Hematopoietic Stem Cell Transposon Therapy for Severe Combined Immunodeficiency

造血干细胞转座子治疗严重联合免疫缺陷

• 批准号: 7686340
• 负责人: Jakub Tolar
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-11 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7686340
• 关键词: Activities of Daily Living; Address; Adenoviruses; Affect; Allogenic; Ataxia; Autologous; Bone Marrow; Bone Marrow Cells; Caring; Catalytic Domain; Cell Line; Cells; Charge; Chemicals; Clinical; Complementary DNA; DNA; DNA Binding Domain; DNA delivery; DNA-PKcs; Data; Defect; Disease; Electromagnetics; Epidermolysis Bullosa; Exons; Gene Delivery; Gene Transfer; Gene Transfer Techniques; Generations; Genes; Genomics; Goals; Hand; Hematological Disease; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Immune; Immune System Diseases; Immune response; Immunobiology; Immunologic Deficiency Syndromes; In Vitro; Insertional Mutagenesis; Investigation; Length; Liposomes; Mammalian Cell; Mechanics; Mediating; Methods; Modeling; Monitor; Morbidity - disease rate; Mus; Muscular Dystrophies; Mutate; Non-Viral Vector; Patients; Point Mutation; Positioning Attribute; Preclinical Testing; Protein Kinase; Retroviridae; Risk; Safety; Severe Combined Immunodeficiency; Simulate; Site; Stem Cell Factor; Stem cells; Subfamily lentivirinae; Syndrome; Testing; Toxic effect; Translations; Treatment Protocols; Trust; Viral; Viral Genes; Viral Vector; Virus; Zinc Fingers; base; cell injury; clinically relevant; conditioning; cytotoxicity; design; endonuclease; gene correction; gene replacement; gene therapy; graft failure; graft vs host disease; homologous recombination; immune function; immunogenicity; insight; leukemogenesis; meetings; mortality; non-viral gene therapy; nuclease; pre-clinical; public health relevance; restoration; success; tumorigenesis; vector

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.

描述（由申请人提供）：我们的目标，以及当今严重联合免疫缺陷（SCID）的主要挑战，是为了发展异元造血干细胞（HSC）移植的可靠替代品，并与病毒载体进行了自由型HSC的基因矫正，因为这两种策略都与不受欢迎的质地相关联，因此与这些策略相关。非病毒基因疗法代表了HSC移植和病毒基因疗法的真正替代方法。为了纠正SCID（由DNA蛋白激酶催化亚基，DNA-PKC，Gene，长度约为13 kb的EXON 85中的点突变引起，我们将重点介绍非病毒TOL2 TORN2转座子介导的DNA-PKC的递送，将DNA-PKC递送到肿瘤细胞系和新鲜的HSC衍生的HSC和新鲜的野生型和Scid scid小鼠中。转座子介导的基因输送具有多个优点。其中最重要的是：简单无错误的设计，降低免疫原性和使用复制能力的病毒污染的风险，以及批判性的高货物TOL2转座子稳定地输送大基因（> 10 kb）到哺乳动物细胞中的能力。作为当前可用的裸体DNA递送方法，例如核反射，导致细胞损伤对干细胞的毒性明显比其更分化的后代，基于电磁电荷的方法对我们的主要目标，在SCID鼠模型中的干细胞基因治疗无用。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结合的脂质体。在SCID治疗领域保证探索的挑战是将非病毒载体传递到HSC而不损害其功能能力和校正大基因（例如DNA-PKC）。一方面，我们在非病毒干细胞转基因方面的专业知识，另一方面，在免疫生物学读数方面，我们在应对这些挑战方面处于良好状态。我们提出的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（基因组转置的先决条件）； （ii）功能读数（建立相关性）； （iii）插入诱变和肿瘤发生的风险（对于拟议方法的临床前安全评估至关重要）。我们专注于原型免疫疾病，由于所需的校正细胞数量少，因此提供了很高成功的可能性。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总体上临床翻译的主要免疫缺陷基因治疗方法的优先级至关重要。公共卫生相关性：我们的目标是开发临床相关的方法，以促进干细胞基因治疗以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具有与基因治疗和血液疾病治疗有关的广泛含义。