Ami Jo1,2, Veronica M Ringel-Scaia3,4, Dylan K McDaniel4,2, Cassidy A Thomas4, Rui Zhang5,2, Judy S Riffle5,2, Irving C Allen6,7,8,9, Richey M Davis10,11,12. 1. Department of Chemical Engineering, Virginia Tech, Blacksburg, VA, 24061, USA. 2. Macromolecules Innovation Institute, Virginia Tech, Blacksburg, VA, 24061, USA. 3. Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, 24061, USA. 4. Department of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24061, USA. 5. Department of Chemistry, Virginia Tech, Blacksburg, VA, 24061, USA. 6. Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, 24061, USA. icallen@vt.edu. 7. Department of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24061, USA. icallen@vt.edu. 8. Macromolecules Innovation Institute, Virginia Tech, Blacksburg, VA, 24061, USA. icallen@vt.edu. 9. Department of Basic Science Education, Virginia Tech Carilion School of Medicine, Roanoke, VA, USA. icallen@vt.edu. 10. Department of Chemical Engineering, Virginia Tech, Blacksburg, VA, 24061, USA. rmdavis@vt.edu. 11. Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, 24061, USA. rmdavis@vt.edu. 12. Macromolecules Innovation Institute, Virginia Tech, Blacksburg, VA, 24061, USA. rmdavis@vt.edu.
Abstract
BACKGROUND: The 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. RESULTS: Using 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. CONCLUSIONS: In 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.
BACKGROUND: The 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. RESULTS: Using 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. CONCLUSIONS: In 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.
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