RESUMO
Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1ß (IL-1ß). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1ß. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.
Assuntos
Aldeído Redutase/metabolismo , Endométrio/enzimologia , Técnicas de Inativação de Genes/métodos , Hidroxiprostaglandina Desidrogenases/metabolismo , Células Estromais/enzimologia , Aldeído Redutase/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endométrio/citologia , Feminino , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Células Estromais/citologiaRESUMO
AKR1B1 of the polyol pathway was identified as a prostaglandin F2α synthase (PGFS). Using a genomic approach we have identified in the endometrium five bovine and three human AKRs with putative PGFS activity and generated the corresponding recombinant enzymes. The PGFS activity of the recombinant proteins was evaluated using a novel assay based on in situ generation of the precursor of PG biosynthesis PGH2. PGF2α was measured by ELISA and the relative potencies of the different enzymes were compared. We identified AKR1A1 and confirmed AKR1B1 as the most potent PGFS expressing characteristic inhibition patterns in presence of methylglyoxal, ponalrestat and glucose.
Assuntos
Aldeído Redutase/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Animais , Bovinos , Dinoprosta/biossíntese , Endométrio/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Alinhamento de SequênciaRESUMO
The congenital form of myotonic dystrophy type 1 (DM1) is the most severe type of the disease associated with CTG expansions over 1500 repeats and delayed muscle maturation. The mechanistic basis of the congenital form of DM1 is mostly unknown. Here, we show that muscle satellite cells bearing large CTG expansions (>3000) secrete a soluble factor that inhibits the fusion of normal myoblasts in culture. We identified this factor as prostaglandin E2 (PGE(2)). In these DM1 cells, PGE(2) production is increased through up-regulation of cyclooxygenase 2 (Cox-2), mPGES-1 and prostaglandin EP2/EP4 receptors. Elevated levels of PGE(2) inhibit myogenic differentiation by decreasing the intracellular levels of calcium. Exogenous addition of acetylsalicylic acid, an inhibitor of Cox enzymes, abolishes PGE(2) abnormal secretion and restores the differentiation of DM1 muscle cells. These data indicate that the delay in muscle maturation observed in congenital DM1 may result, at least in part, from an altered autocrine mechanism. Inhibitors of prostaglandin synthesis may thus offer a powerful method to restore the differentiation of DM1 muscle cells.
Assuntos
Diferenciação Celular/fisiologia , Dinoprostona/biossíntese , Distrofia Miotônica/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Aspirina/farmacologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/genética , Humanos , Distrofia Miotônica/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Expansão das Repetições de Trinucleotídeos , Regulação para CimaRESUMO
BACKGROUND: Various endonucleases can be engineered to induce double-strand breaks (DSBs) in chosen DNA sequences. These DSBs are spontaneously repaired by nonhomologous-end-joining, resulting in micro-insertions or micro-deletions (INDELs). We detected, characterized and quantified the frequency of INDELs produced by one meganuclease (MGN) targeting the RAG1 gene, six MGNs targeting three introns of the human dystrophin gene and one pair of zinc finger nucleases (ZFNs) targeting exon 50 of the human dystrophin gene. The experiments were performed in human cells (i.e. 293 T cells, myoblasts and myotubes). METHODS: To analyse the INDELs produced by the endonucleases the targeted region was polymerase chain reaction amplified and the amplicons were digested with the Surveyor enzyme, cloned in bacteria or deep sequenced. RESULTS: Endonucleases targeting the dystrophin gene produced INDELs of different sizes but there were clear peaks in the size distributions. The positions of these peaks were similar for MGNs but not for ZFNs in 293 T cells and in myoblasts. The size of the INDELs produced by these endonucleases in the dystrophin gene would have permitted a change in the reading frame. In a subsequent experiment, we observed that the frequency of INDELs was increased by re-exposition of the cells to the same endonuclease. CONCLUSIONS: Endonucleases are able to: (i) restore the normal reading of a gene with a frame shift mutation; (ii) delete a nonsense codon; and (iii) knockout a gene. Endonucleases could thus be used to treat Duchenne muscular dystrophy and other hereditary diseases that are the result of a nonsense codon or a frame shift mutation.
Assuntos
Distrofina/genética , Endonucleases/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Linhagem Celular , Códon sem Sentido , Endonucleases/genética , Éxons , Mutação da Fase de Leitura , Genes RAG-1 , Humanos , Mutação INDEL , Lentivirus/genética , Lentivirus/metabolismo , Mioblastos/fisiologia , Fases de Leitura , Dedos de Zinco/genéticaRESUMO
Myogenic cell transplantation is an experimental approach for the treatment of myopathies. In this approach, transplanted cells need to fuse with pre-existing myofibers, form new myofibers, and generate new muscle precursor cells (MPCs). The last property was fully reported following myoblast transplantation in mice but remains poorly studied with human myoblasts. In this study, we provide evidence that the intramuscular transplantation of postnatal human myoblasts in immunodeficient mice generates donor-derived MPCs and specifically donor-derived satellite cells. In a first experiment, cells isolated from mouse muscles 1 month after the transplantation of human myoblasts proliferated in vitro as human myoblasts. These cells were retransplanted in mice and formed myofibers expressing human dystrophin. In a second experiment, we observed that inducing muscle regeneration 2 months following transplantation of human myoblasts led to myofiber regeneration by human-derived MPCs. In a third experiment, we detected by immunohistochemistry abundant human-derived satellite cells in mouse muscles 1 month after transplantation of postnatal human myoblasts. These human-derived satellite cells may correspond totally or partially to the human-derived MPCs evidenced in the first two experiments. Finally, we present evidence that donor-derived satellite cells may be produced in patients that received myoblast transplantation.
Assuntos
Transplante de Células/métodos , Mioblastos/citologia , Células Satélites de Músculo Esquelético/citologia , Adulto , Animais , Células Cultivadas , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mioblastos/fisiologia , Células Satélites de Músculo Esquelético/fisiologiaRESUMO
Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.
Assuntos
Distrofina/metabolismo , Animais , Western Blotting , Linhagem Celular , Cães , Distrofina/genética , Terapia Genética , Humanos , Lentivirus/genética , Camundongos , Camundongos Mutantes , Músculos/citologia , Músculos/metabolismo , Distrofia Muscular de Duchenne/terapia , Plasmídeos/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
In ruminants, interferon-tau (IFNtau) is the maternal recognition signal inhibiting prostaglandin (PG) F2alpha production by endometrial epithelial cells and stimulating interferon-stimulated genes in the stroma. Stromal cells mediate the action of progesterone on epithelial cells during pregnancy. Our working hypothesis is that IFNtau acts as a molecular switch that turns on PGE(2) production in endometrial stromal cells while suppressing PGF2alpha production from epithelial cells. In this report we document immortalization and functional characterization of a bovine stromal cell line from the caruncular region of the endometrium [caruncular stromal cell (CSC)]. Primary stromal cells were immortalized by nucleofection with simian virus 40 large T antigen and integrase. The resulting cell line, CSC, expresses stromal cell-specific vimentin, estrogen, and progesterone receptors, and is amenable for transient transfection. Basal and stimulated production of PGE2 is higher than PGF2alpha and associated with cyclooxygenase (COX) 2 expression. Phorbol myristate acetate (PMA) and IFNtau up-regulate COX2 and PG production in a dose-dependent manner. When added together, low concentrations of IFNtau inhibit PMA-induced COX2 expression; whereas this inhibition is lost at high concentrations. Expression of signal transducer and activator of transcription 1 is induced by IFNtau at all concentrations studied but is not modulated by PMA. Because expression of signal transducer and activator of transcription 1 does not exhibit the biphasic response to IFNtau, we investigated the p38 MAPK pathway using the selective inhibitor SB203580. Inhibition of the p38 MAPK pathway abolishes IFNtau action on PG production. In summary, CSC appears as a good stromal cell model for investigating the molecular mechanisms related to IFNtau action and PG production in the bovine.
Assuntos
Endométrio/fisiologia , Endométrio/virologia , Vírus 40 dos Símios/fisiologia , Células Estromais/fisiologia , Células Estromais/virologia , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Endométrio/citologia , Receptor alfa de Estrogênio/genética , Feminino , Interferon gama/farmacologia , Prostaglandinas/biossíntese , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/citologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Duchenne muscular dystrophy is a recessive disease due to a mutation in the dystrophin gene. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the myostatin signal with an antagonist, follistatin. Three different approaches were studied to overexpress follistatin in the muscles of mdx mice transplanted with myoblasts. First, transgenic follistatin/mdx mice were generated; second, a follistatin plasmid was electroporated in mdx muscles, and finally, follistatin was induced in mdx mice muscles by a treatment with a histone deacetylase inhibitor. The three approaches improved the success of the myoblast transplantation. Moreover, fiber hypertrophy was also observed in all muscles, demonstrating that myostatin inhibition by follistatin is a good method to improve myoblast transplantation and muscle function. Myostatin inhibition by follistatin in combination with myoblast transplantation is thus a promising novel therapeutic approach for the treatment of muscle wasting in diseases such as Duchenne muscular dystrophy.
Assuntos
Folistatina/fisiologia , Distrofia Muscular Animal/terapia , Mioblastos/transplante , Fator de Crescimento Transformador beta/metabolismo , Animais , Transplante de Células/métodos , Células Cultivadas , Folistatina/genética , Folistatina/metabolismo , Terapia Genética/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , MiostatinaRESUMO
Frataxin gene (FXN) expression is reduced in Friedreich's ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondria's iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA) such as VP64 or P300, were used to increase the expression of that gene. Many effectors, plTALEVP64, plTALEp300, and plTALESunTag, targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted selection of 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19-fold in different Friedreich ataxia (FRDA) primary fibroblasts. Adeno-associated viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies in vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10-fold and in skeletal muscles by up to 35-fold. The aconitase activity was positively modulated by the frataxin level in mitochondria, and it was, thus, increased in vitro and in vivo by the increased frataxin expression.
RESUMO
A 26-years old Duchenne muscular dystrophy (DMD) patient received normal muscle-precursor cells, proliferated in vitro and implanted in a thenar eminence, biceps brachii, and in a portion of a gastrocnemius by injections placed 1mm from each other or less. Saline was injected in the contralateral gastrocnemius. The patient was immunosuppressed with tacrolimus. The protocol of cell transplantation was well tolerated and did not cause permanent sequels. Some injected sites were biopsied at 1, 14 and 18 months post-transplantation. Muscles were replaced by fat and fibrosis. In the cell-grafted site of the gastrocnemius, 27.5% of the myofiber profiles expressed donor-derived dystrophin 1 month post-transplantation and 34.5% 18 months post-transplantation. The contralateral gastrocnemius was dystrophin-negative. Myofibers were virtually absent in the biceps brachii, where only two dystrophin-positive myofibers were observed. In conclusion, a "high-density injection" protocol was feasible for intramuscular cell-transplantation in a DMD patient and long-term expression of donor-derived dystrophin was observed.
Assuntos
Transplante de Células/métodos , Células Musculares/transplante , Distrofia Muscular de Duchenne/cirurgia , Análise de Variância , Distrofina/metabolismo , Seguimentos , Humanos , Imunossupressores/uso terapêutico , Células Musculares/imunologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Fatores de TempoRESUMO
A clinical trial was conducted to test a new protocol of normal muscle precursor cell (MPC) allotransplantation in skeletal muscles of patients with Duchenne muscular dystrophy (DMD). Cultured MPCs obtained from one of the patient's parents were implanted in 0.25 or 1 cm of a Tibialis anterior in 9 patients with DMD. MPC injections were placed 1 to 2 mm from each other, and a similar pattern of saline injections was done in the contralateral muscle. The patients were immunosuppressed with tacrolimus. Muscle biopsies were performed at the injected sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donor's dystrophin in 8 patients. The percentage of myofibers expressing donor's dystrophin varied from 3.5% to 26%. Evidence of small myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by reverse transcriptase-polymerase chain reaction in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of patients with DMD, although this expression is restricted to the sites of MPC injection.
Assuntos
Distrofina/biossíntese , Células Musculares/transplante , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Transplante de Células-Tronco , Adolescente , Animais , Criança , Distrofina/imunologia , Imunofluorescência , Rejeição de Enxerto/prevenção & controle , Antígenos de Histocompatibilidade/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Imunossupressores/uso terapêutico , Hibridização in Situ Fluorescente , Camundongos , Camundongos SCID , Células Musculares/imunologia , Músculo Esquelético/imunologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tacrolimo/uso terapêuticoRESUMO
The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.
RESUMO
Prostaglandins (PGs) are important regulators of reproductive function. The mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Prostaglandin transporter (PGT) has been identified as a functional PG carrier. The aim of our study was to outline the expression of PGT in the human endometrium across the menstrual cycle. Quantitative RT-PCR showed human PGT (hPGT) expression to be strong in the proliferative and early secretory phases and low in the middle to late secretory phase. Northern blot analysis revealed hPGT mRNA transcript of 4 kb in the human endometrium. A peptide-directed polyclonal antibody was generated in rabbits against the 22 amino acids forming the C terminus of hPGT. Antibody specificity was demonstrated by Western blot. Immunoblots of endogenous hPGT in the human endometrium revealed a 70-kDa protein in endometrial cells. Endometrial biopsies collected across the menstrual cycle were used to assess hPGT protein expression by immunohistochemistry. hPGT was immunolocalized to luminal, glandular epithelial, and stromal cells. Because it was observed at the mRNA level, semiquantitative analysis showed a higher protein expression in proliferative and early secretory phases than in the mid-late secretory phase. In conclusion, our study revealed that hPGT expression is modulated in epithelial and stromal cells of the human endometrium at both mRNA and protein levels during the menstrual cycle. These findings support a role for hPGT as an important new player in the regulation of PG action in the human endometrium.
Assuntos
Antiporters/genética , Proteínas de Ligação a DNA/genética , Endométrio/metabolismo , Ciclo Menstrual/metabolismo , Antiporters/análise , Antiporters/fisiologia , Células Cultivadas , Ciclo-Oxigenase 2 , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/fisiologia , Endométrio/química , Feminino , Humanos , Imuno-Histoquímica , Interleucina-1/farmacologia , Proteínas de Membrana , Transportadores de Ânions Orgânicos , Prostaglandina-Endoperóxido Sintases/análise , Prostaglandinas/metabolismo , RNA Mensageiro/análiseRESUMO
Prostaglandins derived from arachidonic acid are involved in a wide variety of physiological and pathological processes. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases. These enzymes have been identified in human, but only partially in the monkey where microsomal PGES-1 and cytosolic PGES have not been characterized. The present study was undertaken to clone these enzymes and to study their tissue distribution, along with mPGES-2. The coding sequence of Macaque mPGES-1 is 98% homologous to human mPGES-1 at the nucleic acid level and the deduced amino acid sequence has 98% homology with the human protein. The Macaque cPGES cDNA is more than 99% homologous to the human and the deduced amino acids sequence is identical to that of the human cPGES. By Northern blot analysis, we found that mPGES-2 and cPGES mRNA were expressed in the endometrium, myometrium, ovary and oviduct, albeit at different levels, while mPGES-1 mRNA was detected at a weak level, mainly in the oviduct. Western Blot analysis revealed that mPGES-2, mPGES-1 and cPGES proteins were present in all tissues tested. These results suggest that production of PGE2 in Macaque may involve more than one PGES and that further studies will be needed to fully understand the conditions under which each PGES contributes to PGE2 production.
Assuntos
Citosol/enzimologia , Oxirredutases Intramoleculares/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar , Feminino , Humanos , Oxirredutases Intramoleculares/genética , Macaca fascicularis , Dados de Sequência Molecular , Prostaglandina-E Sintases , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Prostaglandins (PGs) are important regulators of reproductive functions. In ruminants, interferon (IFN)-tau is the embryonic signal responsible for recognition of pregnancy. This is effected by a reduction of the production of PGF(2alpha) relative to PGE(2.) This may be accomplished by a decrease in PGF(2alpha) production, but a stimulation of PGE(2) via the PGE synthase might also be involved. The purpose of the present study was to confirm the presence of PGE synthase (PGES) in the bovine endometrium, identify the factors affecting its expression, and compare it with that of cyclooxygenase-2 (COX-2). This was done by Northern blot analysis using primary cultures of bovine epithelial and stromal cells of the endometrium and bovine endometrial cell line. PGES mRNA expression was increased in the presence of lipopolysaccharides, TNF-alpha, and IFN-tau in stromal cells and IFN-tau in epithelial cells. In stromal cells, IFN-tau induced a rapid increase of PGES and COX-2 mRNA expression. In bovine endometrial cells, phorbol 12-myristate 13-actetate increased PGES mRNA, COX-2 mRNA and PGE(2) production. These results suggest that in endometrial cells, the expression of PGE synthase is correlated with that of COX-2 and is an important enzyme for the production of PGE(2). Increasing this production will modulate the PGE(2)/PGF(2alpha) ratio and contribute to establishment of pregnancy.
Assuntos
Endométrio/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interferon Tipo I/farmacologia , Oxirredutases Intramoleculares/genética , Isoenzimas/genética , Proteínas da Gravidez/farmacologia , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/análise , Animais , Bovinos , Células Cultivadas , Ciclo-Oxigenase 2 , Dinoprosta/biossíntese , Dinoprostona/biossíntese , Endométrio/citologia , Feminino , Prostaglandina-E SintasesRESUMO
Friedreich ataxia (FRDA) is a genetic disease due to increased repeats of the GAA trinucleotide in intron 1 of the frataxin gene. This mutation leads to a reduced expression of frataxin. We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN). This AAV was delivered by intraperitoneal (IP) injection to young conditionally knockout mice in which the frataxin gene had been knocked-out in some tissues during embryogenesis by breeding them with mice expressing the Cre recombinase gene under the muscle creatine kinase (MCK) or the neuron-specific enolase (NSE) promoter. In the first part of the study, different doses of virus were tested from 6 × 10(11) v.p. to 6 × 10(9) v.p. in NSE-cre mice and all leading to an increase in life spent of the mice. The higher and the lower dose were also tested in MCK-cre mice. A single administration of the AAV9-hFXN at 6 × 10(11) v.p. more than doubled the life of these mice. In fact the MCK-cre mice treated with the AAV9-hFXN were sacrificed for further molecular investigations at the age of 29 weeks without apparent symptoms. Echography analysis of the heart function clearly indicated that the cardiac systolic function was better preserved in the mice that received 6 × 10(11) v.p. of AAV9-hFXN. The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models. Thus, gene therapy with an AAV9-hFXN is a potential treatment of FRDA.
RESUMO
TALEs targeting a promoter sequence and fused with a transcription activation domain (TAD) may be used to specifically induce the expression of a gene as a potential treatment for haploinsufficiency. This potential therapeutic approach was applied to increase the expression of frataxin in fibroblasts of Friedreich ataxia (FRDA) patients. FRDA fibroblast cells were nucleofected with a pCR3.1 expression vector coding for TALEFrat#8 fused with VP64. A twofold increase of the frataxin mRNA (detected by quantitative reverse transcription-PCR (qRT-PCR)) associated with a similar increase of the mature form of the frataxin protein was observed. The frataxin mRNA and protein were also increased by this TALE in the fibroblasts of the YG8R mouse model. The addition of 5-aza-2'-deoxycytidine (5-Aza-dC) or of valproic acid (VPA) to the TALE treatment did not produce significant improvement. Other TADs (i.e., p65, TFAP2α, SRF, SP1, and MyoD) fused with the TALEFrat#8 gene did not produce a significant increase in the frataxin protein. Thus the TALEFrat#8-VP64 recombinant protein targeting the frataxin promoter could eventually be used to increase the frataxin expression and alleviate the FRDA symptoms.Molecular Therapy-Nucleic Acids (2013) 2, e119; doi:10.1038/mtna.2013.41; published online 3 September 2013.
RESUMO
Zinc finger nucleases (ZFN) can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif) receptor 5 (CCR5) locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP) or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs) and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O(6)-methylguanine-DNA methyltransferase(P140K) gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.Molecular Therapy - Nucleic Acids (2013) 2, e68; doi:10.1038/mtna.2012.55; published online 29 January 2013.
RESUMO
Genes encoding transcription activator-like effector (TALE) proteins may be engineered to target specific DNA sequences. TALEs fused with a transcription activator can be used to specifically induce the expression of a gene. This could lead to completely new therapies for several diseases. We have applied this potential therapeutic approach to Friedreich ataxia (FRDA), as an example. FRDA is due to reduced expression of frataxin because of elongation of a trinucleotide (GAA) repeat in intron 1. Our aim was to develop a potential treatment for FRDA by increasing the expression of the frataxin gene. We engineered 12 TALE genes (TALE(Frat)) encoding TALE(Frat) proteins, each specifically targeting different 14-bp DNA sequences within the proximal region of the human frataxin promoter. When the genes encoding these TALE(Frat) proteins were fused with a transcription activator, that is, four VP16 peptides (i.e., VP64), the resulting TALE(Frat)-VP64 proteins induced the expression of an mCherry reporter gene fused to a mini-cytomegalovirus promoter able to be activated by the insertion of the frataxin proximal promoter upstream to the minipromoter. These TALE(Frat)-VP64 proteins also increased, by 2- to 3-fold, frataxin gene expression (detected by qRT-PCR) in the cells. We conclude that TALE(Frat) proteins targeting the frataxin promoter may be used to increase the expression of frataxin mRNA and potentially could alleviate the symptoms of Friedreich ataxia. TALE methodology opens a new field of research, which could be used to develop TALE proteins to treat other diseases by inducing the expression of specific genes.
Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Fatores de Transcrição/genética , Sequência de Bases , Linhagem Celular , Ataxia de Friedreich/genética , Ataxia de Friedreich/terapia , Expressão Gênica , Genes Reporter , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Íntrons , Proteínas de Ligação ao Ferro/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Expansão das Repetições de Trinucleotídeos , Frataxina , Proteína Vermelha FluorescenteRESUMO
Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1ß in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1ß is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1ß particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.