RESUMO
Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.
Assuntos
Anisotropia , Canalículos Biliares/metabolismo , Membrana Celular/metabolismo , Hepatócitos/metabolismo , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organogênese , GravidezRESUMO
Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, protein tyrosine kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , RNA Longo não Codificante/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/fisiologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RNA Longo não Codificante/genéticaRESUMO
Translation is an essential biological process, and dysregulation is associated with a range of diseases including ribosomopathies, diabetes, and cancer. Here, we examine translation dysregulation in vivo using RNAi to knock down the m-subunit of the translation initiation factor eIF3 in the mouse liver. Transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses show that eIF3m deficiency leads to the transcriptional response and changes in cellular translation that yield few detectable differences in the translation of particular mRNAs. The transcriptional response fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins) and cell metabolism (alterations in lipid, amino acid, nucleic acid, and drug metabolism). Analysis of ribosome biogenesis reveals inhibition of rRNA processing, highlighting decoupling of rRNA synthesis and ribosomal protein gene transcription in response to eIF3m knockdown. Interestingly, a similar reduction in eIF3m protein levels is associated with induction of the mTOR pathway in vitro but not in vivo. Overall, this work highlights the utility of a RNAi-based in vivo approach for studying the regulation of mammalian translation in vivo.
RESUMO
Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Paclitaxel/síntese química , Neoplasias da Próstata/tratamento farmacológico , Animais , Humanos , Masculino , CamundongosRESUMO
Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), has recently emerged as a prominent biomarker of prostate cancer (PC) and as an attractive protein trap for drug targeting. At the present time, several drugs and molecular diagnostic tools conjugated with selective PSMA ligands are actively evaluated in different preclinical and clinical trials. In the current work, we discuss design, synthesis and a preliminary biological evaluation of PSMA-specific small-molecule carrier equipped by Doxorubicin (Dox). We have introduced an unstable azo-linker between Dox and the carrier hence the designed compound does release the active substance inside cancer cells thereby providing a relatively high Dox concentration in nuclei and a relevant cytotoxic effect. In contrast, we have also synthesized a similar conjugate with a stable amide linker and it did not release the drug at all. This compound was predominantly accumulated in cytoplasm and did not cause cell death. Preliminary in vivo evaluation has showed good efficiency for the degradable conjugate against PC3-PIP(PSMA+)-containing xenograft mine. Thus, we have demonstrated that the conjugate can be used as a template to design novel analogues with improved targeting, anticancer activity and lower rate of potential side effects. 3D molecular docking study has also been performed to elucidate the underlying mechanism of binding and to further optimization of the linker area for improving the target affinity.
Assuntos
Antígenos de Superfície/química , Antineoplásicos/síntese química , Doxorrubicina/química , Glutamato Carboxipeptidase II/química , Animais , Antígenos de Superfície/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Glutamato Carboxipeptidase II/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Simulação de Acoplamento Molecular , Neoplasias da Próstata/tratamento farmacológico , Estrutura Terciária de Proteína , Transplante HeterólogoRESUMO
CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9-sgRNA complex as a guide, the majority of the 3' end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3'-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA-RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.
Assuntos
Sistemas CRISPR-Cas , DNA/genética , Edição de Genes , RNA Guia de Cinetoplastídeos/genética , Alelos , Linhagem Celular Tumoral , Separação Celular , Citometria de Fluxo , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Células Jurkat , Nucleotídeos/genética , Oligonucleotídeos/genéticaRESUMO
The liver is the only organ in mammals that fully regenerates even after major injury. To identify orchestrators of this regenerative response, we performed quantitative large-scale proteomics analysis of cytoplasmic and nuclear fractions from normal versus regenerating mouse liver. Proteins of the ubiquitin-proteasome pathway were rapidly upregulated after two-third hepatectomy, with the ubiquitin ligase Nedd4-1 being a top hit. In vivo knockdown of Nedd4-1 in hepatocytes through nanoparticle-mediated delivery of small interfering RNA caused severe liver damage and inhibition of cell proliferation after hepatectomy, resulting in liver failure. Mechanistically, we demonstrate that Nedd4-1 is required for efficient internalization of major growth factor receptors involved in liver regeneration and their downstream mitogenic signaling. These results highlight the power of large-scale proteomics to identify key players in liver regeneration and the importance of posttranslational regulation of growth factor signaling in this process. Finally, they identify an essential function of Nedd4-1 in tissue repair.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regeneração Hepática , Proteômica/métodos , Ubiquitina-Proteína Ligases/metabolismo , Animais , Endocitose/efeitos dos fármacos , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Mitógenos/farmacologia , Ubiquitina-Proteína Ligases Nedd4 , Poliubiquitina/metabolismo , Proteoma/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
During the past decade asialoglycoprotein receptor (ASGP-R) expressed predominantly by hepatocytes has attracted a considerable attention as a convenient biomolecular trap for targeted drug delivery. Currently, several selective galactose-containing ligands equipped by drug molecules, e.g. known anticancer therapeutics, as well as diagnostic tools are under active preclinical development. In this paper, we have carried out a rational in silico screening among the molecules available in ChemDiv collection and compounds provided by our colleagues to reveal potential ASGP-R binders. Thus, 3D molecular docking approach provided a set of 100 `high score` molecules that was subsequently evaluated in vitro using an advanced Surface Plasmon Resonance (SPR) technique. As a result, dozens of novel small-molecule ASGP-R ligands with high diversity in structure were identified. Several hits showed the binding affinity much more better than that determined for galactose and Nacetylgalactosamine which were used as reference compounds. The disclosed molecules can be reasonably regarded as promising molecular devices for targeted drug delivery to hepatocytes.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Receptor de Asialoglicoproteína/química , Simulação por Computador , Sistemas de Liberação de Medicamentos , Hepatócitos/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
Long non-coding RNAs constitute the most abundant part of the transcribed mammalian genome. lncRNAs affect all essential processes in the living cell including transcription, splicing, translation, replication, shaping of chromatin and post translational modification of proteins. Alterations in lncRNA expression have been linked to a number of diseases; thus, modulation of lncRNA expression holds a huge potential for gene-based therapy. In this review we summarize published data about lncRNAs in the context of hepatic carcinogenesis and liver fibrosis, and the corresponding potential targets for gene therapy. Recent advancements in targeted delivery to the liver made RNA interference an invaluable tool to decipher hepatic lncRNA function and to develop lncRNA-oriented therapies for liver-involved diseases in the future. Different approaches for RNA delivery that can be used for functional studies in the lab and for clinical lncRNA based applications are critically discussed in this review.
Assuntos
Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Fígado/metabolismo , RNA Longo não Codificante/genética , Animais , Pesquisa Biomédica/métodos , Regulação da Expressão Gênica , Terapia Genética/métodos , Humanos , Fígado/patologia , Cirrose Hepática/terapia , Neoplasias Hepáticas/terapia , Interferência de RNARESUMO
Prostate cancer (PC) is the prevalent malignancy widespread among men in the Western World. Prostate specific membrane antigen (PSMA) is an established PC marker and has been considered as a promising biological target for anti-PC drug delivery and diagnostics. The protein was found to be overexpressed in PC cells, including metastatic, and the neovasculature of solid tumors. These properties make PSMA-based approach quite appropriate for effective PC imaging and specific drug therapy. Through the past decade, a variety of PSMA-targeted agents has been systematically evaluated. Small-molecule compounds have several advantages over other classes, such as improved pharmacokinetics and rapid blood clearance. These low-weight ligands have similar structure and can be divided into three basic categories in accordance with the type of their zinc-binding core-head. Several PSMA binders are currently undergoing clinical trials generally for PC imaging. The main goal of the present review is to describe the recent progress achieved within the title field and structure activity relationships (SAR) disclosed for different PSMA ligands. Recent in vitro and in vivo studies for each type of the compounds described have also been briefly summarized.
Assuntos
Antígenos de Superfície/metabolismo , Portadores de Fármacos/química , Glutamato Carboxipeptidase II/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Portadores de Fármacos/farmacocinética , Humanos , Ligantes , Masculino , Estrutura Molecular , Terapia de Alvo Molecular , Neoplasias da Próstata/metabolismo , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacocinética , Relação Estrutura-AtividadeRESUMO
The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.
Assuntos
Sistemas CRISPR-Cas/genética , Genoma Humano , Edição de RNA , Tirosinemias/terapia , Animais , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Lipídeos/química , Camundongos , Mutação , Nanopartículas/química , Tirosinemias/genética , Vírus/genéticaRESUMO
A set of novel selenohydantoins were synthesized via a convenient and versatile approach involving the reaction of isoselenocyanates with various amines. We also revealed an unexpected ZâE isomerization of pyridin-2-yl-substituted selenohydantoins in the presence of Cu(2+) cations. The detailed mechanism of this transformation was suggested on the basis of quantum-chemical calculations, and the key role of Cu(2+) was elucidated. The obtained compounds were subsequently evaluated against a panel of different cancer cell lines. As a result, several molecules were identified as promising micromolar hits with good selectivity index. Instead of analogous thiohydantoins, which have been synthesized previously, selenohydantoins demonstrated a relatively high antioxidant activity comparable (or greater) to the reference molecule, Ebselen, a clinically approved drug candidate. The most active compounds have been selected for further biological trials.
Assuntos
Antineoplásicos/síntese química , Antioxidantes/síntese química , Hidantoínas/síntese química , Compostos Organosselênicos/síntese química , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Azóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Cianatos/química , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/química , Humanos , Hidantoínas/farmacologia , Concentração Inibidora 50 , Isoindóis , Compostos Organosselênicos/farmacologia , Piridinas/química , Teoria Quântica , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
N6-methyladenosine (m(6)A) is ubiquitously present in the RNA of living organisms from Escherichia coli to humans. Methyltransferases that catalyze adenosine methylation are drastically different in specificity from modification of single residues in bacterial ribosomal or transfer RNA to modification of thousands of residues spread among eukaryotic mRNA. Interactions that are formed by m(6)A residues range from RNA-RNA tertiary contacts to RNA-protein recognition. Consequences of the modification loss might vary from nearly negligible to complete reprogramming of regulatory pathways and lethality. In this review, we summarized current knowledge on enzymes that introduce m(6)A modification, ways to detect m(6)A presence in RNA and the functional role of this modification everywhere it is present, from bacteria to humans.
Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Escherichia coli/metabolismo , RNA/metabolismo , Humanos , Metilação , Metiltransferases/metabolismoRESUMO
BACKGROUND & AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.
Assuntos
Genômica/métodos , Fígado/crescimento & desenvolvimento , Nanopartículas , Interferência de RNA , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Ciclo Celular , Expressão Gênica , Genes da Neurofibromatose 2 , Fator de Crescimento de Hepatócito/genética , Hepatomegalia/etiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Serina-Treonina Quinase 3 , Proteína Supressora de Tumor p53/fisiologia , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Myocarditis is characterized by inflammatory cell infiltration of the heart and subsequent deterioration of cardiac function. Monocytes are the most prominent population of accumulating leucocytes. We investigated whether in vivo administration of nanoparticle-encapsulated siRNA targeting chemokine (C-C motif) receptor 2 (CCR2)-a chemokine receptor crucial for leucocyte migration in humans and mice--reduces inflammation in autoimmune myocarditis. METHODS AND RESULTS: In myocardium of patients with myocarditis, CCL2 mRNA levels and CCR2(+) cells increased (P < 0.05), motivating us to pursue CCR2 silencing. Flow cytometric analysis showed that siRNA silencing of CCR2 (siCCR2) reduced the number of Ly6C(high) monocytes in hearts of mice with acute autoimmune myocarditis by 69% (P < 0.05), corroborated by histological assessment. The nanoparticle-delivered siRNA was not only active in monocytes but also in bone marrow haematopoietic progenitor cells. Treatment with siCCR2 reduced the migration of bone marrow granulocyte macrophage progenitors into the blood. Cellular magnetic resonance imaging (MRI) after injection of macrophage-avid magnetic nanoparticles detected myocarditis and therapeutic effects of RNAi non-invasively. Mice with acute myocarditis showed enhanced macrophage MRI contrast, which was prevented by siCCR2 (P < 0.05). Follow-up MRI volumetry revealed that siCCR2 treatment improved ejection fraction (P < 0.05 vs. control siRNA-treated mice). CONCLUSION: This study highlights the importance of CCR2 in the pathogenesis of myocarditis. In addition, we show that siCCR2 affects leucocyte progenitor trafficking. The data also point to a novel therapeutic strategy for the treatment of myocarditis.
Assuntos
Doenças Autoimunes/terapia , Quimiocina CCL2/genética , Miocardite/terapia , RNA Interferente Pequeno/farmacologia , Adulto , Animais , Movimento Celular , Quimiocina CCL2/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Angiografia por Ressonância Magnética , Masculino , Camundongos , Monócitos/metabolismo , Nanopartículas , Interferência de RNA/fisiologiaRESUMO
The liver has a unique regenerative capability, which involves extensive remodelling of cell-cell and cell-matrix contacts. Here we study the role of integrins in mouse liver regeneration using Cre/loxP-mediated gene deletion or intravenous delivery of ß1-integrin siRNA formulated into nanoparticles that predominantly target hepatocytes. We show that although short-term loss of ß1-integrin has no obvious consequences for normal livers, partial hepatectomy leads to severe liver necrosis and reduced hepatocyte proliferation. Mechanistically, loss of ß1-integrin in hepatocytes impairs ligand-induced phosphorylation of the epidermal growth factor and hepatocyte growth factor receptors, thereby attenuating downstream receptor signalling in vitro and in vivo. These results identify a crucial role and novel mechanism of action of ß1-integrins in liver regeneration and demonstrate that protein depletion by nanoparticle-based delivery of specific siRNA is a powerful strategy to study gene function in the regenerating liver.
Assuntos
Proliferação de Células/genética , Hepatócitos/metabolismo , Integrina beta1/genética , Regeneração Hepática/genética , Fígado/metabolismo , Animais , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Hepatectomia , Fator de Crescimento de Hepatócito , Integrina beta1/metabolismo , Fígado/cirurgia , Camundongos , Camundongos Knockout , Transdução de Sinais/genéticaRESUMO
Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting ß1 and αv integrin subunits, we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (2 weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate on sustained integrin downregulation (7 weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of the MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of small interfering RNA-mediated inhibition of integrins as an anti-cancer therapeutic approach.
Assuntos
Carcinoma Hepatocelular/genética , Hepatócitos/metabolismo , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/genética , Neoplasias Hepáticas Experimentais/genética , Nanopartículas , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Interferente Pequeno , Animais , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Técnicas de Silenciamento de Genes , CamundongosRESUMO
We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in â¼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Terapia Genética/métodos , Hidrolases/genética , Tirosinemias/terapia , Animais , Genoma/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Fenótipo , Resultado do TratamentoRESUMO
Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b(+) Ly-6C(hi) monocytic and CD11b(+) Ly-6G(hi) granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.
Assuntos
Macrófagos/imunologia , Neoplasias/patologia , Neutrófilos/imunologia , Animais , Humanos , Camundongos , Neoplasias/imunologia , Baço/imunologia , Baço/patologiaRESUMO
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.