AAV vectors expressing LDLR gain-of-function variants demonstrate increased efficacy in mouse models of familial hypercholesterolemia.

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.