Gene enhanced cartilage repair in a cytokine permissive environment

描述(申请人提供)：软骨损伤和关节炎仍然是导致疼痛、虚弱和生活质量下降的未解决的原因。我们的研究计划目标包括开发和测试生长因子和基于细胞的疗法，以促进软骨愈合。胰岛素样生长因子-I(IGF-I)促进软骨修复；然而，IGF-I治疗的好处受到配体停留时间短和持续的促炎细胞因子流动的影响。我们假设软骨修复和关节健康可以通过软骨细胞移植，使用稳定表达IGF-I基因的细胞，以及通过沉默IL-1b而形成的不那么恶劣的环境相结合来增强。在一个大型动物模型中，腺相关病毒(AAV)将被用于提高软骨细胞移植软骨缺损的转基因寿命。同时，IL-1信息将通过RNA干扰被破坏，这将通过减少金属蛋白酶和聚集酶的增殖来限制软骨的降解和疼痛。该假说将通过以下项目目标来验证：1)评估移植了过量表达IGF-I的软骨细胞的大型全层软骨缺损区的软骨修复、细胞存活和转基因持久性。原理证明，腺病毒-IGF-I可促进早期软骨修复；AAV应长期延长和增强这一作用。2)在软骨细胞和滑膜细胞培养中，使用RNA干扰基序调节关节细胞因子的流量，以评估IL-1b基因的沉默。初步数据显示，RNA干扰盒具有完美的核苷酸同源性，以靶向IL-1mRNA沉默同源IL-1的表达。这些实验筛选了小干扰RNA的转导效率，并确定了下游信号的影响。3)在滑膜细胞/软骨共培养体系中评价siRNAs介导的IL-1b基因敲除对关节软骨基质质量恢复的影响。滑膜细胞主动合成IL-1，是IL-1b沉默的理想靶点。在这种培养的关节炎模型中，随后对退化软骨的影响应该反映了IL-1b基因敲除改善关节炎软骨基质的能力。4)开发稳定整合的质粒载体，扩大siRNA效应。在体内的应用将需要异构体或整合的质粒DNA传递，这些研究测试这些基序。5)评估将转IGF-I基因的软骨细胞移植到局部表达IL-1b基因敲除元件导致分解代谢活性降低的关节的增强的软骨修复。综合起来，这5个目标的结果应该评估在一个允许通过IL-1mRNA沉默进行长期和持久修复的环境中，合成代谢基因增强的软骨修复。这是AAV-IGF-I首次应用于软骨修复模式，使用RNA干扰有望获得额外的好处，最近在医学上描述了它的治疗作用。将这些技术应用于软骨修复，以及可能的早期关节炎，为控制关节炎的进展提供了巨大的希望，并可能将关节损伤的后果降至最低。与公共卫生相关。软骨损伤和关节炎仍然是导致疼痛、虚弱和生活质量下降的一个尚未解决的原因。我们的研究计划目标包括开发和测试生长因子和基于细胞的疗法，以促进软骨愈合。胰岛素样生长因子-I(IGF-I)促进软骨修复，这项资助测试了由RNA干扰控制降解的关节中长期生长因子的表达。

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.