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1.
Cancer Cell ; 39(6): 779-792.e11, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34087162

RESUMO

The mesenchymal subtype of glioblastoma is thought to be determined by both cancer cell-intrinsic alterations and extrinsic cellular interactions, but remains poorly understood. Here, we dissect glioblastoma-to-microenvironment interactions by single-cell RNA sequencing analysis of human tumors and model systems, combined with functional experiments. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived oncostatin M (OSM) that interacts with its receptors (OSMR or LIFR) in complex with GP130 on glioblastoma cells and activates STAT3. We show that MES-like glioblastoma states are also associated with increased expression of a mesenchymal program in macrophages and with increased cytotoxicity of T cells, highlighting extensive alterations of the immune microenvironment with potential therapeutic implications.


Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Glioblastoma/imunologia , Glioblastoma/patologia , Linfócitos T/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Neoplasias Encefálicas/genética , Células Cultivadas , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Citotoxicidade Imunológica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oncostatina M/metabolismo , Subunidade beta de Receptor de Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/patologia
2.
Cancer Res ; 79(15): 3983-3991, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31315836

RESUMO

Development of animal models to investigate the complex ecosystem of malignant gliomas using the Cre/loxP recombination system has significantly contributed to our understanding of the molecular underpinnings of this deadly disease. In these model systems, once the tumor is induced by activation of Cre-recombinase in a tissue-specific manner, further genetic manipulations to explore the progression of tumorigenesis are limited. To expand the application of mouse models for gliomas, we developed glial fibrillary acidic protein (GFAP)-FLP recombinase (FLPo) mice that express FLPo recombinase specifically in GFAP-positive cells. Lentivirus-based in vivo delivery of cancer genes conditioned by FLP/FRT-mediated recombination initiated gliomas in GFAP-FLPo mice. Using the Cre-mediated multifluorescent protein-expressing system, we demonstrated that the GFAP-FLPo mouse model enables the analysis of various stages of gliomagenesis. Collectively, we present a new mouse model that will expand our ability to dissect developmental processes of gliomagenesis and to provide new avenues for therapeutic approaches. SIGNIFICANCE: This study presents a new glioma mouse model derived using lentiviral vectors and two recombination systems that will expand the ability to dissect developmental processes of gliomagenesis.


Assuntos
Glioma/genética , Recombinases/genética , Animais , Modelos Animais de Doenças , Técnicas de Genotipagem , Humanos , Camundongos , Camundongos Transgênicos
3.
BMC Bioinformatics ; 20(1): 294, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142274

RESUMO

BACKGROUND: Biochemical networks are often described through static or time-averaged measurements of the component macromolecules. Temporal variation in these components plays an important role in both describing the dynamical nature of the network as well as providing insights into causal mechanisms. Few methods exist, specifically for systems with many variables, for analyzing time series data to identify distinct temporal regimes and the corresponding time-varying causal networks and mechanisms. RESULTS: In this study, we use well-constructed temporal transcriptional measurements in a mammalian cell during a cell cycle, to identify dynamical networks and mechanisms describing the cell cycle. The methods we have used and developed in part deal with Granger causality, Vector Autoregression, Estimation Stability with Cross Validation and a nonparametric change point detection algorithm that enable estimating temporally evolving directed networks that provide a comprehensive picture of the crosstalk among different molecular components. We applied our approach to RNA-seq time-course data spanning nearly two cell cycles from Mouse Embryonic Fibroblast (MEF) primary cells. The change-point detection algorithm is able to extract precise information on the duration and timing of cell cycle phases. Using Least Absolute Shrinkage and Selection Operator (LASSO) and Estimation Stability with Cross Validation (ES-CV), we were able to, without any prior biological knowledge, extract information on the phase-specific causal interaction of cell cycle genes, as well as temporal interdependencies of biological mechanisms through a complete cell cycle. CONCLUSIONS: The temporal dependence of cellular components we provide in our model goes beyond what is known in the literature. Furthermore, our inference of dynamic interplay of multiple intracellular mechanisms and their temporal dependence on one another can be used to predict time-varying cellular responses, and provide insight on the design of precise experiments for modulating the regulation of the cell cycle.


Assuntos
Ciclo Celular/genética , Redes Reguladoras de Genes , Algoritmos , Animais , Pontos de Checagem do Ciclo Celular/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fase G1/genética , Genes cdc , Camundongos , Fatores de Tempo
4.
Proc Natl Acad Sci U S A ; 116(15): 7363-7370, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918126

RESUMO

Coordination of growth and genomic stability is critical for normal cell physiology. Although the E3 ubiquitin ligase BRCA1 is a key player in maintenance of genomic stability, its role in growth signaling remains elusive. Here, we show that BRCA1 facilitates stabilization of YAP1 protein and turning "off" the Hippo pathway through ubiquitination of NF2. In BRCA1-deficient cells Hippo pathway is "turned On." Phosphorylation of YAP1 is crucial for this signaling process because a YAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1 kinase recognition sites not only resists degradation but also rescues YAP1 transcriptional activity in BRCA1-deficient cells. Furthermore, an ectopic expression of the active Mt-YAP5SA, but not inactive Mt-YAP6SA, promotes EGF-independent proliferation and tumorigenesis in BRCA1-/- mammary epithelial cells. These findings establish an important role of BRCA1 in regulating stability of YAP1 protein that correlates positively with cell proliferation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Via de Sinalização Hippo , Humanos , Mutação de Sentido Incorreto , Neurofibromina 2/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAP
5.
Mol Cell Biol ; 39(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30397075

RESUMO

The nuclear factor kappa B (NF-κB) family of transcription factors plays a central role in coordinating the expression of genes that control inflammation, immune responses, cell proliferation, and a variety of other biological processes. In an attempt to identify novel regulators of this pathway, we performed whole-genome RNA interference (RNAi) screens in physiologically relevant human macrophages in response to lipopolysaccharide and tumor necrosis factor alpha (TNF-α). The top hit was SNW1, a splicing factor and transcriptional coactivator. SNW1 does not regulate the cytoplasmic components of the NF-κB pathway but complexes with the NF-κB heterodimer in the nucleus for transcriptional activation. We show that SNW1 detaches from its splicing complex (formed with SNRNP200 and SNRNP220) upon NF-κB activation and binds to NF-κB's transcriptional elongation partner p-TEFb. We also show that SNW1 is indispensable for the transcriptional elongation of NF-κB target genes such as the interleukin 8 (IL-8) and TNF genes. SNW1 is a unique protein previously shown to be involved in both splicing and transcription, and in this case, its role involves binding to the NF-κB-p-TEFb complex to facilitate transcriptional elongation of some NF-κB target genes.


Assuntos
NF-kappa B/metabolismo , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Regulação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Células THP-1 , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo
6.
Sci Transl Med ; 10(455)2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135249

RESUMO

Obesity and related morbidities pose a major health threat. Obesity is associated with increased blood concentrations of the anorexigenic hormone leptin; however, obese individuals are resistant to its anorexigenic effects. We examined the phenomenon of reduced leptin signaling in a high-fat diet-induced obesity model in mice. Obesity promoted matrix metalloproteinase-2 (Mmp-2) activation in the hypothalamus, which cleaved the leptin receptor's extracellular domain and impaired leptin-mediated signaling. Deletion of Mmp-2 restored leptin receptor expression and reduced circulating leptin concentrations in obese mice. Lentiviral delivery of short hairpin RNA to silence Mmp-2 in the hypothalamus of wild-type mice prevented leptin receptor cleavage and reduced fat accumulation. In contrast, lentiviral delivery of Mmp-2 in the hypothalamus of Mmp-2-/- mice promoted leptin receptor cleavage and higher body weight. In a genetic mouse model of obesity, transduction of cleavage-resistant leptin receptor in the hypothalamus reduced the rate of weight gain compared to uninfected mice or mice infected with the wild-type receptor. Immunofluorescence analysis showed that astrocytes and agouti-related peptide neurons were responsible for Mmp-2 secretion in mice fed a high-fat diet. These results suggest a mechanism for leptin resistance through activation of Mmp-2 and subsequent cleavage of the extracellular domain of the leptin receptor.


Assuntos
Leptina/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Obesidade/metabolismo , Receptores para Leptina/metabolismo , Animais , Encéfalo/enzimologia , Dieta Hiperlipídica , Ativação Enzimática , Hipotálamo/metabolismo , Leptina/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Ratos Wistar , Transdução de Sinais , Aumento de Peso
7.
Mol Cell ; 70(5): 842-853.e7, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29861157

RESUMO

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Dano ao DNA , Instabilidade Genômica , Heterocromatina/genética , RNA Neoplásico/genética , RNA Satélite/genética , Animais , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células MCF-7 , Camundongos , Ligação Proteica , RNA Neoplásico/metabolismo , RNA Satélite/metabolismo , Carga Tumoral
8.
Cell Rep ; 23(5): 1565-1580, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29719266

RESUMO

Hemophilia B is an ideal target for gene- and cell-based therapies because of its monogenic nature and broad therapeutic index. Here, we demonstrate the use of cell therapy as a potential long-term cure for hemophilia B in our FIX-deficient mouse model. We show that transplanted, cryopreserved, cadaveric human hepatocytes remain functional for more than a year and secrete FIX at therapeutic levels. Hepatocytes from different sources (companies and donors) perform comparably in curing the bleeding defect. We also generated induced pluripotent stem cells (iPSCs) from two hemophilia B patients and corrected the disease-causing mutations in them by two different approaches (mutation specific and universal). These corrected iPSCs were differentiated into hepatocyte-like cells (HLCs) and transplanted into hemophilic mice. We demonstrate these iPSC-HLCs to be viable and functional in mouse models for 9-12 months. This study aims to establish the use of cells from autologous and heterologous sources to treat hemophilia B.


Assuntos
Transplante de Células , Fator IX/metabolismo , Hemofilia B/terapia , Hepatócitos/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Modelos Animais de Doenças , Hemofilia B/genética , Hemofilia B/metabolismo , Hemofilia B/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Xenoenxertos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Camundongos Knockout
9.
Cell Rep ; 23(4): 1220-1229, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29694897

RESUMO

We have developed a cancer model of gliomas in human cerebral organoids that allows direct observation of tumor initiation as well as continuous microscopic observations. We used CRISPR/Cas9 technology to target an HRasG12V-IRES-tdTomato construct by homologous recombination into the TP53 locus. Results show that transformed cells rapidly become invasive and destroy surrounding organoid structures, overwhelming the entire organoid. Tumor cells in the organoids can be orthotopically xenografted into immunodeficient NOD/SCID IL2RG-/- animals, exhibiting an invasive phenotype. Organoid-generated putative tumor cells show gene expression profiles consistent with mesenchymal subtype human glioblastoma. We further demonstrate that human-organoid-derived tumor cell lines or primary human-patient-derived glioblastoma cell lines can be transplanted into human cerebral organoids to establish invasive tumor-like structures. Our results show potential for the use of organoids as a platform to test human cancer phenotypes that recapitulate key aspects of malignancy.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Organoides , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante de Neoplasias , Organoides/metabolismo , Organoides/patologia
10.
Proc Natl Acad Sci U S A ; 115(11): 2557-2560, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29487213

RESUMO

In keeping with the growing movement in scientific publishing toward transparency in data and methods, we propose changes to journal authorship policies and procedures to provide insight into which author is responsible for which contributions, better assurance that the list is complete, and clearly articulated standards to justify earning authorship credit. To accomplish these goals, we recommend that journals adopt common and transparent standards for authorship, outline responsibilities for corresponding authors, adopt the Contributor Roles Taxonomy (CRediT) (docs.casrai.org/CRediT) methodology for attributing contributions, include this information in article metadata, and require authors to use the ORCID persistent digital identifier (https://orcid.org). Additionally, we recommend that universities and research institutions articulate expectations about author roles and responsibilities to provide a point of common understanding for discussion of authorship across research teams. Furthermore, we propose that funding agencies adopt the ORCID identifier and accept the CRediT taxonomy. We encourage scientific societies to further authorship transparency by signing on to these recommendations and promoting them through their meetings and publications programs.

11.
Mol Cancer Res ; 16(5): 825-832, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29523765

RESUMO

Small cell lung cancer (SCLC) is the most deadly subtype of lung cancer due to its dismal prognosis. We have developed a lentiviral vector-mediated SCLC mouse model and have explored the role of both the NF-κB and CREB families of transcription factors in this model. Surprisingly, induction of NF-κB activity, which promotes tumor progression in many cancer types including non-small cell lung carcinoma (NSCLC), is dispensable in SCLC. Instead, suppression of NF-κB activity in SCLC tumors moderately accelerated tumor development. Examination of gene expression signatures of both mouse and human SCLC tumors revealed overall low NF-κB but high CREB activity. Blocking CREB activation by a dominant-negative form of PKA (dnPKA) completely abolished the development of SCLC. Similarly, expression of dnPKA or treatment with PKA inhibitor H89 greatly reduced the growth of SCLC tumors in syngeneic transplantation models. Altogether, our results strongly suggest that targeting CREB is a promising therapeutic strategy against SCLC.Implications: Activity of the transcription factor CREB is elevated in SCLC tumors, which helps to maintain its neuroendocrine signature and cell proliferation. Our results highlight the importance of targeting the CREB pathway to develop new therapeutics to combat SCLC. Mol Cancer Res; 16(5); 825-32. ©2018 AACR.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/patologia
12.
Nature ; 553(7688): 351-355, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29320480

RESUMO

The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer and chronic circadian rhythm disruption predisposes individuals to tumour development. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. REV-ERBs, the nuclear hormone receptors REV-ERBα (also known as NR1D1) and REV-ERBß (also known as NR1D2), are essential components of the circadian clock. Here we show that two agonists of REV-ERBs-SR9009 and SR9011-are specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and have no effect on the viability of normal cells or tissues. The anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as HRAS, BRAF, PIK3CA and others) and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 has a critical role in evoking an apoptotic response in malignant cells. Notably, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective antitumour strategy, identifying a class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are inhibitors of autophagy and de novo lipogenesis, with selective activity towards malignant and benign neoplasms.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/patologia , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/agonistas , Oncogenes/genética , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Lipogênese/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/genética , Nevo/tratamento farmacológico , Nevo/patologia , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia
14.
Proc Natl Acad Sci U S A ; 114(48): 12630, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29078419
15.
Mol Oncol ; 11(8): 1078-1098, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28500786

RESUMO

The multikinase inhibitor and FDA-approved drug dovitinib (Dov) crosses the blood-brain barrier and was recently used as single drug application in clinical trials for GB patients with recurrent disease. The Dov-mediated molecular mechanisms in GB cells are unknown. We used GB patient cells and cell lines to show that Dov downregulated the stem cell protein Lin28 and its target high-mobility group protein A2 (HMGA2). The Dov-induced reduction in pSTAT3Tyr705 phosphorylation demonstrated that Dov negatively affects the STAT3/LIN28/Let-7/HMGA2 regulatory axis in GB cells. Consistent with the known function of LIN28 and HMGA2 in GB self-renewal, Dov reduced GB tumor sphere formation. Dov treatment also caused the downregulation of key base excision repair factors and O6 -methylguanine-DNA-methyltransferase (MGMT), which are known to have important roles in the repair of temozolomide (TMZ)-induced alkylating DNA damage. Combined Dov/TMZ treatment enhanced TMZ-induced DNA damage as quantified by nuclear γH2AX foci and comet assays, and increased GB cell apoptosis. Pretreatment of GB cells with Dov ('Dov priming') prior to TMZ treatment reduced GB cell viability independent of p53 status. Sequential treatment involving 'Dov priming' and alternating treatment cycles with TMZ and Dov substantially reduced long-term GB cell survival in MGMT+ patient GB cells. Our results may have immediate clinical implications to improve TMZ response in patients with LIN28+ /HMGA2+ GB, independent of their MGMT methylation status.


Assuntos
Benzimidazóis/farmacologia , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Quinolonas/farmacologia , Benzimidazóis/agonistas , Linhagem Celular Tumoral , Dacarbazina/agonistas , Dacarbazina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Proteínas de Neoplasias/biossíntese , Quinolonas/agonistas , Temozolomida
16.
Proc Natl Acad Sci U S A ; 114(10): E1941-E1950, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28202722

RESUMO

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.


Assuntos
Portadores de Fármacos/administração & dosagem , Fator IX/farmacocinética , Hemofilia B/terapia , Nanopartículas/administração & dosagem , RNA Mensageiro/farmacocinética , Animais , Colesterol/química , Citocinas/metabolismo , Modelos Animais de Doenças , Composição de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Fator IX/genética , Fator IX/metabolismo , Feminino , Terapia Genética/métodos , Hemofilia B/genética , Hemofilia B/metabolismo , Hemofilia B/patologia , Humanos , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosfatidilcolinas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética
17.
Proc Natl Acad Sci U S A ; 114(1): 3-6, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27999178
18.
Cell Rep ; 17(8): 1907-1914, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27851956

RESUMO

Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/ß (IKK2/ß), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is a trimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways.


Assuntos
Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Relação Estrutura-Atividade
20.
Proc Natl Acad Sci U S A ; 113(14): 3702-3, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-26933217
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