I型胶原变异相关重型成骨不全症的基因治疗研究

Osteogenesis imperfect(OI) is one of the most common genetic disorders in the connective tissue. The vast majority (more than 90%) of affected individuals have mutations in one of the two genes, COL1A1 and COL1A2, which encode the two chains of type I collagen, the major protein of bone. OI resulting from mutations in gene COL1A1 or COL1A2 are characterized by bone fragility, multiple fracture, blue sclera. Our unpublished data and the reported data of other labs show that the nonsense mutations and the frameshift mutations lead to low or absent levels of the products of one allele, generally the result of premature termination codons, and the patients with the truncation mutations only have the mild phenotype in OI type I, such as bone fragility, near-normal stature and blue sclera. However, most missense mutations in COL1A1 and COL1A2 will lead to the severe body deformity, dentinogenesis imperfect, limited mobility and so on. Pharmacotherapy and surgical intervention are applicative in some patients with the mild symptoms. Today，there is no effective treatment for the severe OI. It is critical to set up a practicable therapeutic method for the severe OI related to COL1A1 and COL1A2. There are many single nucleotide polymorphisms(SNP) sites in both COL1A1 and COL1A2 and their upstream, and these SNP sites can be used as the antisense interference targets. Peptide nucleic acids(PNAs) are nucleic acid analogs in which the natural backbone is substituted with a polyamide structure.PNA have received great attention due to their favorable properties,including resistance to nuclease and protease digestion, stability in body fluid and hihg affinity for RNA and single/double stranded DNA targets.In this study, antisense PNA will be used to block the expression of the mutant allele of COL1A1 and COL1A2 in the cultured skin fibroblasts of the OI patients and in the animal models with missense mutation of COL1A1 or COL1A2. We try to explore a valid approaches of therapy and early intervention for severe OI induced by missense mutation in molecule of collagen type I.

成骨不全症是最常见的遗传性运动系统疾患之一。90%的成骨不全症由I型胶原基因（COL1A1和COL1A2）突变导致。I型胶原变异相关的OI患者表现为多发性骨折、身材矮小、蓝巩膜和特殊面容。I型胶原基因的无义突变或移码突变导致的蛋白截短往往只引起临床表现较轻的I型OI，错义突变则导致重型OI，患者严重肢体残疾。目前，重型OI尚无有效的治疗手段，现有的外科手术、药物治疗效果都不显著，迫切需要针对重型OI建立有效的治疗措施。本研究我们以前期工作中在OI群体中发现的COL1A1/COL1A2基因内的高频SNP及重型OI致病突变为切入点，利用反义肽核酸高稳定性及其特异性干扰其靶基因表达的特点，在体外培养的人皮肤细胞和重型OI动物模型中进行I型胶原变异相关OI基因治疗方法的探索，力求建立以早期预防为主的重型OI基因治疗方法。

成骨不全症是最常见的遗传性运动系统疾患之一。90%的成骨不全症由I型胶原（collagen type 1，COL1）基因COL1A1或COL1A2突变导致。COL1变异导致的OI患者表现为多发骨折、身材矮小、蓝巩膜和特殊面容等。COL1A1和COL1A2编码蛋白截短突变往往只引起临床表现较轻的I型OI，错义突变则导致重型OI，患者严重肢体残疾。目前，重型OI尚无有效的治疗手段，现有的外科手术、药物治疗效果都不显著，迫切需要针对重型OI建立有效的治疗措施。本课题基于中国人大样本OI患者群体COL1基因变异特征，进行OI患者细胞水平和动物模型上的突变等位基因的干预，初步探索常染色体显性遗传OI的基因治疗方法。本研究主要取得成果如下：1）在明确COL1A1或COL1A2致病变异的基础上，构建20例重型OI患者皮肤成纤维细胞作为致病机制和基因治疗的细胞模型；2）针对3名重型OI患者的COL1A1/2致病突变，分别构建4种大鼠模型。其中COL1A1:c.175C>T杂合子和纯合子均无OI表现，排除了该变异的致病性；COL1A1:c.187T>A杂合子未见异常，但无纯合子出生；COL1A1: c.2461G>A和COL1A2:c.596G>T杂合子成年个体身体小，生育率低，胎鼠多发育异常，显性致死，该模型可以用于后续的OI宫内基因治疗；3）针对重型OI患者致病等位基因[c.175C>T;c.187T>A]设计反义PNAs，对该患者皮肤成纤维细胞进行转染和突变等位基因表达分析，结果发现两个反向互补的PNA分子分别从转录和翻译水平抑制了突变等位基因表达，改善COL1总体水平。总之，本研究成功构建了OI的细胞模型和动物模型，进一步明确在OI患者中新发现突变的致病性；初步证实反义PNAs能够特异性抑制COL1A1突变等位基因表达，预期PNAs可以作为COL1变异相关重型OI基因治疗的重要手段，抑制变异等位基因表达，改善正常I型胶原的稳定性，减轻致病突变产生的表型效应。本研究为进一步开展OI动物模型的基因治疗研究奠定了基础。

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