Using Nanoprecipitation to Design and Engineer PLGA Nanoparticle Carriers Loaded CRISPR-Cas9 Plasmids for Increased Delivery and Transfection

Authors: 
Jo, A., Macromolecules Innovation Institute
Ringel-Scaia, V., Virginia Tech
Allen, I., Macromolecules Innovation Institute
Davis, R. M., Virginia Tech
One of the principal problems in implementing CRISPR-Cas9 in gene therapy applications is the difficulty in effective delivery of high molecular weight plasmid DNA into cells in vivo. Previous gene transfection approaches have used both chemical and physical stimuli, but these often destabilize cellular membranes resulting in unacceptable cytotoxicity.1 Polymer nanoparticles can protect the plasmid from degradation en route and increase delivery into the cell without the harmful side effects seen using more traditional methods for transfection.

Using an FDA-approved polymer and a modified nanoprecipitation method2, a poly(lactic-co-glycolic acid) (PLGA) nanoparticle carrier was engineered to deliver a CRISPR-Cas9 plasmid designed to induce a single point mutation in the mouse Tlr4 gene with the intention of inducing a loss of immune response to stimuli, such as lipopolysaccharides (LPS). The PLGA carrier was also fluorescently labeled by encapsulation of a commercially available fluorophore, 6,13-Bis(triisopropylsilylethynl) pentacene (TIPS pentacene), previously used in similar polymeric nanoparticles and shown to effectively visualize successful uptake of particles into macrophages.3 This previous work suggests that the PLGA particles will also be readily phagocytosed by macrophages and be an excellent candidate to evaluate the release of payload plasmid within the cell.

The main focus of this presentation will be on the particle processing and engineering that preceded the cell and animal studies. This work addresses the questions of solvent system, size and surface chemistry, carrier composition and DNA loading, as well as cell uptake and transfection efficacy. Initial studies demonstrate a loading of 0.61 wt% of the ~8.5 kb CRISPR-Cas9 plasmid using an amine end capped PLGA at an encapsulation efficiency of 82%. These results are already comparable to the typical 1 wt% loadings seen for DNA with only 4.5 kb.2 Following this promising framework, the next steps include increasing the target DNA loading, and studying the release kinetics of the biologic. This systematic design and characterization of the particles can be translated to a variety of different applications in the area of nanoparticle drug delivery and CRISPR-Cas9 gene therapy.

References

1. Bauer, M.; Kristensen, B. W.; Meyer, M.; Gasser, T.; Widmer, H. R.; Zimmer, J.; Ueffing, M., Toxic effects of lipid-mediated gene transfer in ventral mesencephalic explant cultures. Basic Clin Pharmacol 2006, 98 (4), 395-400.

2. Niu, X. M.; Zou, W. W.; Liu, C. X.; Zhang, N.; Fu, C. H., Modified nanoprecipitation method to fabricate DNA-loaded PLGA nanoparticles. Drug Dev Ind Pharm 2009, 35 (11), 1375-1383.

3. McDaniel, D. K.; Jo, A.; Ringel-Scaia, V. M.; Coutermarsh-Ott, S.; Rothschild, D. E.; Powell, M. D.; Zhang, R.; Long, T. E.; Oestreich, K. J.; Riffle, J. S.; Davis, R. M.; Allen, I. C., TIPS pentacene loaded PEO-PDLLA core-shell nanoparticles have similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in vivo microenvironments. Nanomedicine: Nanotechnology, Biology and Medicine 2017, 13 (3), 1255-1266.