外刊英语每日精读（读外刊科学学英语）

本文节选自2021年12月8日发表在《科学》（SCIENCE ADVANCES Vol 7, Issue 50）的论文，Genome-editing prodrug: Targeted delivery and conditional stabilization of CRISPR-Cas9 for precision therapy of inflammatory disease，基因组编辑前药：CRISPR-Cas9的靶向给药和条件性稳定，用于炎症疾病的精确治疗。

Genome-editing prodrug: Targeted delivery and conditional stabilization of CRISPR-Cas9 for precision therapy of inflammatory disease

基因组编辑前药：CRISPR-Cas9的靶向给药和条件性稳定，用于炎症疾病的精确治疗

重点词汇

conditional 有条件的 ; 条件的 ; 附带条件的 ; 依…而定的 ; 条件句，条件从句(由if或unless引起的) ; 动词的条件式(如should用在If I should die...)

stabilization 固定 ; 币值的稳定

precision 精确 ; 准确 ; 细致 ; 精确的

therapy 治疗 ; 疗法

Regulation of CRISPR-Cas9 functions in vivo is conducive to developing precise therapeutic genome editing.

在体内调控CRISPR-Cas9的功能有助于发展精确的治疗基因组编辑。

Here, we report a CRISPR-Cas9 prodrug nanosystem (termed NanoProCas9), which combines the targeted delivery and the conditional activation of CRISPR-Cas9 for the precision therapy of inflammatory bowel disease.

在这里，我们报告了一种CRISPR-Cas9前药纳米系统（称为NanoProCas9），它结合了CRISPR-Cas9的靶向给药和条件性激活，用于炎症性肠病的精确治疗。

NanoProCas9 is composed of (i) cationic poly(β-amino ester) (PBAE) capable of complexing plasmid DNA encoding destabilized Cas9 (dsCas9) nuclease, (ii) a layer of biomimetic cell membrane coated on PBAE/plasmid nanocomplexes for the targeted delivery of PBAE/dsCas9 complexes, and (iii) the stimuli-responsive precursory molecules anchored on the exofacial membrane.

NanoProCas9由（i）阳离子聚（β-氨基酯）（PBAE）组成，能够络合编码不稳定Cas9（dsCas9）核酸酶的质粒DNA，（ii）在PBAE/质粒纳米复合物上涂覆一层仿生细胞膜，用于定向递送PBAE/dsCas9复合物，以及（iii）刺激响应的前驱分子锚定在外表面膜上。

The systemic administration of NanoProCas9 enables the targeted delivery of dsCas9 plasmid into inflammatory lesions, where the precursory small molecule can be activated by ROS signals to stabilize expressed dsCas9, thereby activating Cas9 function for inflammatory genome editing.

系统给药NanoProCas9可以将dsCas9质粒定向递送到炎症病灶中，在炎症病灶中，前体小分子可以被ROS信号激活，以稳定表达的dsCas9，从而激活Cas9的炎症基因组编辑功能。

The proposed “genome-editing prodrug” presents a proof-of-concept example to precisely regulate CRISPR-Cas9 functions by virtue of particular pathological stimuli in vivo.

提出的“基因组编辑前药”提供了一个概念验证示例，通过体内特定的病理刺激精确调节CRISPR-Cas9功能。

重点词汇

in vivo 在生物体内进行的

inflammatory bowel disease 炎性肠病

composed of 由…组成

capable of 可以…的 ; 能…的 ; 易…的 ; 敢于…的

cell membrane 细胞膜

In this study, we developed a conditionally activatable CRISPR-Cas9 nanoassembly, which serves as prodrug for precise inflammatory genome editing.

在这项研究中，我们开发了一种条件激活的CRISPR-Cas9纳米组装体，作为精确炎症基因组编辑的前药。

As a genome-editing prodrug, whereas dsCas9 is subjected to ubiquitin-dependent proteasomal degradation in nonspecific tissues, it becomes stabilized and functional after being delivered to the inflammatory lesion, where the precursory small-molecule stabilizer can be released under the ROS stimuli to transform into its active form.

作为一种基因组编辑前药，dsCas9在非特异性组织中受到泛素依赖的蛋白酶体降解，但在传递到炎症病变后，其前体小分子稳定剂可在ROS刺激下释放并转化为活性形式，从而稳定并发挥功能。

The conditional activation by NanoProCas9 could afford deep-tissue genome editing, which well avoids previous challenges in the penetration depth that the optical regulation of CRISPR-Cas9 suffers.

NanoProCas9的条件激活可以承担深层组织基因组编辑，这很好地避免了之前CRISPR-Cas9光学调节在渗透深度方面所面临的挑战。

The MM not only plays an important role in directing NanoProCas9 to the inflammatory lesion but also serves as a supporter for anchoring small-molecule stabilizers.

MM不仅在将NanoProCas9引导到炎症病变中起着重要作用，而且还是锚定小分子稳定剂的载体。

Thus, the systemic administration of NanoProCas9 enables the targeted delivery of dsCas9 plasmid into inflammatory lesions, where the precursory small molecule can be transformed into its active form under the ROS signals to stabilize expressed dsCas9.

因此，系统给药NanoProCas9可以使dsCas9质粒靶向传递到炎症病灶中，在炎症病灶中，前体小分子可以在ROS信号下转化为其活性形式，以稳定表达的dsCas9。

This also explains why NanoProCas9-mediated genome editing presents in a site-specific manner and can be activated exclusively in the inflammatory lesions.

这也解释了为什么NanoProCas9介导的基因组编辑以位点特异性的方式出现，并且仅在炎症病变中被激活。

重点词汇

inflammatory lesion 炎性病变

transform into 把…转变成…

active form 【医】激活型

but also 而且

A number of previous investigations suggest that MM-coated delivery vesicles could preferentially target inflammation lesions; however, we still found that a large portion of these biomimetic nanoparticles were trapped by MPS in the liver (followed by lung and spleen) when the plasmid encoding wild-type Cas9 was delivered, causing considerable nontargeted, nonspecific genome editing at these organs.

此前的一些研究表明，MM涂层的传递囊泡可优先靶向炎症病变;然而，我们仍然发现，当编码野生型Cas9的质粒被传递时，这些仿生纳米颗粒中的很大一部分被MPS困在肝脏(其次是肺和脾)，导致这些器官中大量非靶向、非特异性的基因组编辑。

So far, several other effective approaches are reported to alleviate nonspecific genome editing in the liver. For example, Siegwart and coworkers reported a selective organ targeting (SORT) strategy by which lipid-based nanoparticles with the addition of a component (SORT molecule) can selectively deliver Cas9 mRNA to the target tissue, which represents an appealing way to minimize nonspecific editing in the liver.

到目前为止，已经报道了几种其他有效的方法来减轻肝脏中的非特异性基因组编辑。例如，Siegwart及其同事报道了一种选择性器官靶向（SORT）策略，通过该策略，添加成分（SORT分子）的脂质纳米粒可以选择性地将Cas9 mRNA传递到靶组织，这是一种减少肝脏非特异性编辑的有吸引力的方法。

Another possible strategy to avoid nonspecific editing in the liver is to express Cas9 with an inducible promoter, or a promoter that is active only in specific biological contexts.

另一种避免肝脏非特异性编辑的可能策略是用可诱导启动子或仅在特定生物学环境中有效的启动子表达Cas9。

Unfortunately, the nonspecific genome editing in the liver is largely unexplored in these reported systems.

不幸的是，在这些报道的系统中，肝脏中的非特异性基因组编辑在很大程度上尚未被探索。

In the current study, NanoProCas9, as a genome-editing prodrug system, can significantly decrease the nonspecific editing in the liver, with an indel frequency less than 3%.

在目前的研究中，作为基因组编辑前药系统的NanoProCas9可以显著减少肝脏中的非特异性编辑，indel频率低于3%。

These encouraging results suggest that the NanoProCas9 system is generally inert in the liver despite its nonspecific distribution.

这些令人鼓舞的结果表明，NanoProCas9系统在肝脏中通常是惰性的，尽管其分布非特异性。

重点词汇

A number of 一些 ; 许多的

biomimetic 仿生化的

followed by 紧随其后;其次是

nonspecific 非特异性的，有不止一种原因的 ; 不明确的;非特定的

So far 目前为止 ; 如此程度 ; 这个地步 ; 迄今为止

For example 例如 ; 比如

During our study, we did not observe the obvious side effects of local disruption of PHD2 in the colon tissue.

在我们的研究中，我们没有观察到结肠组织中PHD2局部破坏的明显副作用。

Although it was reported that systemic conditional knockout of PHD2 in adult animals could induce angiogenesis, erythrocytosis, and changes in energy metabolism with ultimately lethal consequences, the inflammation-specific genome editing meditated by NanoProCas9 can well evade the systemic disruption of PHD2, thereby greatly ensuring the safety profiles of genome editing at PHD2 locus.

虽然有报道称，在成年动物中系统性条件敲除PHD2可以诱导血管生成、红细胞生成和能量代谢变化，并最终导致致命后果，但NanoProCas9介导的炎症特异性基因组编辑可以很好地规避PHD2的系统性破坏。从而极大地保证了PHD2位点基因组编辑的安全性。

Besides, owing to the restricted nuclease activity to a narrow temporal window, the conditional activation of genome editing mediated by NanoProCas9 also significantly reduces off-target mutations, which is similar to our previous findings and in agreement with other reports.

此外，由于核酸酶活性受限，时间窗口较窄，NanoProCas9介导的条件激活基因组编辑也显著减少了脱靶突变，这与我们之前的发现相似，也与其他报道一致。

Regarding the clinical translation of NanoProCas9, the background activity of dsCas9 needs to be strictly limited. To address this issue, future efforts should be dedicated to screening different variants of dsCas9 by optimizing the number and position of DHFR domains in DHFR-fused Cas9 protein.

关于NanoProCas9的临床转译，需要严格限制dsCas9的背景活性。为了解决这个问题，未来的工作应该致力于通过优化DHFR融合Cas9蛋白中DHFR结构域的数量和位置来筛选dsCas9的不同变体。

Furthermore, the delivery of mRNA encoding dsCas9, instead of the plasmid form, may also contribute to lowering the background activity by shortening the time of dsCas9 expression.

此外，编码dsCas9的mRNA的传递，而不是质粒形式，也可能通过缩短dsCas9的表达时间，有助于降低背景活性。

These strategies are expected to boost the safety profiles of NanoProCas9 that are considered for clinical translation.

这些策略有望提高NanoProCas9的安全性，并被考虑用于临床转译。

重点词汇

did not 没有 ; 未 ; 的缩写

erythrocytosis 红细胞增多症

energy metabolism 能量代谢

meditated 冥想 ; 沉思 ; 暗自策划 ; 考虑 ; 谋划 ; meditate的过去分词和过去式

owing to 因为 ; 由于

nuclease 核酸酶

similar to 类似

in agreement with 同意，与…一致

background activity 自发活动, 本底放射性

In summary, as a proof-of-concept study, the NanoProCas9 system integrates targeted delivery and conditional activation of CRISPR-Cas9, offering a precise, site-specific therapeutic genome editing for inflammatory diseases.

总之，作为一项概念验证研究，NanoProCas9系统整合了CRISPR-Cas9的靶向传递和条件性激活，为炎症性疾病提供了精确的、位点特异性的治疗基因组编辑。

Such a system can avoid off-target mutations at nontargeted sites, thereby minimizing potential genotoxicity in those nonspecific tissues or organs.

这样的系统可以避免非靶点的脱靶突变，从而最大限度地降低这些非特异性组织或器官的潜在遗传毒性。

The NanoProCas9 system represents an innovative genome-editing prodrug and can be expanded to many other inflammatory diseases, such as lung/liver injury, atherosclerosis, and stroke.

NanoProCas9系统代表了一种创新的基因组编辑前药，可以扩展到许多其他炎症性疾病，如肺/肝损伤、动脉粥样硬化和中风。

By virtue of similar engineering principles, such an inducible CRISPR system may also be tailored for the clinical scenarios where the epigenetic regulation or RNA editing is required in addition to genome editing.

凭借类似的工程原理，这种可诱导CRISPR系统也可以针对临床场景进行定制，其中除了基因组编辑外，还需要表观遗传调控或RNA编辑。

Collectively, the current study offers new insights for the rational design of CRISPR-Cas–based systems for safe, precise genome editing to accelerate the clinical translation.

总之，当前的研究为基于CRISPR-Cas的系统的合理设计提供了新的见解，用于安全、精确的基因组编辑，以加速临床转译。

As a number of physiological and pathological signals, such as ATP, redox, and pH, can be leveraged to develop different types of genome-editing prodrugs, our current study opens a new avenue to precisely regulate CRISPR-Cas–based systems in vivo for more complicated and diverse genome-editing contexts.

由于许多生理和病理信号（如ATP、氧化还原和pH）可用于开发不同类型的基因组编辑前药，我们目前的研究为精确调节体内CRISPR-Cas系统提供了一条新途径，以适应更复杂和多样的基因组编辑环境。

重点词汇

nonspecific 非特异性的，有不止一种原因的 ; 不明确的;非特定的

such as 例如 ; 像 ; 象…这样 ; 诸如…之类

in addition to 另外，加之，除…之外

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