Exosome-Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs

Abstract

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.

Keywords: CRISPR/Cas9 system; exosomes; hybrid nanoparticles; targeted delivery.

Figure 1

Characterization of HEK293FT cell‐derived exosomes. A) Representative electron microscopy image of the exosomes isolated from HEK293FT cells. Scale bar, 100 nm. B) Size distribution of HEK293FT cell derived exosomes determined by dynamic light scattering. Data represent 20 measurements of four biological samples. C) The protein profile of HEK293FT cells and the exosomes secreted by HEK293FT cells were analyzed by silver blotting. Representative image of three different experiments. D) The protein levels of Alix, TSG101, CD63, CD81, CD9, and GAPDH in HEK293FT cell lysates and the exosomes secreted by HEK293FT cells were analyzed by western blot. Data presented were representative of three different experiments. E) Particle‐to‐protein ratio of exosomes isolated by ultracentrifugation, 12% PEG 6000 precipitation and 12% precipitation + 5% PEG 6000 reprecipitation. Data were expressed as mean ± standard deviation (SD) of three different experiments. *p < 0.05.

Figure 2

Liposome or exosome alone fails to deliver plasmids into MSC. A) FACS analysis of green fluorescence protein expression in MSCs with transfection of control or pEGFP‐C1 plasmids via liposomes. B) FACS analysis of FAM level in MSCs with or without incubation of exosomes electroporated with control or FAM conjugated small RNA. C) FACS analysis of green fluorescence protein expression in MSCs with incubation of exosomes electroporated with control or pEGFP‐C1 plasmids.

Figure 3

Assessment of hybrid exosomes encapsulating large plasmids. A) Illustration of the procedure to produce hybrid exosomes. The isolated exosomes were incubated with liposomes for 12 h at 37 °C to induce the fusion. B) Representative electron microscopy image of exosomes after incubation with liposomes for 12 h at 37 °C. Scale bar, 100 nm. C) Size distribution of exosomes, liposomes, and hybrid exosomes determined by dynamic light scattering. Data represent 20 measurements of four biological samples. D) Western blot analysis of the protein levels of Alix, TSG101, CD63, and GAPDH in hybrid exosomes. Data presented were representative of three different experiments. E) qRT‐PCR analysis of EGFP mRNA level in the MSCs with or without DNase treatment prior to incubation with pEGFP‐C1 plasmid‐only (NC), exosomes+pEGFP‐C1 plasmid (exosome), Lipofectamine2000+pEGFP‐C1 plasmid (liposome) and hybrid exosomes+pEGFP‐C1 plasmid (hybrid exosome). Data were expressed as mean ± SEM of three different experiments. *p < 0.05. F) qRT‐PCR analysis of EGFP mRNA level in the MSCs with incubation of pEGFP‐C1 plasmid‐only (NC) or hybrid exosome+pEGFP‐C1 (Hybrid exosome) with and without proteinase K treatment. Data were expressed as mean ± standard error of the mean (SEM) of three different experiments. *p < 0.05. G) FACS analysis of green fluorescence protein expression of MSCs with incubation of the nanoparticle same as (E). H) Cell viability of MSCs with incubation of the nanoparticles same as (E). Data were expressed as mean ± SD of three different experiments. *p < 0.05.

Figure 4

Construction of CRISPR/dCas9 based Runx2 expression intervention system. A) Diagram of Runx2 sgRNA expressing lentiviral vector. B) Diagram of dCas9 expressing lentiviral vector. C) Illustration of CRISPR/dCas9 based Runx2 gene expression intervention system. D) Sequencing results of sgRNA construct targeting Runx2 gene. E) qRT‐PCR analysis of Runx2 mRNA level in MSCs infected with the control or Runx2 guided CRISPR/dCas9 system. Data were expressed as mean ± SEM of three different experiments. *p < 0.05.

Figure 5

The hybrid exosomes successfully deliver CRISPR/dCas9 interference system. A) Illustration of procedure how the hybrid exosomes deliver the CRISPR/dCas9 interference system. B) qRT‐PCR analysis of sgRNA level in MSCs with control or Runx2 gRNA expressing hybrid exosomes. Control empty vector served as NC. Data were expressed as mean ± SEM of three different experiments. *p < 0.05. C) qRT‐PCR analysis of dCas9 mRNA level in MSCs with control or dCas9 expressing plasmids loaded hybrid exosomes. Control empty vector served as NC. Data were expressed as mean ± SEM of three different experiments. *p < 0.05. D) Runx2 mRNA level in MSCs with incubation of Runx2 guided CRISPR/dCas9 system‐only (NC), exosomes+ Runx2 guided CRISPR/dCas9 system (exosome), liposomes+ Runx2 guided CRISPR/dCas9 system (liposome), and hybrid exosomes+Runx2 guided CRISPR/dCas9 system (hybrid exosome). Data were expressed as mean ± SEM of three different experiments. *p < 0.05.

Figure 6

Construction of CRISPR/Cas9 based CTNNB1 cleavage system and its efficiency in editing gene via virus and hybrid exosome. A) Diagram of CTNNB1 sgRNA and Cas9 expressing lentiviral vector. B) Illustration of CRISPR/Cas9 based CTNNB1 cleavage system. C) Sequencing results of sgRNA construct targeting CTNNB1 gene. D) T7E1 assay results of MSCs infected with the control or CTNNB1 guided CRISPR/Cas9 system. E) T7E1 assay results of MSCs with incubation of CTNNB1 guided CRISPR/Cas9 system‐only (NC), exosomes+CTNNB1 guided CRISPR/Cas9 system (exosome), liposomes+CTNNB1 guided CRISPR/Cas9 system (liposome), and hybrid exosomes+CTNNB1 guided CRISPR/Cas9 system (hybrid exosome). Images are the representative of three different experiments.