Delivery of antisense oligonucleotides for splice‐correction of androgen receptor pre‐mRNA in castration‐resistant prostate cancer models using cell‐penetrating peptides

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. AONmediated 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

Deciphering Structural Determinants Distinguishing Active from Inactive Cell-Penetrating Peptides for Cytosolic mRNA Delivery

“…We have demonstrated before that PF14-mRNA polyplexes show efficient cellular uptake and cytosolic delivery, thereby yielding protein expression. ,, For hLF, we have previously addressed delivery of siRNA and antisense oligonucleotides for which the peptide failed to yield down-regulation of the target mRNA. , Therefore, we first compared both peptides with respect to cellular uptake and delivery of mRNA. At an N/P ratio of 3, PF14 and the monomeric wild-type hLF (Mono-hLF-WT), which was oxidized at conditions that favor intramolecular disulfide bond formation, readily formed monodisperse nanoparticles with mRNA with diameters of 47.9 ± 1.18 nm and 115 ± 1.03 nm (Table S2), respectively.…”

“…PepFect14 (PF14) is a peptide that yields efficient cytosolic delivery for various ONs in vitro and in vivo. − The peptide is amphipathic in nature and is N -terminally stearylated. PF14 is derived from PepFect3 (PF3), an analog of Transportan 10 (TP10), , with four out of five lysine residues replaced by the nonproteinogenic amino acid ornithine.…”

Introductory Chapter: Muscular Dystrophy and Potential Therapeutic Alternatives

“…With progress in ASO chemistry, reduced toxicity, and increased potency, exonskipping approaches have also been developed for other muscular conditions such as dysferlinopathy [6], sarcoglycanopathies [7], laminopathies [8] as well as for other diseases like cancer [9,10], Parkinson disease [11], and rheumatoid arthritis [12]. However, this approach is estimated to be costly and needs lifelong administration.…”

Alternative mRNA Splicing and Promising Therapies in Cancer

“…Likewise, a naturally occurring (i.e., mutation-independent) splice variant that results in a truncated variant of the androgen receptor ( AR-V7 ) is associated with castration-resistant prostate cancer. An antisense oligonucleotide designed to target an ISE of the AR pre-mRNA can shift the isoform balance toward full-length androgen receptor proteins in prostate cancer cells and can re-sensitize cells to androgen depletion [ 48 ]. ASOs may eventually become valuable components of therapeutic strategies as technologies progress.…”