已获批准的寡核苷酸疗法的化学、结构和功能（五）

《已获批准的寡核苷酸疗法的化学、结构和功能（一）》
《已获批准的寡核苷酸疗法的化学、结构和功能（二）》
《已获批准的寡核苷酸疗法的化学、结构和功能（三）》
接上篇《已获批准的寡核苷酸疗法的化学、结构和功能（四）》

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SPLICE SITE SWITCHING OLIGONUCLEOTIDES 剪接位点转换寡核苷酸

SPINRAZA, MOE and spinal muscular atrophy SPINRAZA、MOE 和脊髓性肌肉萎缩症

The ability to modulate alternative splicing is clearly of therapeutic value, given that this process is a major contributor to proteome diversity. Thus, some 70% of human genes are thought to undergo alternative splicing and up to half of all human genetic diseases may arise as a result of mutations that affect splicing. Unlike ASOs that are designed to elicit RNase H action, splice-switching oligonucleotides (SSOs) need to bind the target sequence with high affinity and sterically block and redirect the spliceosome (Figure 5). SSOs enable highly specific targeting of essential sequence elements such as splicing enhancers and silencers in pre-mRNA. The splicing of a pre-mRNA transcript can be redirected by various means that include exon exclusion, intron retention, exon shuffling, selection of alternative 5′- and 3′-splice sites, shifting of promoter and polyadenylation sites, and so forth. Using an oligo to do so would seem more achievable that identifying small molecules to orchestrate the splicing machinery. However, there are numerous ongoing efforts directed at the discovery of small molecule modulators of alternative splicing.

调节剪接位点的能力在治疗中具有明显的价值，因为这一过程是蛋白质组多样性的主要贡献者。据认为，约 70%的人类基因经历了选择性剪接，并且高达一半的人类遗传疾病可能是由影响剪接的突变引起的。与旨在引发 RNase H 作用的反义寡核苷酸（ASO）不同，剪接位点转换寡核苷酸（SSO）需要高亲和力地结合目标序列，并在立体上阻碍和重定向剪接体（图 5）。SSO 使得对前体 mRNA 中的剪接增强子和沉默子等关键序列元素具有高度特异性的靶向成为可能。通过不同的方式，如外显子排除、内含子保留、外显子重排、选择替代的 5'-和 3'-剪接位点、启动子和聚腺苷酸化位点的移动等，可以重定向前体 mRNA 的剪接。使用寡核苷酸来实现这一目的似乎比寻找小分子来编排剪接机制更可行。然而，目前有许多不断进行的工作致力于发现调节选择性剪接的小分子调节剂。

All-PS, all-PS/MOE-wing and all-MOE oligonucleotides against human h-ras or rat PKCα were delivered to rats by intrathecal injection (IT) in order to reach the cerebrospinal fluid (CSF). The MOE–ASO remained intact both in spinal cord tissue and in the CSF for up to a day after injection. The ASO was taken up by neurons and glia alike and found to downregulate PKCα in the spinal cord. By comparison, the PS–ASO was considerably less stable and metabolites were detected only 30 minutes after IT administration. Data resulting from this study also indicated that IT injection using increased dosages might successfully deliver ASOs to dorsal root ganglia cells and thus enable downregulation of sensory neuron targets. The findings were also exciting because they indicated that the MOE chemistry might open the door to a pharmacological approach for treating neurodegenerative disorders.

为了进入脑脊液（CSF），采用腰髓内注射（IT）将全磷酸盐（all-PS）、全磷酸盐/MOE 翼（all-PS/MOE-wing）和全 MOE 寡核苷酸输送到大鼠体内，针对人 h-ras 或大鼠 PKCα进行作用。在注射后的一天内，MOE-ASO 在脊髓组织和 CSF 中仍保持完整。ASO 被神经元和胶质细胞吸收，并发现在脊髓中下调 PKCα的表达。相比之下，PS-ASO 的稳定性明显较差，仅在 IT 给药后 30 分钟后检测到代谢产物。该研究的数据还表明，使用增加的剂量进行 IT 注射可能成功地将 ASO 输送到脊神经节细胞，从而使感觉神经元靶点下调。这些发现也令人兴奋，因为它们表明 MOE 化学可能为治疗神经退行性疾病打开了药理学途径。

Indeed, in 2016 SPINRAZA (nusinersen), a fully modified 18mer PS/MOE–ASO (Figure 12) was approved by the US FDA for treatment of spinal muscular atrophy (SMA). SMA is a major genetic disorder associated with degeneration of alpha motor neurons and leading to progressive muscular weakness and atrophy. Infantile-onset SMA robs the individual of the ability to move, eat or breathe and was reported as the leading genetic cause of death for infants. The vast majority of SMA cases are due to mutations or deletions in the survival motor neuron 1 (SMN1) gene that result in reduced levels of SMN, a multifunctional protein that is required for survival of all animal cells. Humans possess two nearly identical copies of the SMN gene, SMN1 and SMN2. However, unlike in SMN1, exon 7 of SMN2 is predominantly skipped during splicing in most tissues, thereby resulting in an unstable and only partially functioning protein. SPINRAZA promotes inclusion of exon 7 in SMN2 by blocking an intronic splicing silencer and thus increases the total amount of SMN protein produced (Figure12).

的确，2016 年，美国 FDA 批准了 SPINRAZA（nusinersen），一种完全修饰的 18mer PS/MOE–ASO（图 12），用于治疗脊髓性肌萎缩（SMA）。SMA 是一种与α运动神经元退化相关的重大遗传疾病，导致渐进性肌肉无力和萎缩。婴儿早发型 SMA 使患者失去了运动、进食和呼吸的能力，并被报道为婴儿死亡的主要遗传原因。绝大多数 SMA 病例是由生存运动神经元 1（SMN1）基因的突变或缺失导致 SMN 水平降低所致，SMN 是一种多功能蛋白，对所有动物细胞的生存都是必需的。人类拥有两个几乎相同的SMN基因副本，SMN1和SMN2。然而，与SMN1不同，SMN2的第 7 外显子在大多数组织中在剪接过程中主要被跳过，从而导致产生不稳定且仅部分功能的蛋白质。SPINRAZA 通过阻断一个内含子剪接抑制因子，促进了SMN2中第 7 外显子的包含，从而增加了产生的 SMN 蛋白的总量（图 12）。

图 12.

There is no doubt that SPINRAZA has had a fundamental impact on the treatment of SMA and the lives of infants and adolescents inflicted by this genetic disorder. Not only has treatment resulted in prolonged survival but it has led to dramatic improvements in motor development in children with early and late onset of the disease, such as the abilities to sit up or walk without support. Two additional drugs have been developed for the treatment of SMA. One is ZOLGENSMA® (onasemnogene abeparvovec-xioi), a gene therapy that replaces the defect SMN1 gene that was approved by the US FDA in 2019 for use with all types of SMA in newborns and toddlers up to age 2 via a single i.v. administration. The other is an orally administered small molecule drug, EVRYSDI® (risdiplam), that acts like SPINRAZA at the level of splicing by modulating the SMN2 pre-mRNA splicing pattern and increasing the amount of full-length functional SMN protein. The breakthrough with respect to treatment of a neurodegenerative disorder by an SSO has raised the prospect of successfully expanding oligonucleotide-based therapies to combatting neurological disorders. Examples are Huntington's disease and multiple sclerosis for which MOE-based oligonucleotides are currently being evaluated in various phase clinical trials.

毫无疑问，SPINRAZA 对 SMA 的治疗和受此遗传疾病影响的婴儿和青少年的生活产生了根本性的影响。治疗不仅延长了患者的生存期，还使早发型和晚发型患儿的运动发育出现了显著改善，例如能够在没有支持的情况下坐起或行走。另外还开发了两种用于 SMA 治疗的药物。一种是 ZOLGENSMA®（onasemnogene abeparvovec-xioi），一种基因疗法，用于在新生儿和 2 岁以下的幼儿中替代缺陷的SMN1基因，2019 年获得美国 FDA 批准，通过单次静脉注射使用，适用于所有类型的 SMA。另一种是口服小分子药物 EVRYSDI®（risdiplam），在剪接水平上像 SPINRAZA 一样通过调节SMN2前体 mRNA 的剪接模式并增加全长功能性 SMN 蛋白的数量来发挥作用。SSO 在神经退行性疾病治疗方面的突破为成功扩大基于寡核苷酸的治疗到神经系统疾病领域带来了希望。目前，MOE 基础的寡核苷酸正在对亨廷顿病和多发性硬化等疾病进行各个临床试验阶段的评估。

EXONDYS 51/VYONDYS 53, PMOs and Duchenne muscular dystrophy EXONDYS 51/VYONDYS 53, PMOs和杜氏肌营养不良症

Additional SSOs that received market approval are EXONDYS 51 (eteplirsen, 2016) and VYONDYS 53 (golodirsen, 2019) (Figure 3, Table 1), both used in the treatment of Duchenne muscular dystrophy (DMD) and targeting exon 51 and exon 53, respectively, of dystrophin pre-mRNA (Figure 13). The two exon-skipping therapies aim to produce shortened but functional dystrophin to increase the levels of the protein in muscle. Dystrophin is expressed in skeletal and cardiac muscle as well as in the brain and is key for strengthening and connecting muscle fibers. Unlike SMA, DMD is not a CNS disease but a fatal X-linked disorder that is caused by any of >2000 mutations that affect the DMD gene and result in the loss of functional dystrophin protein. Some 70% of patients exhibit single- or multi-exon deletions that result in aberrant expression of dystrophin or duplications, thus leading to progressive skeletal weakness, muscle wasting and cardiomyopathy. DMD patients show levels of dystrophin that are less than 3% of normal. Symptoms of DMD are not limited to motor difficulties such as getting up or climbing stairs, but also include developmental delay and behavioral issues in children as well as impaired growth, delayed puberty and gastrointestinal problems. Life-threatening respiratory and cardiac complications are common in the second or third decade of life. Delays in disease progression and improved life expectancy have been achieved with corticosteroid treatment, surgical intervention and preventive measures in regard to heart failure related to cardiomyopathy.

其他获得市场批准的 SSO 还包括 EXONDYS 51 (eteplirsen，2016)和 VYONDYS 53 (golodirsen，2019)（图 3，表 1），两者均用于治疗杜氏肌营养不良（DMD），分别靶向 dystrophin 前体 mRNA 的 exon 51 和 exon 53（图 13）。这两种外显子跳跃疗法旨在产生缩短但具有功能的 dystrophin，以增加肌肉中该蛋白质的水平。dystrophin 在骨骼和心脏肌肉以及大脑中表达，并且对于加强和连接肌纤维至关重要。与 SMA 不同，DMD 不是一种中枢神经系统疾病，而是一种致命的 X 连锁隐性遗传疾病，由影响DMD基因的 2000 多种突变之一引起，导致功能性 dystrophin 蛋白的丧失。约 70%的患者表现出单个或多个外显子缺失，导致 dystrophin 异常表达或重复，从而导致进行性骨骼肌无力、肌肉萎缩和心肌病。DMD 患者的 dystrophin 水平不到正常水平的 3%。DMD 的症状不仅限于运动困难，如起身或爬楼梯，还包括儿童发育迟缓和行为问题，以及生长受阻、青春期延迟和胃肠问题。在生命的第二或第三个十年，常见的危及生命的呼吸和心脏并发症。通过皮质类固醇治疗、外科干预和预防心力衰竭相关措施，已经延缓了疾病进展并改善了预期寿命。

图 13.

EXONDYS 51 and VYONDYS 53 are phosphorodiamidate morpholino oligonucleotides (PMOs), often referred to as morpholinos . Morpholinos possess a neutral backbone that is completely resistant to nucleases. However, like the phosphorothioates or the neutral methylphosphonates, the PMO modification generates a mixture of Rp- and Sp-diastereoisomers (Figure 13A). PMOs are water soluble and display good cell permeability without assistance from cellular factors, but hybridized to RNA they do not elicit RNase H. Although there is a large body of data regarding cell-based assays and in vivo applications of morpholinos, and protocols for solid-phase synthesis of PMO oligomers, e.g. , detailed biophysical data and in particular experimental structural information are missing.

其他获得市场批准的 SSO 还包括 EXONDYS 51 (eteplirsen，2016)和 VYONDYS 53 (golodirsen，2019)（图 3，表 1），两者均用于治疗杜氏肌营养不良（DMD），分别靶向 dystrophin 前体 mRNA 的 exon 51 和 exon 53（图 13 A）。这两种外显子跳跃疗法旨在产生缩短但具有功能的 dystrophin，以增加肌肉中该蛋白质的水平。dystrophin 在骨骼和心脏肌肉以及大脑中表达，并且对于加强和连接肌纤维至关重要。与 SMA 不同，DMD 不是一种中枢神经系统疾病，而是一种致命的 X 连锁隐性遗传疾病，由影响DMD基因的 2000 多种突变之一引起，导致功能性 dystrophin 蛋白的丧失。约 70%的患者表现出单个或多个外显子缺失，导致 dystrophin 异常表达或重复，从而导致进行性骨骼肌无力、肌肉萎缩和心肌病。DMD 患者的 dystrophin 水平不到正常水平的 3%。DMD 的症状不仅限于运动困难，如起身或爬楼梯，还包括儿童发育迟缓和行为问题，以及生长受阻、青春期延迟和胃肠问题。在生命的第二或第三个十年，常见的危及生命的呼吸和心脏并发症。通过皮质类固醇治疗、外科干预和预防心力衰竭相关措施，已经延缓了疾病进展并改善了预期寿命。

We modeled a morpholino nucleotide into an RNA A-form oligonucleotide and achieved a virtually seamless fit. Specifically, inserting a morpholino residue maintains the puckers of 5′- and 3′-adjacent riboses and no significant alterations of backbone angles were necessary apart from a flip from sc− to sc+ for ζ in the 5′-ribonucleotide (Figure 13B). All three substituents of the morpholine chair (base, C5′, and 3′-phosphate) adopt an equatorial orientation and stacking is maintained. The model was built with the program UCSF Chimera and energy-minimized with the Amber14 force field, using steepest descent and conjugate gradient methods until convergence. A PMO nucleotide can just as easily be slipped into a DNA B-form backbone and accommodated without significant structural perturbation (not shown).

我们将一个 morpholino 核苷酸模型插入了一个 RNA A-型寡核苷酸，并实现了几乎无缝连接。具体而言，插入 morpholino 残基可以保持 5'-和 3'-相邻核糖的扭曲，并且除了在 5'-核糖的ζ从sc−翻转到sc+之外，骨架角度没有显著的改变（图 13B）。morpholine 椅子的三个取代基（碱基、C5'和 3'-磷酸酯基团）采用赤道方向，并且堆叠结构得以保持。该模型是使用 UCSF Chimera 程序构建的，并使用 Amber14 力场进行了能量最小化，采用最陡下降和共轭梯度方法直到收敛。一个 PMO 核苷酸可以轻松地滑入 DNA B-型骨架中，并在没有显著结构扰动的情况下适应其中（未显示）。

Considering that 5 out of the 16 oligonucleotide therapeutics approved to date are splice-shifting oligos (Figure 3) and that they have paved the road to treatments of devastating diseases for which there are no cures, certainly the development of SSOs should be seen as a success, despite the somewhat controversial FDA approval of EXONDYS 51. Several other targets, however, are being pursued in future treatments of DMD patients, among them therapies to counter muscle degeneration and fibrosis, reduce inflammation, bypass aberrant stop codons and modulate utrophin, a protein that is similar to dystrophin and may replicate some of its functions. Thus, other splice site-targeting drugs are currently undergoing clinical evaluation, and the exon 53 PMO-SSO therapeutic VILTEPSO® (viltolarsen) received FDA approval in 2020. The exon 45 PMO–SSO therapeutic AMONDYS 45® (casimersen) received FDA approval in 2021. In addition, CRISPR/Cas9-based gene replacement approaches may be pursued in the case of DMD treatment as with SMA before, and small-molecule drug discovery might result in the identification of new orally available therapeutics that effectively modulate DMD pre-mRNA splicing to boost dystrophin expression.

考虑到迄今为止已批准的 16 种寡核苷酸治疗药物中有 5 种是剪接转换寡核苷酸（图 3），它们为无可治愈的可怕疾病的治疗铺平了道路，尽管 EXONDYS 51 的 FDA 批准引起了一些争议，但 SSO 的开发应被视为成功。然而，在未来治疗 DMD 患者时，还有其他几个目标正在追求，其中包括对抗肌肉退化和纤维化的疗法、减少炎症、绕过异常终止密码子以及调节乌托芬（一种与肌酸激酶类似并可能复制其部分功能的蛋白质）。因此，其他靶向剪接位点的药物目前正在进行临床评估，外显子 53 的 PMO-SSO 治疗药物 VILTEPSO®（viltolarsen）于 2020 年获得 FDA 批准。外显子 45 的 PMO-SSO 治疗药物 AMONDYS 45®（casimersen）于 2021 年获得 FDA 批准。此外，在 DMD 治疗中，基于 CRISPR/Cas9 的基因替代方法可能会像在 SMA 中一样被追求，小分子药物的发现可能会找到新的口服可用药物，有效调节DMD前体 mRNA 的剪接以增加瘦肌蛋白的表达。