Abstract
Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.
Highlights
Homozygous familial hypercholesterolemia (HoFH) is an autosomal-dominant monogenic disease that affects more than 30 million people worldwide.[1]
low-density lipoprotein receptor (LDLR) activity plays a vital role in mediating proprotein convertase subtilisin/ kexin type 9 (PCSK9) inhibitor efficacy, such that HoFH patients with a bi-allelic mutant LDLR gene or patients with residual LDLR activity
Improve LDLR expression by vector optimization To further improve hLDLR expression from associated virus (AAV) vectors containing the liver-specific thyroxine-binding globulin (TBG) promoter, we evaluated the effects of a different intron sequence, intervening sequence 2 (IVS2) of the human beta-globin gene or the WPRE
Summary
Homozygous familial hypercholesterolemia (HoFH) is an autosomal-dominant monogenic disease that affects more than 30 million people worldwide.[1]. Homozygous patients with mutations affecting both alleles of the LDLR gene have very high low-density lipoprotein cholesterol (LDL-C) concentrations, exhibit premature coronary atherosclerosis, and develop cardiovascular disease before 30 years of age. For most HoFH patients, the current lipid-lowering therapies, such as plasmapheresis, LDL apheresis, or liver transplantation, and cholesterol-lowering drugs (e.g., high-dose statins) are not able to reduce LDL-C to normal levels and have adverse side effects.[4,5,6] In recent years, PCSK9 inhibitors, such as evolocumab and alirocumab, have emerged as the newer generation of lipid-lowering drugs and can significantly reduce LDL-C in HeFH and non-familial hypercholesterolemia (non-FH) patients.[7] the results have been variable in HoFH patients,[8] and new therapeutic strategies are being sought.
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