Hematopoietic Stem Cell Transposon Therapy for Severe Combined Immunodeficiency

描述(申请人提供)：我们的目标，也是目前治疗严重联合免疫缺陷(SCID)患者的主要挑战，是开发一种强有力的替代方案，以替代异基因造血干细胞(HSC)移植和用病毒载体纠正自体HSC的基因，因为这两种策略都与SCID患者不可接受的发病率和死亡率有关。非病毒基因治疗是造血干细胞移植和病毒基因治疗的真正替代方法。为了纠正SCID(由DNA蛋白激酶催化亚单位DNA-PKcs基因外显子85的点突变引起，长度约13kB)，我们将重点研究非病毒Tol2转座子介导的DNA-PKcs导入造血细胞系和来自野生型和SCID小鼠的新鲜HSC。转座子介导的基因传递有几个优点。其中最重要的是：简单的无错误设计、降低的免疫原性和被复制能力强的病毒污染的风险，以及关键的是，高载量Tol2转座子能够稳定地将大基因(&gt；10kB)输送到哺乳动物细胞中。由于目前可用的裸露DNA传递方法，如核切割，会导致细胞损伤，对干细胞的毒性明显大于对其更多分化的后代的毒性，基于电磁电荷的方法不适用于我们的主要目标，即在SCID小鼠模型中进行干细胞基因治疗。因此，将使用脂质体介导的干细胞因子结合脂质体传递HSC特异性基因。在SCID治疗领域值得探索的挑战是在不损害HSC功能的情况下向HSC输送非病毒载体，以及纠正大基因(如DNA-PKcs)。一方面，凭借我们在非病毒干细胞转基因方面的专业知识，另一方面，在免疫生物学方面，我们处于有利地位，可以迎接这些挑战。我们建议进行增量数据驱动的研究，以解决三个最重要的问题：(I)基因传递(基因组转座的先决条件)；(Ii)功能读数(确定相关性)；以及(Iii)插入突变和肿瘤发生的风险(对所建议方法的临床前安全性评估至关重要)。我们专注于一种典型的免疫疾病，由于需要少量的纠正细胞，因此提供了很高的成功可能性。我们相信，我们提出了一个令人信服的论点，即非病毒基因治疗策略的测试对于一般临床翻译的初级免疫缺陷基因治疗方法的优先顺序至关重要。公共卫生相关性：我们的目标是开发临床相关的方法，促进干细胞基因治疗，以治疗原发免疫缺陷患者。从这些研究中获得的基本见解将对基因治疗和血液疾病的治疗产生广泛的影响。

DESCRIPTION (provided by applicant): Our goal, and the main challenge in the treatment of severe combined immunodeficiency (SCID) today, is to develop a robust alternative to allogeneic hematopoietic stem cell (HSC) transplantation and to gene correction of autologous HSC with viral vectors, as both these strategies have been associated with unacceptable morbidity and mortality in SCID patients. Non-viral gene therapy represents a genuine alternative to HSC transplantation and viral gene therapy. To correct SCID (caused by a point mutation in exon 85 of the DNA protein kinase catalytic subunit, DNA-PKcs, gene, ~13 kB in length), we will focus on non-viral Tol2 transposon-mediated delivery of DNA-PKcs into hematopoietic cell line and fresh HSC derived from wild type and SCID mice. Transposon-mediated gene delivery has several advantages. Foremost among these are: simple error-free design, reduced immunogenicity and risk of contamination with replication competent virus, and, critically, the ability of the high cargo Tol2 transposons to stably deliver large genes (>10 kB) into mammalian cells. As currently available methods for naked DNA delivery, such as nucleofection, result in cellular injury that is significantly more toxic to the stem cells than to their more differentiated progeny, electromagnetic charge based methods are not useful for our primary goal, stem cell gene therapy in murine model of SCID. Therefore, a liposome-mediated HSC-specific gene delivery with Stem Cell Factor conjugated liposomes will be used instead. The challenges that warrant exploration in the field of SCID treatment are delivery of non-viral vectors to HSC without compromising its functional abilities, and correction of large genes (such as DNA-PKcs). With our expertise in non-viral stem cell transgenesis on one hand, and in immunobiology readouts on the other, we are well positioned to meet these challenges. We propose incremental data-driven investigations that address the three most important concerns: (i) gene delivery (a prerequisite for genomic transposition); (ii) functional readouts (establishing the relevance); and (iii) risk of insertional mutagenesis and tumorigenesis (critical for pre-clinical safety assessment of the proposed approaches). We focused upon a prototypic immune disorder offering a high likelihood of success due to small numbers of corrected cells needed. We trust that we present a compelling argument that testing of non-viral gene therapy strategy in particular is critical for prioritization of primary immunodeficiency gene therapy approaches for clinical translation in general. PUBLIC HEALTH RELEVANCE: Our goal is to develop clinically relevant approaches that will facilitate stem cell gene therapy to treat patients with primary immunodeficiencies. The fundamental insights gained from these studies will have broad implications relevant to both gene therapy and treatment of blood diseases.