RESUMO
Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.
The biggest problem confronting oligonucleotide therapeutics is a lack of compounds capable of targeting compounds to diseased tissues. This paper reports a major advance targeting the transferrin receptor to increase the delivery of morpholine oligomers to muscle cells in vivo. This work suggests the possibility for improved treatments of muscular dystrophy and other diseases.
Assuntos
Distrofina , Éxons , Morfolinos , Distrofia Muscular de Duchenne , Oligonucleotídeos Antissenso , Animais , Camundongos , Distrofina/genética , Éxons/genética , Camundongos Endogâmicos mdx , Morfolinos/farmacologia , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/farmacologia , Receptores da Transferrina/genéticaRESUMO
Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive ciliopathy affecting multiple organs. The development of potential disease-modifying therapy for BBS will require concurrent targeting of multi-systemic manifestations. Here, we show for the first time that monosialodihexosylganglioside accumulates in Bbs2-/- cilia, indicating impairment of glycosphingolipid (GSL) metabolism in BBS. Consequently, we tested whether BBS pathology in Bbs2-/- mice can be reversed by targeting the underlying ciliary defect via reduction of GSL metabolism. Inhibition of GSL synthesis with the glucosylceramide synthase inhibitor Genz-667161 decreases the obesity, liver disease, retinal degeneration and olfaction defect in Bbs2-/- mice. These effects are secondary to preservation of ciliary structure and signaling, and stimulation of cellular differentiation. In conclusion, reduction of GSL metabolism resolves the multi-organ pathology of Bbs2-/- mice by directly preserving ciliary structure and function towards a normal phenotype. Since this approach does not rely on the correction of the underlying genetic mutation, it might translate successfully as a treatment for other ciliopathies.
Assuntos
Síndrome de Bardet-Biedl/genética , Cílios/genética , Ciliopatias/genética , Proteínas/genética , Animais , Síndrome de Bardet-Biedl/tratamento farmacológico , Síndrome de Bardet-Biedl/patologia , Diferenciação Celular/efeitos dos fármacos , Cílios/patologia , Ciliopatias/tratamento farmacológico , Ciliopatias/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Gangliosídeos/biossíntese , Gangliosídeos/genética , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/genética , Camundongos KnockoutRESUMO
Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.
Assuntos
Cílios/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/genética , Falência Renal Crônica/tratamento farmacológico , Doenças Renais Policísticas/tratamento farmacológico , Animais , Diferenciação Celular/efeitos dos fármacos , Cílios/patologia , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Purinas/administração & dosagem , Roscovitina , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Rim/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Doenças Renais Policísticas/metabolismo , Animais , CamundongosRESUMO
Radical S-adenosyl-L-methionine (SAM) enzymes couple the reductive cleavage of SAM to radical-mediated transformations that have proven to be quite broad in scope. DesII is one such enzyme from the biosynthetic pathway of TDP-desosamine where it catalyzes a radical-mediated deamination. Previous studies have suggested that this reaction proceeds via direct elimination of ammonia from an α-hydroxyalkyl radical or its conjugate base (i.e., a ketyl radical) rather than 1,2-migration of the amino group to form a carbinolamine radical intermediate. However, without a crystal structure, the active site features responsible for this chemistry have remained largely unknown. The crystallographic studies described herein help to fill this gap by providing a structural description of the DesII active site. Computational analyses based on the solved crystal structure are consistent with direct elimination and indicate that an active site glutamate residue likely serves as a general base to promote deprotonation of the α-hydroxyalkyl radical intermediate and elimination of the ammonia group.
RESUMO
Osteogenesis imperfecta (OI), is a genetic disorder of bone fragility caused by mutations in collagen I or proteins involved in collagen processing. Previous studies in mice and human OI bones have shown that excessive activation of TGF-ß signaling plays an important role in dominant and recessive OI disease progression. Inhibition of TGF-ß signaling with a murine pan-specific TGF-ß neutralizing antibody (1D11) was shown to significantly increase trabecular bone volume and long bone strength in mouse models of OI. To investigate the frequency of dosing and dose options of TGF-ß neutralizing antibody therapy, we assessed the effect of 1D11 on disease progression in a dominant OI mouse model (col1a2 gene mutation at G610C). In comparison with OI mice treated with a control antibody, we attempted to define mechanistic effects of 1D11 measured via µCT, biomechanical, dynamic histomorphometry, and serum biomarkers of bone turnover. In addition, osteoblast and osteoclast numbers in histological bone sections were assessed to better understand the mechanism of action of the 1D11 antibody in OI. Here we show that 1D11 treatment resulted in both dose and frequency dependency, increases in trabecular bone volume fraction and ultimate force in lumbar bone, and ultimate force, bending strength, yield force, and yield strength in the femur (p ≤ 0.05). Suppression of serum biomarkers of osteoblast differentiation, osteocalcin, resorption, CTx-1, and bone formation were observed after 1D11 treatment of OI mice. Immunohistochemical analysis showed dose and frequency dependent decreases in runt-related transcription factor, and increase in alkaline phosphatase in lumbar bone sections. In addition, a significant decrease in TRACP and the number of osteoclasts to bone surface area was observed with 1D11 treatment. Our results show that inhibition of the TGF-ß pathway corrects the high-turnover aspects of bone disease and improves biomechanical properties of OI mice. These results highlight the potential for a novel treatment for osteogenesis imperfecta. © 2021 Sanofi-Genzyme. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMO
Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase-mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.
Assuntos
Dioxanos/farmacologia , Glucosilceramidas/biossíntese , Doenças Renais Policísticas/metabolismo , Pirrolidinas/farmacologia , Animais , Ciclo Celular , Modelos Animais de Doenças , Gangliosídeo G(M3)/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glicoesfingolipídeos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Doenças Renais Policísticas/tratamento farmacológico , RatosRESUMO
Development of novel therapies for polycystic kidney disease (PKD) requires assays that adequately reflect disease biology and are adaptable to high-throughput screening. Here we describe an embryonic cystic kidney organ culture model and demonstrate that a new mutant allele of the Pkd1 gene (Pkd1(tm1Bdgz)) modulates cystogenesis in this model. Cyst formation induced by cAMP is influenced by the dosage of the mutant allele: Pkd1(tm1Bdgz) -/- cultures develop a larger cystic area compared with +/+ counterparts, while Pkd1(tm1Bdgz) +/- cultures show an intermediate phenotype. A similar relationship between the degree of cystogenesis and mutant gene dosage is seen in cystic kidney organ cultures derived from mice with a mutated Nek8 gene (Nek8(jck)). Both Pkd1- and Nek8- cultures display altered cell-cell junctions, with reduced E-cadherin expression and altered desmosomal protein expression, similar to ADPKD epithelia. Additionally, characteristic ciliary abnormalities are identified in cystic kidney cultures, with elevated ciliary polycystin 1 expression in Nek8 homozygous cultures and elevated ciliary Nek8 protein expression in Pkd1 homozygotes. These data suggest that the Nek8 and Pkd1 genes function in a common pathway to regulate cystogenesis. Moreover, compound Pkd1 and Nek8 heterozygous adult mice develop a more aggressive cystic disease than animals with a mutation in either gene alone. Finally, we validate the kidney organ culture cystogenesis assay as a therapeutic testing platform using the CDK inhibitor roscovitine. Therefore, embryonic kidney organ culture represents a relevant model for studying molecular cystogenesis and a rapid tool for the screening for therapies that block cystic growth.
Assuntos
Adesão Celular/fisiologia , Cílios/metabolismo , Mutação/genética , Doenças Renais Policísticas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Canais de Cátion TRPP/metabolismo , Alelos , Animais , Caderinas/metabolismo , Adesão Celular/genética , Cílios/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Cistos/metabolismo , Cistos/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Quinases Relacionadas a NIMA , Técnicas de Cultura de Órgãos , Doenças Renais Policísticas/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases , Purinas/farmacologia , RoscovitinaRESUMO
Significant progress in understanding the molecular mechanisms of polycystic kidney disease (PKD) has been made in recent years. Translating this understanding into effective therapeutics will require testing in animal models that closely resemble human PKD by multiple parameters. Similar to autosomal dominant PKD, juvenile cystic kidney (jck) mice develop cysts in multiple nephron segments, including cortical collecting ducts, distal tubules, and loop of Henle. The jck mice display gender dimorphism in kidney disease progression with more aggressive disease in male mice. Gonadectomy experiments show that testosterone aggravates the severity of the disease in jck male mice, while female gonadal hormones have protective effects. EGF receptor is overexpressed and mislocalized in jck cystic epithelia, a hallmark of human disease. Increased cAMP levels in jck kidneys and activation of the B-Raf/extracellular signal-regulated kinase pathway are demonstrated. The effect of jck mutation on the expression of Nek8, a NIMA-related (never in mitosis A) kinase, and polycystins in jck cilia is shown for the first time. Nek8 overexpression and loss of ciliary localization in jck epithelia are accompanied by enhanced expression of polycystins along the cilia. The primary cilia in jck kidneys are significantly more lengthened than the cilia in wild-type mice, suggesting a role for Nek8 in controlling ciliary length. Collectively, these data demonstrate that the jck mice should be useful for testing potential therapies and for studying the molecular mechanisms that link ciliary structure/function and cystogenesis.
Assuntos
Cílios/metabolismo , Transtornos da Motilidade Ciliar/fisiopatologia , Doenças Renais Císticas/patologia , Rim Policístico Autossômico Dominante/etiologia , Animais , Cílios/genética , AMP Cíclico/metabolismo , Progressão da Doença , Feminino , Hormônios Esteroides Gonadais/farmacologia , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Quinases Relacionadas a NIMA , Rim Policístico Autossômico Dominante/fisiopatologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Caracteres Sexuais , Transdução de Sinais , Taxa de Sobrevida , Canais de Cátion TRPP/metabolismoRESUMO
Mutations in polycystin-1 (PC-1) are responsible for autosomal dominant polycystic kidney disease (ADPKD), characterized by formation of fluid-filled tubular cysts. The PC-1 is a multifunctional protein essential for tubular differentiation and maturation found in desmosomal junctions of epithelial cells where its primary function is to mediate cell-cell adhesion. To address the impact of mutated PC-1 on intercellular adhesion, we have analyzed the structure/function of desmosomal junctions in primary cells derived from ADPKD cysts. Primary epithelial cells from normal kidney showed co-localization of PC-1 and desmosomal proteins at cell-cell contacts. A striking difference was seen in ADPKD cells, where PC-1 and desmosomal proteins were lost from the intercellular junction membrane, despite unchanged protein expression levels. Instead, punctate intracellular expression for PC-1 and desmosomal proteins was detected. The N-cadherin, but not E-cadherin was expressed in adherens junctions of ADPKD cells. These data together with co-sedimentation analysis demonstrate that, in the absence of functional PC-1, desmosomal junctions cannot be properly assembled and remain sequestered in cytoplasmic compartments. Taken together, our results demonstrate that PC-1 is crucial for formation of intercellular contacts. We propose that abnormal expression of PC-1 causes disregulation of cellular adhesion complexes leading to increased proliferation, loss of polarity and, ultimately, cystogenesis.
Assuntos
Desmossomos/patologia , Células Epiteliais/patologia , Junções Intercelulares/patologia , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Caderinas/metabolismo , Caderinas de Desmossomos/genética , Caderinas de Desmossomos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Junções Intercelulares/metabolismo , Modelos Biológicos , Mutação , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Proteínas/metabolismo , Sensibilidade e Especificidade , Canais de Cátion TRPPRESUMO
Most cases of autosomal dominant polycystic kidney disease are caused by mutations in the gene PKD1, encoding polycystin-1. To gain insight into the role of polycystin-1 in tubulogenesis and cystogenesis using the well-characterized canine kidney epithelial cell line MDCK, we have now cloned and characterized the exon/intron structure of the canine gene PKD1. FISH analysis showed that the dog genome lacks the multiple PKD1 homologs present in human. Intron 21 of dog PKD1 lacked the polypyrimidine tract characteristic of the human gene, whereas pyrimidine-rich elements were identified in canine intron 30. Canine polycystin-1 showed a higher degree of homology with the human counterpart and lower homology with mouse and rat. A striking degree of conservation (97% identity) was determined for the leucine-rich repeat domain between dog and human. Also, the homology analysis indicated that 4 of 16 Ig-like repeats (IgIII, IgVII, IgX, and IgXV) are likely to be functionally significant. This is particularly important in light of our recent findings demonstrating that Iglike domains form strong homophilic interactions and can mediate cell-cell adhesion. These data enable detailed analysis of the role of polycystin-1 in cystogenesis and tubulogenesis using the canine MDCK cell line.
Assuntos
Cães/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Éxons , Dosagem de Genes , Humanos , Íntrons , Dados de Sequência Molecular , Proteínas/química , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPPRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene, but cellular mechanisms of cystogenesis remain unclear. In an attempt to display the array of cyst-specific molecules and to elucidate the disease pathway, we have performed comprehensive high-throughput expression analysis of normal and ADPKD epithelia in a two-step fashion. First, we generated expression profiles of normal and cystic epithelia derived from kidney and liver using serial analysis of gene expression (SAGE). We found 472 and 499 differentially expressed genes with fivefold difference in liver and kidney libraries, respectively. These genes encode growth factors, transcription factors, proteases, apoptotic factors, molecules involved in cell-extracellular matrix interactions, and ion channels. As a second step, we constructed a custom cDNA microarray using a subset of the differentially regulated genes identified by SAGE and interrogated ADPKD patient samples. Subsequently, a set of differentially expressed genes was refined to 26 up-regulated and 48 down-regulated genes with ap value of <0.01. This study may provide valuable insights into the pathophysiology of ADPKD and suggest potential therapeutic targets.