Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

Abstract

High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional therapeutics are still needed because many people are intolerant or nonresponsive to these therapies. We previously showed that increasing cellular levels of microRNA-30c (miR-30c) using viral vectors or liposomes reduces plasma lipids and atherosclerosis. In this study, we aimed to synthesize potent miR-30c analogs that can be delivered to hepatoma cells without the aid of viral vectors and lipid emulsions. We hypothesized that modification of the passenger strand of miR-30c would increase the stability of miR-30c and augment its delivery to liver cells. Here, we report the successful synthesis of a series of miR-30c analogs by using different chemically modified nucleosides. In these analogs, we left the active sense strand untouched so that its biological activity remained unaltered, and we modified the passenger strand of miR-30c to enhance the stability and uptake of miR-30c by hepatoma cells through phosphorothiorate linkages and the addition of GalNAc. We show that these analogs significantly reduced apolipoprotein B secretion in Huh-7 human hepatoma cells and human primary hepatocytes without affecting apolipoprotein A1 secretion and cellular lipid levels. Our results provide a proof of concept that the passenger strand of miR-30c can be modified to increase its stability and delivery to cells while retaining the potency of the sense strand. We anticipate these miR-30c analogs will be useful in the development of more efficacious analogs for the treatment of hyperlipidemias and cardiovascular diseases. High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional therapeutics are still needed because many people are intolerant or nonresponsive to these therapies. We previously showed that increasing cellular levels of microRNA-30c (miR-30c) using viral vectors or liposomes reduces plasma lipids and atherosclerosis. In this study, we aimed to synthesize potent miR-30c analogs that can be delivered to hepatoma cells without the aid of viral vectors and lipid emulsions. We hypothesized that modification of the passenger strand of miR-30c would increase the stability of miR-30c and augment its delivery to liver cells. Here, we report the successful synthesis of a series of miR-30c analogs by using different chemically modified nucleosides. In these analogs, we left the active sense strand untouched so that its biological activity remained unaltered, and we modified the passenger strand of miR-30c to enhance the stability and uptake of miR-30c by hepatoma cells through phosphorothiorate linkages and the addition of GalNAc. We show that these analogs significantly reduced apolipoprotein B secretion in Huh-7 human hepatoma cells and human primary hepatocytes without affecting apolipoprotein A1 secretion and cellular lipid levels. Our results provide a proof of concept that the passenger strand of miR-30c can be modified to increase its stability and delivery to cells while retaining the potency of the sense strand. We anticipate these miR-30c analogs will be useful in the development of more efficacious analogs for the treatment of hyperlipidemias and cardiovascular diseases. Atherosclerosis, hardening of the arteries after lipid deposition, is the leading cause of morbidity and mortality in the United States and worldwide. High plasma cholesterol levels are a major risk factor for atherosclerosis. Cholesterol in the circulation is carried primarily by apolipoprotein B (apoB) containing lipoproteins. Remarkable advances have been made in lowering plasma cholesterol and reducing death by 30 to 40% through treatments with statins and proprotein convertase subtilis/kexin type 9 inhibitors (1Blumenthal R.S. Statins: Effective antiatherosclerotic therapy.Am. Heart J. 2000; 139: 577-583Crossref PubMed Scopus (153) Google Scholar, 2Ehrenstein M.R. Jury E.C. Mauri C. Statins for atherosclerosis--as good as it gets?.N. Engl. J. Med. 2005; 352: 73-75Crossref PubMed Scopus (122) Google Scholar, 3LaRosa J.C. Grundy S.M. Waters D.D. Shear C. Barter P. Fruchart J.C. Gotto A.M. Greten H. Kastelein J.J. Shepherd J. Wenger N.K. Intensive lipid lowering with atorvastatin in patients with stable coronary disease.N. Engl. J. Med. 2005; 352: 1425-1435Crossref PubMed Scopus (2963) Google Scholar, 4Stoekenbroek R.M. Kastelein J.J. Huijgen R. PCSK9 inhibition: The way forward in the treatment of dyslipidemia.BMC. Med. 2015; 13: 258Crossref PubMed Scopus (30) Google Scholar). Despite the availability of these drugs, an unmet need for new lipid-lowering therapies remains, because some patients do not achieve the desirable cholesterol lowering with statins (5Voora D. Shah S.H. Reed C.R. Zhai J. Crosslin D.R. Messer C. Salisbury B.A. Ginsburg G.S. Pharmacogenetic predictors of statin-mediated low-density lipoprotein cholesterol reduction and dose response.Circ. Cardiovasc. Genet. 2008; 1: 100-106Crossref PubMed Scopus (74) Google Scholar); a substantial proportion of patients experience unmanageable adverse effects (6Ahmad Z. Statin intolerance.Am. J. Cardiol. 2014; 113: 1765-1771Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 7Saxon D.R. Eckel R.H. Statin intolerance: A literature review and management strategies.Prog. Cardiovasc. Dis. 2016; 59: 153-164Crossref PubMed Scopus (40) Google Scholar); and statins and proprotein convertase subtilis/kexin type 9 antibodies are not useful in treating patients with homozygous familial hypercholesterolemia and low-density lipoprotein receptor null mutations (8Rader D.J. Kastelein J.J. Lomitapide and mipomersen: Two first-in-class drugs for reducing low-density lipoprotein cholesterol in patients with homozygous familial hypercholesterolemia.Circulation. 2014; 129: 1022-1032Crossref PubMed Scopus (190) Google Scholar, 9Raal F.J. Stein E.A. Dufour R. Turner T. Civeira F. Burgess L. Langslet G. Scott R. Olsson A.G. Sullivan D. Hovingh G.K. Cariou B. Gouni-Berthold I. Somaratne R. Bridges I. et al.PCSK9 inhibition with evolocumab (AMG 145) in heterozygous familial hypercholesterolaemia (RUTHERFORD-2): A randomised, double-blind, placebo-controlled trial.Lancet. 2015; 385: 331-340Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). Therefore, a prevailing need exists to identify safer methods of lowering plasma lipids that can be used independently of or in combination with statins and other available drugs. Statins, inhibitors of hydroxyl-methyl-glutaryl-coenzyme A reductase, lower plasma lipids by increasing the hepatic expression of low-density lipoprotein receptors and decreasing cholesterol synthesis. A complementary approach involves inhibiting the assembly and secretion of lipoproteins to limit their entry into the circulation. Lipoprotein assembly requires two proteins: the structural protein apoB and the chaperone microsomal triglyceride transfer protein (MTP). MTP physically interacts with and transfers lipids in the endoplasmic reticulum to nascent apoB and assists in the formation and maturation of lipoprotein particles for secretion (10Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apolipoprotein B-lipoprotein assembly.J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 11Sirwi A. Hussain M.M. Lipid transfer proteins in the assembly of apoB-containing lipoproteins.J. Lipid Res. 2018; 59: 1094-1102Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). MTP has long been a drug target for lowering plasma lipids, as its biochemical activity of transferring lipids can be easily measured in laboratory settings. Several pharmaceutical companies have developed drugs that potently inhibit MTP activity and lower plasma lipids (12Cuchel M. Meagher E.A. du Toit T.H. Blom D.J. Marais A.D. Hegele R.A. Averna M.R. Sirtori C.R. Shah P.K. Gaudet D. Stefanutti C. Vigna G.B. Du Plessis A.M. Propert K.J. Sasiela W.J. et al.Efficacy and safety of a microsomal triglyceride transfer protein inhibitor in patients with homozygous familial hypercholesterolaemia: A single-arm, open-label, phase 3 study.Lancet. 2013; 381: 40-46Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 13Samaha F.F. McKenney J. Bloedon L.T. Sasiela W.J. Rader D.J. Inhibition of microsomal triglyceride transfer protein alone or with ezetimibe in patients with moderate hypercholesterolemia.Nat. Clin. Pract. Cardiovasc. Med. 2008; 5: 497-505Crossref PubMed Scopus (185) Google Scholar). However, these drugs increase hepatic lipids and plasma transaminases (14Burnett J.R. Watts G.F. MTP inhibition as a treatment for dyslipidaemias: Time to deliver or empty promises?.Expert Opin. Ther. Targets. 2007; 11: 181-189Crossref PubMed Scopus (61) Google Scholar, 15Joy T.R. Hegele R.A. Microsomal triglyceride transfer protein inhibition-friend or foe?.Nat. Clin. Pract. Cardiovasc. Med. 2008; 5: 506-508Crossref PubMed Scopus (16) Google Scholar, 16Hussain M.M. Bakillah A. New approaches to target microsomal triglyceride transfer protein.Curr. Opin. Lipidol. 2008; 19: 572-578Crossref PubMed Scopus (60) Google Scholar). One MTP inhibitor, lomitapide, has been approved for the treatment of homozygous familial hypercholesterolemia on a restricted protocol and carries label for hepatic M. Rader D.J. Microsomal transfer protein inhibition in Opin. Lipidol. 2013; PubMed Scopus (46) Google Scholar, M. New lipid The role of microsomal protein PubMed Scopus Google Scholar). a need for that can levels of MTP and plasma lipids without are in that expression the with the of target and decrease protein synthesis by with and Full Text Full Text PDF PubMed Scopus Google Scholar, J. I. The of and Genet. 11: PubMed Scopus Google Scholar). drugs are in for the treatment of and viral and are to be treatments in the R. F.J. a new for the management of and other PubMed Scopus Google Scholar, of therapeutics is of Med. 2014; PubMed Scopus Google Scholar). A major in the development of therapeutics is the of methods of delivery have been viral vectors and lipid R. F.J. a new for the management of and other PubMed Scopus Google Scholar). is a and The active sense strand interacts with different and the synthesis of proteins Hussain M.M. of microRNA-30c in lipid and Opin. Lipidol. 2015; PubMed Scopus Google Scholar, L. A. Hussain M.M. apolipoprotein lipoprotein 2016; PubMed Scopus Google Scholar). is the of two and in and Hussain M.M. of microRNA-30c in lipid and Opin. Lipidol. 2015; PubMed Scopus Google Scholar). The primary of these and show and and are in the and to the as and is and in is are in the in the of miR-30c with that are in and The the two are different are in and We have that of miR-30c significantly reduces MTP of its MTP activity in Huh-7 human hepatoma cells and human primary hepatocytes J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar, J. Hussain M.M. are for the of microsomal triglyceride transfer protein by 2013; PubMed Scopus Google Scholar, L. Hussain M.M. hepatic apolipoprotein B secretion and lipid PubMed Scopus Google Scholar). miR-30c significantly reduces apoB apoB secretion without affecting apolipoprotein A1 secretion in these cells. that miR-30c MTP activity by with and the J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar, J. P. Hussain M.M. hypercholesterolemia and in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, J. W.J. L. Hussain M.M. reduces plasma cholesterol in familial and Lipid Res. 2018; 59: Full Text Full Text PDF PubMed Scopus Google Scholar). interacts with MTP the and J. Hussain M.M. are for the of microsomal triglyceride transfer protein by 2013; PubMed Scopus Google Scholar). miR-30c interacts with MTP and and reduces MTP inhibiting apoB secretion in liver cells. miR-30c MTP activity and plasma lipids in we with for the expression of or and a J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar). hepatic MTP plasma and hepatic lipoprotein Despite in plasma we not in hepatic lipids and plasma transaminases in that miR-30c hepatic by in lipid as viral is we miR-30c analogs with lipid J. P. Hussain M.M. hypercholesterolemia and in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). delivery of miR-30c to the liver and hypercholesterolemia in the we that miR-30c significantly reduces hypercholesterolemia and in J. P. Hussain M.M. hypercholesterolemia and in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). demonstrated that miR-30c plasma cholesterol in and and in has on plasma and transaminases J. W.J. L. Hussain M.M. reduces plasma cholesterol in familial and Lipid Res. 2018; 59: Full Text Full Text PDF PubMed Scopus Google Scholar). have that the hepatic expression of miR-30c plasma hepatic lipid and without with MTP Therefore, miR-30c be a to and in patients with and homozygous familial In the previously J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar, J. P. Hussain M.M. hypercholesterolemia and in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, J. W.J. L. Hussain M.M. reduces plasma cholesterol in familial and Lipid Res. 2018; 59: Full Text Full Text PDF PubMed Scopus Google miR-30c as or with lipid emulsions. these approaches can be and for we aimed to synthesize miR-30c analogs to cells without using lipid or viral We show that the passenger strand can be modified and with to augment delivery to hepatoma cells and to apoB secretion without affecting is a and approach for treating that are not easily through drug The of has been to in the and safety of modified and and 2007; PubMed Scopus Google Scholar, D.R. The to of Clin. 2007; PubMed Scopus Google Scholar, A. J. J. with A report on 2007; PubMed Scopus Google Scholar). on advances made in delivery we analogs of miR-30c to these approaches be to we to the strand and while the active sense strand untouched to its to with the and of target in A and two of miR-30c and are the of two and and an strand different passenger and we modified and passenger by using these with a sense or strand to the of the a we with of with modified or modified showed However, the stability of with modified by stability of the The showed for and modified a in the of to the of miR-30c strand containing modified not its with the miR-30c sense strand in formation and the stability and of modified miR-30c and of and and with strand. that the synthesis of with modified not the of these modified passenger to the sense strand or the stability of the Therefore, we the of in reducing apoB secretion in Huh-7 human hepatoma cells by to the and their effects on apoB The on apoB secretion to cells without results have been because the to be delivered to the cells or not the for their we with modified or analogs into cells with modified significantly reduced apoB secretion A and without affecting secretion and that the modified active as lipid The of these to apoB secretion in the of delivery because the to the cells. that are delivered to cells and the of synthesis of more potent miR-30c analogs by the of we in of the and different series A the of and the of with and its stability H. J. H. D. of into with and biological Ther. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, F. M. a 2014; 11: PubMed Scopus Google Scholar). analogs into Huh-7 cells using the we used the we used an available with the miR-30c and analogs significantly apoB secretion secretion that delivered to these new analogs to apoB secretion to as with we of these analogs apoB secretion without lipid emulsions. this we of analogs to cells without analogs and miR-30c on apoB and secretion and that these analogs to cells on their to apoB to apoB secretion into cells with analogs and to lower in a new lower In the receptor has been used to deliver and to liver cells A. J. M. M. J.R. L. T. A. et delivery of therapeutics with and Ther. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M.R. M. P. J. L. et in hepatocytes and 2014; PubMed Scopus Google Scholar, The delivery of Res. 2016; 44: PubMed Scopus Google Scholar, A. T. J. P. D. 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PubMed Scopus Google of these these analogs into cells with analogs potently apoB secretion in Huh-7 cells and secretion by these analogs we the of these series B analogs in reducing apoB secretion to cells without to and on apoB secretion with to the However, other analogs reduced apoB secretion 40% to of these analogs secretion The potent analogs inhibiting apoB secretion and these analogs of the or of as in and in a of biological analogs with are for cellular delivery without analogs and containing to lower in a new of miR-30c analogs on apoB secretion in Huh-7 human hepatoma cells. apoB secretion and secretion in Huh-7 cells with and different miR-30c series B analogs with used as a apoB and secretion in Huh-7 cells with and different miR-30c analogs without The apoB and in the measured with are of and with and apolipoprotein apolipoprotein lower analogs and potently apoB we to their analogs showed a decrease in apoB secretion with an of miR-30c different of and on secretion we cells to of and to the delivery of miR-30c to cells and its effects on The levels of miR-30c with miR-30c significantly in the cells and in the cells We previously showed that miR-30c apoB secretion by reducing MTP activity Hussain M.M. of microRNA-30c in lipid and Opin. Lipidol. 2015; PubMed Scopus Google Scholar, L. A. Hussain M.M. apolipoprotein lipoprotein 2016; PubMed Scopus Google Scholar, J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar, J. P. Hussain M.M. hypercholesterolemia and in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, J. W.J. L. Hussain M.M. reduces plasma cholesterol in familial and Lipid Res. 2018; 59: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we the effects of analogs and on MTP analogs and with reduced MTP and activity by more and showed a decrease in MTP that analogs and are to apoB secretion in Huh-7 cells by lowering MTP expression to In a reduction in apoB secretion with of analogs and is to analogs as drugs, we on analogs that are more have that the of linkages the and activity of The delivery of Res. 2016; 44: PubMed Scopus Google Scholar, 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we new series analogs of and by using linkages analogs into cells with and to inhibit apoB secretion without affecting secretion in Huh-7 that are we their to cells without analogs potently apoB secretion on secretion In inhibition of apoB secretion with of these analogs A of the in A and that these analogs be more potent delivered without lipid emulsions. we the effects of different of the potent on apoB secretion in Huh-7 cells The a decrease in apoB with an of the reduction in apoB secretion on secretion that the addition of linkages the of different analogs in decreasing apoB secretion and that is a potent inhibitor of apoB analogs and containing and different to lower in a new lower is that in apoB secretion are with However, we have previously that miR-30c reduces apoB secretion without J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar). Here, we or not in Huh-7 cells. significantly reduced apoB secretion without affecting secretion A and on cellular triglyceride and cholesterol levels and showed that reduces apoB secretion not increase cellular lipid levels. the so in Huh-7 cells. We apoB secretion in human primary significantly reduced apoB secretion on secretion in human primary hepatocytes and in apoB secretion not with in cellular triglyceride and cholesterol levels and that the is a potent inhibitor of apoB secretion in hepatoma cells as as primary and this not increase cellular lipid levels. Our have that miR-30c is a good to lower plasma lipids without Therefore, we to potent miR-30c analogs that be used as for lowering plasma lipid levels. The successful synthesis of an to the of the modified with an synthesis the to the modified passenger strand of miR-30c with the be to other a of and we the of the miR-30c analogs in reducing apoB secretion in Huh-7 cells. of the need for lipid for delivery to hepatoma cells. showed that these analogs reduced MTP and to reduced apoB secretion in human primary provide that the passenger strand of miR-30c can be modified to enhance cellular delivery without lipid emulsions. be more delivery with lipid emulsions. cellular we in in the analogs on Therefore, in apoB secretion are a to these analogs and not to cellular in apoB secretion are with in cellular However, on cellular triglyceride and cholesterol levels in Huh-7 cells and human primary Therefore, these In we report the successful synthesis of miR-30c analogs that potently inhibit MTP activity and apoB the of miR-30c in reducing apoB secretion by liver cells and provide that modification of miR-30c in the passenger strand can be useful to its stability and delivery into hepatoma cells. these analogs can be used as to lower plasma lipids and in to be used and through a with used for The measured on a are in with a the by We used an to synthesize modified with and The modified and in to a of M. in used for and the with in for The with and in on the with in with of the in the the of The synthesis of modified miR-30c passenger with the of the The on and the the with for The with a and the with and with of for The by addition of of 3 and and of the for 3 The in and to in The of by with and The modified with in containing The for 3 to a of and by and to a of with a with a The of strand by the in Turner of the structural for of of by and PubMed Scopus Google Scholar). on a a of to with a with a of of and of and the H. H. R. H. B. J. A A for the synthesis of 2005; PubMed Scopus Google to synthesize and a of in The for The and the by as a in and in by the and The for The and the by 3 as a in and The to the phase synthesis with available through of the with of in a In a of in and and to the The for The with 3 and after for 3 The by for The in and the with The with and with an The with with containing The Huh-7 cells in modified containing and in with and in a Two of to the of different analogs to decrease apoB analogs into cells with cells to different analogs without the of In these Huh-7 cells a of in a in of the The of reduced or containing to the cells. The Huh-7 cells in the reduced with available miR-30c or miR-30c analogs with to the with a of and for 30 to cells. In delivery of analogs, cells in to these analogs without in of to the cells. after the of the and of to the cells. the for apoB and and in the of inhibitor for protein and of the activity of MTP as previously H. J. Hussain M.M. A and for microsomal triglyceride transfer Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). The apoB levels in the with a human apoB development in with The apoB with apoB by the in in the The apoB to the protein in the cellular protein with a protein The apoB in the of cells secretion by cells to miR-30c or miR-30c analogs is as a of this The levels in the with a human apolipoprotein in and The with an in with by the levels in the to protein in the The of in the cells to and in miR-30c cells to this miR-30c by complementary cells with a The miR-30c and miR-30c the with to and the are as MTP by with a The human MTP and and and MTP the with to and are as with to Huh-7 cells with and the in and containing inhibitor through through a and the protein measured with a protein by A primary to human MTP and a to used used The developed with a the MTP of proteins triglyceride transfer as previously J. J. J. C. Hussain M.M. reduces and by decreasing lipid synthesis and lipoprotein Med. 2013; 19: PubMed Scopus Google Scholar, J. P. Hussain M.M. hypercholesterolemia and in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, J. W.J. L. Hussain M.M. reduces plasma cholesterol in familial and Lipid Res. 2018; 59: Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Hussain M.M. A and for microsomal triglyceride transfer Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). the of different miR-30c analogs human hepatoma Huh-7 we human primary hepatocytes using protocol and in cells with the to apoB and levels using in to protein levels. of apoB and to cellular are as of cellular lipid cells with of and we and in of to and cholesterol using of to the protein for Lipid levels to protein and cells used as We in and 9 with and are as The and and the are in the The that have of with the of this M. M. H. P. H. and T. P. and I. A. A. P. M. M. H. P. P. T. A. J. and M. M. H. J. and M. M. H. J. and M. M. H. in by the of and and of of New M. M. by the J. and and the Heart to P. The of this do not the of the of or the The is the of the and not the of the of

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