Voltar
Revisão Acesso aberto Revisado por pares
BIBTEX RIS

Antisense inhibition of apolipoprotein (a) to lower plasma lipoprotein (a) levels in humans

2016; Elsevier BV; Volume: 57; Issue: 3 Linguagem: Inglês

10.1194/jlr.r052258

ISSN

1539-7262

Autores

Mark J. Graham, N. Viney, Rosanne M. Crooke, Sotirios Tsimikas,

Tópico(s)

Protease and Inhibitor Mechanisms

Resumo

Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that lipoprotein (a) [Lp(a)] is an independent causal risk factor for CVD, including myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis. Lp(a) levels >50 mg/dl are highly prevalent (20% of the general population) and are overrepresented in patients with CVD and aortic stenosis. These data support the notion that Lp(a) should be a target of therapy for CVD event reduction and to reduce progression of aortic stenosis. However, effective therapies to specifically reduce plasma Lp(a) levels are lacking. Recent animal and human studies have shown that Lp(a) can be specifically targeted with second generation antisense oligonucleotides (ASOs) that inhibit apo(a) mRNA translation. In apo(a) transgenic mice, an apo(a) ASO reduced plasma apo(a)/Lp(a) levels and their associated oxidized phospholipid (OxPL) levels by 86 and 93%, respectively. In cynomolgus monkeys, a second generation apo(a) ASO, ISIS-APO(a)Rx, significantly reduced hepatic apo(a) mRNA expression and plasma Lp(a) levels by >80%. Finally, in a phase I study in normal volunteers, ISIS-APO(a)Rx ASO reduced Lp(a) levels and their associated OxPL levels up to 89 and 93%, respectively, with minimal effects on other lipoproteins. ISIS-APO(a)Rx represents the first specific and potent drug in clinical development to lower Lp(a) levels and may be beneficial in reducing CVD events and progression of calcific aortic valve stenosis. Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that lipoprotein (a) [Lp(a)] is an independent causal risk factor for CVD, including myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis. Lp(a) levels >50 mg/dl are highly prevalent (20% of the general population) and are overrepresented in patients with CVD and aortic stenosis. These data support the notion that Lp(a) should be a target of therapy for CVD event reduction and to reduce progression of aortic stenosis. However, effective therapies to specifically reduce plasma Lp(a) levels are lacking. Recent animal and human studies have shown that Lp(a) can be specifically targeted with second generation antisense oligonucleotides (ASOs) that inhibit apo(a) mRNA translation. In apo(a) transgenic mice, an apo(a) ASO reduced plasma apo(a)/Lp(a) levels and their associated oxidized phospholipid (OxPL) levels by 86 and 93%, respectively. In cynomolgus monkeys, a second generation apo(a) ASO, ISIS-APO(a)Rx, significantly reduced hepatic apo(a) mRNA expression and plasma Lp(a) levels by >80%. Finally, in a phase I study in normal volunteers, ISIS-APO(a)Rx ASO reduced Lp(a) levels and their associated OxPL levels up to 89 and 93%, respectively, with minimal effects on other lipoproteins. ISIS-APO(a)Rx represents the first specific and potent drug in clinical development to lower Lp(a) levels and may be beneficial in reducing CVD events and progression of calcific aortic valve stenosis. ERRATUMJournal of Lipid ResearchVol. 57Issue 12PreviewThe authors of "Antisense inhibition of apolipoprotein(a) to lower plasma lipoprotein(a) levels in humans" (J. Lipid Res. 2016. 57: 340–351) have advised the Journal that there was an error in the legend to Table 1. The corrected table legend should read "ISIS-APO(a)Rx complementary binding sites within the human apo(a) transcript (GenBank accession NM_005577.2) at position 3901-3920. ISIS-APO(a)Rx was designed to perfectly match only the exon 24-25 splice site (indicated with bold type) but may also bind at 11 other apo(a) exon splice sites containing one to three mismatched nucleotides (indicated by underlined letters)." Additionally, on page 343 under the "Identification of a Second Generation Antisense Drug to Human apo(a)" section, "ISIS_APO(a)Rx also has the potential to bind to 11 alternative sites within the transcript containing one to four mismatched nucleotides" should read "ISIS_APO(a)Rx also has the potential to bind to 11 alternative sites within the transcript containing one to three mismatched nucleotides." Full-Text PDF Open Access Lipoprotein (a) [Lp(a)] is a highly polymorphic lipoprotein found in human plasma in levels ranging from 250 mg/dl. Lp(a) consists of an LDL-like particle and apo(a), which are covalently bound via a disulfide bond between Cys4326 of apoB-100 and Cys4057 of apo(a) located in kringle IV (KIV) type 9 (KIV9). The apo(a) comprises 10 KIV subunits, of which KIV2 is present in variable identically sized repeats, kringle V (KV), and an inactive protease domain. The apo(a) protein shows a high degree of homology (75–100%) to plasminogen at both the nucleotide and the amino acid level (1Kronenberg F. Utermann G. Lipoprotein(a): resurrected by genetics.J. Intern. Med. 2013; 273: 6-30Crossref PubMed Scopus (336) Google Scholar). However, the apo(a) gene transcript is much larger due to the repetitive KIV2 domain (3 to >40 repeats) in the LPA gene. The majority of apo(a) mRNA is expressed in the liver, with minor amounts present in the testes, brain, adrenals, lung, and pituitary. Lp(a) levels are primarily genetically determined by the LPA alleles present within an individual (2McLean J.W. Tomlinson J.E. Kuang W.J. Eaton D.L. Chen E.Y. Fless G.M. Scanu A.M. Lawn R.M. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen.Nature. 1987; 330: 132-137Crossref PubMed Scopus (1594) Google Scholar, 3Boerwinkle E. Leffert C.C. Lin J. Lackner C. Chiesa G. Hobbs H.H. Apolipoprotein(a) gene accounts for greater than 90% of the variation in plasma lipoprotein(a) concentrations.J. Clin. Invest. 1992; 90: 52-60Crossref PubMed Scopus (821) Google Scholar). Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that Lp(a) is an independent causal risk factor for CVD, including myocardial infarction, stroke, and peripheral arterial disease, as well as calcific aortic valve stenosis (4Erqou S. Kaptoge S. Perry P.L. Di Angelantonio E. Thompson A. White I.R. Marcovina S.M. Collins R. Thompson S.G. Danesh J. Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality.JAMA. 2009; 302: 412-423Crossref PubMed Scopus (1156) Google Scholar, 5Clarke R. Peden J.F. Hopewell J.C. Kyriakou T. Goel A. Heath S.C. Parish S. Barlera S. Franzosi M.G. Rust S. PROCARDIS Consortium et al.Genetic variants associated with Lp(a) lipoprotein level and coronary disease.N. Engl. J. Med. 2009; 361: 2518-2528Crossref PubMed Scopus (1031) Google Scholar, 6Thanassoulis G. Campbell C.Y. Owens D.S. Smith J.G. Smith A.V. Peloso G.M. Kerr K.F. Pechlivanis S. Budoff M.J. Harris T.B. et al.Genetic associations with valvular calcification and aortic stenosis.N. Engl. J. Med. 2013; 368: 503-512Crossref PubMed Scopus (626) Google Scholar, 7Kamstrup P.R. Benn M. Tybjaerg-Hansen A. Nordestgaard B.G. Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population: the Copenhagen City Heart Study.Circulation. 2008; 117: 176-184Crossref PubMed Scopus (350) Google Scholar, 8Kamstrup P.R. Tybjaerg-Hansen A. Nordestgaard B.G. Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population.J. Am. Coll. Cardiol. 2014; 63: 470-477Crossref PubMed Scopus (351) Google Scholar, 9Capoulade R. Chan K.L. Yeang C. Mathieu P. Bossé Y. Dumesnil J.G. Tam J.W. Teo K.K. Mahmut A. Yang X. et al.Oxidized phospholipids, lipoprotein(a), and progression of calcific aortic valve stenosis.J. Am. Coll. Cardiol. 2015; 66: 1236-1246Crossref PubMed Scopus (239) Google Scholar). While extensive epidemiological data suggest that elevated plasma Lp(a) levels are pro-atherogenic (10Dubé J.B. Boffa M.B. Hegele R.A. Koschinsky M.L. Lipoprotein(a): more interesting than ever after 50 years.Curr. Opin. Lipidol. 2012; 23: 133-140Crossref PubMed Scopus (95) Google Scholar), the molecular mechanisms by which it contributes to the atherosclerotic process remain enigmatic. There is evidence that apo(a) potentiates atherothrombosis through its LDL moiety and by additional apo(a)-driven mechanisms, including impairing fibrinolysis, mediating pro-inflammatory effects, activating endothelial cells, recruiting monocytes to the vessel wall, accelerating macrophage foam cell formation, and transporting pro-inflammatory oxidized phospholipids (OxPLs) (1Kronenberg F. Utermann G. Lipoprotein(a): resurrected by genetics.J. Intern. Med. 2013; 273: 6-30Crossref PubMed Scopus (336) Google Scholar, 11Bergmark C. Dewan A. Orsoni A. Merki E. Miller E.R. Shin M.J. Binder C.J. Horkko S. Krauss R.M. Chapman M.J. et al.A novel function of lipoprotein [a] as a preferential carrier of oxidized phospholipids in human plasma.J. Lipid Res. 2008; 49: 2230-2239Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 12Merki E. Graham M. Taleb A. Leibundgut G. Yang X. Miller E.R. Fu W. Mullick A.E. Lee R. Willeit P. et al.Antisense oligonucleotide lowers plasma levels of apolipoprotein (a) and lipoprotein (a) in transgenic mice.J. Am. Coll. Cardiol. 2011; 57: 1611-1621Crossref PubMed Scopus (105) Google Scholar, 13Spence J.D. Koschinsky M.L. Mechanisms of lipoprotein(a) pathogenicity.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1550-1551Crossref PubMed Scopus (71) Google Scholar, 14van Dijk R.A. Kolodgie F. Ravandi A. Leibundgut G. Hu P.P. Prasad A. Mahmud E. Dennis E. Curtiss L.K. Witztum J.L. et al.Differential expression of oxidation-specific epitopes and apolipoprotein(a) in progressing and ruptured human coronary and carotid atherosclerotic lesions.J. Lipid Res. 2012; 53: 2773-2790Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15Leibundgut G. Scipione C. Yin H. Schneider M. Boffa M.B. Green S. Yang X. Dennis E.A. Witztum J.L. Koschinsky M.L. et al.Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a).J. Lipid Res. 2013; 54: 2815-2830Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Wiesner P. Tafelmeier M. Chittka D. Choi S.H. Zhang L. Byun Y.S. Almazan F. Yang X. Iqbal N. Chowdhury P. et al.MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma.J. Lipid Res. 2013; 54: 1877-1883Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Due to the expanding evidence indicating that Lp(a) contributes to CVD, a search for potent and specific inhibitors of apo(a) was initiated. Among the various therapeutic platforms amenable to selective inhibition of Lp(a), antisense oligonucleotide (ASO) drugs have emerged as a promising approach in lowering Lp(a) levels in the clinical setting. ASOs represent the third major therapeutic discovery platform, distinct from small molecule and monoclonal antibody approaches, and have shown great promise in specific targeting of disease-associated genes in dyslipidemia, oncology, neurological dysfunction, and metabolic disorders. Due to their mode of action, by binding complementary mRNA targets via Watson-Crick base pairing, isoform-specific targeting is possible. In the case of Lp(a), this very important feature allows direct targeting of the apo(a) transcript without altering plasminogen transcript levels Second generation ASOs are single-stranded chimeric molecules generally 20 nucleotides in length, containing 2′-O-(2-methoxyethyl) (MOE) modifications at the 5′ and 3′ termini (positions 1-5 and 16-20) and DNA-like nucleotides in the central region (position 6-15) with a phosphorothioate (P=S) backbone throughout to enhance nuclease resistance (17Henry S.P. Geary R.S. Yu R. Levin A.A. Drug properties of second-generation antisense oligonucleotides: how do they measure up to their predecessors?.Curr. Opin. Investig. Drugs. 2001; 2: 1444-1449PubMed Google Scholar, 18Crooke S.T. Progress in antisense technology.Annu. Rev. Med. 2004; 55: 61-95Crossref PubMed Scopus (395) Google Scholar, 19Tsimikas S. Viney N.J. Hughes S.G. Singleton W. Graham M.J. Baker B.F. Burkey J.L. Yang Q. Marcovina S.M. Geary R.S. et al.Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.Lancet. 2015; 386: 1472-1483Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). These molecules are up to 15-fold more potent than first-generation P=S only-modified ASO drugs, due to their enhanced mRNA affinity via the MOE moiety (19Tsimikas S. Viney N.J. Hughes S.G. Singleton W. Graham M.J. Baker B.F. Burkey J.L. Yang Q. Marcovina S.M. Geary R.S. et al.Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.Lancet. 2015; 386: 1472-1483Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 20Geary R.S. Norris D. Yu R. Bennett C.F. Pharmaco­kinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Scopus (499) Google Scholar, 21Geary R.S. Baker B.F. Crooke S.T. Clinical and preclinical pharmacokinetics and pharmacodynamics of mipomersen (kynamro(®)): a second-generation antisense oligonucleotide inhibitor of apolipoprotein B.Clin. Pharmacokinet. 2015; 54: 133-146Crossref PubMed Scopus (121) Google Scholar), their gapmer design supporting an RNase H1 enzymatic termination mechanism, and their improved pharmacokinetic properties that permit weekly, monthly, or potentially quarterly dosing (Fig. 1). These drugs also have an improved therapeutic index due to reduced pro-inflammatory properties (22Yu R.Z. Grundy J.S. Geary R.S. Clinical pharmacokinetics of second generation antisense oligonucleotides.Expert Opin. Drug Metab. Toxicol. 2013; 9: 169-182Crossref PubMed Scopus (99) Google Scholar, 23Crooke S.T. Geary R.S. Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B.Br. J. Clin. Pharmacol. 2013; 76: 269-276Crossref PubMed Scopus (119) Google Scholar, 24Gaudet D. Brisson D. Tremblay K. Alexander V.J. Singleton W. Hughes S.G. Geary R.S. Baker B.F. Graham M.J. Crooke R.M. et al.Targeting APOC3 in the familial chylomicronemia syndrome.N. Engl. J. Med. 2014; 371: 2200-2206Crossref PubMed Scopus (337) Google Scholar, 25Büller H.R. Bethune C. Bhanot S. Gailani D. Monia B.P. Raskob G.E. Segers A. Verhamme P. Weitz J.I. FXI-ASO Investigators Factor XI antisense oligonucleotide for prevention of venous thrombosis.N. Engl. J. Med. 2015; 372: 232-240Crossref PubMed Scopus (389) Google Scholar, 26Gaudet D. Alexander V.J. Baker B.F. Brisson D. Tremblay K. Singleton W. Geary R.S. Hughes S.G. Viney N.J. Graham M.J. et al.Antisense inhibition of apolipoprotein C-III in patients with hypertriglyceridemia.N. Engl. J. Med. 2015; 373: 438-447Crossref PubMed Scopus (394) Google Scholar). Because ASO drugs are metabolized by cellular nucleases and not the cytochrome P450 system, they can be safely co-administered with traditional therapeutic agents with differing modes of action (20Geary R.S. Norris D. Yu R. Bennett C.F. Pharmaco­kinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Scopus (499) Google Scholar). Additionally, as they are hydrophilic, they may be administered in saline without special formulation via subcutaneous, intravenous, topical, aerosol, enema, intravitreal, intraventricular, intrathecal, and oral routes (27Geary R.S. Antisense oligonucleotide pharmacokinetics and metabolism.Expert Opin. Drug Metab. Toxicol. 2009; 5: 381-391Crossref PubMed Scopus (227) Google Scholar). The pharmacokinetic properties of ASOs have been extensively quantified in multiple species and in man (22Yu R.Z. Grundy J.S. Geary R.S. Clinical pharmacokinetics of second generation antisense oligonucleotides.Expert Opin. Drug Metab. Toxicol. 2013; 9: 169-182Crossref PubMed Scopus (99) Google Scholar). Following systemic administration, the liver, kidney, bone marrow, adipose tissue, spleen, and lymph nodes accumulate the highest drug concentrations, while distribution is poor to the intestine, skeletal muscle, heart, lung, reproductive organs, pancreas, and brain. MOE-modified ASOs are resistant to exonuclease degradation, resulting in prolonged tissue half-lives, ranging from 10 to 30 days. In general, ASOs are cleared from tissue by endonucleolytic degradation, producing lower molecular weight metabolites (8–12 nucleotides) that are eliminated by urinary excretion. In the first described antisense study performed in vitro, a human apo(a) ribozyme oligonucleotide containing P=S DNA and RNA was designed to specifically target KIV of the apo(a) mRNA without altering plasminogen transcript levels (28Morishita R. Yamada S. Yamamoto K. Tomita N. Kida I. Sakurabayashi I. Kikuchi A. Kaneda Y. Lawn R. Higaki J. et al.Novel therapeutic strategy for atherosclerosis: ribozyme oligonucleotides against apolipoprotein(a) selectively inhibit apolipoprotein(a) but not plasminogen gene expression.Circu­lation. 1998; 98: 1898-1904Crossref PubMed Scopus (35) Google Scholar). Because human cell lines do not adequately express the apo(a) gene, an apo(a) expression vector containing the 5′-untranslated region, the signal sequence, the first five KIV-like repeats, and 291 bp of the kringle repeat of apo(a) driven by the cytomegalovirus promoter was cotransfected into HepG2 cells with a hemagglutinating virus of Japan (HVJ)-liposome-apo(a) DNA complex and either the apo(a) ribozyme or control oligonucleotide. After 72 h, the apo(a) ribozyme was shown to reduce HepG2 protein secretion by approximately 60%, while no significant change in plasminogen protein was observed. While these initial results were encouraging, the requirement of liposomal formulation of the apo(a) targeting ribozyme prior to delivery would have made in vivo pharmacology studies more challenging. Mipomersen, a second generation ASO directed to apoB-100 and approved for clinical use in the United States for lowering LDL cholesterol (LDL-C) in patients with homozygous familial hypercholesterolemia (29Sniderman A.D. Tsimikas S. Fazio S. The severe hypercholesterolemia phenotype: clinical diagnosis, management, and emerging therapies.J. Am. Coll. Cardiol. 2014; 63: 1935-1947Crossref PubMed Scopus (130) Google Scholar), has been shown to lower Lp(a) levels in Lp(a)-transgenic mice (30Merki E. Graham M.J. Mullick A.E. Miller E.R. Crooke R.M. Pitas R.E. Witztum J.L. Tsimikas S. Antisense oligonucleotide directed to human apolipoprotein B-100 reduces lipoprotein(a) levels and oxidized phospholipids on human apolipoprotein B-100 particles in lipoprotein(a) transgenic mice.Circulation. 2008; 118: 743-753Crossref PubMed Scopus (129) Google Scholar) and in humans (31Raal F.J. Santos R.D. Blom D.J. Marais A.D. Charng M.J. Cromwell W.C. Lachmann R.H. Gaudet D. Tan J.L. Chasan-Taber S. et al.Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial.Lancet. 2010; 375: 998-1006Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, 32McGowan M.P. Tardif J.C. Ceska R. Burgess L.J. Soran H. Gouni-Berthold I. Wagener G. Chasan-Taber S. Randomized, placebo-controlled trial of mipomersen in patients with severe hypercholesterolemia receiving maximally tolerated lipid-lowering therapy.PLoS One. 2012; 7: e49006Crossref PubMed Scopus (185) Google Scholar, 33Stein E.A. Dufour R. Gagne C. Gaudet D. East C. Donovan J.M. Chin W. Tribble D.L. McGowan M. Apolipoprotein B synthesis inhibition with mipomersen in heterozygous familial hypercholesterolemia: results of a randomized, double-blind, placebo-controlled trial to assess efficacy and safety as add-on therapy in patients with coronary artery disease.Circulation. 2012; 126: 2283-2292Crossref PubMed Scopus (250) Google Scholar, 34Thomas G.S. Cromwell W.C. Ali S. Chin W. Flaim J.D. Davidson M. Mipomersen, an apolipoprotein B synthesis inhibitor, reduces atherogenic lipoproteins in patients with severe hypercholesterolemia at high cardiovascular risk: a randomized, double-blind, placebo-controlled trial.J. Am. Coll. Cardiol. 2013; 62: 2178-2184Crossref PubMed Scopus (185) Google Scholar). In humans, four phase 3 trials were performed in 382 patients on maximally tolerated lipid-lowering therapy and randomized 2:1 to weekly subcutaneous mipomersen (200 mg) (n = 256) or placebo (n = 126) for 26 weeks. Mipomersen reduced plasma Lp(a) levels by 21–39%; whereas, no significant change was noted in the placebo groups (35Santos R.D. Raal F.J. Catapano A.L. Witztum J.L. Steinhagen-Thiessen E. Tsimikas S. Mipomersen, an antisense oligonucleotide to apolipoprotein B-100, reduces lipoprotein(a) in various populations with hypercholesterolemia: results of 4 phase III trials.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 689-699Crossref PubMed Scopus (143) Google Scholar). Interestingly, in the mipomersen group, only modest correlations were present between percent changes in Lp(a) and apoB (r = 0.43, P < 0.001) and Lp(a) and LDL-C (r = 0.36, P < 0.001), suggesting mechanisms of Lp(a) lowering related to liver synthesis of apoB that are not apparent by evaluating plasma apoB levels. A study in Lp(a)-transgenic mice by Merki et al. (30Merki E. Graham M.J. Mullick A.E. Miller E.R. Crooke R.M. Pitas R.E. Witztum J.L. Tsimikas S. Antisense oligonucleotide directed to human apolipoprotein B-100 reduces lipoprotein(a) levels and oxidized phospholipids on human apolipoprotein B-100 particles in lipoprotein(a) transgenic mice.Circulation. 2008; 118: 743-753Crossref PubMed Scopus (129) Google Scholar) suggested that one potential mechanism of Lp(a) reduction by mipomersen may be through limiting hepatic production of newly formed apoB concomitantly when apo(a) is available to create an Lp(a) particle. Transgenic mice overexpressing both human APOB (h-apoB)-100 plus human LPA to generate genuine Lp(a) particles [human apo(a) does not form a covalent bond with mouse apoB-100] were treated with mipomersen. Mipomersen reduced hepatic apoB production and plasma levels of h-apoB-100 to very low levels ( 10 μM in both primary cell isolates (data not shown).TABLE 1ISIS-APO(a)Rx complementary binding sites within the human apo(a) transcriptISIS-APO(a)Rx Binding SitePosition on NM_005577.2 apo(a) mRNA transcriptBinding Site on First ExonBinding Site on Second Exonkringle IV2 repeat 2Exon 4-5505-524 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 3Exon 6-7847-866 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 4Exon 8-91189-1208 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 5Exon 10-111531-1550 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 6Exon 12-131873-1892 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 7Exon 14-152215-2234 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 8Exon 16-172557-2576 bpCTTGTTCTGCTCAGTCGGTGkringle IV2 repeat 9Exon 18-192899-2918 bpCTTGTTCTGCTCAGTTGGTGkringle IV2 repeat 11Exon 22-2335

Referência(s)