Acta Pharmaceutica Sinica B
·
2024
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IF 14.7
Manyu Chen,Yuan Liu,Quanying Liu,Siyan Deng,Yuhan Liu,Jiehao Chen,Yaojia Zhou,Xiaolin Cui,Jie Liang,Xingdong Zhang,Yujiang Fan,Qiguang Wang,Bin Shen
Mitochondrial dysfunction in chondrocytes is a key pathogenic factor in osteoarthritis (OA), but directly modulating mitochondria in vivo remains a significant challenge. This study is the first to verify a correlation between mitochondrial dysfunction and the downregulation of the FOXO3 gene in the cartilage of OA patients, highlighting the potential for regulating mitophagy via FOXO3 gene modulation to alleviate OA. Consequently, we developed a chondrocyte-targeting CRISPR/Cas9-based FOXO3 gene-editing tool (FoxO3) and integrated it within a nanoengineered ‘truck’ (NETT, FoxO3-NETT). This was further encapsulated in injectable hydrogel microspheres (FoxO3-NETT@SMs) to harness the antioxidant properties of sodium alginate and the enhanced lubrication of hybrid exosomes. Collectively, these FoxO3-NETT@SMs successfully activate mitophagy and rebalance mitochondrial function in OA chondrocytes through the Foxo3 gene-modulated PINK1/Parkin pathway. As a result, FoxO3-NETT@SMs stimulate chondrocytes proliferation, migration, and ECM production in vitro , and effectively alleviate OA progression in vivo , demonstrating significant potential for clinical applications.
研究领域:
生物医学工程
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基因治疗
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骨科
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分子生物学
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组织工程
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产品关联
DOI
PubMed
高影响因子
ADVANCED MATERIALS
·
2023
·
IF 29.4
Suleixin Yang,Yi Wu,Wenzhao Zhong,Ruie Chen,Meilin Wang,Meiwan Chen
Ferroptosis-related cancer therapy is limited by insufficient Fe 2+ /Fe 3+ redox pair and hydrogen peroxide (H 2 O 2 ) for producing lethal hydroxyl radicals (•OH). Although exogenous iron or ROS-producing drugs can enhance ferroptosis, exploiting endogenous iron (labile iron pool, LIP) stored in ferritin and promoting ROS generation may be safer. Herein, a metal/drug-free nanomedicine was developed for responsive LIP release and H 2 O 2 generation on the mitochondria membranes, amplifying hydroxyl radical production to enhance ferroptosis-mediated anti-tumor effects. A GSH/pH dual activatable fluorinated and crosslinked polyethyleneimine with dialdehyde polyethylene glycol layer nanocomplex loaded with MTS-KR-SOD (Mitochondria-targeting-sequence-KillerRed-Superoxide Dismutase) and CRISPR/Cas9-CA IX (Carbonic anhydrase IX) plasmids (FP@MC) were developed for enhanced ferroptosis through endogenous iron de-hijacking and in situ ROS amplification. Two plasmids were constructed to knock down carbonic anhydrase IX and translate KillerRed-SuperOxide Dismutase recombinant protein specifically on mitochondria membranes, respectively. The CA IX knockdown acidified the intracellular environment, leading the release of LIP from ferritin as a “flare” to initiate endogenous chemodynamic therapy. Meanwhile, MTS-KR-SOD generated H 2 O 2 when irradiated by a 590 nm laser to assist chemodynamic therapy, leading to ROS amplification for mitochondria damage and lipid peroxide accumulation. The combined therapeutic effects aggravated cancer ferroptosis and suppressed tumor growth, providing a new paradigm for amplifying ROS and iron ions to promote ferroptosis-related cancer therapy. This article is protected by copyright. All rights reserved
研究领域:
癌症治疗
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基因治疗
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纳米医学
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生物化学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
iScience
·
2025
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IF 4.1
Kuan Yoow Chan,Yini Yu,Yidi Kong,Ling Cheng,Renzhi Yao,Phoebe Sha Yin Chair,Ping Wang,Rong Wang,Wan-Yang Sun,Rong-Rong He,Junxia Min,Fudi Wang,Mikael Björklund
Despite wide variation, each cell type has an optimal size. Maintaining optimal size is essential for cellular fitness and function but the biological basis for this remains elusive. Here, we performed fitness analysis involving genome-wide CRISPR/Cas9 knockout data from tens of human cell lines and identified that cell size influences the essentiality of genes related to mitochondria and membrane repair. These genes also included glutathione peroxidase 4 (GPX4), which safeguards membranes from oxidative damage and prevents ferroptosis — iron-dependent death. Growth beyond normal size, with or without cell cycle arrest, increased lipid peroxidation resulting in a ferroptosis-sensitive state. Proteomic analysis revealed cell cycle -independent superscaling of endoplasmic reticulum, accumulation of iron and lipidome remodeling. Even slight increases from normal cell size sensitized proliferating cells to ferroptosis as evidenced by deep-learning-based single-cell analysis. Thus, lipid peroxidation may be a fitness trade-off which constrains cell enlargement and contributes to the establishment of an optimal cell size.
研究领域:
细胞生物学
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基因组学
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系统生物学
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癌症研究
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产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2019
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IF 3.553
Liuyan Gu,Haibo Yuan,Xueqin Lv,Guangsheng Li,Rigang Cong,Jianghua Li,Guocheng Du,Long Liu
5-Methylpyrazine-2-carboxylic acid (MPCA) is an important pharmaceutical intermediate and is used in the production of hypoglycemic agents and lipid-lowering drugs. This work aimed to develop a whole-cell biocatalytic process for the efficient synthesis of MPCA from 2, 5-dimethylpyrazine (DMP). Firstly, a whole-cell biocatalyst Escherichia coli strain was constructed by plasmid-based expression of xylene monooxygenase (XMO), benzyl alcohol dehydrogenase (BADH), and benzaldehyde dehydrogenase (BZDH) from Pseudomonas putida ATCC 33015, resulting in MPCA titer of 5.0 g/L. Then, the reaction conditions were optimized and the MPCA titer was further increased to 9.1 g/L. Next, the Ribosome Binding Site (RBS) Calculator v2.0 was used to predict and compare the translation initiation rates of the RBS sequences preceding xylM and xylA genes, encoding the two subunits of XMO. By optimizing the RBS sequences preceding xylA , the MPCA titer was increased to 10.2 g/L and the yield of MPCA on DMP reached 0.665 mol/mol. Finally, to achieve plasmid-free production of MPCA, we integrated the genes encoding for XMO, BADH and BZDH in the genome by using CRISPR/Cas9 and further fine-tuned the copy number ratios of xylM and xylA in the genome, improving the MPCA titer to 15.6 g/L and the yield of MPCA on DMP to 1.0 mol/mol. This work developed a high-yield and plasmid-free biocatalysis process for the environmentally friendly production of MPCA with 100% substrate conversion, and paved the way for the commercial production of MPCA in the future.
研究领域:
生物化工
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合成生物学
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代谢工程
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基因组编辑
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产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2024
·
IF 3.4
Xinqi Xu,Yaping Meng,Bingmei Su,Juan Lin
Esomeprazole is the most popular proton pump inhibitor for treating gastroesophageal reflux disease. Previously, a phenylacetone monooxygenase mutant LnPAMOmu15 (LM15) was obtained by protein engineering for asymmetric synthesis of esomeprazole using pyrmetazole as substrate. To scale up the whole cell asymmetric synthesis of esomeprazole and reduce the cost, in this work, an Escherichia coli whole-cell catalyst harboring LM15 and formate dehydrogenase from Burkholderia stabilis 15516 ( Bst FDH) were constructed through optimized gene assembly patterns. CRISPR/Cas9 mediated insertion of P trc promoter in genome was done to enhance the expression of key genes to increase the cellular NADP supply in the whole cell catalyst, by which the amount of externally added NADP + for the asymmetric synthesis of esomeprazole decreased to 0.05 mM from 0.3 mM for reducing the cost. After the optimization of reaction conditions in the reactor, the scalable synthesis of esomeprazole was performed using the efficient LM15- Bst FDH whole-cell as catalyst, which showed the highest reported space-time yield of 3.28 g/L/h with 50 mM of pyrmetazole loading. Isolation procedure was conducted to obtain esomeprazole sodium of 99.55 % purity and > 99.9 % ee with 90.1 % isolation yield. This work provides the basis for production of enantio-pure esomeprazole via cost-effective whole cell biocatalysis.
研究领域:
生物催化
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药物化学
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生物技术
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代谢工程
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产品关联
DOI
PubMed
高影响因子
Cell Metabolism
·
2025
·
IF 30.9
Tiezhu Shi,Jialiang Shao,Yufeng Ding,Hong Tang,Xiangyin Tan,Sisi Zhou,Shaoqing Yu,Xiang Wang,Guanzhen Yu,Ninghan Feng,Xiongjun Wang
Metabolic adaptations involved in tumor metastasis and immune evasion merit investigation. Here, using in vivo metabolic CRISPR/Cas9 knockout screening, we identified xylulokinase (XYLB) as a tumor suppressor that impairs lung colonialization by producing xylulose 5-phosphate (Xu5P), which promotes CD8 + T cell cytotoxicity. Mechanistically, CD8 + T cells express relatively high levels of solute carrier family 35 member E2 (SLC35E2), a homolog of the plant Xu5P transporter, to facilitate Xu5P uptake and subsequently intensify the pentose phosphate pathway and glycolysis for energy/redox balance. Furthermore, we revealed that Xu5P potentiates CD8 + T cell response by promoting Xu5P-responsive progenitor-like SLC35E2 + CD8 + exhausted T cells via tet methylcytosine dioxygenase 3 (TET3)-mediated DNA demethylation of the Tcf7 promoter. Clinically, elevated XYLB or blood Xu5P correlates with enhanced CD8 + T cell efficacy and reduced metastasis. In murine models, Xu5P supplementation or adopting Xu5P-rich diets synergizes with anti-PD-1 therapy to enhance antitumor immunity. These findings offer insights into the potentiality of dietary interventions for metastatic cancer.
研究领域:
癌症生物学
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免疫学
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代谢工程
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分子生物学
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遗传学
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肿瘤免疫学
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营养科学
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细胞信号传导
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DOI
PubMed
高影响因子
Acta Biomaterialia
·
2024
·
IF 9.4
Xiaopeng Yang,Songli Zhou,Jingyi Zeng,Suqin Zhang,Meng Li,Feifan Yue,Zhaoyi Chen,Yanming Dong,Yingchun Zeng,Jingwen Luo
ABSTRACT The development of melanoma is closely related to Braf gene, which is a suitable target for CRISPR/Cas9 based gene therapy. CRISPR/Cas9-sgRNA ribonucleoprotein complexes (RNPs) stand out as the safest format compared to plasmid and mRNA delivery. Similarly, lipid nanoparticles (LNPs) emerge as a safer alternative to viral vectors for delivering the CRISPR/Cas9-sgRNA gene editing system. Herein, we have designed multifunctional cationic LNPs specifically tailored for the efficient delivery of Cas9 RNPs targeting the mouse Braf gene through transdermal delivery, aiming to treat mouse melanoma. LNPs are given a positive charge by the addition of a newly synthesized polymer, deoxycholic acid modified polyethyleneimine (PEI-DOCA). Positive charge enables LNPs to be delivered in vivo by binding to negatively charged cell membranes and proteins, thereby facilitating efficient skin penetration and enhancing the delivery of RNPs into melanoma cells for gene editing purposes. Our research demonstrates that these LNPs enhance drug penetration through the skin, successfully delivering the Cas9 RNPs system and specifically targeting the Braf gene. Cas9 RNPs loaded LNPs exert a notable impact on gene editing in melanoma cells, significantly suppressing their proliferation. Furthermore, in mice experiments, the LNPs exhibited skin penetration and tumor targeting capabilities. This innovative LNPs delivery system offers a promising gene therapy approach for melanoma treatment and provides fresh insights into the development of safe and effective delivery systems for Cas9 RNPs in vivo . Statement of significance CRISPR/Cas9 technology brings new hope for cancer treatment. Cas9 ribonucleoprotein offers direct genome editing, yet delivery challenges persist. For melanoma, transdermal delivery minimizes toxicity but faces skin barrier issues. We designed multifunctional lipid nanoparticles (LNPs) for Cas9 RNP delivery targeting the Braf gene. With metal microneedle pretreatment, our LNPs effectively edited melanoma cells, reducing Braf expression and inhibiting tumor growth. Our study demonstrates LNPs' potential for melanoma therapy and paves the way for efficient in vivo Cas9 RNP delivery systems in cancer therapy.
研究领域:
癌症生物学
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基因治疗
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纳米技术
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分子生物学
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药学
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产品关联
DOI
PubMed
高影响因子
BIOMACROMOLECULES
·
2025
·
IF 5.4
Qianru Li,Yuhe Zhu,Jiahui Ding,Weizhen Pan,Yuwei Hu,Jiao Lu,Sihai Gao,Hongyang Jiang,Zhongyuan Yin,Songwei Tan
Gene therapy is often limited by gene vectors. To reduce the impact of intracellular reactive oxygen species and improve transfection efficiency, thioether bonds were introduced in poly(β-amino ester) (PBAE), a well-known nonviral gene vector, to synthesize PBAE-poly(β-thioether ester) copolymer (PBAE-TE). By varying the monomer feeding ratio and reaction time, the optimized copolymer (PBAE-TE2–5) showed the highest transfection efficiency in SiHa and HeLa cells (75.00 and 78.70%) with a 75:1 copolymer/GFP ratio. PBAE-TE2–5 also demonstrated superior transfection performance compared to PBAE in multiple cell lines (HeLa, Me180, C666–1, and HEK 293T) and better penetration capability in 3D spheroid cell models (SiHa and HEK 293T). The PBAE-TE2–5-CRISPR/Cas9 plasmid polyplexes could effectively edit target genes in HEK 293T (MUC2) and SiHa (HPV16 E7) cells, and inhibit cancer cell proliferation both in vitro and in vivo. These findings highlight the promising potential of PBAE-TE as an effective gene delivery system.
研究领域:
生物材料
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基因治疗
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高分子化学
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生物医学工程
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分子生物学
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纳米技术
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产品关联
DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2025
·
IF 7.3
Xinqi Xu,Shumin Wang,Yaqun Zhong,Juanjuan Yang,Lian Xu,Bingmei Su,Juan Lin
Whole-cell redox-neutral cascade reactions are promising for the sustainable synthesis of bulky lactones. In this study, the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAc) was rationally designed based on enhancing product release and consensus sequence design to enhance the activity and stability of the enzyme. A four-point mutant MU4 (G14A/A43G/M400L/F432I) showed improved activity and stability for the oxidation of cyclohexanone to ε-caprolactone at a high substrate loading. A newly NADPH-dependent alcohol dehydrogenase from Pseudomonas monteilii (PmADH) with high cyclohexanol oxidation activity was introduced for constructing the redox-neutral cascade for the synthesis of ε-caprolactone from cyclohexanol. To improve the intracellular environment for cascade synthesis of ε-caprolactone, CRISPR/Cas9-mediated strong promoter insertion to the endogenous catalase gene katE in the NADP self-sufficient Escherichia coli strain BK-1 (Ptrc-nadK/Ptrc-pncB) was done for mitigating the oxidative stress to obtain the strain E. coli BKE-3 harboring MU4 and PmADH. Finally, 776 mM cyclohexanol could be converted into ε-caprolactone by the BKE-3 whole-cell catalytic system with a >99% conversion rate without cyclohexanone accumulation, which was the highest record for the redox-neutral cascade synthesis of ε-caprolactone. The BKE-3 whole-cell catalytic system also showed high efficiency for the synthesis of various lactones. This work provides an efficient redox-neutral catalytic platform for the sustainable synthesis of lactones featuring high substrate loading and atomic economy.
研究领域:
生物催化
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酶工程
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合成生物学
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生物过程工程
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蛋白质工程
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代谢工程
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工业生物技术
✓ 已验证
DOI
高影响因子
Signal Transduction and Targeted Therapy
·
2025
·Vol. 10
·期 1
·395
·
IF 52.7
Liu Yue,Wang Huirui,Zhan Jie,Sun Jiabo,Sun Yan,Fu Xiaojie,Lv Dongxue,Li Xiuyun,Dong Ting,Lou Hongxiang
The widespread clinical application of paclitaxel (PTX) in cancer treatment has been significantly limited by the emergence of drug resistance and the presence of drug-tolerant persister cells. To systematically identify key regulators of this resistance, we conducted a genome-wide CRISPR/Cas9 knockout screen, which revealed that cell division cycle 6 (CDC6) is a critical determinant of cell adhesion-mediated PTX resistance. Furthermore, our results illustrate that CDC6, an essential DNA replication licensing factor, functions through a pathway distinct from previously well-characterized resistance mechanisms. Genetic depletion of CDC6 considerably sensitizes cells, markedly increasing PTX-induced cell death. In addition to its established role in chromosome stability, CDC6 physically interacts with tropomodulin-3 (Tmod3) in the cytoplasmic compartment. This interaction enhances CDC6 protein stability and drives drug resistance phenotypes through the regulation of actin cytoskeleton remodeling and facilitating focal adhesion assembly. In addition, combination treatment with PTX and actin filament inhibitors synergistically enhanced the antitumor efficacy both in vitro and in vivo. Overall, our studies elucidate the mechanisms through which CDC6 functions as a key regulator of PTX resistance and provide a potential therapeutic strategy to increase PTX efficacy through the modulation of the cytoskeletal-adhesion axis.
研究领域:
癌症生物学
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分子肿瘤学
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细胞信号传导
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药物耐药性
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细胞骨架动力学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
·
2024
·Vol. 22
·期 1
·1-29
·
IF 10.6
Yu Xiao-Jun,Zhao Yuan-Ting,Abudouaini Haimiti,Zou Peng,Li Tian-Qi,Bai Xiao-Fan,Wang Shan-Xi,Guan Jian-Bin,Li Meng-wei,Wang Xiao-dong,Wang Ying-guang,Hao Ding-Jun
Methods Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1β. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. Results CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1β, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). Conclusion The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs. Graphical Abstract Molecular mechanism of GCA targeting CFIm25 in NP cells to promote regeneration of degenerated intervertebral disc NP
研究领域:
生物医学工程
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再生医学
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组织工程
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分子生物学
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细胞生物学
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骨科
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Journal of Advanced Research
·
2024
·
IF 11.4
Songwei Tan,Caiyan Yuan,Yuhe Zhu,Shuangyan Chang,Qianru Li,Jiahui Ding,Xueqin Gao,Rui Tian,Zhiqiang Han,Zheng Hu
Introduction CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.). Objectives To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro . Methods We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro , and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis. Results The particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %–40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein–Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.
研究领域:
基因治疗
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纳米技术
·
分子生物学
·
药学
·
癌症研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）