近日，加拿大多伦多病童医院的Ronald D. Cohn研究组通过上调一个疾病改善基因以无需突变的方式实现对肌营养不良症的治疗。该研究于2019年8月1日发表于国际学术期刊《自然》上。

研究人员报道了一种无需突变的策略，其在小鼠中使用CRISPR激活系统来上调一个与1A型先天性肌营养不良（MDC1A）相关的疾病改善基因的表达。MDC1A是由LAMA2突变产生无功能的laminin-α2蛋白所导致，这使得肌纤维的稳定性和周围神经的髓鞘形成受损。编码结构相似laminin-α1蛋白的Lama1基因过表达可在小鼠MDC1A模型中改善肌肉萎缩和瘫痪，这提示其在这种疾病中补偿性改善的重要性。然而，Lama1的上调受限于其基因长度过大，超过了临床上使用的基因治疗载体的包装容量。研究人员在dy2j/dy2j遗传背景的MDC1A小鼠模型中，通过使用腺相关病毒（AAV9）实现了调控Lama1的表达，这个载体携带了无酶活的Cas9（dCas9）、VP64反式激活因子以及一条靶向Lama1启动子的单链RNA。当小鼠在出现症状前接受了治疗，Lama1在骨骼肌和周围神经中的表达被上调，从而阻止了肌肉萎缩和瘫痪。然而，对于大多数疾病而言，研究治疗的窗口期和症状的可逆性是很关键的。在肌营养不良症中，骨骼肌中的纤维化改变被认为是不可逆的。然而，研究人员发现，在出现明显的后肢麻痹和肌肉纤维化症状的小鼠中，治疗的开始能够明显地改善和缓解营养不良的特征和疾病的进展。总之，这些结果表明通过CRISPR-dCas9介导Lama1的上调具有可行性和治疗效果，这可能用于以无需突变的方式治疗所有MDC1A患者。对于大量疾病改善基因而言，这个方法具有广泛的适用性，并且能够作为许多遗传性和获得性疾病的治疗策略。

研究人员表示，神经肌肉疾病通常因为一些大型结构复杂的基因中的异源突变引起。靶向补偿性修饰基因可能有利于改善疾病表型。
 

附：英文原文

Title: A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene

Author: Dwi U. Kemaladewi, Prabhpreet S. Bassi, Steven Erwood, Dhekra Al-Basha, Kinga I. Gawlik, Kyle Lindsay, Elzbieta Hyatt, Rebekah Kember, Kara M. Place, Ryan M. Marks, Madeleine Durbeej, Steven A. Prescott, Evgueni A. Ivakine, Ronald D. Cohn

Issue&Volume: Volume 572 Issue 7767

Abstract: Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPRdCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

DOI: 10.1038/s41586-019-1430-x

Source:https://www.nature.com/articles/s41586-019-1430-x