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

描述（由申请人提供）：骨关节炎是一种导致大约2700万25岁及以上美国人软骨退化的疾病，影响男性和女性。据估计，到2030年，将有6700万人患有经医生诊断的关节炎。虽然患者在生命后期才出现症状，但这种疾病在生命早期就开始出现病变，可能早在20岁出头就发生了。然而，由于关节软骨不能自我修复，这些病变无法愈合。近二十年来，局灶性软骨病变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而得到加强。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因子之一。因此，通过基因治疗来控制软骨细胞的igf - 1合成已经成为研究的重点。然而，在这些系统中，增强的igf - 1生产仅持续一到两周。增加细胞暴露于IGF-I的时间的另一种策略是基于制造新的材料来控制IGF-I的运输。具体来说，通过碳二亚胺化学将IGF-I结合蛋白（igfbp） 18、22的肽接枝到海藻酸盐上，可以延缓IGF-I的释放，而这种释放取决于肽的结合亲和力和浓度。本建议的目标是将这两种技术结合起来，通过基因治疗控制软骨细胞产生IGF-I，并使用海藻酸盐凝胶修饰含有与IGF-I结合的肽，通过延迟其释放来延长其暴露于IGF-I的时间。目前的建议包含有助于实现这一目标的三个具体目标。具体目标1是在三维海藻酸盐支架中创建基于腺相关病毒的质粒(pAAV)/IGF-I，并在培养36天内测量IGF-I的释放、ECM积累和凝胶的机械性能。具体目的2是表征含有pAAV/IGF-I转染软骨细胞的对照和kplhall修饰的海藻酸盐的IGF-I释放、基质合成和力学性能。最后，特异性目的3是比较用来自mini-IGFBP-5的较长和较短的序列（PRQDEEKPLHALL、KPLHALL和ALL）修饰的海藻酸盐凝胶中，用pAAV/IGF-I转染的软骨细胞接种IGF-I的释放、基质合成和机械性能。我们认为，控制它们的细胞生成和igf - 1的细胞外结合将协同增强软骨细胞的生物合成，促进组织工程软骨的生成。

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.