杜氏肌营养不良综合征中的外显子跳跃外文翻译资料

 2023-01-03 11:01

杜氏肌营养不良综合征中的外显子跳跃

原文作者 Dwi U. Kemaladewi1,2 and Ronald D. Cohn1,2,3,4,5,*

摘要：杜氏肌萎缩症（DMD）是DMD基因编码的抗肌萎缩蛋白突变引起的神经肌肉障碍。我们将讨论最近研究使用的CRISPR / Cas9技术“剪掉”在DMD中的变异外显子,使基因恢复表达。同时在临床实践方面使用这项激动人心的技术时我们也给予警示。

杜氏肌营养不良是一种X染色体隐性遗传疾病，主要发病于男孩。不同类型的突变从删除,复制、点突变或其他更小的基因重组可以生成一个缺陷的DMD转录物导致异常的翻译。因此，抗肌萎缩蛋白缺失。抗肌萎缩蛋白是一个结构蛋白，在肌动蛋白的细胞骨架和骨骼肌的细胞质基质之间提供一个机械的信号连接。[1]抗肌萎缩蛋白的缺失使肌肉细胞易受损伤，导致杜氏肌营养不良症的病人肌肉退化，失去行走能力，呼吸虚弱，患扩张型心肌病。

相比之下，贝克肌营养不良起因于DMD转录物的缺失，产生一个被删减的抗肌萎缩功能蛋白，受突变影响的外显子序列缺乏相应的序列。贝克肌营养不良的个体临床表现没有杜氏肌营养不良严重。因为抗肌萎缩蛋白在内部缩短的能力可以部分实现它的分子功能。确实，大约30%抗肌萎缩蛋白被认为可以充分的阻止人类肌肉营养障碍[2]。

杜氏肌营养不良症和贝克肌营养不良症在临床症状的严重程度上的不同启发了治疗策略的发展，通过调节拼接的DMD基因模型，反义寡核苷酸被利用修改DMD前mRNA的加工，恢复DMD读码框架，产生内部被缩短的抗肌萎缩蛋白；一个策略被认为是外显子跳跃。反义寡核苷酸在前mRNA区域或极为贴近相关的DMD外显子跳跃基因。反义寡核苷酸药物靶向外显子现在已经在高级临床试验检测[3,4]，当前被管制机构例如食品药品监督管理局考虑批准。

CRISPR技术的发现提供了一种可选择的方法，DMD基因的再构造。一个叫Cas9的核酸内切酶当加上一列gRNA时可以用精确的方法分裂基因组[5]。裂开的DNA经由非同源末端连接重新聚合，促进了细胞的自我修复，或者经由同源靶向修复，如果一个修复模板是被提供的。然而同源靶向修复在有丝分裂后的细胞中是极其低效的，例如骨骼肌细胞，非同源末端连接是一种有效的方式。令人信服的是CRISPR/Cas9系统恢复读码框架作为治疗的一种潜在可能性，我们把它称为外显子碎片，最近三个独立的团队已经被报道使用肌营养不良模型鼠做实验(图1)[6–8]。

在第一个研究中，纳尔逊等人包裹金黄色葡萄球菌在腺相关病毒8(AAV8)衍生出Cas9,同两个gRNAs一样。目标在另一个腺相关病毒8的内含子22和23，它们证明了介于中间的外显子23，其中含有点突变肌营养不良蛋白缺陷型杂合子小鼠，可能有效地剪出[6]。结果DMD转录物是框架内的，导致恢复肌营养不良蛋白的表达和肌营养不良蛋白糖蛋白的组分复杂。另外，恢复适当的神经元一氧化氮合酶（iNOS）定位和活性已经被实现。在全身给药后，这种方法的效率很好，恢复的肌营养不良蛋白在腹部，隔膜，和心肌，但不在远端肢体肌肉。因此，改善肌肉未观察到运动功能。作者在结论中指出，评估各种腺相关病毒衣壳和组织特异性启动子将是重要的潜在的临床翻译方法。

这在第二部分中被Long等人的研究部分地解决，他们使用基于工程的AAV血清型9的载体得到化脓性链球菌Cas9在最小巨细胞病毒启动子序列下表达，与两个gRNA偶联封装在另一个AAV9中[7]。全身给药后，阅读框架和肌营养不良蛋白在骨骼和心肌中表达的杂合子小鼠被成功解救。同时观察到抓握力的提高。重要的是，随着时间的推移肌营养不良蛋白水平增加，这可能是由于CRISPR / Cas9组分在出生后骨骼肌中的持续表达或“编辑”肌肉前体细胞。

营养不良的肌肉早期退化迅速对该疾病有影响。肌肉驻留干细胞，被称为卫星细胞，负责产生前体细胞最终成为成熟的肌肉纤维，因此，是干预的重要目标。Tabebordbar等人的第三项研究表明除了成熟肌肉纤维外，CRISPR / Cas9介导的肌营养不良蛋白的恢复可以在卫星细胞中有效实现而不妨碍它们的再生能力[8]。

总的来说，这些研究有效证明了CRISPR / Cas9技术可以递送到终末分化骨骼肌纤维，心肌细胞和肌肉卫星细胞在新生儿以及成年小鼠中。 此外，该系统可用于调节靶向删除突变的外显子，恢复肌营养不良蛋白表达，并恢复营养不良的功能缺陷肌肉，提供一个关键的证据的概念研究使这项技术更接近临床。此外，所有研究都报道了小鼠基因组的最小脱靶活性。

由于其序列特异性，这个策略是有希望实现的。但是，在小鼠中没有脱靶活性基因的小鼠可能不一定翻译患有肌营养不良蛋白突变的患者基因。在未来的研究中，“外显子剪切”的疗效需要在相关的患者细胞中探索，其次是全面的，无偏见的，在人类中评估全基因组的脱靶效应。这种类型的评估可能提供相关的理由进一步探索这种拥有治疗DMD的潜力技术。总之，它将会为CRISPR / Cas9治疗的临床安全建立指导方针和提供一个基础。

图1. 杂合子小鼠中CRISPR / Cas9介导的外显子剪切方法。腺相关载体用于将Cas9与指导RNA（gRNA）一起递送到肌营养不良蛋白缺陷型杂合子鼠中，在DMD外显子23中具有点突变（星号），导致过早的终止密码子。 Cas9在分别由gRNA1和gRNA2靶向的内含子22和23中产生双链断裂（DSB），剪切侧翼基因组序列，包括突变的外显子23。裂解的DNA通过以下机制修复：非同源末端连接（NHEJ）和以下前mRNA剪接，导致框架内转录物包含外显子22和24，在编辑的杂合子小鼠中恢复肌营养不良蛋白蛋白。 缩写：CRISPR，聚簇规则间隔短回文重复序列。

然而，“剪切”的方法使用两个gRNA已经表明成功删除外显子覆盖在DMD基因中的突变“热点”区，例如外显子45-55，其将适用于治疗40-45％的DMD人群[9]。此外，该技术还可以使具有包括约12-15％的DMD突变谱的基因重复的患者受益。我们实验室的前期工作表明，外显子18-30的重复可以在患者的肌红蛋白转化成纤维细胞中成功去除，导致恢复一个全长的肌营养不良蛋白[10]。重要的是，给定重复区域的头对尾方向，一个gRNA足以去除突变，这可以证明是有益的，给定腺相关病毒载体的包装限制。

使用AAV载体作为基因传递方法在基因治疗领域具有巨大的潜力。目前正在进行涉及针对庞贝病（NCT02240407）和脊髓性肌萎缩（NCT02122952）的骨骼肌中AAV9介导的基因转移的几种临床试验。前面讨论过在最近的三个利用AAV载体传递CRISPR / Cas9组件的研究中，一次性注射导致肌营养不良蛋白的恢复长达14周。但是，确定这种治疗是否需要在一定时间后重新给药以维持肌营养不良蛋白的治疗水平仍有待确定。基于AAV的基因治疗的施用可以与病毒特异性免疫应答，例如中和抗体的产生，可以使存在于循环中的AAV无效，并降低其的有效性矢量，特别是如果需要重新管理。此外，Cas9是来自细菌系统的蛋白质，其在人体中的免疫原性的程度仍然是未知的。解决这些潜在的挑战性免疫障碍的临床前研究对推动这些疗法进入临床将是至关重要的。

要考虑的一个积极方面是这种技术的多功能性允许编辑几乎所有DMD导致的突变，提供巨大的潜在可能用于DMD患者的个体化治疗。一个重要的问题，需要监管机构和行业的彻底评估是对每个gRNA设计进行广泛的临床前评估是否是必要的。为每个单独突变创建动物模型以进行临床前评估是昂贵的，耗时的，并且很大程度上是不切实际的。相比之下，容易获得患者衍生的细胞，并且足以评估该方法的效力及其脱靶活性。此外，具有组织特异性启动子的构建体的发展将有利于减少脱靶效应，以及最小化或消除了潜在靶向胚系/胚胎阶段的担忧。

总而言之，CRISPR / Cas9介导的基因组编辑的演示可以发生在体内并导致肌营养不良蛋白阴性杂合子小鼠功能的好处提供了一个关键和令人兴奋的证明，是采取进一步的步骤微调使这种技术在成人体细胞的发展，并认为这是作为DMD的潜在的有希望的治疗。

参考文献

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附原文

Exon Snipping in Duchenne Muscular Dystrophy

Dwi U. Kemaladewi1,2 and Ronald D. Cohn1,2,3,4,5,*

Duchenne muscular dystrophy (DMD) is a life-limiting neuromus- cular disorder caused by mutations in the DMD gene encoding dystro- phin. We discuss very recent stud-ies that used CRISPR/Cas9 technology to lsquo;snip outrsquo; mutated exons in DMD, restoring the read-ing frame of the gene. We also present cautionary aspects of translating this exciting technology into clinical practice.

DMD is an X-linked disorder caused by mutations in the DMD gene and affecting approximately 1 in 3500 males. Different types of mutations ranging from deletions, duplications, point mutations, or other sm

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Exon Snipping in Duchenne Muscular Dystrophy

Dwi U. Kemaladewi1,2 and Ronald D. Cohn1,2,3,4,5,*

Duchenne muscular dystrophy (DMD) is a life-limiting neuromus- cular disorder caused by mutations in the DMD gene encoding dystro- phin. We discuss very recent stud-ies that used CRISPR/Cas9 technology to lsquo;snip outrsquo; mutated exons in DMD, restoring the read-ing frame of the gene. We also present cautionary aspects of translating this exciting technology into clinical practice.

DMD is an X-linked disorder caused by mutations in the DMD gene and affecting approximately 1 in 3500 males. Different types of mutations ranging from deletions, duplications, point mutations, or other smaller gene rearrangements can generate an out-of-frame DMD transcript that leads to aberrant translation. Consequently, the protein dystrophin is absent. Dystrophin, a structural protein, provides a mechanical and signaling link between the actin cyto- skeleton and the extracellular matrix in skel- etal muscle [1]. The lack of dystrophin renders muscle cells susceptible to damage, leading to degeneration, loss of ambulation, respiratory weakness, and dilated cardiomyopathy in DMD patients.

By contrast, Becker muscular dystrophy (BMD) arises from deletions in which the DMD transcript is maintained in-frame, yielding a truncated, yet functional dystro-phin that lacks the domains correspond-ing to exonic sequences affected by the mutation. Individuals affected with BMD show less severe clinical presentation than those with DMD, due to the ability of inter-nally shortened dystrophin to partially fulfill its molecular function. Indeed, approxi-mately 30% of dystrophin expression is deemed sufficient to prevent muscular dystrophy in humans [2].

The disparity in clinical severities between DMD and BMD has inspired the develop-ment of therapeutic strategies to modulate splicing patterns of the DMD gene, where antisense oligonucleotides (AOs) have been utilized to manipulate DMD pre-mRNA processing, and restore the DMD open reading frame to produce internally truncated dystrophin; a strategy known as lsquo;exon skippingrsquo;. The AOs are complimen-tary to regions of the pre-mRNA, at, or in close proximity to the relevant DMD exon targeted for skipping. AO drugs targeting exon 51 have now been tested in advanced clinical trials [3,4] and are cur-rently being considered for approval by regulatory agencies, such as the Food and Drug Administration.

An alternative approach to reframing the DMD gene has been offered by the dis-covery of clustered regularly-interspaced short palindromic repeats (CRISPR) tech-nology, in which an endonuclease called Cas9 can cleave the genome in a precise manner when coupled with a strand of guide RNA (gRNA) [5]. The cleaved DNA will be rejoined via a non-homologous end joining (NHEJ) mechanism facilitated by the cellsrsquo; own repair machinery or, alter-natively, via homologous-directed repair (HDR), if a repair template is provided. While HDR is extremely inefficient in postmitotic cells such as skeletal muscle, NHEJ appears to be achieved in an effi-cient manner. Convincing evidence for the therapeutic potential of CRISPR/Cas9- mediated restoration of the DMD open reading frame, as we have dubbed lsquo;exon snippingrsquo;, has been recently reported by three independent groups who used dys- trophin-deficient mdx mice (Figure 1) [6–8].

In the first study, Nelson et al. packaged Staphylococcus aureus-derived Cas9 in an adeno-associated vector serotype 8 (AAV8), as well as two gRNAs, targeted at introns 22 and 23 in another AAV8; they demonstrated that the intervening exon 23, which harbors a point mutation in dystrophin-deficient mdx mice, could be efficiently snipped out [6]. The resulting DMD transcript was in-frame, leading to restoration in the expression of dystrophin and components of the dystrophin glyco-protein complex. In addition, restoration of appropriate neuronal nitric oxide synthase (nNOS) localization and activity was achieved. Following systemic delivery, this approach was encouragingly efficient, res- cuing dystrophin in abdominal, diaphragm, and cardiac muscles, yet not in distal limb muscles. Consequently, improved muscle motor function was not observed. As the authors stated in their conclusion, evalua- tion of various AAV capsids and tissue- specific promoters will be important for potential clinical translation of this approach.

This is partially addressed in the second study by Long et al., who used an engi- neered AAV serotype 9-based vector con- taining Streptococcus pyogenes-derived Cas9 expressed under a minimal CMV pro- moter sequence, coupled to two gRNAs encapsulated in another AAV9 [7]. Follow- ing systemic administration, the reading frame and the expression of dystrophin in skeletal and cardiac muscles in mdx mice were successfully rescued. Improved grip strength activity was also observed. Impor- tantly, dystrophin protein levels increased over time, which likely resulted from persis- tent expression of the CRISPR/Cas9 components in postnatal skeletal muscles and/or a selective advantage of lsquo;editedrsquo; muscle precursor cells.

Dystrophic muscles degenerate early and rapidly in response to the disease. Muscle resident stem cells, called satellite cells, are responsible for generating precursor cells that eventually arise to become mature muscle fibers and, therefore, are important targets for intervention. The third study by Tabebordbar et al. showed that in addition to mature muscle fibers, the CRISPR/Cas9-mediated dystrophin restoration could be efficiently achieved in satellite cells without hampering their regenerative capacity [8].

Collectively, these studies have effectively demonstrated that CRISPR/Cas9 tech-nology can be delivered to terminally dif-ferentiate skeletal muscle fibers, cardiomyocytes, and muscle satellite cells in neonatal as well as adult mice. In addi-tion, the system can be used to mediate targete

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