GENE THERAPY FOR ARTHRITIS IN THE TNFA-TRANSGENIC MOUSE

TNFA 转基因小鼠的关节炎基因治疗

• 批准号: 6511963
• 负责人: Edward M. Schwarz
• 金额: $ 22.21万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-20 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511963
• 关键词: Adenoviridae; Lentivirus; RNase protection assay; SDS polyacrylamide gel electrophoresis; adeno associated virus group; arthritis therapy; cell adhesion molecules; chondrocytes; collagenase; confocal scanning microscopy; cytokine; enzyme linked immunosorbent assay; flow cytometry; gene expression; gene therapy; genetically modified animals; in situ hybridization; laboratory mouse; nonhuman therapy evaluation; northern blottings; nuclear factor kappa beta; polyarthritis; polymerase chain reaction; transfection /expression vector; tumor necrosis factor alpha; western blottings

Arthritis is the most prevalent cause of chronic morbidity among those over 65 years of age, with over 30 million Americans suffering from the various arthritedes. The actual cost of treating these orthopaedic conditions represents billions of dollars in health care costs. The cost in human suffering is impossible to quantify. Despite tremendous research efforts, our understanding of the etiology and pathogenesis of these diseases is still incomplete. This proposal is designed to utilize gene therapy to systematically study molecular questions about arthritis, as well as test new treatments for this disabling disease. Specifically, this project focuses on a novel anti- TNFalpha gene therapy protocol to treat the polyarthritis of the hTNFalpha-transgenic mouse. A current paradigm is that TNFalpha is at the apex of the pro- inflammatory cytokine cascade which is responsible for joint destruction in arthritis. At the cellular level, the transcription factor NFkappaB is of paramount importance because it is both directly involved in the transcription of many inflammatory cytokines genes like TNFalpha and is activated by cell stimulation with these proteins. In turn NFkappaB amplifies these stimuli by initiating the transcription of a large number of genes, including matrix metaloproteases (MMPs), which cause the pathology. Our hypothesis is that the TNFalpha/NFkappaB signal transduction pathway is central to the genesis of arthritis and by using a specific inhibitor of this pathway we will prevent the disease process. In our studies of NFkappaB signal transduction, we have generated a dominant-negative IkappaBa (mIkappaB) which represents the specific inhibitor we desire. When over-expressed in mammalian cells, the mIkappaB renders them void of NFkappaB activity. Here we propose the following specific aims: i) identify additional cytokines, adhesion molecules and MMPs which promote disease in the hTNFalpha-transgenic mouse, and determine their cellular source, ii) determine the efficiency of our recombinant adenovirus, adenoassociated virus (AAV) and lentivirus (HIV) vector systems to transduce the cells of the joint in vitro and in vivo; and to modify these vectors with specific promoters to achieve the desired tissue expression, iii) test the ability of our mIkappaB to eliminate NFkappaB gene expression and monitor this effect on anabolic and catabolic mechanisms relevant to joint homeostasis, and iv) evaluate the usefulness of our mIkappaB to treat the arthritis of the hTNFalpha-transgenic mouse by in vivo gene therapy.

关节炎是 65 岁以上人群慢性发病的最普遍原因，超过 3000 万美国人患有各种关节炎。 治疗这些骨科疾病的实际费用相当于数十亿美元的医疗保健费用。 人类痛苦的代价是无法量化的。 尽管付出了巨大的研究努力，我们对这些疾病的病因和发病机制的了解仍然不完整。 该提案旨在利用基因疗法系统地研究有关关节炎的分子问题，并测试针对这种致残疾病的新疗法。 具体来说，该项目侧重于一种新型抗 TNFα 基因治疗方案，用于治疗 hTNFα 转基因小鼠的多关节炎。目前的范例是 TNFα 处于促炎细胞因子级联的顶端，该级联负责关节炎的关节破坏。 在细胞水平上，转录因子 NFkappaB 至关重要，因为它直接参与许多炎症细胞因子基因（如 TNFα）的转录，并通过这些蛋白质的细胞刺激而被激活。 反过来，NFkappaB 通过启动大量基因的转录来放大这些刺激，其中包括引起病理的基质金属蛋白酶 (MMP)。 我们的假设是，TNFα/NFkappaB 信号转导途径是关节炎发生的核心，通过使用该途径的特定抑制剂，我们将阻止疾病进程。 在我们对 NFkappaB 信号转导的研究中，我们生成了显性失活 IkappaBa (mIkappaB)，它代表了我们想要的特定抑制剂。当在哺乳动物细胞中过度表达时，mIkappaB 使其失去 NFkappaB 活性。 在这里，我们提出以下具体目标：i) 鉴定在 hTNFα 转基因小鼠中促进疾病的其他细胞因子、粘附分子和 MMP，并确定它们的细胞来源，ii) 确定我们的重组腺病毒、腺相关病毒 (AAV) 和慢病毒（HIV）载体系统可在体外和体内转导关节细胞；并用特定启动子修饰这些载体以实现所需的组织表达，iii) 测试我们的 mIkappaB 消除 NFkappaB 基因表达的能力，并监测这种对与关节稳态相关的合成代谢和分解代谢机制的影响，以及 iv) 评估我们的 mIkappaB 的有用性mIkappaB 通过体内基因疗法治疗 hTNFα 转基因小鼠的关节炎。

项目成果

In vivo gene delivery to articular chondrocytes mediated by an adeno-associated virus vector.

由腺相关病毒载体介导的体内基因递送至关节软骨细胞。

• DOI: 10.1016/j.orthres.2003.12.003
• 发表时间: 2004
• 期刊: Journal of orthopaedic research : official publication of the Orthopaedic Research Society.
• 作者: Ulrich-Vinther,M;Duch,MR;Soballe,K;O'Keefe,RJ;Schwarz,EM;Pedersen,FS
• 通讯作者: Pedersen,FS

Anti-TNF-alpha therapy as a clinical intervention for periprosthetic osteolysis.

• DOI: 10.1186/ar81
• 发表时间: 2000
• 期刊: Arthritis research
• 作者: Schwarz EM;Looney RJ;O'Keefe RJ
• 通讯作者: O'Keefe RJ

Empirical advantages of adeno associated viral vectors in vivo gene therapy for arthritis.

• 发表时间: 2000-04
• 期刊: The Journal of rheumatology
• 作者: J. Goater;R. Müller;G. Kollias;G. Firestein;I. Sanz;R. O’Keefe;E. Schwarz

The adeno-associated virus vector for orthopaedic gene therapy.

用于骨科基因治疗的腺相关病毒载体。

• DOI: 10.1097/00003086-200010001-00005
• 发表时间: 2000
• 期刊: Clinical orthopaedics and related research
• 影响因子: 4.2
• 作者: Schwarz,EM

Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair.

• DOI: 10.1172/jci15681
• 发表时间: 2002-06
• 期刊: The Journal of clinical investigation
• 作者: Xinping Zhang;E. Schwarz;D. Young;J. Puzas;R. Rosier;R. O’Keefe