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 (BTK) 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 BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK 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 BTK gene. Successful integration of the intron-containing BTK 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 BTK transgene expression. Combining these modifications led to a BTK 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 in vitro. 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.

中文翻译：

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优化转基因布鲁顿酪氨酸激酶的整合和表达，用于 X 连锁无丙种球蛋白血症的 CRISPR-Cas9 介导的基因编辑

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，其特征是免疫球蛋白水平非常低，并且复发和严重感染的风险大大增加。XLA 患者的布鲁顿酪氨酸激酶 ( BTK ) 基因发生功能丧失性突变，无法产生成熟的 B 淋巴细胞。在 XLA 患者的造血干细胞中进行基因编辑以纠正或替换缺陷基因应该可以恢复 B 细胞发育和体液免疫反应。我们使用成簇的规则间隔短回文重复序列 (CRISPR)-Cas9 平台来精确定位校正、密码子优化的BTK 的整合互补 DNA (cDNA) 盒插入其内源基因座。这个过程是由同源重组驱动的，应该将转基因BTK置于其内源性调控元件的转录控制之下。该 cDNA 在 BTK 缺陷型 K562 细胞中的每个整合拷贝产生的 BTK 蛋白仅为野生型基因的 11%。供体 cDNA 被修饰为包括BTK基因的末端内含子。成功整合含内含子的BTK供体导致每个细胞的 BTK 表达比基础供体增加近两倍。然而，这种供体变体太大而无法包装到腺相关病毒载体中以输送到原代细胞中。含有末端内含子截短变体的供体也产生了升高的表达，但程度低于完整内含子。添加土拨鼠肝炎病毒转录后调控元件导致BTK转基因表达大幅增加。结合这些修改导致BTK供体模板在细胞系中产生接近生理水平的 BTK 表达。然后对这些试剂进行优化，以最大限度地提高与人类造血干细胞和祖细胞的整合率，这些细胞已达到潜在的治疗水平体外。新的供体修饰支持 XLA 的有效基因治疗，并可能有助于开发其他基于基因编辑的遗传疾病疗法。