RESUMEN
Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.
Asunto(s)
Sistemas CRISPR-Cas , Ribonucleasas , Sistemas CRISPR-Cas/genética , Endorribonucleasas/genética , Edición Génica , Técnicas de Inactivación de Genes , Ingeniería Genética , Humanos , Monocitos , ARN Mensajero/genética , Ribonucleasas/genéticaRESUMEN
CRISPR-Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases have revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting single guide RNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs. Existing plasmid-based reporters lack flexibility, being fixed to specific DNA sequences, hindering direct comparisons between various editing approaches. To address this, we developed the versatile genome editing application reporter (V-GEAR) system. V-GEAR comprises genes detectable after desired editing via base editing, prime editing, or homology-directed repair within relevant genomic contexts. It employs a detectable synthetic cell surface protein (RQR8) followed by a customizable target sequence resembling genomic regions of interest. These genes allow for reliable identification of corrective editing and cell enrichment. We validated the V-GEAR system with base editors, prime editors, and Cas9-mediated homology-directed repair. Furthermore, the V-GEAR system offers versatility by allowing transient screening or stable integration at the AAVS1 safe harbor loci, rapidly achieved through immunomagnetic isolation. This innovative system enables direct comparisons among editing technologies, accelerating the development and testing of genome editing approaches.
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Enzymopathy disorders are the result of missing or defective enzymes. Amongst these enzymopathies, mucopolysaccharidosis type I, is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), ultimately causes toxic build-up of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS). Human memory T cells (Tm) migrate throughout the body's tissues and can persist for years, making them an attractive approach for cellular-based, systemic enzyme replacement therapy. Here, we tested genetically engineered, IDUA-expressing Tm as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm take up residence in nearly all tested tissues, producing IDUA and leading to metabolic correction of GAG levels in the heart, lung, liver, spleen, kidney, bone marrow, and the CNS. Our study indicates that genetically engineered Tm holds great promise as a platform for cellular-based enzyme replacement therapy for the treatment of mucopolysaccharidosis type I and potentially many other enzymopathies and protein deficiencies.
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A method for simultaneously patterning and functionalizing thin poly(2-hydroxyethyl methacrylate) films through a reactive silane infusion based wrinkling is developed. Wrinkled patterns with tunable wavelengths on submicrometer size are easily produced over large area surfaces and can express a wide variety of chemical functional groups on the surface. The characteristic wavelength of wrinkling scales linearly with initial film thickness, in agreement with a gradationally swollen film model. Results from X-ray photoelectron spectroscopy confirm that the wrinkled film is composed of two layers: a gradient cross-linked top layer and a uniform un-cross-linked bottom layer. The surface chemical properties of wrinkles can be easily tuned by infusion of different functional silanes. Hierarchical wrinkled patterns with micro/nano structure can be achieved by combining wrinkling with other simple lithography methods. Wrinkled nanopatterns can be used as a mold to transfer the topology to a variety of other materials using nanoimprint lithography.
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B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.
Asunto(s)
Linfocitos B/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Ingeniería Genética/métodos , Genoma Humano , Antígenos CD19/metabolismo , Linfocitos B/citología , Secuencia de Bases , Proliferación Celular , Células Cultivadas , Dependovirus/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mutación INDEL/genéticaRESUMEN
Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.
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Factor Estimulante de Colonias de Macrófagos , Osteosarcoma , Aminopiridinas , Animales , Carcinogénesis , Ratones , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Pirroles , Receptores de Factor Estimulante de Colonias de Granulocitos y MacrófagosRESUMEN
Semaphorins, specifically type IV, are important regulators of axonal guidance and have been increasingly implicated in poor prognoses in a number of different solid cancers. In conjunction with their cognate PLXNB family receptors, type IV members have been increasingly shown to mediate oncogenic functions necessary for tumor development and malignant spread. In this study, we investigated the role of semaphorin 4C (SEMA4C) in osteosarcoma growth, progression, and metastasis. We investigated the expression and localization of SEMA4C in primary osteosarcoma patient tissues and its tumorigenic functions in these malignancies. We demonstrate that overexpression of SEMA4C promotes properties of cellular transformation, while RNAi knockdown of SEMA4C promotes adhesion and reduces cellular proliferation, colony formation, migration, wound healing, tumor growth, and lung metastasis. These phenotypic changes were accompanied by reductions in activated AKT signaling, G1 cell cycle delay, and decreases in expression of mesenchymal marker genes SNAI1, SNAI2, and TWIST1. Lastly, monoclonal antibody blockade of SEMA4C in vitro mirrored that of the genetic studies. Together, our results indicate a multi-dimensional oncogenic role for SEMA4C in metastatic osteosarcoma and more importantly that SEMA4C has actionable clinical potential.
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Neoplasias Óseas/patología , Progresión de la Enfermedad , Osteosarcoma/patología , Semaforinas/metabolismo , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia , Semaforinas/deficiencia , Semaforinas/genéticaRESUMEN
We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas using the Sleeping Beauty (SB) transposon system. Here, we followed up by investigating the genetic role of ZNF217 in osteosarcoma initiation and progression through the establishment of a novel genetically engineered mouse model, in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small-molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of osteosarcoma transformation, including proliferation, cell motility, and anchorage independent growth, and ultimately promoting osteosarcoma growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling. Pharmacologic blockade of AKT signaling with nucleoside analogue triciribine in ZNF217+ orthotopically injected osteosarcoma cell lines reduced tumor growth and metastasis. Our data demonstrate that triciribine treatment may be a relevant and efficacious therapeutic strategy for patients with osteosarcoma with ZNF217+ and p-AKT rich tumors. With the recent revitalization of triciribine for clinical studies in other solid cancers, our study provides a rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ osteosarcoma.
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Biomarcadores de Tumor , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Transactivadores/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Modelos Biológicos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/etiología , Osteosarcoma/metabolismo , Osteosarcoma/patología , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Carboranes have been previously noted to distinctively affect the luminescent properties of semiconducting polymers when incorporated into the conjugated backbone. In this report, we use carborane-based poly(fluorene) derivatives as active materials for polymer light-emitting diodes and transistors. Optoelectronic analysis unequivocally shows that carborane does not participate in the π-conjugated network, yet their presence causes major red-shifting in device electroluminescence as well as in thin film photoluminescence. In field effect transistors, they also improve charge carrier mobility by an order of magnitude despite disrupting π-conjugation. This use of carborane-containing conjugated polymers in active devices holds promise as new responsive materials in electronic polymer applications.
RESUMEN
Hybrid conjugated polymers containing carborane directly bonded in the aromatic backbone repeat structure have interesting electronic bonding structures and are potentially useful new materials in organic electronics. Conjugated polymers based on o-carborane are particularly interesting for applications in sensing and detection because of the cage's unique bonding scheme and its bent geometry. Poly(fluorene) containing o-carborane displays multiple emission pathways that can be modulated through interactions with small molecules. In this paper, we report that films of poly(fluorene) with o-carborane in the backbone function as vapochromatic photoluminescent sensors toward volatile organic molecules.
Asunto(s)
Aminas/química , Boranos/química , Fluorenos/química , Polímeros/química , Solventes/química , Estructura MolecularRESUMEN
Poly(9,9-dihexyl fluorene) (PF) layers have been grown from modified silicon and quartz surfaces using Ni(0)-mediated coupling polymerization reactions. The surfaces were functionalized using a silane coupling reagent containing a dibromofluorene unit. 9,9-Diallyl-2,7-dibromo-9H-fluorene (1) was synthesized followed by a facile hydrosilylation with trichlorosilane yielding the desired coupling agent (3,3'-(2,7-dibromo-9H-fluorene-9,9-diyl)bis(propane-3,1-diyl))bis(trichlorosilane) (2). After treatment of the substrate surface with 2, a surface-directed polymerization of 2,7-dibromo-9,9-dihexylfluorene was performed from the functional siloxane surface to producing surfaces coated with a covalently grafted PF layer. The various surfaces were characterized using surface contact angle measurements, film thickness measurements, AFM studies, and fluorescence spectrometer measurements. This simple technique presents a novel and effective means of synthesizing a brush-like layer of a conjugated polymer on silicon and quartz substrates.
RESUMEN
Polyfluorene with p-carborane in the backbone was synthesized from the new monomer 1,12-bis(7-bromo-9,9-dihexyl-9H-fluoren-2-yl)-closo-1,12-dicarbodecaborane to give a high MW, soluble, blue-emitting material characterized by NMR, UV-vis, and fluorescence spectroscopy.