Novel molecular therapies for the treatment of familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is an autosomal dominant genetic disorder characterised by elevated levels of plasma low density lipoprotein (LDL) cholesterol. Loss-of-function mutations in the Low-Density Lipoprotein Receptor (LDLR) gene are responsible for ~85% of all FH cases. Heterozygous FH (HeFH) affects 1 in 200-500 individuals world-wide, while homozygous FH (HoFH) has a prevalence of one in a 160000-1000000. Elevated levels of LDL-cholesterol in the blood lead to an accelerated development of atherosclerosis and progression to coronary heart disease. The work presented in this thesis investigates two novel molecular therapies for the treatment of FH. A small molecule approach for the treatment of HeFH and a non-viral gene therapy for the treatment of HoFH. Small molecule: Through a compound screen, we identified a series of small molecules able to upregulate the LDLR at nanomolar potencies, via a mechanism distinct from statins. We then went on to elucidate the mechanism of action as inhibitors of squalene synthase and demonstrated, that when used in combination with statins, these compounds give a much greater increase in LDLR expression than can be achieved with statins alone. By carrying out extensive structure-activity relationship studies we improved the potency and pharmacokinetics of our lead compound and demonstrated in vivo efficacy. Non-viral gene therapy: We have previously generated mini-gene vectors carrying the human LDLR cDNA, driven by 10 kb of genomic DNA from the native human LDLR locus, encompassing the promoter region. To further enhance LDLR transgene expression, a miRNA, targeting Hmgcr (miR82) was cloned into the same vector, generating a combinatorial RNAi-LDLR vector. In vivo, we show the RNAi-LDLR vector is able to significantly reduce total and LDL-cholesterol in Ldlr^-/- mice fed a high cholesterol diet twelve weeks post-delivery. The LDL-cholesterol lowering resulted in reduced atherosclerosis in the RNAi-LDLR vector treated mice. Here we demonstrate for the first time, that an episomal non-viral vector is able to significantly reduce LDL-cholesterol and slow the progression of atherosclerosis in a mouse model of FH.