Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR\u2013Cas9 plasmid

Ami Jo,Judy S Riffle,Cassidy A Thomas,Dylan K Mcdaniel,Irving C Allen,Richey M Davis,Rui Zhang,Veronica M Ringel-Scaia

doi:10.1186/s12951-019-0564-1

Abstract

BackgroundThe clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR–Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells.ResultsUsing poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR–Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2–3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells.ConclusionsIn this work, plasmids for the CRISPR–Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR–Cas9 system.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy
Solvent mixture used in particle formation Since 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene), DNA, and poly(lactic-co-glycolic acid) (PLGA) have very different solubility characteristics, a solvent mixture was needed to solubilize all three components and form welldefined nanoparticles when mixed with the aqueous Pluronic F-127 solution
Ke et al [15] used a mixture of 5 vol% tris-Ethylenediaminetetraacetic acid (TE) buffer and 95 vol% DMF and showed that plasmid DNA was Evaluation of nanoparticles made with different PLGA end groups show main difference to be in DNA loading For particles made with the ester-end capped PLGA, the intensity-average hydrodynamic diameters (Di) were ~ 160 nm while the diameters of particles made with the amine-end capped PLGA were slightly larger, Di ~ 180 nm with no significant change when DNA was encapsulated (Table 1)

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas protein system is a revolutionary tool for gene therapy. The clustered regularly interspaced short palindromic repeats (CRISPR)–Cas system has received much attention recently due to its potential for revolutionizing targeted genome editing with unprecedented precision and control. Efficient transfection of DNA or RNA into the cell for transcription is a significant hurdle. Both chemically- and mechanically-based transfection methods have been successfully utilized in vitro, but effective, clinically relevant, in vivo transfection methods are significantly lacking. Electroporation and sonoporation have been examined in vivo [6, 7] These approaches are not always feasible when attempting to transfect animals or humans due to accessibility limitations and invasiveness of the treatments. Adenoviral vectors are being studied as potential carriers for the CRISPR–Cas system and have shown great success in vitro [8, 9] yet the relatively large size of the CRISPR–Cas system and potential immunogenicity of adenoviral vectors have, for the moment, limited in vivo applications

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