Adeno-associated virus-mediated gene therapy for patient's fibroblasts, induced pluripotent stem sells, and a mouse model of congenital adrenal hyperplasia
Adeno-associated virus-mediated gene therapy for patient's fibroblasts, induced pluripotent stem sells, and a mouse model of congenital adrenal hyperplasia
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腺相关病毒介导的对患者成纤维细胞、诱导多能干细胞和先天性肾上腺增生小鼠模型的基因治疗
DOI：
10.1089/hum.2022.005
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发表时间：
2022
影响因子：
4.2
通讯作者：
Fukami M
中科院分区：
文献类型：
--
作者：
Naiki Y;Miyado M;Shindo M;Horikawa R;Hasegawa Y;Katsumata N;Takada S;Akutsu H;Onodera M;Fukami M
先天性肾上腺皮质增生症（CAH）是一种常染色体隐性遗传疾病所造成的类固醇合成酶含有单基因缺陷。大多数类固醇生成酶是细胞色素P450基团，其可归类为微粒体P450，包括21-羟化酶和17α-羟化酶/17，20裂解酶，以及线粒体P450，包括11β-羟化酶。已经表明，Cyp 21 a1的异位给药改善21-羟化酶缺陷小鼠的类固醇代谢。然而，这种方法对线粒体P450的有效性尚未得到评估。在这项研究中，用腺相关病毒2型（AAV 2）载体感染来自21-羟化酶缺乏症（CYP 21 A2 D）（n= 4）、17α-羟化酶/17，20裂解酶缺乏症（CYP 17 A1 D）（n= 1）和11β-羟化酶缺乏症（CYP 11 B1 D）（n= 1）患者的原代成纤维细胞。在AAV 2感染的CYP 11B 1D成纤维细胞中未检测到类固醇生成酶活性。建立了CYP 11B 1D的诱导多能干细胞（iPSCs），并通过NR 5A 1基因的诱导向肾上腺皮质细胞分化。用含有CYP 11B 1的AAV 9型（AAV 9）载体感染从CYP 11B 1D的iPSCs建立的肾上腺皮质细胞（CYP 11B 1D-iPSCs），并表现出11β-羟化酶活性。为了进行体内评价，我们通过使用CRISPR/Cas9方法敲除小鼠中的Cyp 11b 1。将含有Cyp 11b 1的AAV 9载体直接注射到Cyp 11b 1缺陷小鼠的肾上腺中，在注射后4周显著降低血清11-脱氧皮质酮/皮质酮比率，并且效果延长至12个月。这项研究表明，CYP 11B 1D可以通过肾上腺中的基因诱导来改善，这表明CAH需要缺陷酶依赖性治疗策略。微粒体P450缺陷，包括CYP 21 A2 D和CYP 17 A1 D，可以通过肾上腺外基因诱导治疗。然而，线粒体P450的缺陷，如CYP 11B 1D所代表的，可能需要肾上腺基因诱导。
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by steroidogenic enzymes containing monogenetic defects. Most steroidogenic enzymes are cytochrome P450 groups that can be categorized as microsomal P450s, including 21-hydroxylase and 17α-hydroxylase/17,20 lyase, and mitochondrial P450s, including 11β-hydroxylase. It has been shown that ectopic administration ofCyp21a1ameliorates steroid metabolism in 21-hydroxylase-deficient mice. However, the effectiveness of this approach for mitochondrial P450 has not yet been evaluated. In this study, primary fibroblasts from patients with 21-hydroxylase deficiency (CYP21A2D) (n= 4), 17α-hydroxylase/17,20 lyase deficiency (CYP17A1D) (n= 1), and 11β-hydroxylase deficiency (CYP11B1D) (n= 1) were infected with adeno-associated virus type 2 (AAV2) vectors. Steroidogenic enzymatic activity was not detected in the AAV2-infected CYP11B1D fibroblasts. Induced pluripotent stem cells (iPSCs) of CYP11B1D were established and differentiated into adrenocortical cells by induction of theNR5A1gene. Adrenocortical cells established from iPSCs of CYP11B1D (CYP11B1D-iPSCs) were infected with an AAV type 9 (AAV9) vector containingCYP11B1and exhibited 11β-hydroxylase activity. For anin vivoevaluation, we knocked outCyp11b1in mice by using the CRISPR/Cas9 method. Direct injection ofCyp11b1-containing AAV9 vectors into the adrenal gland ofCyp11b1-deficient mice significantly reduced serum 11-deoxycorticosterone/corticosterone ratios at 4 weeks after injection and the effect was prolonged for up to 12 months. This study indicated that CYP11B1D could be ameliorated by gene induction in the adrenal glands, which suggests that a defective-enzyme-dependent therapeutic strategy for CAH would be required. Defects in microsomal P450, including CYP21A2D and CYP17A1D, can be treated with extra-adrenal gene induction. However, defects in mitochondrial P450, as represented by CYP11B1D, may require adrenal gene induction.