Collagen Gene Targeting with AAV Vectors

描述(申请人提供)：胶原基因打靶 腺相关病毒载体成骨不全是一种遗传性疾病 由I型胶原基因COLIAI或COL1 42突变引起的疾病 会导致严重的骨骼畸形，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 破坏胶原蛋白三螺旋的突变，所以有效的治疗方法 将需要移除或纠正显性、突变的等位基因。长期的 这项建议的目的是开发一种新的治疗方法 基于转基因自体间充质干细胞移植的OI 干细胞(MSCs)有望在体内产生成骨细胞 活着。腺相关病毒载体已被证明是有效的 在同源染色体序列中引入特定的遗传修饰， 在这里，它们将被用来敲除和纠正突变的人类1A1I 等位基因。将使用现有的OI集合执行实验 具有明确的COLIA1突变的成纤维细胞，并建立了OI MSC库 并以此为特征。在一种策略中，单个AAV靶向载体将是 用于敲除任何Col 1A等位基因，目的是将严重的OI 由于单个零等位基因导致轻微的OI形式的螺旋中断突变。 在其他战略中，AAV目标载体将被设计为纠正特定的 COL1A1突变并产生野生型等位基因。几种方法将是 为筛选已接受AAV介导的基因打靶的细胞而开发 并表达正常的col11a基因，目的是简化体外实验 将在未来的临床试验中使用的操作。增殖者 基因靶向MSCs的多系分化潜能将通过体外实验进行评估 植入后进行体内骨锻造试验和骨密度测定。 免疫缺陷小鼠。标记有报告基因的MSCs的混合物将被 共同检测以评估MSCs与 不同的大肠埃希氏菌]基因型别共存。为了促进植树造林 转基因间充质干细胞，我们将使用二聚化的化学诱导剂 多聚化工程化生长因子受体并提供可诱导的细胞 增殖转向间充质干细胞。此开关将提供一种方法 移植后MSCs体内扩增的药理控制。 这些研究旨在为未来的临床试验奠定基础。 OI和其他可能受益于自体移植的疾病 转基因间充质干细胞。

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.