Hematopoietic Stem Cell Transposon Therapy for Severe Combined Immunodeficiency

描述（由申请人提供）：我们的目标和目前治疗严重联合免疫缺陷（SCID）的主要挑战是开发一种替代异基因造血干细胞（HSC）移植和用病毒载体对自体HSC进行基因校正的稳健替代方案，因为这两种策略均与SCID患者不可接受的发病率和死亡率相关。非病毒基因治疗代表了HSC移植和病毒基因治疗的真正替代方案。为了纠正SCID（由DNA蛋白激酶催化亚基DNA-PKcs基因的外显子85中的点突变引起，长度约13 kB），我们将专注于非病毒Tol 2转座子介导的DNA-PKcs递送到来自野生型和SCID小鼠的造血细胞系和新鲜HSC中。转座子介导的基因递送具有若干优点。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型病毒污染的风险，以及关键的是，高负载Tol 2转座子将大基因（>10 kB）稳定递送到哺乳动物细胞中的能力。由于目前可用的裸DNA递送方法，如核转染，导致细胞损伤，其对干细胞的毒性明显大于对它们更分化的后代的毒性，因此基于电磁电荷的方法对于我们的主要目标，SCID鼠模型中的干细胞基因治疗是无用的。因此，将使用脂质体介导的HSC特异性基因递送与干细胞因子缀合的脂质体。在SCID治疗领域值得探索的挑战是将非病毒载体递送至HSC而不损害其功能能力，以及校正大基因（如DNA-PKcs）。凭借我们在非病毒干细胞转基因方面的专业知识，以及另一方面在免疫生物学读数方面的专业知识，我们有能力应对这些挑战。我们提出了增量数据驱动的研究，解决了三个最重要的问题：（i）基因递送（基因组转座的先决条件）;（ii）功能读数（建立相关性）;（iii）插入诱变和肿瘤发生的风险（对所提出的方法的临床前安全性评估至关重要）。我们专注于一种原型免疫疾病，由于所需的纠正细胞数量较少，因此成功的可能性很高。我们相信，我们提出了一个令人信服的论点，特别是非病毒基因治疗策略的测试是至关重要的优先级的主要免疫缺陷基因治疗方法的临床翻译一般。公共卫生相关性：我们的目标是开发临床相关的方法，这将有助于干细胞基因疗法治疗原发性免疫缺陷患者。从这些研究中获得的基本见解将对基因治疗和血液病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.