RESUMO
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 patient's fibroblasts transdifferentiated into myoblasts. In this system, we showed that the selected antisense was able to produce 50% skipping of exon 51.
Assuntos
Distrofina/genética , Éxons/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , RNA Antissenso/genética , RNA Nuclear Pequeno/genética , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Células Cultivadas , Distrofina/metabolismo , Humanos , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for "stem" cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor beta. Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor beta, and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Proteínas Musculares/biossíntese , Miócitos de Músculo Liso/citologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/fisiologia , Animais , Aorta/citologia , Aorta/embriologia , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Músculo Liso Vascular , Miócitos Cardíacos/citologia , Miócitos de Músculo Liso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Ativação Transcricional , TransfecçãoRESUMO
The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment.