Collagen Gene Targeting with AAV Vectors

描述（由申请人提供）：胶原蛋白基因靶向， 成骨不全症（Osteogenesis Imperfecta，OI）是一种遗传性的 由I型胶原基因COLIAI或COL1 42突变引起的疾病， 会导致严重的骨骼异常，畸形，频繁骨折， 疼痛和过早死亡严重形式的OI通常是由显性遗传引起的。 破坏胶原蛋白三螺旋的突变，因此有效的治疗方法 将需要去除或纠正显性突变等位基因。长期 该提案的目的是开发一种新的治疗方法， 转基因自体骨髓间充质干细胞移植治疗骨质疏松症 干细胞（MSC），预计将产生骨形成成骨细胞， vivo.腺相关病毒（AAV）载体已被证明有效地 将特定的遗传修饰引入同源染色体序列， 在这里，它们将用于敲除和纠正突变体，人类COL 1A I， 等位基因将使用现有OI集合进行实验 具有确定的COLIA1突变的成纤维细胞，并建立OI MSC库 并在此描述。在一种策略中，将使用单个AAV靶向载体。 用于敲除任何COL 1A等位基因，目的是将严重的OI转化为 到螺旋破坏突变到由于单个无效等位基因导致的轻度OI。 在其他策略中，AAV靶向载体将被设计成校正特异性靶向载体。 COL 1A1突变并产生野生型等位基因。将采取几种方法 开发用于选择经历了AAV介导的基因靶向的细胞 并表达正常的COL1 1A基因，目的是简化离体 这些操作将用于未来的临床试验。增殖 基因靶向MSC的多系潜能将通过体外实验进行评估。 通过体内骨形成测定以及植入后的体内骨形成测定， 免疫缺陷小鼠标记有报告基因的MSC的混合物将被 当MSC与骨形成细胞结合时， 不同的[COLIA]基因型共存。为了促进移植， 基因修饰的MSC，我们将使用二聚化的化学诱导剂， 使工程化生长因子受体多聚化并提供可诱导细胞 增殖切换到MSC。此开关将提供一种方法， 骨髓间充质干细胞在移植后的体内扩增受组织学控制。 这些研究旨在为未来的临床试验奠定基础， OI和其他可以从自体移植中受益的疾病 基因修饰的MSC。

DESCRIPTION (provided by applicant): Collagen Gene Targeting with Adeno-Associated Virus Vectors Osteogenesis Imperfecta (OI) is a genetic disease caused by mutations in the type I collagen genes COLIAI or COL1 42 that can result in major skeletal abnormalities, deformities, frequent fractures, pain, and premature death. Severe forms of OI are typically caused by dominant mutations that disrupt the Collagen triple helix, so an effective treatment will require removal or correction of dominant, mutant alleles. The long-term objective of this proposal is to develop a novel approach for the treatment of OI based on the transplantation of genetically modified autologous mesenchymal stem cells (MSCs) that are expected to produce bone-forming osteoblasts in vivo. Adenoassociated virus (AAV) vectors have been shown to efficiently introduce specific genetic modifications into homologous chromosomal sequences, and here they will be used both to knockout and correct mutant, human COL 1A I alleles. Experiments will be performed with an existing collection of OI fibroblasts with defined COLIA1 mutations, and with an OI MSC bank established and characterized here. In one strategy, a single AAV targeting vector will be used to knockout any COL 1A allele, with the goal of converting severe OI due to helix-disrupting mutations to a mild form of OI due to a single null allele. In other strategies, AAV targeting vectors will be designed to correct specific COL 1A1 mutations and create wild-type alleles. Several approaches will be developed to select for cells that have undergone AAV-mediated gene targeting and express normal COL1 1A genes, with the goal of simplifying the ex vivo manipulations that would be used in future clinical trials. The proliferative and muItilineage potential of gene-targeted MSCs will be assessed by in vitro assays and also by an in vivo bone-forniing assay after implantation in immunodeficient mice. Mixtures of MSCs marked with reporter genes will be assayed together to assess proliferation and bone formation when MSCs with different COLIA] genotypes coexist. In order to promote the engraftment of genetically modified MSCs, we will use chemical inducers of dimerization to multimerize engineered growth factor receptors and provide an inducible cell proliferation switch to MSCs. This switch will provide a method for pharmacologically controlled in vivo expansion of MSCs after transplantation. These studies are intended to lay the groundwork for future clinical trials for OI and other diseases that could benefit from the transnlantation of autologous genetically modified MSCs.