Therapeutic Antisense Oligonucleotides for the Increase of HDL-Cholesterol Levels and Improvement of HDL Function

HDL-cholesterol levels and function can be modified directly or indirectly by several pathways, some of which have been targeted by ASOs.

1.3.1 Cholesteryl Ester Transfer Protein

One of the first ASO experimentally approached to target HDL levels was directed against CETP. Sugano's lab first demonstrated that a single injection of the ASO, coupled with the complex asialoglycoprotein-poly-L-lysine, into cholesterol-fed rabbits, reduced CETP activity and increased plasma HDL-cholesterol levels (Sugano and Makino 1996). This effect was due to reduced liver CETP mRNA levels, which was accompanied by a reduction in LDLand/or VLDL-cholesterol levels. In a longer study, 8-week treatment with the same molecule (30 μg/kg twice a week) reduced both CETP mass and atherosclerosis in cholesterol-fed rabbits (Sugano et al. 1998). While triglyceride levels did not change, LDLand VLDLcholesterol levels were significantly decreased by the ASO treatment (Sugano et al. 1998). Despite these promising preclinical antiatherogenic findings, the controversial clinical development of CETP inhibitors (Barter et al. 2007; Schaefer 2013) increased the caution for the future development of this type of therapy. In this context, recent preliminary finding suggests that inhibition of CETP by ASOs may differ from CETP inhibition by small-molecule inhibitors (Bell et al. 2013). Indeed, the 20-mer phosphorothioate ASO containing 20-O-methoxyethyl (20MOE) targeted to human CETP (ISIS Pharmaceutical) did not only reduce CETP activity and increase HDL-C levels but also enhance macrophage reverse cholesterol transport and reduce the accumulation of aortic cholesterol in a CETP transgenic LDLR-/mice (Bell et al. 2013). This finding together with a previous study regarding the lack of association of genetic inhibition of CETP (Johannsen et al. 2012) and possible side effects previously reported for torcetrapib suggests that not all inhibitors of CETP are equal. Thus inhibition of CETP still holds promise as a beneficial therapeutic target, but as for other drugs, this needs to be experimentally and clinically validated.

1.3.2 Endothelial Lipase

Endothelial lipase plays an important role in HDL metabolism (Kuusi et al. 1980; Voight et al. 2012), and it has been suggested that its inhibition may improve cardioprotection (Singaraja et al. 2013). In a preliminary study, a 20-mer ASO containing 20-O-(methoxy)-ethyl (20MOE) modifications on the first five and last five bases (ISIS Pharmaceutical) to target the rabbit endothelial lipase was tested in rabbits for 6 weeks (Zhang et al. 2012b). Even though the experimental protocol did not show a clear increase in HDL-cholesterol levels, the cholesterol content of large HDL (>12.1 nm) was increased (Zhang et al. 2012b). Whether other ASO chemistries may increase the impact on HDL-C levels and function is not known, but this deserves further investigation.

1.3.3 ACAT2

The sterol O-acyltransferase 2, encoded by the SOAT2 gene and originally named ACAT2 (referred here as ACAT2), is a membrane-bound enzyme, with an acylCoA cholesterol acyltransferase activity, localized in the endoplasmic reticulum. SOAT2/ACAT2 catalyzes the synthesis of cholesteryl esters from long-chain fatty acyl-CoA and cholesterol and is involved in cholesterol absorption and the secretion of cholesteryl esters into apoB-containing lipoproteins. ACAT2 is expressed exclusively in lipoprotein-producing cells, the enterocytes and hepatocytes (Anderson et al. 1998). While both hepatic and intestinal deletion of ACAT2 improves atherogenic hyperlipidemia and limits hepatic cholesteryl ester accumulation (Zhang et al. 2012a), it has been proposed that specific tissue ACAT inhibition would be beneficial for atheroprotection (Nissen et al. 2006; Brown et al. 2008). However, ACAT inhibition is not free of controversies in clinical development (Nissen et al. 2006). A 20-mer antisense phosphorothioate oligonucleotide containing 2-0-methoxyethyl groups at positions 1–5 and 15–20 was originally found to reduce hepatic ACAT2 levels and mediate protection against diet-induced hypercholesterolemia and aortic cholesteryl ester deposition (Bell et al. 2006). Interestingly, in mice this antisense oligonucleotide (ISIS Pharmaceutical) therapy (25 mg/kg biweekly for 8 weeks) promoted fecal neutral sterol excretion without altering biliary sterol secretion (Brown et al. 2008). This potentially important finding indicates that the antisense oligonucleotide promotes non-biliary fecal sterol loss and thus reverses cholesterol transport enhancement. Pharmacological inhibition of liver ACAT2 by using ASOs has also been shown to reduce cholesterol-associated hepatic steatosis (Alger et al. 2010) which may explain the hypertriglyceridemia observed in mice lacking ACAT2, probably by enhancing hepatic TG mobilization. In overall, ASO treatment against hepatic ACAT2 has uncovered other novel benefits distinct from that of HDL function and increased its therapeutic potential.

Although we are expecting the results of several ongoing clinical trials with ASOs for different pathologies (Sehgal et al. 2013), the recent approval for commercialization of Kynamro will really increase our interest to follow this therapeutic arena. Even when the long-term toxicity effects and other forms of delivery need to be evaluated, the promising preclinical results on ASOs to treat HDL levels and function make them an interesting alternative to small-molecule inhibitors. After all, opening new possible avenues to treat HDL dysfunction using ASOs makes more sense than simply awaiting for the discovery of potential small-molecule inhibitors.