Date Published: January 30, 2018
Publisher: John Wiley and Sons Inc.
Author(s): Yao Lin, Jiahua Wu, Weihuai Gu, Yulei Huang, Zhongchun Tong, Lijia Huang, Jiali Tan.
Targeted delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system to the receptor cells is essential for in vivo gene editing. Exosomes are intensively studied as a promising targeted drug delivery carrier recently, while limited by their low efficiency in encapsulating of large nucleic acids. Here, a kind of hybrid exosomes with liposomes is developed via simple incubation. Different from the original exosomes, the resultant hybrid nanoparticles efficiently encapsulate large plasmids, including the CRISPR–Cas9 expression vectors, similarly as the liposomes. Moreover, the resultant hybrid nanoparticles can be endocytosed by and express the encapsulated genes in the mesenchymal stem cells (MSCs), which cannot be transfected by the liposome alone. Taken together, the exosome–liposome hybrid nanoparticles can deliver CRISPR–Cas9 system in MSCs and thus be promising in in vivo gene manipulation.
Gene therapy is considered as a promising and radical treatment for diseases like cancers and inherited disorders.1 Since its innovation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system has been recognized as the most promising gene‐editing and gene‐regulation technique.2 The CRISPR/Cas9 system is an adaptive immunological response found in archaea and bacteria preventing invasion like viruses and plasmids.3 The engineered CRISPR/Cas system works as a Cas9 nuclease‐single guide RNA (sgRNA) complex. The sgRNA recognizes the complementary 20‐nucleotide genomic sequence, and Cas9 nuclease generates double‐strand DNA breaks three bases upper stream of the protospacer adjacent motif of the target gene, resulting in gene deletion, insertion, and mutation by error‐prone nonhomologous end‐joining or precise homology‐directed repair.[[qv: 2a,4]] Besides, CRISPR/Cas9 system can also be engineered to regulate gene expression via fusing the gene regulator to the dead Cas9 (dCas9), in which the two catalytic domains of Cas9, RuvC and HNH, are inactivated and thus have no cleavage activity.5
In summary, we produced hybrid exosomes via simple incubating exosomes with liposomes and successfully delivered CRISPR–Cas9 system in MSCs via hybrid exosomes for the first time. The application of CRISPR/Cas9 system has been a significant breakthrough in gene therapy remaining intractable over decades. Researchers have demonstrated the possibility of using CRISPR/Cas9 system to cure various genetic diseases such as cancers and inherited disorders via repairing, deleting, or silencing certain genetic mutations relating to the diseases in vivo or even to clinical trials in the future.27 For example, one study revealed that CRISPR/Cas9 system could treat Duchenne muscular dystrophy via deleting exon 23 of the dystrophin gene and improving muscle function in a mouse model.[[qv: 27c]] However, there still exist some challenges when it comes to applying in clinical. One major difficulty is the lack of more efficient delivery and safer delivery system. Although virus vectors like AAV have shown great efficacy in CRISPR/Cas9 system delivery, people still concern about the immunogenic response and long‐term expression of virus vectors, which prevent the wider applications of virus vectors in clinical. In this study, we attempted different approaches to encapsulate CRISPR–Cas9 system into exosomes and found out that the proposed hybrid exosome via incubating with liposomes could be a new strategy for drug encapsulating and delivering CRISPR–Cas9 system in vivo or in transfection resistant cells in vitro.
Cell Culture: HEK293FT cells and human MSCs were cultured in high‐glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under 37 °C and 5% CO2. The cells were passaged at a ratio of 1:8 at 90% confluency.
The authors declare no conflict of interest.