反义治疗剂如剪接调节反义寡核苷酸(ASO)是治疗由剪接改变内含子变体引起的疾病的有前途的工具。然而,它们在动物模型中的测试受到人类和其他物种之间间插序列通常较差的序列保守性的阻碍。在这里,我们的目标是在小鼠中建立一个反复出现的模型,深内含子,接合激活,COL6A1变体,与严重形式的胶原VI相关性肌营养不良(COL6-RD)相关,用于体内测试人类准备的反义治疗剂。变种,c.930+189C>T,创建一个供体剪接位点并插入一个72nt长的假外显子,which,翻译时,以主导否定的方式行事,但可以跳过ASO。我们创造了一个独特的人源化小鼠等位基因(命名为“h”),其中将1.9kb的编码胶原蛋白α1(VI)的三螺旋(TH)结构域的氨基末端(N-)的小鼠基因组区交换为人直向同源序列。此外,我们还在相同的人源化敲入序列(命名为“h+189T”)上创建了携带c.930+189C>T变体的等位基因。我们表明,在这两个模型中,人外显子与小鼠外显子无缝剪接,以产生嵌合的小鼠-人胶原蛋白α1(VI)蛋白。在纯合Col6a1h+189T/h+189T小鼠中,假外显子的表达水平与杂合患者肌肉活检中观察到的水平相当。虽然与野生型动物相比,Col6a1h/h小鼠没有任何表型,Col6a1h/h189T和Col6a1h189T/h189T小鼠的肌肉质量较小,并且早在4周龄时就可以检测到握力不足。致病性h189T人源化敲入小鼠等位基因因此概括了在患者活检中看到的致病性剪接缺陷,并允许测试旨在跳过假外显子的人类准备的精确反义疗法。鉴于COL6A1N-TH区域是COL6-RD变体的热点,人源化敲入小鼠模型可用作模板以引入其他COL6A1致病变体.因此,这种独特的人源化小鼠模型代表了开发COL6-RD的反义治疗剂的有价值的工具。
Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating, COL6A1 variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics in vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long
pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as \"h\"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as \"h+189T\"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous Col6a1 h+189T/h+189T mice, the
pseudoexon is expressed at levels comparable to those observed in heterozygous patients\' muscle biopsies. While Col6a1h/h mice do not show any phenotype compared to wildtype animals, Col6a1 h/h+189T and Col6a1 h+189T/h+189T mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients\' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the
pseudoexon. Given that the COL6A1 N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other COL6A1 pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.