Homologous Recombination Mediated Gene Correction for the Hemoglobinopathies

项目摘要 镰状细胞病是一种遗传性疾病，导致产生一种功能失调的 血红蛋白。在美国大约有10万人患有镰状细胞病， 每年有30万名受影响的儿童出生。地中海贫血也是一种遗传性疾病， 血红蛋白的b-球蛋白链合成减少或完全缺失。预计年 地中海贫血的发病率是500，000最常见的父母来自地中海国家，北 非洲、中东、印度、中亚和东南亚。这两种疾病都是由基因突变引起的。 b-珠蛋白（HBB）基因，并可通过异基因造血干细胞移植（allo-HSCT）治愈 或简称为“同种异体基因疗法”）。缺乏免疫匹配的供体 然而，allo-HSCT的严重并发症意味着同种异体基因治疗 仅用于治疗一小部分可能受益的患者。目前没有其他 为这些疾病中的任何一种提供明确和治愈的方法。 我们已经开发出一种替代方法，有可能规避所有这些问题 介绍了对于镰状细胞病，我们正在开发的功能性基因校正过程 CRISPR/Cas9通过精确纠正镰状化直接在患者自身的HSPC中编辑HBB基因 点突变但是b型地中海贫血是由整个基因突变引起的。为此，我们要么 将野生型cDNA敲入HBB基因的外显子1，使得cDNA利用内源性ATG 起始密码子或将全长HBB基因敲入HBA 1。通过这种方式，HBB基因将被表达 使用内源性起始密码子和使用所有内源性天然调节元件。通过 敲入HBA 1基因，我们也将同时产生α-地中海贫血性状，一种已知 降低B型地中海贫血的严重程度。在目标1中-我们将纠正内源性突变中的镰状突变。 使用基因组编辑HBB基因;在目标2中，我们专注于开发敲入策略以允许表达 b-地中海贫血患者HSPC中HBB cDNA的表达。最终目标（目标3）是使用一系列 功能测定，以测试优化过程的总体毒性，从而确定 基因组编辑过程。作为这项资助的一部分，我们组建了一个合作研究小组， 拥有创建GMP兼容细胞制造工艺的专业知识的顾问。我们共同的目标是 开发优化HBB基因校正的试剂，从而提供关键IND，使数据能够移动 将其推进到针对b-血红蛋白病的首次人体临床I/II期临床试验。 !

Project Summary Sickle cell disease is a genetic disorder that results in the production of a dysfunctional form of hemoglobin. In the United States about 100,000 people have sickle cell disease and worldwide almost 300,000 affected children are born every year. b-thalassemia is also an inherited disorder that results in the decreased synthesis or complete absence of the b-globin chains of hemoglobin. The estimated annual incidence of b-thalassemia is 500,000 most commonly to parents from Mediterranean countries, North Africa, the Middle East, India, Central Asia, and Southeast Asia. Both diseases are caused by mutations in the b-globin (HBB) gene and can be cured by allogeneic hematopoietic stem cell transplantation (allo-HSCT or provocatively called “allogeneic gene therapy”). The lack of available immunologically matched donors and the significant morbid complications from allo-HSCT, however, means that allogeneic gene therapy has only been used to treat a small fraction of the patients who could benefit. There are currently no other definitive and curative approaches for either of these diseases. We have developed an alternative approach that has the potential to circumvent all of these issues described. For sickle cell disease, the functional gene correction process we are developing uses CRISPR/Cas9 to edit the HBB gene directly in patient’s own HSPCs by precisely correcting the sickling point mutation. But b-thalassemia is caused by mutations throughout the gene. For this we will either knock-in a wild-type cDNA into exon 1 of the HBB gene such that the cDNA utilizes the endogenous ATG initiation codon or knock-in a full length HBB gene into HBA1. In this way the HBB gene will be expressed using the endogenous initiation start codon and using all of the endogenous natural regulatory elements. By knocking into HBA1 we will also simultaneously create a-thalassemia trait, a genotype that is known to decrease the severity of b-thalassemia. In Aim 1- we will correct the sickle mutation in the endogenous HBB gene using genome editing; in Aim 2 we focus on developing the knock-in strategy to allow expression of the HBB cDNA in HSPCs from b-thalassemia patients. The final aim (Aim 3) is to use a series of functional assays to test the overall toxicity of the optimized process and thereby determine the safety of the genome editing process. As part of this grant we have assembled a team of co-Investigators and consultants with expertise in creating a GMP compatible cell manufacturing process. Collectively our goal is to develop reagents that optimize HBB gene correction thereby providing key IND enabling data to move forward to first-in-human clinical phase I/II clinical trials for the b-hemoglobinopathies. !