Delivery of antisense oligonucleotides for splice‐correction of androgen receptor pre‐mRNA in castration‐resistant prostate cancer models using cell‐penetrating peptides. Issue 6 (31st January 2022)

• Record Type: Journal Article
• Authors: Luna Velez, Maria V.; Paulino da Silva Filho, Omar; Verhaegh, Gerald W.; van Hooij, Onno; El Boujnouni, Najoua; Brock, Roland; Schalken, Jack A.
• Is Part Of: Prostate. Volume 82:Issue 6(2022)
• Page Start: 657
• Page End: 665
• Publication Date: 2022-01-31
• Subjects: antisense oligonucleotides -- AR‐V7 -- castration‐resistant prostate cancer -- cell‐penetrating peptide -- PepFect 14; Prostate -- Diseases -- Periodicals; 616
• DOI: 10.1002/pros.24309
• Languages: English
• ISSNs: 0270-4137

Abstract

Abstract: Background: Cell‐penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor ( AR ) pre‐mRNA, thereby preventing the generation of the splice variant AR‐V7 mRNA. AON‐mediated knockdown of AR‐V7 resulted in inhibition of androgen‐independent cell proliferation. In this study, we evaluated the CPP‐mediated delivery of this AON into castration‐resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). Methods: Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter‐Glo cell viability assay. The effectivity of CPP‐mediated delivery of the splice‐correcting AON‐intronic splicing enhancer (ISE) targeting the ISE in the castration‐resistant prostate cancer (CRPC)‐derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR‐V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 ( HP1BP3 ). Western blot analysis was used to confirm AR‐V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. Results: The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine‐rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier‐associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 μM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON‐ISE directed against AR pre‐mRNA achieved significant downregulation of AR‐V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen‐independent cell proliferation of DuCaP and VCaP cells. Conclusions: This proof‐of‐principle study constitutes the basis for further development of CPP‐mediated delivery of AONs for targeted therapy in prostate cancer.