RESUMO
Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species.
Assuntos
Células Satélites de Músculo Esquelético , Peixe-Zebra , Camundongos , Animais , Músculo Esquelético/fisiologia , Transplante de Células-Tronco , Lipídeos/farmacologia , Diferenciação Celular , Desenvolvimento MuscularRESUMO
Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.
Assuntos
Capsídeo/metabolismo , Dependovirus/metabolismo , Evolução Molecular Direcionada , Técnicas de Transferência de Genes , Músculo Esquelético/metabolismo , Sequência de Aminoácidos , Animais , Capsídeo/química , Células Cultivadas , Modelos Animais de Doenças , Células HEK293 , Humanos , Integrinas/metabolismo , Macaca fascicularis , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/terapia , Multimerização Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/uso terapêutico , RNA Guia de Cinetoplastídeos/metabolismo , Recombinação Genética/genética , Especificidade da Espécie , TransgenesRESUMO
Muscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of "pro-regenerative" target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses.
Assuntos
Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Genes fos , CamundongosRESUMO
Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.
Assuntos
Alelos , Proteínas Morfogenéticas Ósseas/genética , Sistemas CRISPR-Cas , Deleção de Genes , Edição de Genes , Fatores de Diferenciação de Crescimento/genética , Animais , Feminino , Genes Reporter , Engenharia Genética , Genoma , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Homozigoto , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células Mieloides/metabolismo , Fenótipo , Domínios Proteicos , Triptofano/metabolismoRESUMO
Tightly regulated production of mature blood cells is essential for health and survival in vertebrates and dependent on discrete populations of blood-forming (hematopoietic) stem and progenitor cells. Prior studies suggested that inhibition of growth differentiation factor 11 (GDF11) through soluble activin receptor type II (ActRII) ligand traps or neutralizing antibodies promotes erythroid precursor cell maturation and red blood cell formation in contexts of homeostasis and anemia. As Gdf11 is expressed by mature hematopoietic cells, and erythroid precursor cell expression of Gdf11 has been implicated in regulating erythropoiesis, we hypothesized that genetic disruption of Gdf11 in blood cells might perturb normal hematopoiesis or recovery from hematopoietic insult. Contrary to these predictions, we found that deletion of Gdf11 in the hematopoietic lineage in mice does not alter erythropoiesis or erythroid precursor cell frequency under normal conditions or during hematopoietic recovery after irradiation and transplantation. In addition, although hematopoietic cell-derived Gdf11 may contribute to the pool of circulating GDF11 protein during adult homeostasis, loss of Gdf11 specifically in the blood system does not impair hematopoietic stem cell function or induce overt pathological consequences. Taken together, these results reveal that hematopoietic cell-derived Gdf11 is largely dispensable for native and transplant-induced blood formation.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Deleção de Genes , Fatores de Diferenciação de Crescimento/genética , Hematopoese , Animais , Células Cultivadas , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , CamundongosRESUMO
In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.