Poster J7 - Polymersomes with siRNA Targeting PSEN1 as Non-invasive Alzheimer’s Disease Therapy

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kati Lorenz

Undergraduate Student
Clemson University
Taylors, South Carolina, 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: Alzheimer’s disease (AD) causes rapid, degenerative memory loss which decreases quality of life. The average diagnosis leads to fatality after 4 to 8 years. Currently there are 7 million+ people in the United States with AD, and this number is increasing. Current treatments are focused on delaying symptoms and not curative.

Within the brain, the PSEN1 gene acts within the γ-secretase pathway that when mutated, creates amyloid-beta plaque accumulations within the brain. This leads to early onset of AD. PSEN1 has been seen to enhance tumors within the brain as well. The overarching goal of this project is to deliver siRNA to targeted cerebral tissues to induce a silencing effect on PSEN1 as a therapeutic approach. Targeting the neural tissue is difficult due to the presence of the blood-brain barrier (BBB), which prevents non-invasive transport of >98% of small molecules into the brain. Polymersomes, amphiphilic in nature, are versatile structures that can be modified to respond to pathologic stimuli and are capable of encapsulating peptides, proteins, mRNA, and siRNA. We can also modify polymersomes to deliver therapeutics across the BBB through the addition of brain endothelial cell targeting ligands. In this case, polymersomes can be modified to cross the BBB to release a siRNA payload within brain tissue to induce the silencing effect.

Materials and

Methods: Polymersomes made from polyethylene glycol (PEG, MW=1000 Da)-b-polylactic acid (PLA, MW= 5000 Da) were synthesized using the solvent injection method and lyophilized with 2 wt% mannitol. 100nM of siGLO, a non-targeting fluorescent siRNA, was encapsulated within 10 mg of synthesized polymersomes, and encapsulation efficiency and loaded content were calculated using measured unencapsulated fluorescence. U87 MG cells were grown in T75 flasks, and a dose escalation study was done to quantify the amount of siGLO and polymer needed for sufficient uptake within the cells. Flow cytometry was completed to quantify the amount of U87 cells that were successfully dosed. Early results indicate that we can deliver active siRNA to PSEN1.

Results, Conclusions, and Discussions: PEG-b-PLA polymersomes were synthesized in repeatable quantities, with a diameter of 117.6 ± 13.5 nm, a stable surface charge of -21.8 ± 5.0, and a uniform polydispersity index of 0.06 ± 0.02. Due to the promising characterization of PEG-PLA polymersomes, they were used to encapsulate both fluorescein and siGLO within the hydrophilic core. Encapsulation efficiency of 100 nM siGLO within the synthesized polymersomes can be seen within Table 1. Since the amount of siGLO encapsulated was smaller than the amount of fluorescein, finding optimal dosage amounts of polymersomes is needed. Dosing with 2.6E-5 mg of siGLO only led to 3% fluorescence within U87 cells, so dose escalation was conducted to find the optimal dosage of siGLO for uptake. After completing dose escalation of loaded polymersomes, it was found that the amount of siGLO encapsulated within the polymersomes needs to be revisited.

Given high encapsulation efficiency of the small amount of siGLO, it is seen that polymersomes can be used to transport siRNA to cells. Polymersomes can be uptaken into U87 cells; however, loaded concentration within polymersomes must be higher to see optimal results. After optimizing siGLO internalization, siRNA for the PSEN1 gene will be encapsulated and delivered, confirmed via western blot and RT-qPCR.

Acknowledgements (Optional):