Basic study on a gene therapy for allergic diseases

肥大细胞上表达的FCR在变态反应中起关键作用。抑制IgE与FCR的结合可能是变态反应治疗的重要靶点。从小鼠脾基因中克隆了FceR基因，并将其导入带有免疫球蛋白分泌信号的CAG(pCAG-GS)和CMV(PSecTag2A)两种启动子和MYC-Tag中表达可溶性FceR蛋白，表达载体导入HEK293T细胞。用抗myc抗体对条件培养液进行Western印迹分析，成功地鉴定出假定相对分子质量的蛋白质。转导CAG启动子的细胞比转导CMV启动子1的细胞产生的蛋白质多10倍。与IgE孵育后，该条件培养液成功地抑制了PCa的活性。这一效应依赖于条件培养液的剂量。体内直接注射表达载体成功地抑制了大鼠被动皮肤过敏反应。这一发现为人类变态反应性疾病的基因治疗提供了可能。然而，这种效果在注射96小时后失效。给药途径和给药部位可能需要改进。除了这些研究外，我们还研究了肥大细胞的免疫调节作用，IgE通过FceR增敏肥大细胞。研究表明，肥大细胞在粘膜免疫中对Th2的发展至关重要。因此，我们现在正在进行通过FCR表达载体管理来调控基于Th2受体的免疫疾病，这可能为以Th2免疫反应为基础的疾病提供另一种基因治疗的应用，如系统性红斑狼疮。

FcR expressed on mast cells have a critical role in allergic reaction. Inhibition of IgE binding to FcR could be a crucial target of allergy therapy. We cloned a mouse FceR gene from mouse spleen cDNA and introduced under two different promoters, CAG (pCAG-GS) and CMV (pSecTag2A) with immunoglobulin secretory signal and a MYC-tag to express soluble FceR protein.The expression plasmids were introduced to HEK293T cells. The protein of presumed molecular weight was successfully identified from the conditioned medium with anti-myc antibody by Western blot. The CAG promoter plasmid transfected cells produced ten times more proteins than CMV promoter one transfected cells. Incubation with IgE, the conditioned medium successfully inhibited the activity in PCA. This effect depended on the dose of the conditioned medium. The in vivo direct injection of expression plasmid successfully inhibited passive cutaneous anaphylaxis (PCA) response in rats. This finding provided us the promising possibility of gene therapy for human allergic diseases. However, the effect expired 96 hours after the injection. The route and site of administration may require improvement.In addition to those studies we investigated an immunoregulatory role of mast cells which IgE sensitize through FceR. The study revealed that mast cells are critical for Th2 development in mucosal immunity. Thus we now are conducting to modulate Th2 repnese based immune disease by FcR expression plasmid administration, which may provide another application of gene therapy to Th2 immune response based disorders, such as systemic lupus erythematosus.