RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form.

Abstract

Hypercholesterolemia is a medical condition often characterized by high levels of low-density lipoprotein cholesterol (LDL-C) in the blood. Despite the available therapies, not all patients show sufficient responses, especially those with very high levels of LDL-C or those with familial hypercholesterolemia. Regulation of plasma cholesterol levels is very complex and several proteins are involved (both receptors and enzymes). From these, the proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising pharmacologic target. The objective of this work is to develop a new approach to inactivate PCSK9 by splice-switching oligonucleotides (SSOs), converting the normal splice form to a natural, less abundant and inactive, splice variant. For this purpose, a new RNA therapeutic approach for hypercholesterolemia based on SSOs was developed for modulation of the splice pattern of human PCSK9 pre-mRNA. Our results show an increase of the selected splice form at both the mRNA and protein level when compared to non-treated Huh7 and HepG2 cell lines, with concomitant increase of the protein level of the low-density lipoprotein receptor (LDLR) demonstrating the specificity and efficiency of the system. In vivo, full conversion to the splice form was achieved in a reporter system when mice were treated with the specific oligonucleotide, thus further indicating the therapeutic potential of the approach. In conclusion, PCSK9 activity can be modulated by splice-switching through an RNA therapeutic approach. The tuning of the natural active to non-active isoforms represents a physiological way of regulating the cholesterol metabolism, by controlling the amount of LDL receptor available and the rate of LDL-cholesterol clearance.

• Authors: Cristina S J Rocha, Oscar P B Wiklander, Lilian Larsson, Pedro M D Moreno, Paolo Parini, Karin E Lundin, C I Edvard Smith
• Journal: Journal of molecular and cellular cardiology (J Mol Cell Cardiol) Vol. 82 Pg. 186-93 (May 2015) ISSN: 1095-8584 [Electronic] England
• PMID: 25791168 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015. Published by Elsevier Ltd.

Chemical References

• Oligonucleotides
• Receptors, LDL
• RNA
• PCSK9 protein, human
• Proprotein Convertase 9
• Proprotein Convertases
• Serine Endopeptidases

Topics

• Animals
• Cell Line
• Cell Survival (genetics)
• Gene Expression
• Gene Silencing
• Genes, Reporter
• Hepatocytes (metabolism)
• Humans
• Intracellular Space (metabolism)
• Mice
• Oligonucleotides (genetics)
• Proprotein Convertase 9
• Proprotein Convertases (genetics, metabolism)
• Protein Transport
• RNA (genetics)
• RNA Splicing
• Receptors, LDL (metabolism)
• Serine Endopeptidases (genetics, metabolism)
• Transfection