Gene enhanced cartilage repair in a cytokine permissive environment

描述（由申请人提供）：软骨损伤和关节炎仍然是疼痛、虚弱和生活质量下降的一个未解决的原因。我们的研究项目目标包括开发和测试生长因子和细胞疗法，以改善软骨愈合。胰岛素样生长因子- i （IGF-I）促进软骨修复；然而，igf - 1治疗的益处受到短配体驻留和持续的促炎细胞因子通量的影响。我们假设软骨修复和关节健康可以通过软骨细胞植入、稳定表达igf -1基因的细胞以及通过沉默IL-1b形成的不那么敌对的环境相结合来增强。腺相关病毒（AAV）将用于在大型动物模型中提高软骨细胞移植软骨缺陷的转基因寿命。同时，IL-1信息将通过RNA干扰被敲除，这将通过减少金属蛋白酶（MMP）和聚合酶增殖来限制软骨降解和疼痛。该假设将通过以下项目目标进行验证：1)评估软骨修复、细胞存活和转基因持续在表达过IGF-I的软骨细胞移植的大全层软骨缺损中。原理证明腺病毒- igf - i增强早期软骨修复；AAV应长期延长和加强这种效果。2)利用RNA干扰基序评估IL-1b基因沉默在软骨细胞和滑膜细胞培养中调节关节细胞因子通量的作用。初步数据显示，具有完美核苷酸同源性的RNA干扰盒可靶向IL-1 mRNA沉默同源IL-1表达。这些实验筛选了小干扰rna的转导效率并确定了其下游信号效应。3)评估滑膜细胞与sirna转导导致IL-1b敲低对滑膜细胞/软骨共培养中关节炎软骨基质质量恢复的影响。滑膜细胞积极合成IL-1，是IL- 1b沉默的理想靶点。在这种关节炎培养模型中，对退化软骨的后续影响应该反映了IL-1b敲低改善关节炎软骨基质的能力。4)通过构建稳定的整合质粒载体来扩展siRNA效应。体内应用将需要外体或整合质粒DNA传递，这些研究测试了这些基序。5)评估IGF-I基因转导的软骨细胞移植到关节的软骨修复增强，在关节中，分解代谢活性通过局部表达IL-1b敲低元件而降低。综上所述，这5个目标的结果应该评估合成代谢基因在允许IL-1 mRNA沉默进行长期和持久修复的环境中增强软骨修复。这是AAV-IGF-I首次应用于软骨修复模式，并且预计使用RNA干扰会带来额外的好处，RNA干扰最近被描述为在医学上的治疗作用。将这些技术应用于软骨修复，以及潜在的早期关节炎，为控制关节炎的进展和潜在地减少关节损伤的后果提供了重要的希望。公共卫生相关性。软骨损伤和关节炎仍然是疼痛、虚弱和生活质量下降的未解决的原因。我们的研究项目目标包括开发和测试生长因子和基于细胞的疗法来改善软骨愈合。胰岛素样生长因子- i （IGF-I）增强软骨修复，本研究测试了生长因子在降解受RNA干扰控制的关节中的长期表达。

DESCRIPTION (provided by applicant): Cartilage injuries and arthritis remain an unresolved cause of pain, debility, and diminished quality of life. Our research program goals include developing and testing growth factor and cell-based therapies to improve cartilage healing. Insulin-like growth factor-I (IGF-I) enhances cartilage repair; however, the benefits of IGF-I therapy are tempered by short ligand residence and ongoing pro-inflammatory cytokine flux. We hypothesize that cartilage repair and joint health can be enhanced by a combination of chondrocyte implantation, using cells stably expressing the IGF-I gene, in concert with a less hostile environment developed by silencing IL-1b. Adeno-associated virus (AAV) will be used to enhance transgene longevity in chondrocyte-transplanted cartilage defects in a large animal model. Concurrently, IL-1 message will be knocked down using RNA interference, which will limit cartilage degradation and pain through reduced metalloprotease (MMP) and aggrecanase proliferation. The hypothesis will be tested by these project aims: 1) Evaluate cartilage repair, cell survival, and transgene persistence in large full-thickness cartilage defects grafted with chondrocytes over expressing IGF-I. Proof of principle shows adenovirus-IGF-I enhances early cartilage repair; AAV should extend and enhance this effect long-term. 2) Evaluate IL-1b gene silencing using RNA interference motifs to moderate articular cytokine fluxes in chondrocyte and synovial cell cultures. Preliminary data shows RNA interference cassettes with perfect nucleotide identity to target IL-1 mRNA silence cognate IL-1 expression. These experiments screen the transduction efficiency and determine downstream signaling effects of small interfering RNAs. 3) Assess the effects of IL-1b knockdown resulting from synoviocyte transduction with siRNAs on the restoration of arthritic cartilage matrix quality in synoviocyte/ cartilage co-cultures. Synoviocytes actively synthesize IL-1 and are ideal primary targets for IL- 1b silencing. Subsequent effects on degraded cartilage in this culture model of arthritis should reflect the ability of IL-1b knockdown to improve arthritic cartilage matrix. 4) Extend siRNA effects by developing stably integrating plasmid vectors. In vivo application will require episomal or integrated plasmid DNA delivery and these studies test these motifs. 5) Assess enhanced cartilage repair from transplantation of IGF-I gene transduced chondrocytes to joints where catabolic activity is reduced by locally expressing IL-1b knockdown elements. Combined, the results of these 5 aims should assess anabolic gene enhanced cartilage repair in an environment made permissive to long-term and durable repair by IL-1 mRNA silencing. This is the first time AAV-IGF-I will be applied in a cartilage repair mode, and additional benefits are expected using RNA interference, which has recently been described in a therapeutic role in medicine. Application of these technologies to cartilage repair, and potentially early stages of arthritis, provide significant hope for control of the progression of arthritis and potentially minimize the consequences of joint injury. PUBLIC HEALTH RELEVANCE. Cartilage injuries and arthritis remain an unresolved cause of pain, debility, and diminished quality of life. Our research program goals include developing and testing growth factor and cell- based therapies to improve cartilage healing. Insulin-like growth factor-I (IGF-I) enhances cartilage repair and this grant tests long-term growth factor expression in joints where degradation is controlled by RNA interference.