Chiara Zanetta

Direct reprogramming of human astrocytes into neural stem cells and neurons

Generating neural stem cells and neurons from reprogrammed human astrocytes is a potential strategy for neurological repair. Here we show dedifferentiation of human cortical astrocytes into the neural stem/progenitor phenotype to obtain progenitor and mature cells with a neural fate. Ectopic expression of the reprogramming factors OCT4, SOX2, or NANOG into astrocytes in specific cytokine/culture conditions activated the neural stem gene program and induced generation of cells expressing neural stem/precursor markers. Pure CD44 + mature astrocytes also exhibited this lineage commitment change and did not require passing through a pluripotent state. These astrocyte-derived neural stem cells gave rise to neurons, astrocytes, and oligodendrocytes and showed in vivo engraftment properties. ASCL1 expression further promoted neuronal phenotype acquisition in vitro and in vivo. Methylation analysis showed that epigenetic modifications underlie this process. The restoration of multipotency from human astrocytes has potential in cellular reprogramming of endogenous central nervous system cells in neurological disorders.

Therapeutic applications of the cell-penetrating HIV-1 Tat peptide

Over the past decades, many new therapeutic approaches have been developed for several conditions, including neurodegenerative diseases. However, efficient biodistribution and delivery at biological target sites are hampered by the presence of cell and tissue barriers, and a clinical therapy is prevented by the requirement of invasive administration routes. Candidate drug conjugation to cell-penetrating peptides, which are able to cross cellular membranes and reach biological targets even when administered systemically, represents a promising tool to overcome this issue. Here, we review the biology, classification and mechanisms of internalization of cell-penetrating peptides. We focus our attention on the cell-penetrating peptide: HIV-derived Tat peptide, and discuss its efficient but controversial use in basic, preclinical and clinical research from its discovery to the present day.

Minimally invasive transplantation of iPSC-derived ALDHhiSSCloVLA4+ neural stem cells effectively improves the phenotype of an amyotrophic lateral sclerosis model

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently, there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS, and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study, we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity, low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS, treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span, in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms, including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases.

Molecular Therapeutic Strategies for Spinal Muscular Atrophies: Current and Future Clinical Trials

BACKGROUND Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by mutations in the survival motor neuron gene (SMN1) and the leading genetic cause of infant mortality. Currently, there is no effective treatment other than supportive care. OBJECTIVE This article provides a general overview of the main aspects that need to be taken into account to design a more efficient clinical trial and to summarize the most promising molecular trials that are currently in development or are being planned for the treatment of SMA. METHODS A systematic review of the literature was performed, identifying key clinical trials involving novel molecular therapies in SMA. In addition, abstracts presented at the meetings of the Families of Spinal Muscular Atrophy were searched and the Families of Spinal Muscular Atrophy Web site was carefully analyzed. Finally, a selection of SMA clinical trials registered at clinical-trials.gov has been included in the article. RESULTS The past decade has seen a marked advancement in the understanding of both SMA genetics and molecular mechanisms. New molecules targeting SMN have shown promise in preclinical studies, and various clinical trials have started to test the drugs that were discovered through basic research. CONCLUSIONS Both preclinical and early clinical trial results involving novel molecular therapies suggest that the clinical care paradigm in SMA will soon change.

Antisense Oligonucleotide Therapy for the Treatment of C9ORF72 ALS/FTD Diseases

Motor neuron disorders, and particularly amyotrophic lateral sclerosis (ALS), are fatal diseases that are due to the loss of motor neurons in the brain and spinal cord, with progressive paralysis and premature death. It has been recently shown that the most frequent genetic cause of ALS, frontotemporal dementia (FTD), and other neurological diseases is the expansion of a hexanucleotide repeat (GGGGCC) in the non-coding region of the C9ORF72 gene. The pathogenic mechanisms that produce cell death in the presence of this expansion are still unclear. One of the most likely hypotheses seems to be the gain-of-function that is achieved through the production of toxic RNA (able to sequester RNA-binding protein) and/or toxic proteins. In recent works, different authors have reported that antisense oligonucleotides complementary to the C9ORF72 RNA transcript sequence were able to significantly reduce RNA foci generated by the expanded RNA, in affected cells. Here, we summarize the recent findings that support the idea that the buildup of “toxic” RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS and also suggest that the use of antisense oligonucleotides targeting this transcript is a promising strategy for treating ALS/frontotemporal lobe dementia (FTLD) patients with the C9ORF72 repeat expansion. These data are particularly important, given the state of the art antisense technology, and they allow researchers to believe that a clinical application of these discoveries will be possible soon.

Effect of combined systemic and local morpholino treatment on the spinal muscular atrophy δ7 mouse model phenotype

BACKGROUND Spinal muscular atrophy (SMA) is a fatal motor neuron disease of childhood that is caused by mutations in the SMN1 gene. Currently, no effective treatment is available. One possible therapeutic approach is the use of antisense oligos (ASOs) to redirect the splicing of the paralogous gene SMN2, thus increasing functional SMN protein production. Various ASOs with different chemical properties are suitable for these applications, including a morpholino oligomer (MO) variant with a particularly excellent safety and efficacy profile. OBJECTIVE We investigated a 25-nt MO sequence targeting the negative intronic splicing silencer (ISS-N1) 10 to 34 region. METHODS We administered a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) in the SMAΔ7 mouse model and evaluated the effect and neuropathologic phenotype. We tested different concentrations (from 2 to 24 nM) and delivery protocols (intracerebroventricular injection, systemic injection, or both). We evaluated the treatment efficacy regarding SMN levels, survival, neuromuscular phenotype, and neuropathologic features. RESULTS We found that a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) exhibited superior efficacy in transgenic SMAΔ7 mice compared with previously described sequences. In our experiments, the combination of local and systemic administration of MO (bare or conjugated to octaguanidine) was the most effective approach for increasing full-length SMN expression, leading to robust improvement in neuropathologic features and survival. Moreover, we found that several small nuclear RNAs were deregulated in SMA mice and that their levels were restored by MO treatment. CONCLUSION These results indicate that MO-mediated SMA therapy is efficacious and can result in phenotypic rescue, providing important insights for further development of ASO-based therapeutic strategies in SMA patients.

Ongoing therapeutic trials and outcome measures for Duchenne muscular dystrophy

Muscular dystrophy is a heterogeneous group of genetic disorders characterised by progressive muscle tissue degeneration. No effective treatment has been discovered for these diseases. Preclinical and clinical studies aimed at the development of new therapeutic approaches have been carried out, primarily in subjects affected with dystrophinopathies (Duchenne and Becker muscular dystrophy). In this review, we outline the current therapeutic approaches and past and ongoing clinical trials, highlighting both the advantages and limits of each one. The experimental designs of these trials were based on different rationales, including immunomodulation, readthrough strategies, exon skipping, gene therapy, and cell therapy. We also provide an overview of available outcome measures, focusing on their reliability in estimating meaningful clinical improvement in order to aid in the design of future trials. This perspective is extremely relevant to the field considering the recent development of novel therapeutic approaches that will result in an increasing number of clinical studies over the next few years.

Pluripotent stem cell-based models of spinal muscular atrophy.

Motor neuron diseases, as the vast majority of neurodegenerative disorders in humans, are incurable conditions that are challenging to study in vitro, owing to the obstacles in obtaining the cell types majorly involved in the pathogenesis. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, have opened up the possibility of generating a substantial amount of disease-specific neuronal cells, including motor neurons and glial cells. The present review analyzes the practical implications of iPSCs, generated from fibroblasts of patients affected by spinal muscular atrophy (SMA), and discusses the challenges in the development and optimization of in vitro disease models. Research on patient-derived disease-specific cells may shed light on the pathological processes behind neuronal dysfunction and death in SMA, thus providing new insights for the development of novel effective therapies.

The wide spectrum of clinical phenotypes of spinal muscular atrophy with respiratory distress type 1: a systematic review.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1), also known as distal spinal-muscular atrophy 1 (DSMA10), is an autosomal recessive type of spinal muscular atrophy that is related to mutations in the IGHMBP2 gene, which encodes for the immunoglobulin μ-binding protein. SMARD1 patients usually present low birth weight, diaphragmatic palsy and distal muscular atrophy. Clinical features are still the most important factor that leads to the diagnosis of SMARD1, due to the fact that IGHMBP2 gene mutations are characterized by significant phenotypic heterogeneity. In the present review, we will systematically discuss the genetic, clinical and neuropathological features of SMARD1 in order to provide a complete overview of SMARD1 variable clinical presentations and of the most important diagnostic tools which can be used to identify and properly manage affected individuals. This background is crucial also in the perspective of the development of novel therapeutic strategies for this still orphan disorder.

Molecular, genetic and stem cell-mediated therapeutic strategies for spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell‐mediated approaches for the treatment of SMA.