indicating that these metabolites may serve as valid neurospectroscopic biomarkers of disease and treatment. This study makes important contributions to the design of new clinical approaches for MJD and other SCAs/polyglutamine disorders.