RESUMO
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.
Assuntos
Processamento Alternativo , Dependovirus/genética , Distrofina/genética , Distrofia Muscular Animal/terapia , Oligorribonucleotídeos Antissenso/genética , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Cálcio/metabolismo , Cães , Éxons , Membro Anterior/fisiopatologia , Terapia Genética , Vetores Genéticos/administração & dosagem , Injeções Intramusculares , Dados de Sequência Molecular , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Força Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/fisiopatologia , Transcrição Gênica , Utrofina/genética , Utrofina/metabolismoRESUMO
Once escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray-based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [3H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth.
Assuntos
Apoptose , Macrófagos/fisiologia , Monócitos/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Células Satélites de Músculo Esquelético/fisiologia , Células Cultivadas , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/metabolismo , Quimiotaxia , Técnicas de Cocultura , Meios de Cultivo Condicionados , Humanos , Macrófagos/citologia , Dados de Sequência Molecular , Monócitos/citologia , Músculo Esquelético/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Células Satélites de Músculo Esquelético/citologiaRESUMO
Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.
Assuntos
Diferenciação Celular , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/embriologia , Proteínas ADAM , Proteína ADAM12 , Anticorpos Bloqueadores/farmacologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Interações Medicamentosas , Eletroforese em Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato , Imunofluorescência , Corantes Fluorescentes , Humanos , Immunoblotting , Indóis , Integrinas/antagonistas & inibidores , Integrinas/efeitos dos fármacos , Integrinas/genética , Cinética , Fusão de Membrana/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/efeitos dos fármacos , Metaloendopeptidases/genética , Microscopia Confocal , Fibras Musculares Esqueléticas/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Testes de Precipitina , Propídio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RodaminasRESUMO
Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.
Assuntos
Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/terapia , Proteínas de Transporte/genética , Éxons , Oligorribonucleotídeos Antissenso/uso terapêutico , RNA Nuclear Pequeno/uso terapêutico , Adenoviridae/genética , Processamento Alternativo , Animais , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/fisiopatologia , Técnicas de Introdução de Genes , Terapia Genética , Células HEK293 , Coração/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/prevenção & controle , Camundongos , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Oligorribonucleotídeos Antissenso/administração & dosagem , Oligorribonucleotídeos Antissenso/genética , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/administração & dosagem , RNA Nuclear Pequeno/genética , Transdução GenéticaRESUMO
To investigate the therapeutic potential of bone marrow transplantation in Duchenne muscular dystrophy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into irradiated wild-type and dystrophin-deficient mdx mice. Tibialis anterior muscles showed fivefold to sixfold more GFP+ mononucleated cells and threefold to fourfold more GFP+ myofibers in mdx than in wild-type mice. In contrast, dystrophin expression in mdx mice remained within the level of nontransplanted mdx mice, and co-expression with GFP was rare. Longitudinal sections of 5000 myofibers showed 160 GFP+ fibers, including 9 that co-expressed dystrophin. GFP was always visualized as full-length sarcoplasmic fluorescence that exceeded the span of sample length (up to 1500 microm), whereas dystrophin expression was restricted to 11 to 28% of this length. Dystrophin expression span was much shorter in GFP+ fibers (116 +/- 46 microm) than in revertant fibers (654 +/- 409 microm). These data suggest that soluble GFP diffuses far from the fusion site with a pre-existing dystrophin(-) myofiber whereas dystrophin remains mainly expressed close to the site of fusion. Because restoration of dystrophin in whole muscle fiber length is required to expect functional improvement and clinical benefits for Duchenne muscular dystrophy, future applications of cell therapies to neuromuscular disorders could be more appropriately envisaged for replacement of defective soluble sarcoplasmic proteins.
Assuntos
Transplante de Medula Óssea , Distrofina/biossíntese , Proteínas de Fluorescência Verde/biossíntese , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Animal/terapia , Sarcolema/metabolismo , Animais , Citoplasma/química , Citoplasma/metabolismo , Distrofina/deficiência , Citometria de Fluxo , Terapia Genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/química , Sarcolema/química , TransgenesRESUMO
Muscle satellite cells are heterogeneous and present functional disparities, some of them behaving as multipotent stem cells. Yet their phenotype is obscure and their isolation remains elusive. The ability to purify stem cells from a wide variety of tissues by using Hoechst 33342 staining/FACS methods has permitted access to this category of cells (side population, or SP) in a manner independent of antibodies. Here, we show that the C2C12 myogenic line comprises a minor population of cells with SP phenotype. These cells are growth-arrested and delayed in their ability to differentiate. Dye efflux in C2C12-derived SPs is likely mediated by mdr1a, whose overexpression results in increased dedifferentiation. Interestingly, growth-arrested SPs rapidly appear in purified MP populations, thus suggesting a dynamic equilibrium among different states of differentiation. Finally, transcriptional profiling of C2C12-derived SP and MP cells corroborates the many similarities of SP to stem cells.
Assuntos
Linhagem Celular , Mioblastos Esqueléticos/citologia , Células Satélites de Músculo Esquelético/citologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Benzimidazóis , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Corantes Fluorescentes , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/ultraestrutura , Fenótipo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/ultraestruturaRESUMO
Skeletal muscle includes satellite cells, which reside beneath the muscle fiber basal lamina and mainly represent committed myogenic precursor cells, and multipotent stem cells of unknown origin that are present in muscle connective tissue, express the stem cell markers Sca-1 and CD34, and can differentiate into different cell types. We tracked bone marrow (BM)-derived stem cells in both muscle connective tissue and satellite cell niches of irradiated mice transplanted with green fluorescent protein (GFP)-expressing BM cells. An increasing number of GFP+ mononucleated cells, located both inside and outside of the muscle fiber basal lamina, were observed 1, 3, and 6 months after transplantation. Sublaminal cells expressed unambiguous satellite cell markers (M-cadherin, Pax7, NCAM) and fused into scattered GFP+ muscle fibers. In muscle connective tissue there were GFP+ cells located close to blood vessels that expressed the ScaI or CD34 stem-cell antigens. The rate of settlement of extra- and intralaminal compartments by BM-derived cells was compatible with the view that extralaminal cells constitute a reservoir of satellite cells. We conclude that both muscle satellite cells and stem cell marker-expressing cells located in muscle connective tissue can derive from BM in adulthood.