Hematopoietic Stem Cell Transposon Therapy for Severe Combined Immunodeficiency

描述（由申请人提供）：我们的目标，也是目前治疗严重联合免疫缺陷（SCID）的主要挑战，是开发一种强大的替代异体造血干细胞（HSC）移植和用病毒载体对自体HSC进行基因校正的方法，因为这两种策略都与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一个真正的替代HSC移植和病毒基因治疗。为了纠正SCID（由DNA蛋白激酶催化亚基DNA- pkcs基因外显子85点突变引起，长度约13 kB），我们将重点关注非病毒Tol2转座子介导的DNA- pkcs进入造血细胞系和野生型和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，降低了免疫原性和被复制能力强的病毒污染的风险，最重要的是，高载货Tol2转座子能够稳定地将大基因（bbb10 kB）递送到哺乳动物细胞中。由于目前可用的裸DNA递送方法，如细胞核感染，会导致细胞损伤，对干细胞的毒性明显大于对其分化程度更高的后代，因此基于电磁电荷的方法对我们的主要目标——SCID小鼠模型的干细胞基因治疗无效。因此，脂质体介导的hsc特异性基因传递将使用干细胞因子偶联脂质体代替。在SCID治疗领域值得探索的挑战是在不损害其功能的情况下将非病毒载体输送到HSC，以及校正大基因（如DNA-PKcs）。凭借我们在非病毒干细胞转化和免疫生物学方面的专业知识，我们已经做好了迎接这些挑战的准备。我们提出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（基因组转位的先决条件）；（ii）功能性读数（建立相关性）；（iii）插入性突变和肿瘤发生的风险（对于拟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紊乱，由于需要少量的校正细胞，它有很高的成功可能性。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初级免疫缺陷基因治疗方法的临床转化的优先级至关重要。公共卫生相关性：我们的目标是开发临床相关方法，促进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疾病的治疗具有广泛的意义。

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.