Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy

Abstract

Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

3D Cell Cultures as Prospective Models to Study Extracellular Vesicles in Cancer

“…This strategy has described an increase of the avidity and specificity of cells to uptake the decorated EVs. For instance, HepG2 cells and human primary liver cancer-derived organoids accumulate more efficiently EVs that have been decorated with tetrahedral DNA nanostructures conjugated with DNA aptamer [ 80 ]. These EVs can effectively deliver an engineered cargo, which consists of CRISPR-Cas9 RNA-guided endonucleases, aiming to silence the expression of the protein Wnt-10b.…”

“…These EVs can effectively deliver an engineered cargo, which consists of CRISPR-Cas9 RNA-guided endonucleases, aiming to silence the expression of the protein Wnt-10b. In fact, these EVs inhibited the growth of tumoral cells in vitro [ 80 ]. Another strategy was to decorate methotrexate-loaded EVs with Lys-Leu-Ala bound to low-density lipoprotein peptides.…”

Nanocarriers, Progenitor Cells, Combinational Approaches, and New Insights on the Retinal Therapy

“…In recent years, studies on therapeutic effects of exosomes as carriers for gene delivery have been evaluated to maintain retinal homeostasis via immune-modulation for retinal degenerative diseases [ 36 ]. In the future, the use of exosomes as the carrier for CRISPR/Cas9 technology will remarkably promote the research of genetic disorders and retinal therapy [ 37 ].…”

Nanovesicle-Mediated Delivery Systems for CRISPR/Cas Genome Editing

“…The surfaces of exosomes have been modified for targeted delivery to specific cancer cell types ( Figure 4 B) [ 41 ]. In one example, the surfaces of HEK293T cell-derived exosomes were decorated with a cholesterol-tagged DNA aptamer.…”

“…( B ) DNA aptamer-conjugated extracellular vesicles for Cas9/sgRNA delivery. Adapted from [ 41 ], Oxford University Press, 2020. ( C ) Chimeric antigen receptor-extracellular vesicles for Cas9 ribonucleoprotein with complexed sgRNA.…”