Mosaicism for the smooth muscle cell (SMC)-specific knock-in of the Acta2 R179C pathogenic variant: Implications for gene editing therapies.
Mosaicism for the smooth muscle cell (SMC)-specific knock-in of the Acta2 R179C pathogenic variant: Implications for gene editing therapies.
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Acta2 R179C 致病性变异的平滑肌细胞 (SMC) 特异性敲入的嵌合现象：对基因编辑疗法的影响。
DOI：
10.1016/j.yjmcc.2022.07.004
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发表时间：
2022
影响因子：
5
通讯作者：
Milewicz,DiannaM
中科院分区：
文献类型：
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作者：
Kaw,Anita;Pedroza,AlbertJ;Chattopadhyay,Abhijnan;Pinard,Amelie;Guo,Dongchuan;Kaw,Kaveeta;Zhou,Zhen;Shad,Rohan;Fischbein,MichaelP;Kwartler,CallieS;Milewicz,DiannaM
亲爱的编辑，ACTA2的17个致病变异编码了平滑肌α-肌动蛋白(α-SMA)，易患18例胸主动脉瘤和夹层(TAD)。ACTA2杂合突变体19干扰精氨酸179可导致血管平滑肌功能障碍综合征(SMDS)，以20岁儿童起病的TAD和脑血管疾病为特征，并伴有其他SMC依赖系统的破坏。1、2由于人和小鼠基因编码的α-SmA具有相同的22个氨基酸序列，我们试图建立一个SMDS小鼠模型，以确定这种23个突变是如何导致比其他ACTA2突变更严重的表型的。24根据机构准则批准和进行动物用途。通过有条件的SMC特异性插入Acta2R179C26(c.535C>；T；T)突变，设计了25只小鼠模型。外显子5和6(包含c.535C和gt；T突变)反向克隆到内含子27 6中，loxP位点包围整个区域，Lox2272位点28仅围绕C57BL/6J背景中的突变盒(Acta2R179C-FLOX/FLOX)(图29 1A)。在Cre重组酶的作用下，一对Lox位点反转盒式磁带，然后30秒的一对切除野生型外显子。Acta2R179C-FLOX/FLOX小鼠与SMC特异的31只Tagln-Cre+/-小鼠杂交，产生表达SMC杂合子Acta2R179C突变32的突变小鼠，命名为Acta2SMC-R179C/+。用34个引物P1和P3扩增Acta2 R179C突变，并用基因组DNA中的P2和P3进行测序(图1A)，以及来自主动脉组织和移植的主动脉SMC的35个Acta2转录本测序(图1B，顶部)，证实了Acta2 R179C突变的33个Knock-in。36此外，在翻译的Acta2SMC-R179C/+SMC的批量RNA测序的856个读数中，37 50.6%的转录本具有C>；T变体，49.4%的转录本具有WT。(图1B，中间)。2胜38负
Dear Editor, 17 Pathogenic variants in ACTA2, encoding smooth muscle α-actin (α-SMA), predispose to 18 thoracic aortic aneurysms and dissections (TAD). De novo heterozygous ACTA2 variants 19 disrupting arginine 179 cause Smooth Muscle Dysfunction Syndrome (SMDS), characterized by 20 childhood onset TAD and cerebrovascular disease, with disruption of other smooth muscle cell 21 (SMC)-dependent systems. 1, 2 Because the human and mouse genes encode α-SMA with identical 22 amino acid sequence, we sought to establish a SMDS mouse model to determine how this 23 alteration causes a more severe phenotype than other ACTA2 mutations. 24 Animal use was approved and conducted in accordance with institutional guidelines. A 25 mouse model was engineered with a conditional SMC-specific insertion of the Acta2 R179C 26 (c. 535C> T) mutation. Exons 5 and 6 (containing the c. 535C> T mutation) were cloned into intron 27 6 in reverse orientation with LoxP sites surrounding the entire region and Lox2272 sites 28 surrounding only the mutant cassette in on the C57BL/6J background (Acta2R179C-flox/flox)(Fig. 29 1A). With exposure to Cre recombinase, one pair of Lox sites inverts the cassette, then the 30 second pair excises the wildtype exons. The Acta2R179C-flox/flox mice were bred with SMC-specific 31 Tagln-Cre+/-mice to generate mutant mice expressing the heterozygous Acta2 R179C mutation 32 in SMCs, designated as Acta2SMC-R179C/+. 33Knock-in of the Acta2 R179C mutation in SMCs was confirmed by amplification using 34 primers P1 and P3 and sequencing using primers P2 and P3 in genomic DNA (Fig 1A) and 35 sequencing Acta2 transcripts from aortic tissue and explanted aortic SMCs (Fig. 1B, top). 36 Furthermore, of 856 reads from bulk RNA-sequencing of explanted Acta2SMC-R179C/+ SMCs, 37 50.6% of transcripts had the C> T variant and 49.4% were WT.(Fig. 1B, middle). Two-38