Targeting LDL Cholesterol With LNA

1RaNA Therapeutics, Cambridge, Massachusetts, USA Correspondence: Arthur M Krieg, RaNA Therapeutics, 25 First Street, Suite 303, Cambridge, Massachusetts 02141, USA. E-mail: [email protected] Cardiovascular disease is the number one cause of death in the industrialized world. Much of the morbidity and mortality from heart disease can be linked to elevated low-density lipoprotein (LDL) cholesterol, and one of the key genes that increases LDL cholesterol is Proprotein convertase subtilisin/ kexin type 9 (PCSK9). In a recent study in nonhuman primates, Lindholm et al. demonstrate that a 1 month course of weekly subcutaneous therapy with either of two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 rapidly decreases the expression of both PCSK9 and circulating LDL cholesterol without reported toxicity.1 The reduction in serum LDL is sustained for more than a month after the last dose of the LNA antisense. LDL is normally removed from the circulation on binding the LDL receptor, LDLR. PCSK9 functions by increasing the rate of degradation of the LDLR, secondarily preventing LDL clearance. PCSK9 thus increases circulating LDL, thereby also increasing the risk of atherosclerosis. Intriguingly, PCSK9 does not appear to be required for any other function, as humans completely lacking PCSK9 are perfectly healthy, and in fact have elevated expression of LDLR and much lower circulating LDL, with reduced risk of cardiovascular disease.2 Based on these and other genetic data PCSK9 is widely considered to be a validated target, and inhibitors of PCSK9 are excellent therapeutic candidates. Given that nearly all patients with elevated LDL will be treated with statins, the gold standard for therapy of hyperlipidemias, it is extremely important that PCSK9 inhibitors have been shown in primate models to provide further reductions in LDL beyond what can be achieved with statins alone.3 Several PCSK9 inhibitors are in preclinical and clinical development by biotech and pharmaceutical companies, using approaches including small molecules, monoclonal antibodies, antisense and RNA interference.4 Against this backdrop of clear need for PCSK9 antagonists, the results of Lindholm et al. reveal the great progress over recent years in the development of antisense oligonucleotides against targets expressed in the liver. Two different antisense oligonucleotides, SPC4061 and SPC5001, are compared to a control, SPC3088 (the control was used for the in vitro studies, but not in vivo). All three of these oligonucleotides had phosphorothioate backbones and were designed as LNA gapmers 13–16 bases long with two or three LNA modifications at the 5′ end, three LNA modifications at the 3′ end, and a DNA core (to facilitate RNAse H-mediated cleavage of the complementary PCSK9 mRNA target). Both of the antisense oligonucleotides showed similar activity in vitro, whether by transfection or unassisted uptake (also referred to as “gymnotic delivery”) into HepG2 cells. There has been some speculation that perhaps unassisted uptake would better predict in vivo efficacy than transfection experiments, but such was not the case in the results of Lindhom et al.: despite similar in vitro activity of the two antisense oligonucleotides, SPC5001 proved to be significantly more potent than SPC4061 in the multiple dose monkey study, reducing circulating PCSK9 protein by 85% compared to 50% in the monkeys dosed with SPC4061. Both antisense gapmers were present within liver tissue at similar concentrations (~50 μg oligonucleotide/gram of tissue), excluding sequence-dependent differences in liver uptake. At present, there appears to be no in vitro correlate for in vivo efficacy with antisense oligonucleotides, and experimenters wishing to develop the most potent compound with the best therapeutic index may be well advised to proceed into primate testing with multiple candidate compounds before selecting a lead for clinical development. The in vivo efficacy of the SPC5001 was both rapid and sustained. Serum PCSK9 was reduced by >50% as early as 24 hours after the first dose. The target protein concentration was ~85% reduced during the dosing period, with a gradual recovery during the subsequent 2 months. Even 1 month after the last dose of SPC5001, monkeys still showed approximately a 50% reduction from baseline PCSK9 concentration.1 In a fairly typical previous study using 20 base-long 2′-O-methoxyethyl antisense gapmers the oligonucleotide concentration in the liver required for 50% inhibition of the target ApoB mRNA was ~300 μg/g of liver tissue.5 Although one cannot really compare potency across different targets, and of course the Lindholm et al. study is not a pharmacokinetic analysis allowing the derivation of a liver EC50, the fact that Lindholm et al. show a liver concentration of just 50 μg oligonucleotide SPC5001/gram of tissue is associated with a >80% reduction in target RNA suggests a much greater in vivo potency of the relatively short gapmer LNA designs compared to the earlier 20mer gapmer designs using 2′-O-methoxyethyl. The safety profile is paramount for any compound under consideration for chronic administration, such as a PCSK9 Commentary