(598g) Engineering CRISPR-Cas9 Plasmid Loaded PLGA Nanoparticles to Repair Mutations in the Tlr4 Gene in Mice
Previous gene transfection approaches have used both chemical and physical stimuli, but these often destabilize cellular membranes resulting in unacceptable cytotoxicity especially in test animals.1 We hypothesize that using polymer nanoparticles will protect the plasmid from degradation en route and increase delivery into the cell without the harmful side effects seen using other 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 correct a single point mutation in the Tlr4 gene in mice that cause them to not react to endotoxins, such as lipopolysaccharides (LPS). The PLGA carrier is also fluorescently labeled by encapsulation of a commercially available fluorophore, 6,13-Bis(triisopropylsilylethynl) pentacene (TIPS pentacene), that had not been used before in the biomedical field until recently.3
However, before even getting to cell studies, the particles must first be appropriately designed, fabricated, and characterized to ensure that the requirements of the application are met. The focus of the research to be presented is on the particle processing and engineering that precedes the cell and animal studies. This work addresses the questions: What is the best solvent system to use for particle fabrication? What are the resulting sizes and zeta potentials? What is the composition of the polymeric carrier as well as what was successfully loaded? How can the particles be processed for long-term storage stability? Once these are first answered, then the method can be optimized to increase DNA loading. Initial studies showed a loading of 0.61 wt% of the ~8500 bp 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 4500 bp.2 From the promising framework set up, 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.
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.