title: 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: Division Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden. Electronic address: cristina.rocha@ki.se.
authors: Cristina S J
authors: CS
authors: Rocha
authors: Division Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
authors: Oscar P B
authors: OP
authors: Wiklander
authors: Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
authors: Lilian
authors: L
authors: Larsson
authors: Division Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
authors: Pedro M D
authors: PM
authors: Moreno
authors: Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
authors: Paolo
authors: P
authors: Parini
authors: Division Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
authors: Karin E
authors: KE
authors: Lundin
authors: Division Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden. Electronic address: edvard.smith@ki.se.
authors: C I Edvard
authors: CI
authors: Smith
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D015870:Gene Expression; D020868:Gene Silencing; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D009841:Oligonucleotides / Q000235:genetics*; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D012313:RNA / Q000235:genetics*; D012326:RNA Splicing; D011973:Receptors, LDL / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D009841:Oligonucleotides; D011973:Receptors, LDL; D012313:RNA; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: Gene therapy; Hypercholesterolemia; PCSK9; RNA therapeutics; Splice modulation