Controlling IGF-I/Scaffold Binding to Enhance Gene Therapy for Cartilage Repair

描述(由申请人提供)：骨性关节炎是一种在大约2700万25岁及以上的美国人中导致软骨退化的疾病，影响男性和女性。据估计，到2030年，将有6700万人患有医生诊断的关节炎。虽然患者在生命后期出现症状，但这种疾病在生命早期就开始出现，病变最早可能发生在20岁出头。然而，由于关节软骨不能自我修复，这些损伤无法愈合。近二十年来，局部软骨损伤一直使用软骨细胞移植治疗。这种治疗将通过增加软骨细胞基质的合成来加强。胰岛素样生长因子(IGF-I)是软骨细胞代谢最有效的合成代谢调节剂之一。因此，通过基因治疗控制软骨细胞IGF-I的合成一直是人们关注的焦点。然而，在这些系统中，增强型IGF-I的产生只持续一到两周。增加细胞对IGF-I暴露时间的另一种策略是基于制造新材料来控制IGF-I的运输。具体地说，通过碳二亚胺化学将IGF-I结合蛋白(IGFBPs)18、22的多肽接枝到海藻酸盐上，可以延缓IGF-I的释放，这种释放取决于多肽的结合亲和力和浓度。这项提议的目标是将这两种技术结合起来，通过基因疗法控制软骨细胞产生IGF-I，并通过使用经过修饰的含有与IGF-I结合的多肽的海藻酸凝胶来推迟其释放，从而延长软骨细胞接触IGF-I的时间。目前的提案包含将有助于实现这一目标的三个具体目标。具体目标1是在3D藻酸盐支架中创建腺相关病毒载体(PAAV)/IGF-I，并在培养36天内测量IGF-I的释放、ECM积累和凝胶的力学性能。具体目的2是研究含有pAAV/IGF-I转基因软骨细胞的对照和KPLHALL修饰海藻酸盐的IGF-I释放、基质合成和力学性能。最后，具体目标3是比较在用来自迷你IGFBP-5的更长和更短的序列(PRQDEEKPLHALL，KPLHALL，和ALL)修饰的海藻酸凝胶中种植pAAV/IGF-I转基因软骨细胞的构建物的IGF-I释放、基质合成和机械性能。我们认为，控制它们的细胞生产和IGF-I的细胞外结合将协同增强软骨细胞的生物合成，促进组织工程化软骨的生成。 公共卫生相关性：这项建议对改善公共健康至关重要，因为它有巨大的潜力帮助美国大约2700万受骨性关节炎(OA)影响的人1、2。我目前的研究是两项技术的结合：组织工程学和基因疗法。我有兴趣通过将生物材料结合到一种结构中来改进目前的基因治疗技术，这种结构允许组成软骨的细胞用它们组成的材料填充结构；因此，该结构将具有软骨，使我们能够制造包含软骨的身体部分，如膝盖，这通常会受到骨关节炎的影响。

DESCRIPTION (provided by applicant): Osteoarthritis is a disease that causes cartilage degradation in approximately 27 million Americans age 25 and older, affecting both men and women. It is estimated that by 2030, 67 million people will have doctor-diagnosed arthritis. Although patients become symptomatic later in life, the disease initiates earlier in life with a lesion that could happened as early as in the early 20s. However, because articular cartilage does not self-repair, these lesions are unable to heal. For almost two decades focal cartilage lesions have been treated using chondrocyte transplantation therapy. Such therapy would be enhanced by increasing the chondrocyte matrix synthesis. One of the most potent anabolic regulators of chondrocyte metabolism is insulin-like growth factor (IGF-I). As such, much effort has been focused on controlling IGF-I synthesis by chondrocytes via gene therapy. However, in these systems enhanced IGF-I production persists only one to two weeks. An alternative strategy to increase the time of cell's exposure to IGF-I is based on fabricating new materials to control IGF-I transport. Specifically, by grafting peptides from IGF-I binding proteins (IGFBPs) 18,22 to alginate, through carbodiimide chemistry, can delay release of IGF-I, and that this release depends on the binding affinity and the concentration of the peptide. The goal of this proposal is to combine these two technologies to control production of IGF-I by chondrocytes via gene therapy and extend the time of their exposure to IGF-I by delaying its release using alginate gels modified to contain peptides that bind to IGF-I. The current proposal contains three specific aims that will contribute to the goal. Specific aim 1 is to create an adeno-associated virus based plasmid (pAAV)/IGF-I in 3D alginate scaffolds and to measure IGF-I release, ECM accumulation, and mechanical properties of gel over 36 days in culture. Specific aim 2 is to characterize IGF-I release, matrix synthesis, and mechanical properties of control and KPLHALL-modified alginate containing chondrocytes transfected with pAAV/IGF-I. Finally, Specific aim 3 is to compare IGF-I release, matrix synthesis, and mechanical properties of constructs seeded with pAAV/IGF-I transfected chondrocytes in alginate gels modified with longer and shorter the sequences (PRQDEEKPLHALL, KPLHALL, and ALL) from the mini-IGFBP-5. We believe that controlling both their cellular production and extracellular binding of IGF-I will synergistically enhance chondrocyte biosynthesis and facilitate generation of tissue engineered cartilage. PUBLIC HEALTH RELEVANCE: This proposal is essential to improve the public health because it has great potential to help the approximately 27 million people in America who are affected by Osteoarthritis (OA)1,2. My current research is a combination of two technologies: tissue engineering and gene therapy. I am interested in improving the current gene therapy technology by combining biomaterials into a structure that allows the cells that make up cartilage to fill out the structure with materials that they make up; therefore the construct will have cartilage enabling us to fabricate body parts that contain cartilage such as knees, which are commonly affected by OA.