Abstract 5816: An effective delivery platform for nucleic acid therapeutics: Enhanced cancer cell internalization and endosomal escape
Authors: Li Siqi , Tietz Ole , El-Sagheer Afaf H. , Brown Tom , Vallis Katherine A. 
Published: March 2024

Abstract
Nucleic acid therapeutics (NATs), such as siRNA and mRNA, show great potential as cancer medicines. However, translation of these compounds has been held back by their limited bioavailability in target tissues. The aim of this study was to test the use of a novel cell-penetrating peptide (CPP)-based delivery platform to enhance uptake of NAT into cancer cells. This platform consists of a highly efficient CPP that combines the advantages of multimerization and cyclization of the archetypal CPP, TAT peptide1, plus a photochemical (PC) strategy to promote endosomal release of NAT into the cytoplasm2. Negatively charged NATs form complexes with cationic CPPs through charge:charge interaction. NATs (siRNA or mRNA) were incubated with CPP at different molar ratios and assessed in gel retardation assays. Efficiency of internalization of Cy3-NAT:CPP complexes was evaluated using live cell confocal imaging with quantification of intracellular fluorescence. The mechanism of cellular uptake of NAT:CPP was investigated using inhibitors of endocytosis (chlorpromazine, NaN3, amiloride and methyl-β-cyclodextrin). To trigger NAT release from intracellular vesicles, 420 nm/50 mA LED light irradiation was applied following pre-treatment of cells with a photosensitizer, fimaprofin. CPP-PC mediated internalization and endosomal release was used for delivery of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA. GAPDH enzymatic activity assays (Sigma, USA) were conducted to confirm inhibition of GAPDH in HeLa, HEK293T and MDA-MB-231 cells. Transfection efficiency of mRNA encoding enhanced green fluorescence protein (eGFP) was evaluated by flow cytometry in HeLa cells. NAT:CPP complexes were found to be maximally efficient at a 10:1 molar ratio. Analysis of live cell confocal images indicated that GAPDH Cy3-siRNA:CPP complexes were more efficiently internalized compared to Cy3-siRNA + monomeric TAT or Cy3-siRNA + lipofectamine (****p&lt;0.0001, one-way ANOVA). The combined CPP-PC platform delivers NAT via a clathrin-mediated endocytosis pathway. CPP-PC delivery of GAPDH siRNA caused marked GAPDH enzyme activity downregulation in HeLa (91%), MDA-MB-231 (51%) and HEK293T (44%) cells. These results were similar to the level of downregulation achieved with lipofectamine + GAPDH siRNA. Flow cytometric analysis revealed that 52.4% of HeLa cells expressed eGFP at 1 h following delivery of mRNA-eGFP by CPP-PC. Functional siRNA and mRNA were efficiently delivered into the cytoplasm of cancer cells through combined use of a multimeric/cyclized CPP plus photochemical-mediated endosomal release. This approach holds promise for clinical application, particularly in the treatment of intraluminal tumors. 1. Tietz, O. et al. Nat Chem. 14: 284-93, 2022. 2. Selbo, P. K. et al. J Control Release. 148, 2-12, 2010.
Citation Format: Siqi Li, Ole Tietz, Afaf H. El-Sagheer, Tom Brown, Katherine A. Vallis. An effective delivery platform for nucleic acid therapeutics: Enhanced cancer cell internalization and endosomal escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5816.