Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase family of proteases that is thought to play a role in regulating lipid metabolism (1Kotowski I. K. Pertsemlidis A. Luke A. Cooper R. S. Vega G. L. Cohen J. C. Hobbs H. H. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am. J. Hum. Genet. 2006; 78: 410-422Google Scholar). Recent epidemiological studies have suggested that loss-of-function mutations in PCSK9 result in lifelong reductions in LDL that are associated with significant reductions in the incidence of coronary heart disease (1Kotowski I. K. Pertsemlidis A. Luke A. Cooper R. S. Vega G. L. Cohen J. C. Hobbs H. H. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am. J. Hum. Genet. 2006; 78: 410-422Google Scholar, 2Yue P. Averna M. Lin X. Schonfeld G. The c. 43₄4insCTG variation in PCSK9 is associated with low plasma LDL-cholesterol in a Caucasian population. Hum. Mutat. 2006; 17: 460-466Google Scholar, 3Rashid S. Curtis D. Garuti R. Anderson N. Bashmakov Y. Ho Y. K. Hammer R. Moon Y. Horton J. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc. Natl. Acad. Sci. USA. 2005; 112: 5374-5379Google Scholar, 4Kowal R. C. Herz J. Weisgraber K. H. Mahley R. W. Brown M. S. Goldstein J. L. Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein. J. Biol. Chem. 1990; 265: 10771-10779Google Scholar, 5Cohen J. C. Boerwinkle E. Mosley T. H. Hobbs H. H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 2006; 354: 1310-1312PubMed Google Scholar). Mechanistic studies in Pcsk9-deficient mice have shown that inactivation of this gene produces ∼2-fold increases in hepatic low density lipoprotein receptors (LDLRs), resulting in significant reductions in LDL via an enhanced hepatic clearance mechanism (4Kowal R. C. Herz J. Weisgraber K. H. Mahley R. W. Brown M. S. Goldstein J. L. Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein. J. Biol. Chem. 1990; 265: 10771-10779Google Scholar). Although these studies have demonstrated an important role for PCSK9 in lipid homeostasis, the mechanism of these effects has not been elucidated, nor has the pharmacological intervention of PCSK9 been described, as no such agent currently exists. To determine whether pharmacological inhibition of PCSK9 decreases LDL through upregulation of LDLR protein, an antisense oligonucleotide (ASO) complementary to the mouse PCSK9 gene was identified and administered to hyperlipidemic mice. Inhibition of that target resulted in significant reductions in hepatic PCSK9 mRNA levels, with concomitant reductions in total cholesterol and LDL. Consistent with the results observed in Pcsk9-deficient mice, hepatic LDLR protein expression was increased significantly (∼2-fold). Thus, lipid lowering was dependent on a functional LDLR. These findings validate PCSK9 as a pharmacological target for LDL lowering and suggest that the specific and selective inhibition of PCSK9 mRNA using ASOs may be an effective approach for decreasing LDL in human. A series of chimeric 20-mer phosphorothioate oligonucleotides containing 2′-O-methoxyethyl groups at positions 1–3 and 17–20 targeted to mouse PCSK9 were synthesized and purified as described (6Baker B. F. Lot S. S. Condon T. P. Cheng-Flournoy S. Lesnik E. A. Sasmor H. M. Bennett C. F. 2′-O- (2-Methoxy) ethyl-modified anti-cellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem. 1997; 272: 11994-12000Google Scholar) with an automated DNA synthesizer (380B; Perkin-Elmer Applied Biosystems, Foster City, CA). The most potent ASO, ISIS 394814 (5′-GGGCTCATAGCACATTATCC-3′), was used for all subsequent in vivo pharmacological assessments. ISIS 141923 (5′-CCTTCCCTGAAGGTTCCTCC-3′), which is in the same chemical and mechanistic class as the PCSK9 compound but not complementary to any known gene sequences, was used as a control ASO. Total RNA was extracted from whole liver tissue and primary hepatocytes with Qiagen RNeasy isolation kits, as described (7Butler M. McKay R. A. Popoff I. J. Gaarde W. A. Witchell D. Murray S. F. Dean N. M. Bhanot S. Monia B. P. Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice. Diabetes. 2002; 51: 1028-1034Google Scholar). Samples (50 ng) were subjected to quantitative RT-PCR analysis using commercial reagents (Invitrogen, Carlsbad, CA) and analyzed using a Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosciences). Values were normalized to glyceraldehyde-3-phosphate dehydrogenase and/or Ribogreen levels. In all cases, the probes were labeled with 5′ FAM (a 6-carboxyfluorescein reporter) and 3′ TAMRA a 5 (6) -carboxytetra-methyl-rhodamine quencher. After 40 amplification cycles, absolute values were obtained with SDS analysis software (Applied Biosystems). Cells or tissues were harvested in lysis buffer (50 mM Tris, pH 7. 5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0. 5% Nonidet P-40, 1% Triton, 0. 25% sodium deoxycholate, 0. 1% SDS, protease inhibitor cocktail 1: 100, and 0. 2 mM sodium orthovanadate), and protein concentrations were measured with the detergent compatible (DC) kit from Bio-Rad (Hercules, CA). Mouse plasma (10 μl, 1: 100) was subjected to electrophoresis on 4–12% Tris-glycine gels and transferred to polyvinylidene fluoride membranes (Invitrogen). Mouse hepatic LDLR Western blotting was performed overnight at 4°C with a primary anti-mouse LDLR antibody (1: 1, 000; R 46: 872-884Google Scholar) using a Beckman System Gold 126 HPLC system, a 507e refrigerated autosampler, a 126 photodiode array detector (Beckman Instruments, Fullerton, CA), and a Superose 6 HR 10/30 column (Pfizer, Chicago, IL). HDL, LDL, and VLDL fractions were measured at a wavelength of 505 nm and validated with a cholesterol calibration kit (Sigma). All animal experiments were conducted in accordance with Institutional American Association for the Accreditation of Laboratory Animal Care guidelines. C57BL/6 mice were obtained from Jackson Laboratory (http: //www. jax. org). Ldlr-deficient/apoB-100 mice were kindly provided by Dr. Larry Rudel (Wake Forest University). A majority of the mice were male, and studies were initiated when animals were 4–5 weeks of age. The mice were maintained on a 12 h light/12 h dark cycle and fed ad libitum. C57BL/6 mice were fed a Western diet consisting of 60% lard (Research Diets, New Brunswick, NJ), whereas all Ldlr-deficient/apoB-100 mice were fed regular chow. Oligonucleotides were administered twice weekly (50 mg/kg) for 6 weeks by intraperitoneal injection (10 mg/ml dosing solution formulated in saline). Mice were anesthetized, and whole blood was obtained through cardiac puncture. Serum was analyzed as described above. Whole liver was processed for RNA, protein, and histological examination. Hepatic TG levels were assayed as described (9Desai U. J. Slosberg E. D. Boettcher B. R. Caplan S. L. Fabelli B. Stephan Z. Gunther V. J. Kaleko M. Connelly S. Phenotypic correction of diabetic mice by adenovirus-mediated glucokinase expression. Diabetes. 2001; 50: 2287-2295Google Scholar). Pharmacological studies were performed at least twice using six mice per treatment group. A nonparametric, two-tailed t-test comparison was performed for all serum lipid parameters (saline or control ASO vs. PCSK9 ASO treated). Statistics for mRNA and protein changes were deemed significant at P 2-fold increase in LDLR protein levels (Fig. 2), whereas control ASO (ISIS 141923) -treated animals were unaffected. ISIS 394814 effects were also specific and selective. For example, PCSK9 ASO treatment had no effect on the levels of SR-BI, a hepatic receptor involved in HDL clearance (Fig. 2A). Administration of ISIS 394814 for 6 weeks reduced total cholesterol (52%; P < 0. 0001), LDL (32%; P < 0. 005), and HDL (54%; P < 0. 005) (Table 1). A reduction in HDL has also been observed in Pcsk9-deficient mice (3Rashid S. Curtis D. Garuti R. Anderson N. Bashmakov Y. Ho Y. K. Hammer R. Moon Y. Horton J. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc. Natl. Acad. Sci. USA. 2005; 112: 5374-5379Google Scholar), and those authors suggested that this results from enhanced clearance of apoE-containing lipoproteins facilitated by increased hepatic LDLR abundance. Furthermore, Lieu et al. (10Lieu H. D. Withycombe S. K. Walker Q. Rong J. X. Walzem R. L. Wong J. S. Hamilton R. L. Fisher E. A. Young S. J. Eliminating atherogenesis in mice by switching off hepatic lipoprotein secretion. Circulation. 2003; 107: 1315-1321Google Scholar) reported similar findings in Reversa mice and suggested that HDL particle formation requires components derived from apoB-associated lipoproteins. Serum free cholesterol and phospholipids were reduced as well 25% (P < 0. 005) and 54% (P < 0. 0001), respectively. HPLC analysis of pooled samples from treated mice corroborated the alterations in serum lipids measured using the clinical analyzer (Fig. 3A). TABLE 1PCSK9 mRNA, lipid, and liver TG levels after 6 weeks of ISIS 394814 treatment (100 mg/kg/week) in HF-fed and Ldlr-deficient/apoB-100 micePhenotypeSaline PCSK9 mRNATotal CholesterolFree CholesterolHDLLDLTGKetonePhospholipidLiver TG%mg/dlmg/dlmg/dlmg/dlmg/dlμmol/lmg/dlmg/gHF-fedSaline100 ± 13183 ± 1841 ± 10133 ± 1622 ± 4102 ± 18141 ± 52354 ± 2997 ± 10ISIS 141923135 ± 11194 ± 1458 ± 3145 ± 1025 ± 289 ± 18146 ± 48383 ± 2172 ± 28ISIS 3948148 ± 4aP < 0. 0001. 87 ± 19aP < 0. 0001. 31 ± 5bP < 0. 005. 61 ± 16aP < 0. 0001. 14 ± 2bP < 0. 005. 87 ± 12101 ± 25169 ± 35aP < 0. 0001. 36 ± 18bP < 0. 005. Ldlr-deficient/apoB-100Saline100 ± 9358 ± 12159 ± 492 ± 4261 ± 18118 ± 14265 ± 101304 ± 1022 ± 7ISIS 14192396 ± 13375 ± 15163 ± 399 ± 1262 ± 11155 ± 17321 ± 187314 ± 2126 ± 10ISIS 3948149 ± 2aP < 0. 0001. 445 ± 25bP < 0. 005. 186 ± 7bP < 0. 005. 109 ± 7bP < 0. 005. 329 ± 11bP < 0. 005. 127 ± 26524 ± 102354 ± 1221 ± 4apoB, apolipoprotein B; HF, high fat; LDLR, low density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; TG, triglyceride. All values indicated are group means (n = 5) and SEM. a P < 0. 0001. b P < 0. 005. Open table in a new tab apoB, apolipoprotein B; HF, high fat; LDLR, low density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; TG, triglyceride. All values indicated are group means (n = 5) and SEM. Based upon the increased expression of hepatic apobec-1 mRNA, Western analysis was performed to determine whether serum apoB-48 and apoB-100 would be differentially affected after PCSK9 ASO administration. Consistent with those data, serum apoB-48 protein levels were increased (3-fold) but apoB-100 levels were reduced by 50% (Fig. 4A, B). In contrast, PCSK9 ASO treatment did not produce any significant change in apoA-I protein levels relative to those observed in ISIS 141923 (control ASO) -treated mice. Treatment with ISIS 394814 for 6 weeks reduced liver TG content by ∼65% (P = 0. 01) relative to saline controls (Table 1). Although there was a modest reduction in hepatic TG content with the control ASO (ISIS 141923), these changes were not statistically significant. These results are consistent with liver TG reductions observed previously in Pcsk9-deficient mice (3Rashid S. Curtis D. Garuti R. Anderson N. Bashmakov Y. Ho Y. K. Hammer R. Moon Y. Horton J. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc. Natl. Acad. Sci. USA. 2005; 112: 5374-5379Google Scholar), and further studies are to determine the mechanism. To determine whether the pharmacological effects by the PCSK9 ASO the of a functional LDLR, the PCSK9 ASO was administered to Ldlr-deficient/apoB-100 mice. Consistent with the mechanism of PCSK9 on cholesterol no reduction in serum cholesterol was observed in mice lacking the LDLR (Table a reduction in hepatic mRNA levels. In a increase in total cholesterol and LDL was observed in these mice (P = and HPLC analysis these results (Fig. in to HF-fed mice, hepatic TG levels were by PCSK9 inhibition in Ldlr-deficient/apoB-100 mice (Table 1). an increase in hepatic mRNA levels was observed (data not shown). In this for the that of a second generation PCSK9 ASO to hyperlipidemic mice reduced hepatic PCSK9 mRNA expression by 92% and resulted in significant reductions in total cholesterol and LDL. second generation antisense inhibitors (8Crooke R. M. Graham M. J. Lemonidis K. M. Whipple C. Koo S. Perera R. J. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis. J. Lipid Res. 2005; 46: 872-884Google Scholar, U. J. Slosberg E. D. Boettcher B. R. Caplan S. L. Fabelli B. Stephan Z. Gunther V. J. Kaleko M. Connelly S. Phenotypic correction of diabetic mice by adenovirus-mediated glucokinase expression. Diabetes. 2001; 50: 2287-2295Google Scholar), ISIS 394814 was well in mice. These results that pharmacological suppression of PCSK9 significantly reduce LDL in a consistent with role in the of cholesterol homeostasis, as suggested by epidemiological and studies in Pcsk9-deficient mice (1Kotowski I. K. Pertsemlidis A. Luke A. Cooper R. S. Vega G. L. Cohen J. C. Hobbs H. H. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am. J. Hum. Genet. 2006; 78: 410-422Google Scholar, 3Rashid S. Curtis D. Garuti R. Anderson N. Bashmakov Y. Ho Y. K. Hammer R. Moon Y. Horton J. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc. Natl. Acad. Sci. USA. 2005; 112: 5374-5379Google Scholar, 5Cohen J. C. Boerwinkle E. Mosley T. H. Hobbs H. H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 2006; 354: 1310-1312PubMed Google Scholar). Based upon these studies are in to further the potential of PCSK9 as a targeted intervention for cholesterol lowering in animals that have similar serum lipid to such as and The expression of key involved in cholesterol and fatty acid were also evaluated in mice after PCSK9 ASO administration. the significant effects of PCSK9 inhibition to be an increase in the expression of mRNA to with a in serum apoB-48 protein levels is the enzyme for the of lipoproteins the is also in but not in liver J. J. E. B mRNA editing in 12 hepatic expression is in low concentrations of plasma lipoproteins. J. Lipid Res. Scholar). As PCSK9 is a protease of in to on LDLR, may upon Thus, to these is that for PCSK9 are the involved in editing apoB mRNA. is to this is also important to the for PCSK9 and the potential pharmacological and effects of this protein. A specific and selective antisense inhibitor of this protein to determine potential mechanism of as Additional studies are to determine whether alterations in mRNA expression in liver and of treated animals with any To specific inhibition of PCSK9 via has Antisense has been shown to target not to molecule or antibody R. J. N. D. P. antisense oligonucleotide lowers protein, blood glucose, and in diabetic mice. Proc. Natl. Acad. Sci. USA. 2002; Scholar, A. P. S. D. Graham M. J. R. M. hepatic expression of hepatic in a mouse of and hepatic steatosis. J. Biol. Chem. 2006; Scholar, Brown Graham M. J. Lemonidis K. M. R. M. Rudel inhibition of 2 with antisense oligonucleotides in apolipoprotein low-density lipoprotein mice. Biol. 2006; Scholar). generation ASOs are that be administered in saline without via injection and of R. S. S. Lesnik E. A. Sasmor M. of oligonucleotide in J. 2001; Scholar, R. S. M. J. J. of and tissue of an antisense phosphorothioate oligonucleotide targeting human mRNA in mouse and J. Sci. 2001; Scholar). The of these is well and after a of the to the a of PCSK9 expression. of their and a these be administered weekly or R. S. J. J. of antisense oligonucleotides in J. Sci. Scholar, D. of antisense In Antisense and S. New 2001; Scholar). For example, a second generation antisense inhibitor to lipid-lowering resulted in significant effects on apoB-100 and all as a agent and in with statins with an B. F. J. E. Graham M. J. R. M. reduction of apolipoprotein B and low-density lipoprotein cholesterol by of an antisense inhibitor of apolipoprotein B. Circulation. 2006; Scholar). Thus, a PCSK9 of this class be effective and well in the and may therapeutic in at for disease. The authors for the of the oligonucleotides used in these studies and for the of the apolipoprotein B apolipoprotein B mRNA editing enzyme catalytic polypeptide 1 antisense oligonucleotide high fat low density lipoprotein receptor proprotein convertase subtilisin/kexin type 9 scavenger receptor class B type I