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BACKGROUND: The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined. METHODS: The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale. RESULTS: An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter). CONCLUSION: These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization. 相似文献
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Focal defects of articular cartilage are an unsolved problem in clinical orthopaedics. These lesions do not heal spontaneously and no treatment leads to complete and durable cartilage regeneration. Although the concept of gene therapy for cartilage damage appears elegant and straightforward, current research indicates that an adaptation of gene transfer techniques to the problem of a circumscribed cartilage defect is required in order to successfully implement this approach. In particular, the localised delivery into the defect of therapeutic gene constructs is desirable. Current strategies aim at inducing chondrogenic pathways in the repair tissue that fills such defects. These include the stimulation of chondrocyte proliferation, maturation, and matrix synthesis via direct or cell transplantation-mediated approaches. Among the most studied candidates, polypeptide growth factors have shown promise to enhance the structural quality of the repair tissue. A better understanding of the basic scientific aspects of cartilage defect repair, together with the identification of additional molecular targets and the development of improved gene-delivery techniques, may allow a clinical translation of gene therapy for cartilage defects. The first experimental steps provide reason for cautious optimism. 相似文献
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采用PCR和体外连接的方法构建了带有信号肽的脑源性神经营养因子(BDNF)的融合基因, 将此基因插入到腺相关病毒载体穿梭质粒pSNAV, 然后用重组质粒pSNAV-Ig-BDNF转染包装细胞, 筛选出永久细胞株后, 用携带腺相关病毒rep及cap基因的单纯疱疹病毒超感染包装细胞, 成功包装出含有目的基因的一型血清型腺相关病毒。经PCR鉴定该病毒含有目的基因片段, 被该病毒感染的细胞裂解液经Western blotting 证实有BDNF表达, 其培养液经ELISA检测证实含有高水平的BDNF蛋白。该病毒与小量的非复制型腺病毒Ad-null联合应用时, 其BDNF蛋白的表达水平显著提高。可弥补腺相关病毒起效慢、对细胞转导效率低的弱点, 为今后应用AAV作基因治疗提供了实验证据。 相似文献
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采用PCR和体外连接的方法构建了带有信号肽的脑源性神经营养因子(BDNF)的融合基因, 将此基因插入到腺相关病毒载体穿梭质粒pSNAV, 然后用重组质粒pSNAV-Ig-BDNF转染包装细胞, 筛选出永久细胞株后, 用携带腺相关病毒rep及cap基因的单纯疱疹病毒超感染包装细胞, 成功包装出含有目的基因的一型血清型腺相关病毒。经PCR鉴定该病毒含有目的基因片段, 被该病毒感染的细胞裂解液经Western blotting 证实有BDNF表达, 其培养液经ELISA检测证实含有高水平的BDNF蛋白。该病毒与小量的非复制型腺病毒Ad-null联合应用时, 其BDNF蛋白的表达水平显著提高。可弥补腺相关病毒起效慢、对细胞转导效率低的弱点, 为今后应用AAV作基因治疗提供了实验证据。 相似文献
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Aurélien Jacob Laurie Brun Paloma Jiménez Gil Lucie Ménard Mohammed Bouzelha Frédéric Broucque Aline Roblin Luk H. Vandenberghe Oumeya Adjali Cécile Robin Achille François Véronique Blouin Magalie Penaud-Budloo Eduard Ayuso 《Biotechnology journal》2021,16(1):2000014
Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints. Moreover, baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raises concerns about safety due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, a faster and safer method based on homologous recombination (HR) is investigated. First, the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes are confirmed. Strikingly, it is found that the exogenous cassette showed increased stability over passages when using the HR system. Finally, both materials generated high rAAV vector genome titers, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for its manufacturing. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products. 相似文献
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BACKGROUND: The development of DNA-based therapies holds great promise for the treatment of diseases that remain difficult to manage using conventional pharmaceuticals. Whilst there are considerable data regarding chemical-induced DNA damage, there are limited reports published studying the potential of exogenous DNA to damage genomic DNA. METHODS: To investigate this problem, the differential gene expression (DGE) of DNA repair genes was examined to identify biomarkers, based on the hypothesis that DNA damage, including double-strand breaks (DSBs) and insertional mutagenesis, would be expected to induce biological pathways associated with repair. Human HepG2 cells were exposed to the chemical genotoxins, etoposide (ETOP) and methylmethanesulphonate (MMS), as positive controls, or biological agents (i.e. exogenous DNA with and without the use of transfection complexes or via various viral vectors). Following transfection (6-72 h) the cells were harvested for RNA and DGE was determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The expression of genes involved in the repair of DSBs were significantly increased after treatment with ETOP (>4-fold) or MMS (>5-fold). Transfection using Effectene and ExGen 500 resulted in no significant changes; however, transfection with ExGen 500 resulted in an increase in the expression levels of GADD45 mRNA, consistent with global cellular stress. Viral vectors increased (3-6-fold) expression of genes associated with DSBs and cellular stress responses and, as expected, the effect was the most marked with the retroviral vector. CONCLUSIONS: The DGE profiles observed in HepG2 cells following transduction/transfection suggest that a subset of DNA repair genes may provide novel biomarkers to rapidly detect DNA damage induced by DNA products at the level of the genome, rather than at selected genes. 相似文献
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The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully. 相似文献
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Insulin‐like growth factor (IGF)‐I is important in the acquisition and maintenance of both soft and hard tissues. Skeletal remodeling requires energy and recent work has demonstrated that bone can influence insulin sensitivity and thereby regulate metabolic processes. New insights from mouse models into the role of IGF‐binding proteins (IGFBPs) as more than mere depots for the IGFs has reignited investigations into the metabolic targets influenced by the IGF regulatory system and the pathways that link bone to adipose tissue. Although there remains continued uncertainty about the relative balance between the effects of circulating versus tissue IGF‐I actions, the role of the IGFBPs has been redefined both as modulators of IGF‐I action and as independent signaling factors. This review highlights several recent findings that shed new light on the physiologic role of the IGF regulatory system and its influence on skeletal and fat metabolism. J. Cell. Biochem. 111: 14–19, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
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Kim S Lee SH Kim JH Jeong YW Hashem MA Koo OJ Park SM Lee EG Hossein MS Kang SK Lee BC Hwang WS 《Molecular reproduction and development》2006,73(12):1523-1530
Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos. 相似文献
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Wightman L Kircheis R Rössler V Carotta S Ruzicka R Kursa M Wagner E 《The journal of gene medicine》2001,3(4):362-372
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Hall SL Chen ST Wergedal JE Gridley DS Mohan S Lau KH 《The journal of gene medicine》2011,13(2):77-88
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本文概述了当前肿瘤基因治疗研究中存在的一些主要问题,如绝大多数治疗方案中目的基因只有一个,肿瘤基因治疗缺乏靶向性,基因转移载体的效率、安全性及容量等问题。讨论了解决这些问题的主要途径,即肿瘤多基因联合治疗、直接体内途径基因治疗与靶向基因治疗、基因转移载体的改造。 相似文献
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Huang CY Buchanan DL Gordon RL Sherman MJ Razzaq J White K Buetow DE 《Cell biochemistry and function》2003,21(4):355-361
Chronic pressure overload leads to an increase in the size, i.e. hypertrophy, of cardiomyocytes in the heart. However, the molecular mechanisms underlying this hypertrophy are not understood. Insulin-like growth factor-I (IGF-I) synthesized locally in the heart is known to be associated with the hypertrophic process. So far, however, cardiac IGF-I gene expression in the widely used rat model system has only been shown to be increased when the hypertrophy induced by pressure-overload was already established. Therefore, the question of whether IGF-I serves as an initiating or early-enhancing factor for the cardiac hypertrophy remains unanswered. Here, cardiac hypertension and hypertrophy were rapidly induced in the rat by complete constriction of the abdominal aorta between the origins of the renal arteries. Carotid arterial systolic blood pressure remained unchanged in sham rats but increased rapidly in the pressure-overloaded constricted rats with a sustained hypertension established by 3 days. Hypertrophy of left ventricular (LV) cardiomyocytes in constricted rats also occurred by 3 days. However, this hypertrophy was preceded by increases in LV IGF-I mRNA and protein which occurred within 1 day. These results support the hypothesis that cardiac-synthesized IGF-I is an initiating or early-enhancing factor for hypertrophy of LV cardiomyocytes. 相似文献
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