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The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.
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Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.
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Baculoviridae/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Marcação de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transdução Genética , Transgenes , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Vetores Genéticos , Células HEK293 , Humanos , Integrases/metabolismo , Mutação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Human health is seriously endangered by spontaneous intracerebral hemorrhage (ICH) and aneurysmal subarachnoid hemorrhage (aSAH). Because the majority of ICH and aSAH survivors experience disability, increased risk of stroke recurrence, cognitive decline, and systemic vascular disease, ICH and aSAH assume special importance in neurological disease. Early detection and prediction of neurological function and understanding of etiology and correction are the basis of successful treatment. ICH and aSAH cause complex inflammatory cascades in the brain. In order to establish precise staging and prognosis, as well as provide a basis for treatment selection and monitoring, it is imperative to determine appropriate biological markers according to pathological and physiological mechanisms. In this review, we focus on the research progress of S100B, an endogenous danger signaling molecule, as a potential biomarker for ICH and aSAH, assisting in the development of further basic research and clinical translational studies.
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Acidente Vascular Cerebral , Hemorragia Subaracnóidea , Humanos , Hemorragia Cerebral , Fatores de Risco , Biomarcadores , Subunidade beta da Proteína Ligante de Cálcio S100RESUMO
Retinal degeneration (RD), a group of diseases leading to irreversible vision loss, is characterised by retinal pigment epithelium (RPE) or retinal neuron damage and loss. With fewer risks of immune rejection and tumorigenesis, stem cell-secreted extracellular vesicles (EVs) offer a new cell-free therapeutic paradigm for RD, which remains to be investigated. Human retinal organoid-derived retinal progenitor cells (hERO-RPCs) are an easily accessible and advanced cell source for RD treatment. However, hERO-RPCs-derived EVs require further characterisation. Here, we compared the characteristics of EVs from hERO-RPCs (hRPC-EVs) with those of human embryonic stem cell (hESC)-derived EVs (hESC-EVs) as controls. Based on in-depth proteomic analysis, we revealed remarkable differences between hRPC-EVs and hESC-EVs. A comparison between EVs and their respective cells of origin demonstrated that the protein loading of hRPC-EVs was more selective than that of hESC-EVs. In particular, hESC-EVs were enriched with proteins related to angiogenesis and cell cycle, whereas hRPC-EVs were enriched with proteins associated with immune modulation and retinal development. More importantly, compared with that of hESC-EVs, hRPC-EVs exhibited a lower correlation with cell proliferation and a unique capacity to regulate lipid metabolism. It was further confirmed that hRPC-EVs potentially eliminated lipid deposits, inhibited lipotoxicity and oxidative stress, and enhanced phagocytosis and survival of oleic acid-treated ARPE-19 cells. Mechanistically, hRPC-EVs are integrated into the mitochondrial network of oleic acid-treated ARPE-19 cells, and increased the level of mitochondrial fatty acid ß-oxidation-related proteins. Thus, organoid-derived hRPC-EVs represent a promising source of cell-free therapy for RD, especially for blinding diseases related to abnormal lipid metabolism in RPE cells.
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Vesículas Extracelulares , Células-Tronco Embrionárias Humanas , Humanos , Epitélio Pigmentado da Retina/metabolismo , Proteômica , Ácido Oleico/metabolismo , Vesículas Extracelulares/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Organoides/metabolismo , Metabolismo dos LipídeosRESUMO
Immune checkpoint blockade (ICB) therapy, particularly antibodies targeting the programmed death receptor 1 (PD-1) and its ligand (PD-L1), has revolutionized cancer treatment. However, its efficacy as a standalone therapy remains limited. Although ICB therapy in combination with chemotherapy shows promising therapeutic responses, the challenge lies in amplifying chemotherapy-induced antitumor immunity effectively. This relies on efficient drug delivery to tumor cells and robust antigen presentation by dendritic cells (DCs). Here, we developed tumor-repopulating cell (TRC)-derived microparticles with exceptional tumor targeting to deliver doxorubicin (DOX@3D-MPs) for improve anti-PD-1 therapy. DOX@3D-MPs effectively elicit immunogenic tumor cell death to release sufficient tumor antigens. Heat shock protein 70 (HSP70) overexpressed in DOX@3D-MPs contributes to capturing tumor antigens, promoting their phagocytosis by DCs, and facilitating DCs maturation, leading to the activation of CD8+ T cells. DOX@3D-MPs significantly enhance the curative response of anti-PD-1 treatment in large subcutaneous H22 hepatoma, orthotopic 4T1 breast tumor and Panc02 pancreatic tumor models. These results demonstrate that DOX@3D-MPs hold promise as agents to improve the response rate to ICB therapy and generate long-lasting immune memory to prevent tumor relapse.
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Antineoplásicos , Micropartículas Derivadas de Células , Neoplasias Pancreáticas , Humanos , Linfócitos T CD8-Positivos , Doxorrubicina/farmacologia , Neoplasias Pancreáticas/terapia , Antígenos de Neoplasias/genética , Antineoplásicos/uso terapêuticoRESUMO
Objectives: This study aimed to determine whether SII on different days of admission is associated with severity and 180-day functional outcomes after basal ganglia ICH. Methods: In this retrospective study, data on baseline CT imaging characteristics, mRS, hematoma volume, and laboratory variables were included. The SII and NLR, LMR, and PLR were calculated from laboratory data collected on admission day, day 1, and days 5-7. Both univariate and multivariable logistic regression analyses were used to assess the association between the SII and the outcome. The receiver operating characteristic (ROC) analysis and area under the curve (AUC) were also used to evaluate the ability of the SII to predict outcomes. Result: A total of 245 patients were enrolled in the study. On different days, the NLR, PLR, and SII were significantly lower in patients with favorable outcomes than in those with poor outcomes, and the volume of hemorrhage was positively correlated with the SII. These parameters were associated with outcomes in the univariate logistic regression. In the adjusted analyses, the SII and PLR were independent predictors of basal ganglia ICH outcomes. ROC analysis revealed that the SII showed a stronger ability to predict the 6-month outcomes of patients after basal ganglia ICH than the PLR on different days (AUC = 0.642, 0.804, 0.827 vs. 0.592, 0.725, 0.757; all P < 0.001). Conclusion: The SII independently and strongly predicts the outcome of basal ganglia ICH. A high SII was associated with poor 6-month outcomes in patients with basal ganglia ICH.
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Objectives: Serum neurofilament light chain (NfL) is a biomarker for neuroaxonal damage, and S100B is a blood marker for cerebral damage. In the present study, we investigated the relationship between serum NfL and S100B levels, severity, and outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH). Methods: We prospectively recruited aSAH patients and healthy controls between January 2016 and January 2021. Clinical results included mortality and poor outcomes (modified Rankin scale score of 3-6) after 6 months. The ultrasensitive Simoa technique was used to evaluate NfL levels in the blood, and ELISA was used to detect S100B. Results: A total of 91 patients and 25 healthy controls were included in the study, with a death rate of 15.4%. The group of aSAH patients had significantly higher serum levels of NfL and S100B (P < 0.01). Furthermore, the levels of NfL and S100B increased when the Hunt-Hess, World Federation of Neurological Surgeons (WFNS), and Fisher grades increased (P < 0.01). Serum NfL and S100B levels were linked to poor prognoses and low survival rates. The blood levels of NfL and S100B were found to be an independent predictor related to 6-month mortality in multivariable analysis. Additionally, the areas under the curves for NfL and S100B levels in serum were 0.959 and 0.912, respectively; the clinical diagnostic critical thresholds were 14.275 and 26.54 pg/ml, respectively; sensitivities were 0.947 and 0.921, and specificities were 0.849 and 0.811. Conclusions: The NfL and S100B values for aSAH patients within 12 days of admission were considerably associated with Hunt-Hess grade, WFNS, and Fisher grade. The higher the grade, the higher the NfL and S100B value, and the poorer the prognosis. Serum NfL and S100B values could be feasible biomarkers to predict the clinical prognosis of patients with aSAH.
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The main challenges for programmed cell death 1(PD-1)/PD-1 ligand (PD-L1) checkpoint blockade lie in a lack of sufficient T cell infiltration, tumor immunosuppressive microenvironment, and the inadequate tumor accumulation and penetration of anti-PD-1/PD-L1 antibody. Resetting tumor-associated macrophages (TAMs) is a promising strategy to enhance T-cell antitumor immunity and ameliorate tumor immunosuppression. Here, mannose-modified macrophage-derived microparticles (Man-MPs) loading metformin (Met@Man-MPs) are developed to efficiently target to M2-like TAMs to repolarize into M1-like phenotype. Met@Man-MPs-reset TAMs remodel the tumor immune microenvironment by increasing the recruitment of CD8+ T cells into tumor tissues and decreasing immunosuppressive infiltration of myeloid-derived suppressor cells and regulatory T cells. More importantly, the collagen-degrading capacity of Man-MPs contributes to the infiltration of CD8+ T cells into tumor interiors and enhances tumor accumulation and penetration of anti-PD-1 antibody. These unique features of Met@Man-MPs contribute to boost anti-PD-1 antibody therapy, improving anticancer efficacy and long-term memory immunity after combination treatment. Our results support Met@Man-MPs as a potential drug to improve tumor resistance to anti-PD-1 therapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Micropartículas Derivadas de Células/imunologia , Portadores de Fármacos/farmacologia , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Memória Imunológica , Masculino , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Células RAW 264.7 , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Hepatocyte growth factor (HGF) and its receptor play an important role in the formation and progression of glioma and can promote tumor proliferation. In this study, we investigated the ability of HGF to promote the proliferation and invasion of U251n cells; we also tested the effects of HGF on stromal cell-derived factor 1 (SDF1) and CXCR4 mRNA expression. METHODS: We measured the effect of HGF on the proliferation of U251n cells using enzyme-linked immunosorbent assays (ELISAs) to detect incorporated bromodeoxyuridine (BrdU) as a marker of DNA synthesis. The effects of HGF and SDF-1 on U251n cell invasion and proliferation were measured using the inhibitors K252a to c-Met and AMD3100 to CXCR4. SDF-1 and CXCR4 mRNA and protein expression were measured using quantitative polymerase chain reaction (PCR) and fluorescence-activated cell sorter (FACS) analysis. Small interfering (si)RNAs were also used to down-regulate HGF and c-Met expression in U251n cells. RESULTS: HGF significantly increased U251n cell proliferation and invasion in a dose-dependent manner; K252a blocked this. AMD3100 blocked invasion but not proliferation. CXCR4 and SDF-1 mRNAs were up-regulated when cells were treated with HGF. CXCR4 and SDF-1 mRNA levels and HGF and c-Met protein levels were down-regulated after cells were transfected with siRNAs. CONCLUSIONS: HGF has a direct effect on glioma cell proliferation and invasion. HGF up-regulates SDF-1 and CXCR4 mRNA expression and contributes to cell invasion.
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Quimiocina CXCL12/metabolismo , Glioma/metabolismo , Glioma/patologia , Fator de Crescimento de Hepatócito/administração & dosagem , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Invasividade NeoplásicaRESUMO
Extracellular microparticles (MPs) can function as drug-delivery vehicles for anticancer drugs. Here, we show that the softness of MPs derived from tumour-repopulating cells (TRCs) isolated from three-dimensional fibrin gels enhances the MPs' drug-delivery efficiency. We found that, compared with MPs derived from tumour cells cultured in conventional tissue-culture plastic, TRC-derived MPs intravenously injected in tumour-xenograft-bearing mice showed enhanced accumulation in tumour tissues, enhanced blood-vessel crossing and penetration into tumour parenchyma, and preferential uptake by highly tumorigenic TRCs. We also show that the cytoskeleton-related protein cytospin-A plays a critical role in the regulation of TRC-derived MP softness. The modulation of the mechanical properties of TRC-derived MPs could aid the efficiency of delivery of anticancer drugs.
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Antineoplásicos/farmacologia , Micropartículas Derivadas de Células/química , Sistemas de Liberação de Medicamentos/métodos , Animais , Apoptose , Vasos Sanguíneos , Linhagem Celular Tumoral , Sobrevivência Celular , Citoesqueleto , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tecido Parenquimatoso/irrigação sanguínea , Tecido Parenquimatoso/efeitos dos fármacos , Fosfoproteínas/metabolismo , Células RAW 264.7 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We herein report a case of recurrent nasal natural killer (NK)/T-cell lymphoma in a 21-year-old male patient. The patient presented with an esophageal mass, fever and difficulty in swallowing. There were no other obvious sites of recurrence apart from the esophageal lesion. Metastatic esophageal lesions are extremely rare. The histological analysis demonstrated a highly aggressive tumor with a characteristic angiodestructive growth pattern and nasal cavity necrosis. The lymphoma cells were immunopositive for leukocyte common antigen, T-cell intracytoplasmic antigen 1 and CD68, negative for CD56 and CD3, and positive for Epstein-Barr virus. A computed tomography scan revealed mild thickening of the wall of the lower esophagus. The barium swallow revealed stiffness of the esophageal wall, with limited expansion and mucosal damage. The final diagnosis was primary nasal NK/T-cell lymphoma, with metastasis to the esophagus. Clinically, it is important to distinguish nasal-type NK/T-cell lymphoma from other types of tumors, as its prognosis and treatment of secondary metastases differ significantly.
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Hepatocyte growth factor (HGF) has been shown to be overexpressed in gliomas, and high-grade gliomas (glioblastoma multiforme) express more HGF than lower-grade astrocytoma, and HGF enhances their resistance to radiotherapy. To examine the effect of serum HGF levels on the likelihood of response to radiotherapy and the disease-free survival in patients with glioma, the blood samples of the patients were collected before commencing treatment and serum HGF was measured by quantitative ELISA in 48 patients with glioma grade I-IV, and all patients underwent primary conventionally fractionated radiotherapy. For statistical analysis, SPSS Version 13.0 software was used. Thirty-eight of the 48 patients had a response to treatment, and ten patients had persistent disease at 3 months. Overall, the median serum HGF level was 1,219.5 pg/ml (range 650.4-2,264.7 pg/ml). Eight patients with local failure had HGF levels >1,219.5 pg/ml, and 28 patients with response had serum HGF level of ≤ 1,219.5 pg/ml (P = 0.01). The median time to progression was 6 months in patients with HGF level of >1,219.5 pg/ml compared with 17 months in patients with HGF level of ≤ 1,219.5 pg/ml (log-rank, P = 0.041). In multivariate analysis, serum HGF, the KPS, tumour size and pathological grade, but not the patient's age, gender and oligodendroglial component influenced the progression-free survival. Elevated pre-therapeutic serum HGF levels are associated with poor response and a shorter time to progression in patients with glioma undergoing primary radiotherapy.
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Biomarcadores Tumorais/sangue , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/mortalidade , Glioma/sangue , Glioma/mortalidade , Fator de Crescimento de Hepatócito/sangue , Adulto , Idoso , Neoplasias Encefálicas/radioterapia , Intervalo Livre de Doença , Feminino , Glioma/radioterapia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinase-mediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.
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Baculoviridae/genética , Reprogramação Celular , Endonucleases/metabolismo , Fibroblastos/metabolismo , Vetores Genéticos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/metabolismo , Transdução Genética , Dedos de Zinco , Diferenciação Celular , Linhagem Celular , Cromossomos Humanos Par 19 , Endonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Integrases/genética , Integrases/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. We describe a protocol for islet isolation from mouse pancreas. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30-45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.