(J-398) Delivery of CRISPR-Cas9 to HEK293-GFP Cells via Polymersomes for Gene Knockdown

A rapid increase in the prevalence of genetic diseases represents an alarming global burden. Current treatments are limited to addressing patient symptoms rather than treating the root cause. However, with the innovation of gene editing technologies, like gene therapy vectors and non-viral methods, scientists may now be able to develop one-time curative treatments. One such method demonstrating major potential as a gene editing tool is clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9). To overcome limitations associated with delivery of CRISPR-Cas9, we investigate the use of self-assembling polymer-based nanoparticles (polymersomes) as an effective encapsulation method and drug delivery system for the cas9 ribonucleoprotein (RNP) to allow for non-viral gene-editing across the blood-brain barrier (BBB).

Polyethylene glycol and poly(lactic-co-glycolic acid) (PEG-PLGA) polymersomes were synthesized through film rehydration. The nanoparticles were characterized through dynamic light scattering (DLS) to determine size, polydispersity index, and zeta potential. Polymersomes with RNP were tested for encapsulation efficiency with a bicinchoninic acid (BCA) assay. A positive charge, TAT, was included in polymersome synthesis. to enhance endocytosis and endosomal escape. The human embryonic kidney cell line (HEK293-GFP) was utilized in this study as it contains a green fluorescence protein. The effectiveness of the RNP was investigated through an electroporation study. HEK293-GFP and control HEK293 cells were plated in a 24 well-plate and a treatment of 0.5 μl RNP was administered to HEK293-GFP cells in triplicate. The 24-well plates were utilized for fluorescence microscopy which captured visual, qualitative fluorescence knockdown data. Flow cytometry was conducted for quantitative knockdown data.

Polymersome synthesis was optimized using sonication and filtration for desired size and polydispersity index (PDI) to allow delivery across the BBB. The results were uniform for the loaded and unloaded polymersomes. The RNP-loaded with, unloaded with TAT, and unloaded polymersomes were found to have an average size of 94.48 ± 4.55 nm with an average PDI of 0.217 ± 0.047 nm. A PDI < 0.3 indicates monodispersity and consistency of the polymersomes. Encapsulation efficiency of RNP determined through bicinchoninc acid (BCA) assay was found to have an average encapsulation efficiency of 5μg ± 1μg per 1 mL of polymersomes. Electroporation of HEK293-GFP cells showed noticeable fluorescent knockdown when compared to the controls after 72-hour incubation as pictured in Figure 1. Flow cytometry revealed an average fluorescent knockdown of 64%. The results confirm the effectiveness of RNP in disrupting GFP in HEK293-GFP cells. PEG-PLGA polymersomes are able to be formed at diameters capable of penetrating cellular membranes for intracellular delivery. TAT peptide was added to increase the surface charge. The BCA assay proves polymersomes to be an effective encapsulation vector for RNP. While electroporation confirms the effectiveness of RNP in fluorescent knockdown through qualitative images, flow cytometry provides quantitative data showing knockdown. Next steps involve cell studies with polymersome delivery of RNP and flow cytometry to quantify knockdown ad qPCR will be utilized to confirm double-stranded breaks. Additionally in vivo studies will be done to investigate fluorescent knockdown in the brains of zebrafish.

Thank you to Shoaib Iqbal, Sara Edgecomb, and Chlo Forenzo for laying the groundwork of this project. Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM139769. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.