Tania Incitti

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy

Duchenne muscular dystrophy (DMD)—which is caused by mutations in the dystrophin gene—is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA—miR‐31—that represses dystrophin expression by targeting its 3′ untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR‐31 inhibition increases dystrophin rescue. These results indicate that interfering with miR‐31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.

Long-Term Benefit of Adeno-Associated Virus/Antisense-Mediated Exon Skipping in Dystrophic Mice

Many mutations and deletions in the dystrophin gene, responsible for Duchenne muscular dystrophy (DMD), can be corrected at the posttranscriptional level by skipping specific exons. Here we show that long-term benefit can be obtained in the dystrophic mouse model through the use of adeno-associated viral vectors expressing antisense sequences: persistent exon skipping, dystrophin rescue, and functional benefit were observed 74 weeks after a single systemic administration. The therapeutic benefit was sufficient to preserve the muscle integrity of mice up to old age. These results indicate a possible long-term gene therapy treatment of the DMD pathology.

Exon Skipping and Duchenne Muscular Dystrophy Therapy: Selection of the Most Active U1 snRNA-Antisense Able to Induce Dystrophin Exon 51 Skipping

One promising approach for the gene therapy of Duchenne muscular dystrophy (DMD) is exon skipping. When thinking of possible intervention on human, it is very crucial to identify the most appropriate antisense sequences able to provide the highest possible skipping efficiency. In this article, we compared the exon 51 skipping activity of 10 different antisense molecules, raised against splice junctions and/or exonic splicing enhancers (ESEs), expressed as part of the U1 small nuclear RNA (snRNA). The effectiveness of each construct was tested in human DMD myoblasts carrying the deletion of exons 48-50, which can be treated with skipping of exon 51. Our results show that the highest skipping activity and dystrophin rescue is achieved upon expression of a U1 snRNA-derived antisense molecule targeting exon 51 splice sites in combination with an internal exon sequence. The efficacy of this molecule was further proven on an exon 45-50 deletion background, utilizing patients fibroblasts transdifferentiated into myoblasts. In this system, we showed that the selected antisense was able to produce 50% skipping of exon 51.

Gene-mediated Restoration of Normal Myofiber Elasticity in Dystrophic Muscles

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.

Genipin-crosslinked gelatin–silk fibroin hydrogels for modulating the behaviour of pluripotent cells

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin‐crosslinked gelatin–silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin‐crosslinked gelatin–silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin‐crosslinked gelatin–silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright

Viability and neuronal differentiation of neural stem cells encapsulated in silk fibroin hydrogel functionalized with an IKVAV peptide

Three‐dimensional (3D) porous scaffolds combined with therapeutic stem cells play vital roles in tissue engineering. The adult brain has very limited regeneration ability after injuries such as trauma and stroke. In this study, injectable 3D silk fibroin‐based hydrogel scaffolds with encapsulated neural stem cells were developed, aiming at supporting brain regeneration. To improve the function of the hydrogel towards neural stem cells, silk fibroin was modified by an IKVAV peptide through covalent binding. Both unmodified and modified silk fibroin hydrogels were obtained, through sonication, with mechanical stiffness comparable to that of brain tissue. Human neural stem cells were encapsulated in both hydrogels and the effects of IKVAV peptide conjugation on cell viability and neural differentiation were assessed. The silk fibroin hydrogel modified by IKVAV peptide showed increased cell viability and an enhanced neuronal differentiation capability, which contributed to understanding the effects of IKVAV peptide on the behaviour of neural stem cells. For these reasons, IKVAV‐modified silk fibroin is a promising material for brain tissue engineering. Copyright

Sorting of Sox1-GFP Mouse Embryonic Stem Cells Enhances Neuronal Identity Acquisition upon Factor-Free Monolayer Differentiation

Abstract Embryonic stem cells (ESCs) can give rise to all the differentiated cell types of the organism, including neurons. However, the efficiency and specificity of neural differentiation protocols still needs to be improved in order to plan their use in cell replacement therapies. In this study, we modified a monolayer differentiation protocol by selecting green fluorescent protein (GFP) positive neural precursors with fluorescence-activated cell sorting (FACS). The enhancement of neural differentiation was obtained by positively selecting for neural precursors, while specific neuronal subtypes spontaneously differentiated without additional cues; a comparable but delayed behavior was also observed in the GFP negative population, indicating that sorting settings per se eliminated nonneural and undifferentiated ESCs. This highly reproducible approach could be applied as a strategy to enhance neuronal differentiation and could be the first step toward the selection of pure populations of neurons, to be generated by the administration of specific factors in high throughput screening assays.

Myogenic Progenitors from Mouse Pluripotent Stem Cells for Muscle Regeneration.

Muscle homeostasis is maintained by resident stem cells which, in both pathologic and non-pathologic conditions, are able to repair or generate new muscle fibers. Although muscle stem cells have tremendous regenerative potential, their application in cell therapy protocols is prevented by several restrictions, including the limited ability to grow ex vivo. Since pluripotent stem cells have the unique potential to both self-renew and expand almost indefinitely, they have become an attractive source of progenitors for regenerative medicine studies. Our lab has demonstrated that embryonic stem cell (ES)-derived myogenic progenitors retain the ability to repair existing muscle fibers and contribute to the pool of resident stem cells. Because of their relevance in both cell therapy and disease modeling, in this chapter we describe the protocol to derive myogenic progenitors from murine ES cells followed by their intramuscular delivery in a murine muscular dystrophy model.

Noggin-Mediated Retinal Induction Reveals a Novel Interplay Between Bone Morphogenetic Protein Inhibition, Transforming Growth Factor β, and Sonic Hedgehog Signaling

It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose‐dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose‐dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)β signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFβ and SHH signaling. Stem Cells 2015;33:2496–2508

Noggin Expression in the Adult Retina Suggests a Conserved Role during Vertebrate Evolution

Vertebrates share common mechanisms in the control of development and in the maintenance of neural and retinal function. The secreted factor Noggin, a BMP inhibitor, plays a crucial role in neural induction during embryonic development. Moreover, we have shown its involvement in retinal differentiation of pluripotent cells. Here we show Noggin expression in the adult retina in three vertebrate species. Four Noggin genes are present in zebrafish (Danio rerio; ZbNog1, 2, 3, 5), three in frog (Xenopus laevis; XenNog1, 2 and 4), and one in mouse (Mus musculus; mNog). Quantitative RT-PCR experiments show the presence of ZbNog3 and ZbNog5 mRNAs, but not ZbNog1 and ZbNog2, in the adult zebrafish retina. All three genes are expressed in the frog retina, and mNog in the mouse. Immunohistochemistry data show that Noggin proteins are predominantly localized in the Golgi apparatus of photoreceptors and in the fibers of the outer plexiform layer. Lower expression levels are also found in inner plexiform layer fibers, in ganglion cells, in the ciliary marginal zone, and in retinal pigmented epithelium. Our results show that Noggin has a specific cellular and sub-cellular expression in the adult vertebrate retina, which is conserved during evolution. In addition to its established role during embryonic development, we postulate that Noggin also exerts a functional role in the adult retina.