An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells

Ye, Yangyang; Zhang, Xiang; Xie, Fei; Xu, Bin; Xie, Ping; Yang, Ting; Shi, Qian; Zhang, Chenyu; Zhang, Yujing; Chen, Jiangning; Jiang, Xiaohong; Li, Jing

doi:10.1039/d0bm00427h

Cited by 123 publications

(108 citation statements)

References 38 publications

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“…Guidance of signature sequence In this method, also called active loading, mRNA can be entrapped into the exosomes by employing some helper proteins. As we know, some proteins enable to bind to specific RNA sequences, Packaging of mRNA inside exosome can be easily guided through the fusion of the structural and specific proteins of exosome and creating an engineered cell [101,102]. This method increases the efficiency of mRNA loading into the exosome [97].…”

Section: Exosome Loadingmentioning

confidence: 99%

Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope

et al. 2021

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Over the past decade, therapeutic messenger RNAs (mRNAs) have emerged as a highly promising new class of drugs for protein replacement therapies. Due to the recent developments, the incorporation of modified nucleotides in synthetic mRNAs can lead to maximizing protein expression and reducing adverse immunogenicity. Despite these stunning improvements, mRNA therapy is limited by the need for the development of safe and efficient carriers to protect the mRNA integrity for in vivo applications. Recently, leading candidates for in vivo drug delivery vehicles are cell-derived exosomes, which have fewer immunogenic responses. In the current study, the key hurdles facing mRNA-based therapeutics, with an emphasis on recent strategies to overcoming its immunogenicity and instability, were highlighted. Then the immunogenicity and toxicity of exosomes derived from various cell sources were mentioned in detail. Finally, an overview of the recent strategies in using exosomes for mRNA delivery in the treatment of multiple diseases was stated.

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Section: Exosome Loadingmentioning

confidence: 99%

Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope

et al. 2021

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“…CD63 cDNA was amplified and cloned into the pEGFP‐N1 vector (Addgene, MA, USA) as described previously (Ye et al., 2020). Then, plasmid for CD63‐EGFP expression was transfected into cells.…”

Section: Methodsmentioning

confidence: 99%

Pancreatic β cells control glucose homeostasis via the secretion of exosomal miR‐29 family

Zhang

et al. 2021

J of Extracellular Vesicle

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Secreted microRNAs (miRNAs) are novel endocrine factors that play essential pathological and physiological roles. Here, we report that pancreatic β cell-released exosomal miR-29 family members (miR-29s) regulate hepatic insulin sensitivity and control glucose homeostasis. Cultured pancreatic islets were shown to secrete miR-29s in response to high levels of free fatty acids (FFAs) in vitro. In vivo, high levels of FFAs, promoted by either high-fat diet (HFD) feeding (physiopathological) or fasting (physiological), increased the secretion of miR-29s into plasma. Intravenous administration of exosomal miR-29s attenuated insulin sensitivity. The overexpression of miR-29s in the β cells of transgenic (TG) mice promoted the secretion of miR-29s and inhibited the insulin-mediated suppression of glucose output in the liver. We used selective overexpression of traceable heterogenous mutant miR-29s in β cells to confirm that islet-derived exosomal miR-29s target insulin signalling in the liver and blunt hepatic insulin sensitivity. Moreover, in vivo disruption of miR-29s expression in β cells reversed HFD-induced insulin resistance. In vitro experiments demonstrated that isolated exosomes enriched in miR-29s inhibited insulin signalling in the liver and increased hepatic glucose production. These results unveil a novel β cellderived secretory signal-exosomal miR-29s-and provide insight into the roles of miR-29s in manipulating glucose homeostasis.

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“…108 Another study found that sgRNA and Cas9 (although not Cas9 mRNA) can be packaged into exosomes and form a functional sgRNA:-Cas9 RNP complex, which can be delivered to target cells. 109 The researchers also engineered a modified exosome by fusing CD63, a membrane protein often found on exosomes, with GFP and used GFP nanobody-fused Cas9 since it readily binds with the CD63-GFP for more efficient loading of sgRNA:Cas9 RNPs into the exosome. The modified exosome delivered larger quantities of sgRNA and Cas9 proteins without altering the exosome morphology or the function of the CRISPR-Cas9 system and successfully removed the stop sequence in A549 cells containing stop-DsRed sequences, resulting in greater red fluorescent signals than their unmodified counterparts.…”

Section: Delivering Rna Therapies To the Cf Lung Is Challengingmentioning

confidence: 99%

Treating Cystic Fibrosis with mRNA and CRISPR

et al. 2020

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Less than 20% of the protein coding genome is thought to be targetable using small molecules. mRNA therapies are not limited in the same way since in theory, they can silence or edit any gene by encoding CRISPR nucleases, or alternatively, produce any missing protein. Yet not all mRNA therapies are equally likely to succeed. Over the past several years, an increasing number of clinical trials with siRNA-and antisense oligonucleotide-based drugs have revealed three key concepts that will likely extend to mRNA therapies delivered by nonviral systems. First, scientists have come to understand that some genes make better targets for RNA therapies than others. Second, scientists have learned that the type and position of chemical modifications made to an RNA drug can alter its therapeutic window, toxicity, and bioavailability. Third, scientists have found that safe and targeted drug delivery vehicles are required to ferry mRNA therapies into diseased cells. In this study, we apply these learnings to cystic fibrosis (CF). We also describe lessons learned from a subset of CF gene therapies that have already been tested in patients. Finally, we highlight the scientific advances that are still required for nonviral mRNA-or CRISPR-based drugs to treat CF successfully in patients.

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An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells

Abstract: CRISPR-Cas9 components delivered by engineered exosomes achieve genome editing in recipient cells.

Cited by 123 publications

References 38 publications

Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope

Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope

Pancreatic β cells control glucose homeostasis via the secretion of exosomal miR‐29 family

Treating Cystic Fibrosis with mRNA and CRISPR

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