Marianna Falcone

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.

Embryonic stem cell-derived neural stem cells improve spinal muscular atrophy phenotype in mice.

Spinal muscular atrophy, characterized by selective loss of lower motor neurons, is an incurable genetic neurological disease leading to infant mortality. We previously showed that primary neural stem cells derived from spinal cord can ameliorate the spinal muscular atrophy phenotype in mice, but this primary source has limited translational value. Here, we illustrate that pluripotent stem cells from embryonic stem cells show the same potential therapeutic effects as those derived from spinal cord and offer great promise as an unlimited source of neural stem cells for transplantation. We found that embryonic stem cell-derived neural stem cells can differentiate into motor neurons in vitro and in vivo. In addition, following their intrathecal transplantation into spinal muscular atrophy mice, the neural stem cells, like those derived from spinal cord, survived and migrated to appropriate areas, ameliorated behavioural endpoints and lifespan, and exhibited neuroprotective capability. Neural stem cells obtained using a drug-selectable embryonic stem cell line yielded the greatest improvements. As with cells originating from primary tissue, the embryonic stem cell-derived neural stem cells integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in neural stem cell-mediated therapies and highlight potential safety improvements and benefits of drug selection for neuroepithelial cells.

Human motor neuron generation from embryonic stem cells and induced pluripotent stem cells

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years, significant developments in stem cell research have been applied to MNDs, particularly regarding neuroprotection and cell replacement. However, a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types, including motor neurons that could be used for MND therapies. Recently, it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons, since they share ES characteristics, self-renewal, and the potential to differentiate into any somatic cell type. In this review, we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons, and possible clinical applications.

Systemic transplantation of c-kit+ cells exerts a therapeutic effect in a model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disease characterized by the loss of motor neurons. Motor neuron degeneration is probably both a cell autonomous and a non-autonomous event. Therefore, manipulating the diseased microenvironment via non-neural cell replacement could be a therapeutic strategy. We investigated a cell therapy approach using intravascular injection to transplant a specific population of c-kit(+) stem/progenitor cells from bone marrow into the SOD1G93A mouse model of ALS. Transplanted cells engrafted within the host spinal cord. Cell transplantation significantly prolonged disease duration and lifespan in superoxide dismutase 1 mice, promoted the survival of motor neurons and improved neuromuscular function. Neuroprotection was mediated by multiple effects, in particular by the expression of primary astrocyte glutamate transporter GLT1 and by the non-mutant genome. These findings suggest that this type of somatic cell transplantation strategy merits further investigation as a possible effective therapy for ALS and other neurodegenerative diseases.

Beta-lactam antibiotic offers neuroprotection in a spinal muscular atrophy model by multiple mechanisms.

Spinal muscular atrophy (SMA) is a devastating genetic motoneuron disease leading to infant death. No effective therapy is currently available. It has been suggested that β-lactam antibiotics such as ceftriaxone may offer neuroprotection in motoneuron diseases. Here, we investigate the therapeutic effect of ceftriaxone in a murine model of SMA. Treated animals present a modest, but significant ameliorated neuromuscular phenotype and increased survival, which correlate with protection of neuromuscular units. Whole gene expression profiling in treated mice demonstrates modifications in several genes including those involved in RNA metabolism toward wild-type. The neuroprotective effect seems to be mediated by multiple mechanisms that encompass the increase of the glutamate transporter Glt1, the transcription factor Nrf2, as well as SMN protein. This study provides the first evidence of a potential positive effect of this class of molecules in SMA. Further investigation of analogs with increased and more specific therapeutic effects warrants the development of useful therapies for SMA.

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.

ALS genetic modifiers that increase survival of SOD1 mice and are suitable for therapeutic development.

Amyotrophic lateral sclerosis (ALS) is a frequently fatal motor neuron disease without any cure. To find molecular therapeutic targets, several studies crossed transgenic ALS murine models with animals transgenic for some ALS target genes. We aimed to revise the new discoveries and new works in this field. We selected the 10 most promising genes, according to their capability when down-regulated or up-regulated in ALS animal models, for increasing life span and mitigating disease progression: XBP-1, NogoA and NogoB, dynein, heavy and medium neurofilament, NOX1 and NOX2, MLC-mIGF-1, NSE-VEGF, and MMP-9. Interestingly, some crucial modifier genes have been described as being involved in common pathways, the most significant of which are inflammation and cytoskeletal activities. The endoplasmic reticulum also seems to play an important role in ALS pathogenesis, as it is involved in different selected gene pathways. In addition, these genes have evident links to each other, introducing the hypothesis of a single unknown, common pathway involving all of these identified genes and others to be discovered.

Direct Reprogramming of Adult Somatic Cells Into Other Lineages: Past Evidence and Future Perspectives

Direct reprogramming of an adult cell into another differentiated lineage—such as fibroblasts into neurons, cardiomyocytes, or blood cells—without passage through an undifferentiated pluripotent stage is a new area of research that has recently emerged alongside stem cell technology and induced pluripotent stem cell reprogramming; indeed, this avenue of investigation has begun to play a central role in basic biological research and regenerative medicine. Even though the field seems new, its origins go back to the 1980s when it was demonstrated that differentiated adult cells can be converted into another cell lineage through the overexpression of transcription factors, establishing mature cell plasticity. Here, we retrace transdifferentiation experiments from the discovery of master control genes to recent in vivo reprogramming of one somatic cell into another from the perspective of possible applications for the development of new therapeutic approaches for human diseases.

Beta-lactam antibiotic offers neuroprotection in a spinal muscular atrophy model by multiple mechanisms: Experimental Neurology 2011; 229: 214-225.

Spinal muscular atrophy (SMA) is the most common genetic neurodegenerative disease leading to death in childhood. SMA is characterized by the loss of spinal cord anterior horn neurons and progressive denervation of skeletal muscles. SMA is caused by deletion or mutation of the telomeric copy of human survival motor neuron gene 1 (hSMN1) and retention of the hSMN2 gene. SMA animal models are extremely useful in studying the mechanism of SMA-related motoneuronal death, and may provide an in vivo system for testing a potential SMA therapy. doi : 10.5214/ans.0972.7531.1118407