Article
Microbiology
Lionel Galibert, Aurelien Jacob, Adrien Savy, Yohann Dickx, Delphine Bonnin, Christophe Lecomte, Lise Rivollet, Peggy Sanatine, Marjorie Boutin Fontaine, Christine Le Bec, Otto-Wilhelm Merten
Summary: Large-scale manufacturing of rAAV remains a challenge, with the Monobac system showing higher yields of rAAV vectors and lower DNA contamination compared to dual baculovirus systems.
Article
Virology
Renuka Rao, Melad Farraha, Grant J. Logan, Sindhu Igoor, Cindy Y. Kok, James J. H. Chong, Ian E. Alexander, Eddy Kizana
Summary: This study compared two production methods for cardiotropic rAAVs, finding that rAAV produced by the traditional HEK293 method outperformed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This underscores the importance of thorough assessment of vector performance when using diverse rAAV production methods.
Article
Biotechnology & Applied Microbiology
Nikolaus Virgolini, Marco Silvano, Ryan Hagan, Ricardo Correia, Paula M. Alves, Colin Clarke, Antonio Roldao, Ines A. Isidro
Summary: Using single-cell RNA sequencing, this study performed transcriptomics analysis of Sf9 insect cells during the production of rAAV2. The study found an increase in transcriptional heterogeneity during infection, which was linked to the expression of baculovirus genes and rAAV2 transgenes. Additionally, the study highlighted limitations of the dual-baculovirus system in producing rAAV2 particles.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Jose Escandell, Filipa Moura, Sofia B. Carvalho, Ricardo J. S. Silva, Ricardo Correia, Antonio Roldao, Patricia Gomes-Alves, Paula M. Alves
Summary: The majority of rAAVs for clinical use are produced using the HEK293 cell line, but this platform has manufacturing bottlenecks such as low product quality and high cost. The HeLa-based stable cell line rAAV production system offers a scalable alternative, but has barriers due to time and complexity. This study developed a more streamlined and efficient process using the HeLaS3-based cell line, achieving high yield and purity of rAAV2.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Ngoc Tam Tran, Emilie Lecomte, Sylvie Saleun, Suk Namkung, Cecile Robin, Kristina Weber, Eric Devine, Veronique Blouin, Oumeya Adjali, Eduard Ayuso, Guangping Gao, Magalie Penaud-Budloo, Phillip W. L. Tai
Summary: Over the past two decades, significant progress has been made in the manufacturing of adeno-associated virus (AAV) vectors to meet the production demands for preclinical and clinical trials. However, the presence of empty AAV capsids and particles containing inaccurate vector genomes remains a concern. Different methods have been utilized to separate empty capsids from full particles, but no single technique can completely eliminate empty or partial capsids. This study reveals that vectors produced by different manufacturing systems exhibit varying degrees of truncated and unresolved species, and purified empty particles actually contain genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). These findings provide valuable insights into the efficacy, safety, and quantification of clinical vectors.
HUMAN GENE THERAPY
(2022)
Article
Immunology
Luis Giovani Oliveira Guardalini, Paulo Eduardo da Silva Cavalcante, Jaci Leme, Renata Gois de Mello, Thaissa Consoni Bernardino, Simone Goncalves Silva Jared, Marta Maria Antoniazzi, Renato Mancini Astray, Aldo Tonso, Eutimio Gustavo Fernandez Nunez, Soraia Attie Calil Jorge
Summary: This study aimed to evaluate the scalability from Schott flasks to a stirred-tank bioreactor for production of rabies-recombinant baculovirus and rabies virus-like particles (VLP) using Sf9 insect cells as a host. Equivalent assays were performed in both systems and multivariate statistical approach was used to maximize VLP production. The hydrodynamic and chemical differences in the two systems greatly affected the virus and VLP integrity.
Article
Biochemistry & Molecular Biology
Luis Giovani Oliveira Guardalini, Paulo Eduardo da Silva Cavalcante, Jaci Leme, Renata Gois de Mello, Thaissa Consoni Bernardino, Renato Mancini Astray, Aldo Tonso, Soraia Attie Calil Jorge, Eutimio Gustavo Fernandez Nunez
Summary: The study aims to characterize the oxygen uptake and transfer rate parameters during the production of recombinant baculovirus (rBV) and rabies virus-like particles (VLPs) using Sf9 cells in a stirred tank bioreactor (STB). Four different experiments were conducted to monitor the changes in oxygen uptake rate, oxygen transfer coefficient, cell concentration, viability, rBV titers, and protein concentration. The results suggest that the aeration and agitation systems in a larger-scale bioreactor can be designed based on the criterion of constant kLa, without the need for oxygen facilities. Furthermore, a volumetric yield of 2.8 mg/L of rabies VLPs with a typical size of 55-68 nm was obtained, indicating a promising bioprocess for commercial-scale production of rabies VLPs.
PROCESS BIOCHEMISTRY
(2023)
Article
Biochemistry & Molecular Biology
Luis Giovani Oliveira Guardalini, Jaci Leme, Paulo Eduardo da Silva Cavalcante, Renata Gois de Mello, Thaissa Consoni Bernardino, Simone Goncalves Silva Jared, Marta Maria Antoniazzi, Renato Mancini Astray, Aldo Tonso, Eutimio Gustavo Fernandez Nunez, Soraia Attie Calil Jorge
Summary: This study aimed to evaluate the metabolism of Sf9 cells during growth and infection with recombinant baculovirus, to obtain rabies viral-like particles (VLP) in two culture systems: Schott flask (SF) and stirred tank reactor (STR). The specific rates of uptake or release of glucose, glutamine, glutamate, lactate, and ammonium were quantified during exponential growth and infection stages. The findings demonstrated that glucose had the highest uptake rate in SF during exponential growth, while glutamine had the highest uptake rate in STR. A wave pattern was observed for the analyzed compounds during infection, with glucose exhibiting the highest wave amplitude. The results suggest that the virus propagation and VLP generation were not limited by nutrient availability or toxicity under the conditions tested.
MOLECULAR BIOTECHNOLOGY
(2023)
Review
Genetics & Heredity
Sofia Fernandes, Joana Diogo, Ana Sofia Coroadinha
Summary: Adeno-associated viruses (AAV) are widely used in gene therapy due to their non-pathogenicity, ability to transduce both dividing and non-dividing cells, and flexibility in targeting different tissues. The success of AAV gene therapy has been demonstrated by the approval of three products. To meet the requirements of high dosage, safety, and reproducibility in clinical trials, production platforms based on stable mammalian cell lines have been proposed as the best strategy, although the methodologies employed may result in different productivities.
CURRENT GENE THERAPY
(2023)
Article
Immunology
Manon M. J. Cox
Summary: The insect cell expression system, proposed as the preferred biosecurity strategy for vaccine production, has limitations that need to be addressed for effective use in future pandemics.
Article
Biotechnology & Applied Microbiology
Henry de Malmanche, Mazhar Hussain, Esteban Marcellin, Steve Reid, Sassan Asgari
Summary: The Sf9 cell line, widely used for the production of recombinant proteins and virus-like particles, has been found to use RNA interference (RNAi) as an antiviral response to baculovirus infection. Knocking out Dicer-2, a key protein in the RNAi pathway, resulted in increased viral replication in Sf9 cells, indicating a potential role for RNAi in managing chronic viral infections in this cell line.
JOURNAL OF GENERAL VIROLOGY
(2022)
Article
Genetics & Heredity
Yang Wu, Zengpeng Han, Mingzhu Duan, Liangyu Jiang, Tiantian Tian, Dingyu Jin, Qitian Wang, Fuqiang Xu
Summary: The optimized single BEV-derived OneBac system allows for the efficient and scalable production of all serotypes of rAAV1-13, making it popular for laboratory scaling-up production.
CURRENT GENE THERAPY
(2021)
Article
Virology
Oleksandr Kondratov, Sergei Zolotukhin
Summary: This article introduces a new method for evaluating the structural and biological fitness of rAAV manufactured in Sf9 cells. By packaging a combinatorial capsid library in Sf9 cells and screening for high infectivity in Cre-expressing cells, an optimal AAV capsid composition can be determined.
Review
Biotechnology & Applied Microbiology
Qiang Fu, Ashli Polanco, Yong Suk Lee, Seongkyu Yoon
Summary: Gene therapy is a promising treatment for genetic and acquired diseases, and recombinant adeno-associated virus (rAAV) is an effective and safe vector used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing falls short of the necessary dosages for clinical and commercial use, leading to high prices and limited accessibility. This review discusses the challenges associated with rAAV gene therapy bioprocesses and explores recent advances that could enhance productivity and reduce impurities during production.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Cell Biology
Marine Geoffroy, Louna Pili, Valentina Buffa, Maelle Caroff, Anne Bigot, Evelyne Gicquel, Gregory Rouby, Isabelle Richard, Romain Fragnoud
Summary: In this study, FKRP knock-out cells were generated using CRISPR-Cas9 technology to evaluate the functional activity of rAAV-FKRP gene therapy. A high-throughput On-Cell-Western method was developed to assess the restoration of alpha-DG glycosylation and determine the biological activity of the FKRP transgene. The study provides a cell-based potency assay for evaluating rAAV-FKRP gene therapy products.
Article
Biochemistry & Molecular Biology
Lei Chen, Shuang Li, Ying Zhou, Taotao Liu, Aoling Cai, Zongze Zhang, Fuqiang Xu, Anne Manyande, Jie Wang, Mian Peng
Summary: The study demonstrates the important roles played by A(2A) receptor in the loss of consciousness induced by propofol, suggesting that the neural circuit between NAc-DR may be controlled by A(2A)R in the mechanism of anesthesia induced by propofol.
JOURNAL OF NEUROCHEMISTRY
(2021)
Article
Biochemistry & Molecular Biology
Qian Xiao, Xinyi Zhou, Pengfei Wei, Li Xie, Yaning Han, Jie Wang, Aoling Cai, Fuqiang Xu, Jie Tu, Liping Wang
Summary: Parvalbumin interneurons within the nucleus accumbens shell are crucial in modulating anxiety-like avoidance behavior, potentially via GABAergic pathways. Chronic stress increases the excitability of these neurons, leading to excessive maladaptive avoidance responses. The coordination between different types of neurons and GABAergic afferents plays a key role in regulating anxiety-related behaviors, providing a potential neurobiological basis for therapeutic interventions in pathological anxiety.
MOLECULAR PSYCHIATRY
(2021)
Article
Genetics & Heredity
Yang Wu, Zengpeng Han, Mingzhu Duan, Liangyu Jiang, Tiantian Tian, Dingyu Jin, Qitian Wang, Fuqiang Xu
Summary: The optimized single BEV-derived OneBac system allows for the efficient and scalable production of all serotypes of rAAV1-13, making it popular for laboratory scaling-up production.
CURRENT GENE THERAPY
(2021)
Article
Ophthalmology
Fang Yang, Xutao Zhu, Xin Liu, Lin Ma, Zhijian Zhang, Lei Pei, Huadong Wang, Fuqiang Xu, Haixia Liu
Summary: The study identified the efferent pathway from the hypothalamus to the autonomic innervation in the anterior chamber structures. Results suggest both crossed and uncrossed hypothalamo-pre-parasympathetic and pre-sympathetic tracts between the bilateral hypothalamic nuclei and the autonomic innervation of the bilateral anterior chamber structures.
EXPERIMENTAL EYE RESEARCH
(2021)
Article
Neurosciences
Xiaodan Huang, Pengcheng Huang, Lu Huang, Zhengfang Hu, Xianwei Liu, Jiawei Shen, Yue Xi, Yan Yang, Yunwei Fu, Qian Tao, Song Lin, Anding Xu, Fuqiang Xu, Tian Xue, Kwok-Fai So, Haohong Li, Chaoran Ren
Summary: Exposure to bright-light treatment promotes spatial memory through a di-synaptic visual circuit involving specific retinal ganglion cells and thalamic neurons, with the effects dependent on the activation of these pathways.
Article
Neurosciences
Jiao Li, Yiqiong Liu, Qin Li, Xiaolin Huang, Dingxi Zhou, Hanjian Xu, Feng Zhao, Xiaoxiao Mi, Ruoxu Wang, Fan Jia, Fuqiang Xu, Jing Yang, Dong Liu, Xuliang Deng, Yan Zhang
Summary: Myoclonus dystonia syndrome (MDS) is an inherited movement disorder commonly caused by mutations in the epsilon-sarcoglycan (SGCE) gene. Loss of SGCE function results in excessive excitatory synapses, which may contribute to MDS pathology. Vigabatrin has been identified as a potent compound in reversing MDS symptoms in mouse disease models, suggesting its potential therapeutic use for MDS.
NEUROSCIENCE BULLETIN
(2021)
Article
Neurosciences
Junjun Wang, Pei Sun, Xiaohua Lv, Sen Jin, Anan Li, Jianxia Kuang, Ning Li, Yadong Gang, Rui Guo, Shaoqun Zeng, Fuqiang Xu, Yu-Hui Zhang
Summary: By utilizing whole-brain florescence imaging, researchers were able to fully reconstruct the morphologies of 25 OFC pyramidal neurons and compare their projection targets in different cortical layers. The study revealed layer-dependent projections characterized by divergent patterns for information delivery, providing a structural basis for understanding laminar organization principles in the OFC and offering clues for future functional and behavioral studies.
NEUROSCIENCE BULLETIN
(2021)
Article
Multidisciplinary Sciences
Lichao Xu, Yue Zheng, Xuejing Li, Andi Wang, Dawei Huo, Qinglan Li, Shikang Wang, Zhiyuan Luo, Ying Liu, Fuqiang Xu, Xudong Wu, Min Wu, Yan Zhou
Summary: This study highlights the importance of SETD2 in epigenetic mechanisms underlying cortical arealization and the pathogenesis of Sotos-like syndrome. Loss of SETD2 leads to defects in cortical arealization and cognitive functions, similar to patients with SETD2 mutations.
Article
Cell Biology
Xiaojun Wang, Hanqing Xiong, Yurong Liu, Tao Yang, Anan Li, Fei Huang, Fangfang Yin, Lei Su, Ling Liu, Ning Li, Longhui Li, Shenghua Cheng, Xiaoxiang Liu, Xiaohua Lv, Xiuli Liu, Jun Chu, Tonghui Xu, Fuqiang Xu, Hui Gong, Qingming Luo, Jing Yuan, Shaoqun Zeng
Summary: CSFT technique utilizing chemical switching of fluorescent proteins enables high-resolution imaging of brain structures for studying neuron connections and cellular compositions. It also allows quantitative acquisition at the single-neuron level.
Article
Medicine, Research & Experimental
Yuanyuan Qin, Jinfeng Wu, Tao Chen, Jia Li, Guiling Zhang, Di Wu, Yiran Zhou, Ning Zheng, Aoling Cai, Qin Ning, Anne Manyande, Fuqiang Xu, Jie Wang, Wenzhen Zhu
Summary: The study found that COVID-19 patients showed severe changes in gray matter, white matter, and cerebral blood flow, especially in patients with severe disease. These changes were significantly correlated with inflammatory markers, suggesting that the inflammatory storm may cause indirect brain damage.
JOURNAL OF CLINICAL INVESTIGATION
(2021)
Article
Neurosciences
Zengpeng Han, Nengsong Luo, Jiaxin Kou, Lei Li, Zihong Xu, Siyuan Wei, Yang Wu, Jie Wang, Chaohui Ye, Kunzhang Lin, Fuqiang Xu
Summary: Retrograde tracers based on viral vectors are important tools for studying neural circuits. However, current tracers have limitations. We have developed a new method and a cell-type-specific retrograde tracing system to overcome these limitations.
Article
Biochemistry & Molecular Biology
Zengpeng Han, Nengsong Luo, Yang Wu, Jiaxin Kou, Wenyu Ma, Xin Yang, Yuxiang Cai, Lin Ma, Lu Han, Xiujie Wang, Hualing Qin, Qing Shi, Jie Wang, Chaohui Ye, Kunzhang Lin, Fuqiang Xu
Summary: In this study, AAV13 was identified as a new serotype that can precisely transduce local neurons in the brain with a smaller diffusion range and rigorous labeling. AAV13-based single and dual viral strategies were developed for sparse labeling of local neurons. Additionally, the functionality of AAV13 was demonstrated through neurobehavioral tests.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Optics
Nengsong Luo, Zengpeng Han, Jiaxin Kou, Yuxiang Cai, Xin Yang, Jie Wang, Kunzhang Lin, Fuqiang Xu
Summary: Rabies-viruses-based retrograde tracers are powerful tools for studying the neural circuits of the brain. However, they have limitations in terms of human health risks and limited labeling capabilities. In this study, researchers developed a new method using genetically modified viruses to overcome these limitations and analyze the input neural networks in rodents and nonhuman primates.
JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES
(2023)
Article
Virology
Zengpeng Han, Nengsong Luo, Fei Wang, Yuxiang Cai, Xin Yang, Weiwei Feng, Zhenxiang Zhu, Jie Wang, Yang Wu, Chaohui Ye, Kunzhang Lin, Fuqiang Xu
Summary: In this study, two novel AAV variants, AAV2.A1 and AAV2.A2, were generated by engineering the VR-IV region of AAV2. The transduction efficiency of these variants in the central nervous system was found to be 10-15 times higher than that of AAV2, providing new vehicles for gene drug delivery to the brain.
