ZAP-70 Deficiency: A model for gene therapy studies

描述(由申请人提供)： 通过基因转移治疗遗传性和获得性免疫缺陷是基因治疗中最先进的领域之一。使用ZAP-70蛋白酪氨酸激酶缺乏症作为范例，我们先前在敲除小鼠模型中证明了肿瘤逆转录病毒介导的造血干细胞(HSC)基因纠正ZAP-70缺乏症的可行性(Steinberg/Otsu等人，布拉德，2002)。虽然这种方法已经成功地应用于SCID患者，但我们假设，一些相关的缺陷可以通过胸腺中T淋巴祖细胞的原位基因纠正来克服。在体内胸腺内转移一种基因，为转导的前胸腺细胞提供选择优势，应该会导致产生功能正常的T淋巴细胞后代，从而允许长期的免疫重建。事实上，在将表达ZAP-70的T细胞特异性慢病毒载体注射到ZAP-70-/-小鼠的胸腺后，我们观察到一组小鼠的成熟TCRap胸腺细胞长期(12个月)分化，表明该载体已整合到祖细胞中。此外，在这些小鼠中产生了表达ZAP-70的外周T细胞，表现出部分多样化的受体谱系，并对同种抗原有反应(Adjali等人，J Clin Invest，2005)。我们现在建议优化这项技术的有效性和安全性。我们将改善病毒粒子向胸腺的输送；特别是通过注射更多的病毒粒子，重复注射，以及刺激胸腺细胞增殖。此外，我们将改进表达ZAP-70的慢病毒载体(I)以增强来自CD4启动子的基因表达和特异性，以及(Ii)包括HS4绝缘子序列和HSV1-TK自杀基因等安全措施。这些新方法和载体胸腺内注射后的免疫重建和功能将与体外慢病毒介导的HSC矫正后的免疫重建和功能进行比较。最终，我们将使用改进的方法和载体进行实验，以评估来自ZAP-70缺陷婴儿的人CD34来源细胞在NOD/RAG-/-小鼠中的T细胞分化。总之，来自这些实验的数据应该会促进针对ZAP-70缺乏症和其他形式的SCID患者的最佳和安全的基因治疗策略的发展。

DESCRIPTION (provided by applicant): The treatment of genetic and acquired immunodeficiencies by gene transfer is one of the most advanced field of gene therapy. Using the ZAP-70 protein tyrosine kinase deficiency as a paradigm, we previously demonstrated the feasibility of oncoretroviral-mediated hematopotetic stem cell (HSC) gene correction for ZAP-70-deficiency in a knock out mouse model (Steinberg/Otsu et al., Blood, 2002). While this approach has been successfully used in SCID patients, we hypothesized that some of the associated drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. In vivo intrathymic transfer of a gene providing a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny allowing long-term immune reconstitution. Indeed, following intrathymic injection of a T cell-specific ZAP-70-expressing lentiviral vector into thymi of ZAP-70-/- mice, we observed long-term (>12 month) differentiation of mature TCRap thymocytes in a subset of mice, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells were generated in these mice, demonstrated a partially diversified receptor repertoire and were responsive to allo-antigens (Adjali et al., J Clin Invest, 2005). We now propose to optimize the efficacy and safety of this technology. We will improve delivery of virions to the thymus; notably, by injecting higher amounts of virions, performing repeat injections, and stimulating thymocyte proliferation. Additionally, we will improve the ZAP-70-expressing lentiviral vector (i) to enhance gene expression and specificity from the CD4 promoter, and (ii) to include safety measures such as HS4 insulator sequences and the HSV1-TK suicide gene. The immune reconstitution and function following intrathymic injections with these new methods and vectors will be compared with that obtained following ex vivo lentiviral-mediated correction of HSC. Ultimately, we will perform experiments using improved methods and vectors to assess T cell differentiation of human CD34+ derived cells from ZAP-70-deficient infants in NOD/Rag-/- mice. Altogether, the data emerging from these experiments should promote the development of an optimal and safe gene therapy strategy for patients with ZAP-70-deficiency as well as other forms of SCID.