Federica Rizzo

Genetic Correction of Human Induced Pluripotent Stem Cells from Patients with Spinal Muscular Atrophy

Motor neurons generated from genetically corrected iPSCs derived from patients with spinal muscular atrophy show rescue of the disease phenotype. Engineering iPSC-Derived Motor Neurons for Cell Therapy Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in the gene encoding the survival motor neuron 1 (SMN1) protein. The mutant protein causes loss of spinal cord motor neurons, and there is no effective therapy. Humans have a paralogous gene, SMN2, that differs from SMN1 by a single nucleotide variant within exon 7 that results in the production of an incomplete and nonfunctional protein. Now, Corti et al. investigate the feasibility of genetically engineering induced pluripotent stem cells (iPSCs) derived from SMA patients to generate motor neurons that do not show the disease phenotype. The authors generated human SMA-iPSCs using nonviral, nonintegrating episomal vectors and then performed genetic editing with oligonucleotides to modify SMN2 to produce a functional SMN1-like protein. Uncorrected SMA-iPSC–derived motor neurons reproduced disease-specific features, whereas motor neurons derived from genetically corrected SMA-iPSCs showed rescue of the disease phenotype. Upon direct transplantation into a severe SMA mouse model, corrected SMA-iPSC–derived motor neurons engrafted in the spinal cord and improved the disease phenotype. This study demonstrates the feasibility of generating patient-specific iPSCs and their motor neuron progeny that are genetically corrected and free of exogenous sequences and suggests the potential of this approach for clinical translation. Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival motor neuron 2 (SMN2) gene into an SMN1-like gene. Corrected iPSC lines contained no exogenous sequences. Motor neurons formed by differentiation of uncorrected SMA-iPSCs reproduced disease-specific features. These features were ameliorated in motor neurons derived from genetically corrected SMA-iPSCs. The different gene splicing profile in SMA-iPSC motor neurons was rescued after genetic correction. The transplantation of corrected motor neurons derived from SMA-iPSCs into an SMA mouse model extended the life span of the animals and improved the disease phenotype. These results suggest that generating genetically corrected SMA-iPSCs and differentiating them into motor neurons may provide a source of motor neurons for therapeutic transplantation for SMA.

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.

Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis

Neurodegenerative disorders are characterized by the selective vulnerability and progressive loss of discrete neuronal populations. Non-neuronal cells appear to significantly contribute to neuronal loss in diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, and Alzheimer’s disease. In ALS, there is deterioration of motor neurons in the cortex, brainstem, and spinal cord, which control voluntary muscle groups. This results in muscle wasting, paralysis, and death. Neuroinflammation, characterized by the appearance of reactive astrocytes and microglia as well as macrophage and T-lymphocyte infiltration, appears to be highly involved in the disease pathogenesis, highlighting the involvement of non-neuronal cells in neurodegeneration. There appears to be cross-talk between motor neurons, astrocytes, and immune cells, including microglia and T-lymphocytes, which are subsequently activated. Currently, effective therapies for ALS are lacking; however, the non-cell autonomous nature of ALS may indicate potential therapeutic targets. Here, we review the mechanisms of action of astrocytes, microglia, and T-lymphocytes in the nervous system in health and during the pathogenesis of ALS. We also evaluate the therapeutic potential of these cellular populations, after transplantation into ALS patients and animal models of the disease, in modulating the environment surrounding motor neurons from pro-inflammatory to neuroprotective. We also thoroughly discuss the recent advances made in the field and caveats that need to be overcome for clinical translation of cell therapies aimed at modulating non-cell autonomous events to preserve remaining motor neurons in patients.

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.

Mitochondrial Fusion Proteins and Human Diseases

Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1) and 2 (MFN2), located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1), in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimers and Parkinsons diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.

Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies

•  Introduction •  Transdifferentiation of somatic cells in skeletal myoblasts •  Myogenic cell induction from ESCs ‐  The first method: generation of MMPs from ESCs and their differentiation into myogenic cells ‐  Isolation of MMPs ‐  Differentiation into skeletal myoblasts ‐  The second method: EB generation and differentiation into skeletal muscle ‐  Formation of EBs ‐  Differentiation of EBs into skeletal myoblasts •  Protocols for myogenic cell induction from iPSCs •  Mouse and human‐pluripotent derived myocytes as an in vitro model of MDs •  Therapeutic development based on ESC‐ or iPSC‐derived cells •  Conclusion

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.

MFN2-related neuropathies: Clinical features, molecular pathogenesis and therapeutic perspectives

Mitofusin 2 (MFN2) is a GTPase dynamin-like protein of the outer mitochondrial membrane, encoded in the nuclear genome by the MFN2 gene located on the short (p) arm of chromosome 1. MFN2 protein is involved in several intracellular pathways, but is mainly involved in a network that has an essential role in several mitochondrial functions, including fusion, axonal transport, interorganellar communication and mitophagy. Mutations in the gene encoding MFN2 are associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a wide clinical phenotype that involves the central and peripheral nervous system. Here, we present the clinical, genetic and neuropathological features of human diseases associated with MFN2 mutations. We also report proposed pathogenic mechanisms through which MFN2 mutations likely contribute to the development of neurodegeneration. MFN2-related disorders may occur more frequently than previously considered, and they may represent a paradigm for the study of the defective mitochondrial dynamics that seem to play a significant role in the molecular and cellular pathogenesis of common neurodegenerative diseases; thus they may also lead to the identification of related therapeutic targets.

Research advances in gene therapy approaches for the treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons that causes progressive muscle weakness, paralysis, and premature death. No effective therapy is available. Research in the motor neuron field continues to grow, and recent breakthroughs have demonstrated the possibility of completely achieving rescue in animal models of spinal muscular atrophy, a genetic motor neuron disease. With adeno-associated virus (AAV) vectors, gene transfer can be achieved with systemic non-invasive injection and minimal toxicity. In the context of this success, we review gene therapy approaches for ALS, considering what has been done and the possible future directions for effective application of the latest generation of vectors for clinical translation. We focus on recent developments in the areas of RNA/antisense-mediated silencing of specific ALS causative genes like superoxide dismutase-1 and other molecular pathogenetic targets, as well as the administration of neuroprotective factors with viral vectors. We argue that gene therapy offers new opportunities to open the path for clinical progress in treating ALS.

Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches

Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality due to mutations in the Survival Motor Neuron (SMN) 1 gene. No cure is available. Antisense oligonucleotides (ASOs) aimed at increasing SMN levels from the paralogous SMN2 gene represent a possible therapeutic strategy. Here, we tested in SMA human induced pluripotent stem cells (iPSCs) and iPSC-differentiated motor neurons, three different RNA approaches based on morpholino antisense targeting of the ISSN-1, exon-specific U1 small nuclear RNA (ExSpeU1), and Transcription Activator-Like Effector-Transcription Factor (TALE-TF). All strategies act modulating SMN2 RNA: ASO affects exon 7 splicing, TALE-TF increase SMN2 RNA acting on the promoter, while ExSpeU1 improves pre-mRNA processing. These approaches induced up-regulation of full-length SMN mRNA and differentially affected the Delta-7 isoform: ASO reduced this isoform, while ExSpeU1 and TALE-TF increased it. All approaches upregulate the SMN protein and significantly improve the in vitro SMA motor neurons survival. Thus, these findings demonstrate that therapeutic tools that act on SMN2 RNA are able to rescue the SMA disease phenotype. Our data confirm the feasibility of SMA iPSCs as in vitro disease models and we propose novel RNA approaches as potential therapeutic strategies for treating SMA and other genetic neurological disorders.