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，会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，在世界范围内，几乎 每年有30万名受影响的儿童出生。B-地中海贫血也是一种遗传性疾病，会导致 血红蛋白的b-珠蛋白链合成减少或完全缺失。预计每年 B-地中海贫血的发病率为50万，最常见的是来自地中海国家、北方的父母 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)基因可通过异基因造血干细胞移植(allo-HSCT)治愈 或被挑衅地称为“异体基因疗法”)。缺乏可获得的免疫匹配的捐赠者 然而，异基因造血干细胞移植的显著病态并发症意味着异基因治疗已经 仅用于治疗一小部分可以受益的患者。目前没有其他 针对这两种疾病的权威性和治愈性方法。 我们已经开发了一种替代方法，有可能绕过所有这些问题 描述。对于镰状细胞疾病，我们正在开发的功能基因校正过程使用 CRISPR/Cas9通过精确纠正镰状，直接在患者自己的HSPC中编辑HBB基因 点突变。但b-地中海贫血是由整个基因的突变引起的。为此，我们将要么 在HBB基因的外显子1中插入野生型基因，从而使该基因利用内源ATG 起始密码子或将全长HBB基因插入HBA1。通过这种方式，HBB基因将得到表达 使用内源起始密码子和使用所有内源自然调节元件。通过 在敲入HBA1基因后，我们还将同时创造a-地中海贫血特征，这是一种已知的 降低b型地中海贫血的严重程度。在目标1中-我们将纠正内源基因中的镰状突变 使用基因组编辑的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基因的表达。最终目标(目标3)是使用一系列 功能分析，以测试优化工艺的总体毒性，从而确定 基因组编辑过程。作为这笔赠款的一部分，我们组建了一个联合调查组， 在创造符合GMP标准的电池制造工艺方面具有专业知识的顾问。总的来说，我们的目标是 开发优化HBB基因校正的试剂，从而提供关键的IND，使数据能够移动 展望人类第一个针对b型血红蛋白病的临床I/II期临床试验。 好了！

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. !