Nonenzymatic Gene Editing in Treatment of Heredity Spherocytosis

项目摘要/摘要 遗传性球形红细胞增多症是一种常见的遗传性贫血，大约四分之一的患者患有阿尔法贫血。 幽灵蛋白连锁隐性HS(RHS)，是这种疾病最严重的形式。RHS患者在婴儿期患有 危及生命的溶血性贫血，许多是输血依赖的。在这些患者中，脾切除术只是 姑息疗法；唯一的治疗方法是造血干细胞(HSC)移植。基因组工程的发展已经 扩大了遗传性红细胞疾病治疗的潜在策略。我们有 开发了一种使用生物相容和可生物降解的纳米颗粒的非核酸酶基因编辑技术 (Np)包裹化学修饰的肽核酸(PNA)和供体DNA。使用这一战略，我们 通过静脉注射NP-PNA治愈了一种β-地中海贫血小鼠模型，实现了对 6%，在使用未经修改的PNA进行一次治疗后，建立了原则证明并论证了 我们的方法。这项技术避免了体外操作及其相关的挑战，并避免了 大多数遗传毒性与基于核酸酶的方法有关。该项目利用多PI协作 努力开发用于SPH小鼠基因编辑的NP-PNA方法，这是一种由于 阿尔法血影蛋白基因的点突变。它解决了NP-PNA和供体DNA可以是 用于在HS的活体模型中以足够的临床相关频率校正α-血影蛋白突变 以最小的毒性和极低的基因组脱靶效应来改善HS疾病的表型。我们 将建立在简单静脉注射后对造血细胞进行体内DNA修饰的可靠方案 NP-PNA，为HS的治疗提供了一种简便、无毒的策略，不需要复杂的 移植手术或体外操作。目标一的目标是优化三链形成的PNA α-血影蛋白基因的定点基因编辑。相关研究包括基因编辑分析，偏离目标 影响和遗传毒性。目标二的目标是识别和开发用于全身用药的纳米颗粒制剂 体内编辑造血干细胞和祖细胞(HSPC)中的α-血影蛋白基因。相关研究 包括开发和表征具有新尺寸和聚合物组成的纳米颗粒以改进靶标 全身给药后向HSPC递送PNAS以及提高骨髓渗透率 车厢。目标三的目标是建立用于体内修饰的健壮的基因编辑协议 α-血影蛋白缺陷SPH/SPH小鼠HSPC中α-血影蛋白基因的表达及其对HS的改善或治疗作用 表型。结果将通过对HS表型的详细实验室分析进行监测，包括功能 红细胞膜的分析。造血干细胞的基因编辑效率和遗传毒性将 也会被分析。这些方法不仅广泛适用于其他遗传性红血球疾病， 而且还与造血干细胞中出现的许多疾病有关。这项工作中的许多观察结果将 很可能超出了血液学领域。

Project Summary/Abstract Approximately a quarter of patients with hereditary spherocytosis, a common inherited anemia, suffer from alpha- spectrin linked recessive HS (rHS), the most severe form of the disease. rHS patients present in infancy with life-threatening hemolytic anemia, many are transfusion-dependent. In these patients, splenectomy is only palliative; the only cure is hematopoietic stem cell (HSC) transplant. Development of genome engineering has expanded the repertoire of potential strategies for treatment of inherited erythrocyte disorders. We have developed a non-nuclease based gene editing technique using biocompatible and biodegradable nanoparticles (NP) encapsulating chemically modified peptide nucleic acids (PNAs) and donor DNAs. Using this strategy, we have cured a murine model of beta-thalassemia using IV injections of NP-PNAs, achieving gene correction of 6% after a single treatment with unmodified PNA, establishing proof-of-principle and demonstrating feasibility of our approach. This technique avoids ex vivo manipulation and its associated challenges, as well as obviates most of the genotoxicity associated with nuclease-based methods. This project leverages a multi-PI collaborative effort to develop the NP-PNA approach for gene editing in sph mice, a spontaneous murine model of rHS due to a point mutation in the alpha-spectrin gene. It addresses the hypothesis that NP-PNAs and donor DNAs can be used to correct the alpha-spectrin mutation in an in vivo model of HS at clinically relevant frequencies sufficient to ameliorate the HS disease phenotype with minimal toxicity and extremely low genomic off-target effects. We will establish robust protocols for in vivo DNA modification in hematopoietic cells after simple IV administration of NP-PNAs, providing a facile, non-toxic strategy for treatment of HS without the need for complex transplantation procedures or ex vivo manipulation. The goal of aim one is to optimize triplex-forming PNAs for site-specific gene editing of the alpha-spectrin gene. Relevant studies include assays of gene editing, off target effects, and genotoxicity. The goal of aim two is to identify and develop nanoparticle formulations for systemic in vivo editing of the alpha-spectrin gene in hematopoietic stem and progenitor cells (HSPCs). Relevant studies include development and characterization of NPs with novel size and polymer composition to improve target delivery of PNAs after systemic administration to HSPCs as well as improving penetration of the bone marrow compartment. The goal of aim three is the establishment of robust gene editing protocols for in vivo modification of the alpha-spectrin gene in HSPCs in alpha-spectrin deficient sph/sph mice to ameliorate or cure the HS phenotype. Results will be monitored by detailed laboratory analyses of the HS phenotype, including functional analyses of the erythrocyte membrane. Gene editing efficiency in hematopoietic stem cells and genotoxicity will also be analyzed. These approaches are widely applicable not only to other inherited disorders of the erythrocyte, but also to many disorders arising in the hematopoietic stem cell. Many of the observations from this work will likely extend beyond the field of hematology.