Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles

Abstract

The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases. However, the inability to perform specific gene editing in targeted tissues and cells, which may cause off-target effects, is one of the critical bottlenecks for therapeutic application of CRISPR/Cas9. Herein, macrophage-specific promoter-driven Cas9 expression plasmids (pM458 and pM330) were constructed and encapsulated in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN). The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo. More importantly, after further encoding a guide RNA targeting Ntn1 (sgNtn1) into the plasmid, the resultant CLAN successfully disrupted the Ntn1 gene in macrophages and their precursor monocytes in vivo, which reduced expression of netrin-1 (encoded by Ntn1) and subsequently improved type 2 diabetes (T2D) symptoms. Meanwhile, the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by the CD68 promoter. This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/Cas9 system.

Delivery of CRISPR/Cas9 for therapeutic genome editing

“…In this study, cationic lipid‐assisted PEG‐ b ‐PLGA nanoparticles (CLAN) were used to pack the plasmids, pM330 or pM458, that contain the human CD68 promoter to drive gene expression in specific cells (monocytes and macrophages). The mice were treated by intravenous injection of CLANs to disrupt the expression of netrin‐1 genes in monocytes and macrophages specifically, and the results obtained suggested that this could reduce macrophage retention and ameliorate the profiles of glucose tolerance and insulin sensitivity to control T2D (Figure D) …”

“…(D) Macrophage‐specific in vivo gene editing using cationic lipid‐assisted polymeric nanoparticles. Reproduced with permission…”

“…The mice were treated by intravenous injection of CLANs to disrupt the expression of netrin-1 genes in monocytes and macrophages specifically, and the results obtained suggested that this could reduce macrophage retention and ameliorate the profiles of glucose tolerance and insulin sensitivity to control T2D ( Figure 5D). 122 Recently, Liang et al 132 126 Recently, a facile method to construct a multifunctional vector that can deliver CRISPR/Cas9 plasmids into cancer cells was also developed. The gene-editing tools were delivered into cancer cells for the disruption of CTNNB1 genes.…”

Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy

“…With the guidance of the sgRNA-targeting Ntn1 gene, the CLANs specifically edit the Ntn1 gene only in macrophages andr educe the expression of netrin1a nd improve typeIIdiabetes symptomsinv ivo (Figure3C). [35] Al arge number of polymer-based nanoparticles are useful for the deliveryo fg ene-editingt oolboxes.P olyethyleneimine (PEI) is ap opular polymer for CRISPR/Cas9d elivery.F or instance, ag raphene oxide-polyethyleneg lycol-polyethyleneimine nanocarrier can load Cas9/gRNAt hrough physical absorptionand p-stacking interactions. This method achievesefficient gene editing in AGS cells.…”

Advances in CRISPR/Cas9 Technology for <i>in Vivo</i> Translation

“…) A recent study by Finn et al used biodegradable lipid nanoparticles complexed with Cas9-mRNA and sgRNA to knock-out the mouse Transthyretin (TTR) gene in the liver, the murine homolog of a therapeutic target for amyloidosis in humans 50). …”