AAV Vectors Expressing LDLR Gain-of-Function Variants Demonstrate Increased Efficacy in Mouse Models of Familial Hypercholesterolemia

• Published in: Circulation research Vol. 115; no. 6; pp. 591 - 599
• Main Authors: Somanathan, Suryanarayan, Jacobs, Frank, Wang, Qiang, Hanlon, Alexandra L., Wilson, James M., Rader, Daniel J.
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 29.08.2014
• Subjects: Animals; APOBEC-1 Deaminase; Cholesterol - metabolism; Cytidine Deaminase - genetics; Dependovirus - genetics; Disease Models, Animal; Genetic Therapy; Genetic Variation - genetics; Genetic Vectors - genetics; HEK293 Cells; Humans; Hyperlipoproteinemia Type II - genetics; Hyperlipoproteinemia Type II - metabolism; Hyperlipoproteinemia Type II - therapy; In Vitro Techniques; Male; Mice; Mice, Knockout; Proprotein Convertase 9; Proprotein Convertases - genetics; Receptors, LDL - genetics; Serine Endopeptidases - genetics; Treatment Outcome; hyperlipoproteinemia type II; low-density lipoprotein receptor, human; genetic therapy; PCSK9 protein, human
• ISSN: 0009-7330; 1524-4571; 1524-4571
• DOI: 10.1161/CIRCRESAHA.115.304008

RATIONALE:Familial hypercholesterolemia is a genetic disorder that arises because of loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous familial hypercholesterolemia is a candidate for gene therapy using adeno-associated viral vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen adeno-associated viral vector encoded LDLR expression. OBJECTIVE:We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 (hPCSK9) and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction. METHODS AND RESULTS:Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and human IDOL. A panel of mutant human LDLRs was initially screened in vitro for escape from PCSK9. The variant human LDLR-L318D was further evaluated using a mouse model of homozygous familial hypercholesterolemia lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme catalytic, APOBEC-1 (double knockout). Administration of wild-type human LDLR to double knockout mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9-mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\C818A construct avoided human IDOL regulation and achieved stable reductions in serum cholesterol. An adeno-associated viral vector serotype 8.LDLR-L318D\K809R\C818A vector that carried all 3 amino acid substitutions conferred partial resistance to both hPCSK9- and human IDOL–mediated degradation. CONCLUSIONS:Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve on a potential gene therapeutic approach to treat homozygous familial hypercholesterolemia subjects.