Gene Delivery to Cartilage Defects via Marrow Coagulates

描述（由申请人提供）：人们普遍认为，软骨病变的骨髓祖细胞暴露于软骨因子可以促进修复组织的合成。这一建议是基于这样的假设，即基因转移可以作为一种手段来实现软骨病变内特定蛋白质的持续合成，这可以用来增强体内间充质干细胞向软骨形成的分化。我们最近发现，从新鲜凝固的骨髓抽吸液中产生的天然凝块形成了一种简单但令人惊讶的有效基质，用于将基因转移载体和转基因细胞运送到骨软骨缺陷中。形成的基质完全是供体自身的，是自然修复过程的一部分。从我们的初步结果中我们发现，在凝固骨髓中培养间充质干细胞，在适当的细胞因子刺激下，会向软骨组织分化。此外，将外源转基因种子作为载体或转基因MSCs植入血块中，将表达数周。本研究的实验方案旨在拓宽我们对蛋白质因子在软骨形成过程中的作用的认识，同时为开发基于基因和干细胞的方法来改善软骨病变的自然修复奠定基础。为此，我们将解决以下具体目标：(1)确定将软骨基因转移到间充质干细胞的合适载体；(2)评估候选转基因在微球培养系统中刺激间充质干细胞成软骨的能力；(3)体外优化成软骨基因向骨髓凝块的传递；(4)研究转基因骨髓凝块植入骨软骨缺损后的体内转基因表达模式；(5)评估转基因骨髓凝块在体内诱导骨软骨缺损软骨形成的能力；(6)阐明软骨基因在关节内长期表达的生物学效应。

DESCRIPTION (provided by applicant): It is widely thought that the exposure of chondrogenic factors to bone marrow progenitor cells in cartilage lesions can enhance the synthesis of a repair tissue. This proposal is based on the hypotheses that gene transfer can be used as a means to achieve sustained synthesis of specific proteins within a cartilaginous lesion, and that this can be used to augment the differentiation of mesenchymal stem cells toward chondrogenesis in vivo. We have recently found that a natural clot created from freshly coagulated bone marrow aspirate forms a simple, but surprisingly effective, matrix for delivery of gene transfer vectors and genetically modified cells to osteochondral defects. The matrix formed is completely native to the donor and part of the natural reparative process. From our preliminary results we have found that in culture mesenchymal stem cells within coagulated bone marrow aspirates will undergo differentiation into cartilaginous tissue under the proper cytokine stimulation. Furthermore exogenous transgenes seeded into the clot as vectors or genetically modified MSCs will be expressed for several weeks. The experimental scheme of the proposed study is designed to broaden our knowledge of the role of protein factors in the process of chondrogenesis while laying the foundation for development of gene- and stem cell-based approaches to improve the natural repair of cartilage lesions. For this we will address the following Specific Aims: (1) to determine the appropriate vector for delivery of chondrogenic transgenes to MSCs; (2) to evaluate the ability of candidate transgenes to stimulate MSCs toward chondrogenesis within a pellet culture system; (3) to optimize the delivery of chondrogenic genes to bone marrow clots in vitro; (4) to characterize patterns of transgene expression in vivo following implantation of genetically modified bone marrow clots in osteochondral defects; (5) to evaluate the capacity of genetically modified bone marrow clots to induce chondrogenesis in osteochondral defects in vivo; and (6) to elucidate the biological effects of long-term expression of chondrogenic transgenes within the joint.