Mechanisms of alginate/rAAV gene vector encapsulation and release for enhanced, saferAAV-mediated therapeutic gene transfer in human meniscal lesions

半月板是膝关节中的重要组织，半月板的损伤在运动医学中是非常普遍的、尚未解决的问题，如果不治疗，可能会使邻近的关节软骨容易患上骨性关节炎。鉴于半月板完全自愈的能力有限，特别是在其无血供区，人们已经开发了各种方案来改善半月板的修复，但没有一种方案能够完全修复半月板的损伤，也受到临床上同种异体移植物/替代物的可用性、排斥性和/或失败的限制。在半月板损伤处应用有效的、临床适用的重组腺相关病毒(RAAV)载体是在半月板损伤部位临时和空间转移半月板修复基因以扩大基因产物表达的有效方法。然而，rAAV的安全、临床应用仍然受到体内各种障碍的阻碍，特别是在人类群体中自然存在的针对AAV衣壳蛋白的中和抗体。为了解决这个问题，本提案的目标是检验这样一种假设，即编码半月板修复(增殖的，促合成代谢的)基因(碱性成纤维细胞生长因子-成纤维细胞生长因子-2，转化生长因子-β-转化生长因子-β)的治疗性rAAV载体可以通过基于临床批准的海藻酸盐作为保护性的、受控的递送系统的水凝胶来传递，以安全、有效和持久地增强体外培养的原代人半月板纤维软骨细胞的半月板修复相关过程和机制，在人类实验性半月板原位损伤的翻译前模型中，以及在体内临床相关的平移性半月板损伤中，相对于直接应用这些载体的游离形式。该项目可能会在不久的将来提供新的、有效的治疗方法来加强患者的半月板修复。

Lesions to the meniscus, a vital tissue in the knee joint, are highly prevalent, unsolved problems in sports medicine and may predispose the adjacent articular cartilage to osteoarthritis if left untreated. In light of the restricted ability of the meniscus for complete self-healing especially in its avascular zone, various options have been developed to improve meniscal repair, but none can fully heal meniscal lesions, being also limited by the availability, rejection, and/or failure of allograft/substitutes in the clinics. Administration of the potent, clinically adapted recombinant adeno-associated virus (rAAV) vectors in meniscal lesions is a strong approach to temporarily and spatially transfer meniscal reparative genes in sites of injury for an extended expression of the gene products. Yet, the safe, clinical use of rAAV is still hindered by various obstacles in vivo, especially by the natural presence of neutralizing antibodies against the AAV capsid proteins in the human population. To address this issue, the goal of the present proposal is to test the hypothesis that therapeutic rAAV vectors coding for meniscal reparative (proliferative, pro-anabolic) genes (basic fibroblast growth factor - FGF-2, transforming growth factor beta - TGF- ß) may be delivered via hydrogels based on clinically approved alginate as protective, controlled delivery systems to safely, effectively, and durably enhance the processes and mechanisms relevant of meniscal repair in primary human meniscal fibrochondrocytes in vitro, in a pre-translational model of human experimental meniscal lesion in situ, and in clinically relevant, translational meniscal lesions in vivo relative to direct application of the vectors in their free form. This project may offer new, effective therapies to enhance meniscal repair in patients in a close future.