Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA; University of California, Los Angeles, Los Angeles, California, USA.
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA; University of California, Los Angeles, Los Angeles, California, USA.
CRISPR J. 2021 Apr;4(2):191-206. doi: 10.1089/crispr.2020.0080.
X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase () gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the gene. Successful integration of the intron-containing donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in transgene expression. Combining these modifications led to a donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels . The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.
X 连锁无丙种球蛋白血症（XLA）是一种单基因原发性免疫缺陷病，其特征是免疫球蛋白水平极低，且复发性和严重性感染的风险大大增加。XLA 患者在 Bruton 酪氨酸激酶（BTK）基因中存在功能丧失突变，无法产生成熟的 B 淋巴细胞。对 XLA 患者造血干细胞中的基因进行编辑，以纠正或替换有缺陷的基因，应能恢复 B 细胞发育和体液免疫反应。我们使用成簇规律间隔短回文重复（CRISPR）-Cas9 平台，精确地将一个纠正的、密码子优化的 cDNA 盒靶向整合到其内源基因座中。这个过程由同源重组驱动，应使转基因在其内源调控元件的转录控制下。在 BTK 缺陷的 K562 细胞中，每个整合的 cDNA 拷贝仅产生 11%的 BTK 蛋白，而野生型基因则产生 11%的 BTK 蛋白。供体 cDNA 被修饰，以包含 BTK 基因的末端内含子。成功整合含内含子的 BTK 供体，使每个细胞的 BTK 表达增加近两倍，超过基础供体。然而，这种供体变体太大，无法包装到腺相关病毒载体中，用于递送至原代细胞。含有末端内含子截断变体的供体也产生了较高的表达，尽管程度低于全长内含子。添加 Woodchuck 肝炎病毒转录后调控元件可大大提高转基因表达。结合这些修饰，产生了一个 BTK 表达接近生理水平的供体模板。然后对这些试剂进行优化，以最大限度地提高整合到人类造血干细胞和祖细胞中的效率，其整合效率已达到潜在的治疗水平。这些新的供体修饰支持 XLA 的有效基因治疗，并且可能有助于开发其他基于基因编辑的遗传疾病治疗方法。
