Multistage Delivery Nanoparticle Facilitates Efficient CRISPR/dCas9 Activation and Tumor Growth Suppression In Vivo

Abstract

CRISPR/dCas9 systems can precisely control endogenous gene expression without interrupting host genomic sequence and have provided a novel and feasible strategy for the treatment of cancers at the transcriptional level. However, development of CRISPR/dCas9‐based anti‐cancer therapeutics remains challenging due to the conflicting requirements for the design of the delivery system: a cationic and membrane‐binding surface facilitates the tumor accumulation and cellular uptake of the CRISPR/dCas9 system, but hinders the circulating stability in vivo. Here, a multistage delivery nanoparticle (MDNP) that can achieve tumor‐targeted delivery of CRISPR/dCas9 systems and restore endogenous microRNA (miRNA) expression in vivo is described. MDNP is designed as a core‐shell structure in which the shell is made of a responsive polymer that endows MDNP with the capability to present different surface properties in response to its surrounding microenvironment, allowing the MNDP overcoming multiple physiological barriers and delivering the payload to tumor tissues with an optimal efficiency. Systemic administration of MDNP/dCas9‐miR‐524 to tumor‐bearing mice achieved effective upregulation of miR‐524 in tumors, leading to the simultaneous interferences of multiple signal pathways related to cancer cell proliferation and presenting remarkable tumor growth retardation, suggesting the feasibility of utilizing MDNP to achieve tumor‐targeting delivery of CRISPR/dCas9 with sufficient levels to realize its therapeutic effects.

Engineering a CRISPR Interference System To Repress a Class 1 Integron in Escherichia coli

“…For instance, based on experimentally validated sgRNA R3, a vehicle encompassing sgRNA R3 and the dCas9 could be prepared and delivered to desired settings and subsequently respond to cues such as pH (30), light (29,32), or magnetism (33) for eliciting a CRISPRi system. In fact, CRISPR delivery systems have been under construction recently (29,30,32,33). For instance, one group from Nanjing University attempted to cure a tumor by a near-infrared (NIR) light-responsive nano carrier of CRISPR-Cas9 (32).…”

“…Considering that integron-assisted MDR involves a series of genes, CRISPRi may simultaneously interfere with multiple ARGs and thus hold great potential for prevention of bacterial infection and MDR treatment. For instance, simply by encapsulating the dCas9 and a panel of sgRNAs into nanoparticles (29,30) and incubating these with bacteria for transformation, a wide range of ARGs could be inhibited, achieving desired therapeutic effects.…”

Perturbing Enhancer Activity in Cancer Therapy

“…Firstly, the lack of effective, safe and specific delivery options of the Cas9 system remains a major challenge in applying these machineries in therapy. One of the novel approaches currently under study to deliver the Cas9 system includes multistage delivery nanoparticle (MDNP), which still requires further validation [27]. Additionally, the use of permanent gene editing in humans faces numerous ethical complications.…”

Research progress on non-viral vectors of CRISPR/Cas9 system

“…这些研究均强 的优异性(图2(d)) [84] . Liu等 [85] [77,78,83,84] (网络版彩图) Figure 2 Non-viral vector for delivery of pDNA encoding the CRISPR/Cas9 system [77,78,83,84] . 同时, Zhang等 [95] 和Miller等 [96] 对常规脂质体进行了功能化改性, 实现 了对Cas9蛋白的mRNA在小鼠体内的高效投递的同 时, 有效降低了脂质体的生物毒性 (图3(a, b) .…”