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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster H20 - Cas9-Mediated Gene Knockdown through Optimized Endosomal Escape of Polymeric Micelles

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sierra Niskanen

Undergraduate student
Clemson University
Greer, South Carolina, United States

Co-Author(s)

Lucian Williams (he/him/his)

Ph.D. Candidate
Clemson University
Central, South Carolina, United States
GB

George Barbery

Student
Clemson University, United States

Primary Investigator(s)

Jessica Larsen, PhD

Carol and John Cromer '63 Family Endowed Associate Professor
Clemson University Department of Chemical and Biomolecular Engineering, South Carolina, United States

Introduction: Gene-editing therapies have been growing in significance in recent years due to their potential to cure genetic diseases. Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) has been found to be the most popular gene editing technology due to its ability to efficiently gene edit with high specificity. However, the clinical application of these therapies has been limited by concerns regarding mutagenesis and immune responses during delivery. Non-viral vectors have shown legitimate treatment results in clinical applications, such as the non-viral COVID-19 vaccine. The aim of this project is to find a delivery vehicle that can safely encapsulate Cas9 ribonucleoprotein (RNP) and overcome translation limitations. We use self-assembling polymeric micelle nanoparticles to deliver Cas9 RNP to the precise location for gene editing. To enable endosomal escape, we have worked to optimize the addition of a positively charged peptide, Transactivator of transcription for the human immunodeficiency virus (TAT), on the surface of these micelles, testing their delivery and gene editing efficiency in HEK293-GFP cells. The overall objective is to advance the field of gene editing for therapeutic applications and breakthroughs in treating genetic diseases.

Materials and

Methods: We used a film rehydration method to self-assemble the block copolymer micelles from polyethylene glycol (PEG)-b-polylactic acid (PLA) either with or without TAT. When using maleimide bioconjugation chemistry, a positively charged peptide, TAT, was bound to enhance endocytosis and lysosomal escape. We varied the amount of TAT added to increase the positive charge. We also created Cas9 RNPs by combining the guide RNA with Cas9 in a 1:1 ratio. The polymers were dissolved in organic solvent which was evaporated using rotary evaporation. The polymer left a thin film around the vial and was rehydrated with DI water or PBS, containing Cas9 RNP when needed, for 18 to 24 hours. Micelles were characterized through dynamic light scattering (DLS) to determine the size, polydispersity index (PDI) and zeta potential. Transmission electron microscopy (TEM) was used to confirm micelle formation. The ability of the Cas9 RNP complex to knock out green fluorescent protein (GFP) in human embryonic kidney (HEK) 293-GFP cells was first evaluated using electroporation. Micelles were encapsulated with Cas9 RNP, and a gel shift assay was used to visualize and calculate the encapsulation efficiency.

Results, Conclusions, and Discussions: Micelle synthesis was optimized to obtain the desired size and PDI to maximize entry into cells. Table 1 summarizes the key optimization parameters of PEG-b-PLA micelles. Untagged PEG-b-PLA has an average diameter of 52.1 ± 0.37 nm with an average PDI of 0.121 ± 0.015, which was confirmed by TEM imaging (Figure 1). The addition of TAT increases the size of micelles when not all maleimide groups are bound; this is likely due to the aggregation of particles due to uneven surface charge. Untagged PEG-b-PLA had a surface charge of approximately -16 mV, whereas adding TAT increased the charge significantly. Table 1 shows that as the ratio of the PEG-b-PLA-maleimide in our micelle system increased, the charge did as well. On average, a ratio of 75% PEG-b-PLA-maleimide complex increased the charge to up to around 5 mV, and a ratio of 100% PEG-b-PLA-maleimide could increase the charge to 9 mV. In addition to a higher maleimide ratio increasing charge, higher volumes of TAT increased the charge because the maleimide then became fully saturated with TAT. Electroporation of gRNA(1)-GFP in the Cas9 RNP led to the greatest knockdown at 64% after three days, so we used this Cas9 in further studies. Using gel electrophoresis, we evaluated the encapsulation efficiency of Cas9 RNP (Figure 2). The TAT-tagged loaded micelles showed higher encapsulation efficiency of the Cas9 RNP, suggesting a TAT-tagged system increases electrostatic interactions due to the large positive charge of the TAT peptide and negative charge of the Cas9 RNP complex. In future studies, we would like to further explore cellular uptake of the micelle system using Nile Red within cells to visualize uptake, focusing on lysosomal escape. We will further quantify these results with flow cytometry for both TAT-tagged and untagged systems. In addition, we will study the fluorescent knockdown of HEK293-GFP after a three-day incubation period. This study suggests a strong trajectory towards enhancing the effectiveness of non-viral delivery methods for encapsulation and release of Cas9 RNP for gene editing therapies. The system shows favorable results regarding size and cellular uptake.

Acknowledgements (Optional):