RESUMO
Wiskott-Aldrich syndrome (WAS) is an immune deficiency with thrombopenia resulting from mutations in the WASP gene. This gene normally encodes the Wiskott-Aldrich syndrome protein (WASP), a major cytoskeletal regulator expressed in hematopoietic cells. Gene therapy is a promising option for the treatment of WAS, requiring that clinically applicable WASP gene transfer vectors demonstrate efficacy in preclinical studies. Here, we describe a self-inactivating HIV-1-derived lentiviral vector encoding human WASP and show that it effectively transduced bone marrow progenitor cells of WASP knockout (WKO) mice. Transplantation of these transduced cells into lethally irradiated WKO recipients led to stable expression of WASP and correction of immune, inflammatory and cytoskeletal defects. Splenic T-cell proliferation was restored, podosomes were reinstated on bone-marrow-derived dendritic cells and colon inflammation was reduced. This shows for the first time (a) that cytoskeletal defects can be corrected in WKO mice, (b) that human WASP is biologically active in mice and (c) that a lentiviral vector is effective to express human WASP in vivo over several months. These data support further development of such lentiviral vectors for the gene therapy of WAS.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , HIV-1/genética , Proteínas/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Transplante de Medula Óssea/métodos , Colite/terapia , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Expressão Gênica , Terapia Genética/efeitos adversos , Humanos , Camundongos , Camundongos Knockout , Proteínas/metabolismo , RNA Mensageiro/genética , Linfócitos T/imunologia , Transdução Genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-AldrichRESUMO
The molecular events by which eccentric muscle contractions induce muscle damage and remodelling remain largely unknown. We assessed whether eccentric exercise modulates the expression of proteinases (calpains 1, 2 and 3, proteasome, cathepsin B+L), muscle structural proteins (alpha-sarcoglycan and desmin), and the expression of the heat shock proteins Hsp27 and alphaB-crystallin. Vastus lateralis muscle biopsies from twelve healthy male volunteers were obtained before, immediately after, and 1 and 14 days after a 30 min downhill treadmill running exercise. Eccentric exercise induced muscle damage as evidenced by the analysis of muscle pain and weakness, creatine kinase serum activity, myoglobinaemia and ultrastructural analysis of muscle biopsies. The calpain 3 mRNA level was decreased immediately after exercise whereas calpain 2 mRNA level was increased at day 1. Both mRNA levels returned to control values by day 14. By contrast, cathepsin B+L and proteasome enzyme activities were increased at day 14. The alpha-sarcoglycan protein level was decreased immediately after exercise and at day 1, whereas the desmin level peaked at day 14. alphaB-crystallin and Hsp27 protein levels were increased at days 1 and 14. Our results suggest that the differential expression of calpain 2 and 3 mRNA levels may be important in the process of exercise-induced muscle damage, whereas expression of alpha-sarcoglycan, desmin, alphaB-crystallin and Hsp27 may be essentially involved in the subsequent remodelling of myofibrillar structure. This remodelling response may limit the extent of muscle damage upon a subsequent mechanical stress.
Assuntos
Adaptação Fisiológica/fisiologia , Exercício Físico/fisiologia , Proteínas de Choque Térmico , Contração Muscular/fisiologia , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Actinas/análise , Adulto , Calpaína/genética , Cisteína Endopeptidases/metabolismo , Proteínas do Citoesqueleto/análise , Desmina/análise , Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP27 , Humanos , Lisossomos/enzimologia , Masculino , Glicoproteínas de Membrana/análise , Chaperonas Moleculares , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/química , Músculo Esquelético/lesões , Proteínas de Neoplasias/análise , Doenças Neuromusculares/fisiopatologia , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/análise , Corrida/fisiologia , Sarcoglicanas , Estresse Mecânico , alfa-Cristalinas/análise , beta-Cristalinas/análiseRESUMO
Lack of functional calpain 3 in humans is a cause of limb girdle muscular dystrophy, but the function(s) of calpain 3 remain(s) unknown. Special muscle conditions in which calpain 3 is downregulated could yield valuable clues to the understanding of its function(s). We monitored calpain 3 mRNA amounts by quantitative RT-PCR and compared them with those of alpha-skeletal actin mRNA in mouse leg muscles for different types of denervation and muscle injury. Intact muscle denervation reduced calpain 3 mRNA expression by a factor of 5 to 10, while alpha-skeletal actin mRNA was reduced in a slower and less extensive manner. Muscle injury (denervation-devascularization), which leads to muscle degeneration and regeneration, induced a 20-fold decrease in the mRNA level of both calpain 3 and alpha-skeletal actin. Furthermore, whereas in normal muscle and intact denervated muscle, the full-length transcript is the major calpain 3 mRNA, in injured muscle, isoforms lacking exon 6 are predominant during the early regeneration process. These data suggest that muscle condition determines the specific calpain 3 isoform pattern of expression and that calpain 3 expression is downregulated by denervation.
Assuntos
Calpaína/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Musculares , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Actinas/genética , Processamento Alternativo/fisiologia , Animais , Apoptose/fisiologia , Primers do DNA , Masculino , Camundongos , Denervação Muscular , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nervo Isquiático/fisiologia , Nervo Isquiático/cirurgiaRESUMO
Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.
Assuntos
DNA Complementar/genética , Eritropoese/genética , Eritropoetina/genética , Globinas/genética , Músculo Esquelético/metabolismo , RNA Mensageiro/biossíntese , Reticulócitos/metabolismo , Talassemia beta/genética , Animais , Proteína 1 de Troca de Ânion do Eritrócito/análise , Compartimento Celular , Diferenciação Celular , DNA Complementar/administração & dosagem , Modelos Animais de Doenças , Eletroporação , Eritrócitos/metabolismo , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Eritropoetina/sangue , Teste de Complementação Genética , Injeções Intramusculares , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Espectrina/análise , TransfecçãoRESUMO
The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus. Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A(1), brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form. MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.
Assuntos
Cisteína Endopeptidases/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Endossomos/virologia , Vetores Genéticos/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , DNA Viral/análise , Endocitose , Endossomos/metabolismo , Vetores Genéticos/genética , Humanos , Concentração de Íons de Hidrogênio , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma , Transdução Genética , Células Tumorais Cultivadas , Vírion/metabolismoRESUMO
Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.
Assuntos
Apoptose , Calpaína/deficiência , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Distrofias Musculares/enzimologia , Distrofias Musculares/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Calpaína/química , Calpaína/genética , Calpaína/metabolismo , Creatina Quinase/metabolismo , Cruzamentos Genéticos , Azul Evans , Feminino , Fertilidade , Deleção de Genes , Marcação de Genes , Genótipo , Masculino , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Inibidor de NF-kappaB alfa , Fenótipo , RNA Mensageiro/análise , RNA Mensageiro/genética , Sarcolema/patologiaRESUMO
We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.
Assuntos
Calpaína/biossíntese , Fibrinolíticos/farmacologia , Heparina/farmacologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fibrinolíticos/química , Heparina/análogos & derivados , RatosRESUMO
Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.
Assuntos
Calpaína/genética , Calpaína/metabolismo , Isoenzimas , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Processamento Alternativo , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Clonagem Molecular , Conectina , Primers do DNA , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/metabolismo , Humanos , Hibridização In Situ , Íntrons , Cristalino/anatomia & histologia , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/metabolismo , Modelos Genéticos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Miocárdio/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Distribuição TecidualRESUMO
BACKGROUND: Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. METHODS: We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. RESULTS: We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. CONCLUSION: We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.
Assuntos
Vetores Genéticos , Reação em Cadeia da Polimerase/métodos , Retroviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Terapia Genética , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , TransfecçãoRESUMO
The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti-insect selective toxins, was investigated. The ET50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET50 value of the wild-type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.