Development of immune supression against gene product in gene therapy

治疗隐性遗传性皮肤病的基因疗法可能会引起对引入基因产物的不必要的免疫反应，因为患者对这种新蛋白缺乏耐受性。本研究以desmoglin 3敲除（Dsg3-/-）小鼠作为Dsg3遗传缺陷的疾病模型，研究非病毒基因治疗是否会引起对Dsg3的免疫反应，以及这种反应是否可以预防。通过裸DNA注射将小鼠Dsg3 CDNA引入Dsg3-/-小鼠皮肤，部分Dsg3-/-小鼠体内产生抗Dsg3 IgG，并与Dsg3结合。为了克服这一问题，我们使用抗cd40l单克隆抗体（MR1）来阻断CD40-CD40L之间的共刺激相互作用，这在免疫应答的触发和维持中是重要的。在这项实验中，我们将Dsg3+/+皮肤移植到Dsg3-/-小鼠身上，以代表Dsg3稳定的基因转移。皮肤移植后，所有Dsg3-/-受体小鼠定期用MR1或仓鼠IgG处理。所有鼠IgG处理小鼠在皮肤移植后2-3周产生循环抗dsg3 IgG。IgG的产生持续4周以上，IgG在移植物角质形成细胞表面沉积；然而，当受体小鼠接受MR1处理时，这种抗dsg3 IgG的产生被有效抑制。这些发现表明，有效预防这种不良免疫反应是隐性遗传性皮肤病基因治疗成功的必要条件，而阻断CD40-CD40L相互作用可能是实现这种预防的有价值的方法。

Gene therapy to treat recessive genodermatoses may provoke unwanted immune response against the introduced gene product because tolerance against this novel protein is absent in patients. In this study, using desmoglein 3 knockout (Dsg3-/-) mice as a disease model for genetic defect of DSG3, we investigated whether immune response against Dsg3 is provoked by nonviral gene therapy and whether such reaction could be prevented. When mouse Dsg3 CDNA was introduced in the skin of Dsg3-/- mice by naked DNA injection, some Dsg3-/- mice developed anti-Dsg3 IgG which bound to Dsg3 expressed by the therapy in vivo. To overcome this problem, we used anti-CD40L monoclonal antibody (MR1) to block co-stimulatory interaction between CD40-CD40L, which is important in the triggering and maintenance of immune responses. For this assay, we employed the Dsg3+/+ skin graft on Dsg3-/- mice to represent stable gene transfer of Dsg3. After skin grafting, all recipient Dsg3-/- mice were treated either with MR1 or with hamster IgG regularly. All of hamster IgG treated mice developed circulating anti-Dsg3 IgG 2-3 weeks after the skin graft. This IgG production lasted: more than 4 weeks and IgG deposition was observed on the surfaces of the keratinocytes in grafts.; However, this anti-Dsg3 IgG production was efficiently suppressed when the recipient mice were treated with MR1. These findings indicate that effective prevention of such undesirable immune response is required for a successful gene therapy for recessive genodermatoses and that the blockade of CD40-CD40L interaction may be a valuable way to achieve this prevention.