Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle

Abstract

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

References 48

1. 1.

   Khvorova A., Watts J.K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotech.2017; 35:238–248.

   Not in our library10.1038/nbt.3765

2. 2.

   Stein C.A., Castanotto D. FDA-Approved Oligonucleotide Therapies in 2017. Mol. Ther.2017; 25:1069–1075.

   Not in our library10.1016/j.ymthe.2017.03.023

3. 3.

   Crooke S.T., Witztum J.L., Bennett C.F., Baker B.F. RNA-targeted therapeutics. Cell Metab.2018; 27:714–739.

   Not in our library10.1016/j.cmet.2018.03.004

4. 4.
   Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N -acetyl galactosamine improves potency 10-fold in mice · 2014

   No notice
5. 5.

   Ammala C., Drury W.J. 3rd, Knerr L., Ahlstedt I., Stillemark-Billton P., Wennberg-Huldt C., Andersson E.M., Valeur E., Jansson-Lofmark R., Janzen D. et al. Targeted delivery of antisense oligonucleotides to pancreatic β-cells. Sci Adv.2018; 4:eaat3386.

   Not in our library10.1126/sciadv.aat3386

6. 6.

   Hung G., Xiao X., Peralta R., Bhattacharjee G., Murray S., Norris D., Guo S., Monia B.P. Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals. Nucleic Acid Therap.2013; 23:369–378.

   Not in our library10.1089/nat.2013.0443

7. 7.

   Overby S.J., Cerro-Herreros E., Llamusi B., Artero R. RNA-mediated therapies in myotonic dystrophy. Drug Discov. Today. 2018; 23:2013–2022.

   Not in our library10.1016/j.drudis.2018.08.004

8. 8.

   Stein C.A., Yakubov L., Zhang L.M., Tonkinson J. Mode of uptake of 5′-cholesteryl-linked phosphodiester oligodeoxynucleotides in HL60 cells. Nucleic Acids Symp. Ser.1991; 24:155–156.

   Not in our library

9. 9.

   De Smidt P.C., Trung L.D., De Falco S., Van Berkel T.J.C. Association of antisense oligonucleotides with lipoproteins prolongs the plasma half-life and modifies the tissue distribution. Nucleic Acids Res.1991; 19:4695–4700.

   Not in our library10.1093/nar/19.17.4695

10. 10.

    Wolfrum C., Shi S., Jayaprakash K.N., Jayaraman M., Wang G., Pandey R.K., Rajeev K.G., Nakayama T., Charrise K., Ndungo E.M. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol.2007; 25:1149–1157.

    Not in our library10.1038/nbt1339

11. 11.

    Petrova N.S., Chernikov I.V., Meschaninova M.I., Dovydenko I.S., Venyaminova A.G., Zenkova M.A., Vlassov V.V., Chernolovskaya E.L. Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group. Nucleic Acids Res.2012; 40:2330–2344.

    Not in our library10.1093/nar/gkr1002

12. 12.

    Bijsterbosch M.K., Rump E.T., De Vrueh R.L.A., Dorland R., Van Veghel R., Tivel K.L., Biessen E.A.L., Van Berkel T.J.C., Manoharan M. Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation. Nucleic Acids Res.2000; 28:2717–2725.

    Not in our library10.1093/nar/28.14.2717

13. 13.

    Moroz E., Lee S.H., Yamada K., Halloy F., Martinez-Montero S., Jahns H., Hall J., Damha M.J., Castagner B., Leroux J.-C. Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differentiated intestinal cells. Mol. Ther.–Nucleic Acids. 2016; 5:e364.

    Not in our library10.1038/mtna.2016.69

14. 14.
    Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways · 2018

    No notice
15. 15.

    Biscans A., Coles A., Echeverria D., Khvorova A. The valency of fatty acid conjugates impacts siRNA pharmacokinetics, distribution, and efficacy in vivo. J. Controlled Release. 2019; 302:116–125.

    Not in our library10.1016/j.jconrel.2019.03.028

16. 16.

    Khan T., Weber H., DiMuzio J., Matter A., Dogdas B., Shah T., Thankappan A., Disa J., Jadhav V., Lubbers L. et al. Silencing myostatin using Cholesterol-conjugated siRNAs induces muscle growth. Mol. Ther.–Nucleic Acids. 2016; 5:e342.

    Not in our library10.1038/mtna.2016.55

17. 17.

    Van Der Vusse G.J., Glatz J.F.C., Van Nieuwenhoven F.A., Reneman R.S., Bassingthwaighte J.B. Transport of long-chain fatty acids across the muscular endothelium. Adv. Exp. Med. Biol.1998; 441:181–191.

    Not in our library10.1007/978-1-4899-1928-1_17

18. 18.

    Kratz F. Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J. Controlled Release. 2008; 132:171–183.

    Not in our library10.1016/j.jconrel.2008.05.010

19. 19.

    Garcovich M., Zocco M.A., Gasbarrini A. Clinical use of albumin in hepatology. Blood Transfus.2009; 7:268–277.

    Not in our library10.2450/2008.0080-08

20. 20.
    Albumin-based drug delivery: harnessing nature to cure disease · 2016

    No notice
21. 21.

    Plum A., Jensen L.B., Kristensen J.B. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis. J. Pharm. Sci.2013; 102:2882–2888.

    Not in our library10.1002/jps.23648

22. 22.

    Home P., Kurtzhals P. Insulin detemir: from concept to clinical experience. Expert Opin. Pharmacother.2006; 7:325–343.

    Not in our library10.1517/14656566.7.3.325

23. 23.
    Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay · 2018

    No notice
24. 24.

    Seth P.P., Tanowitz M., Bennett C.F. Selective tissue targeting of synthetic nucleic acid drugs. J. Clin. Invest.2019; 129:915–925.

    Not in our library10.1172/jci125228

25. 25.

    Leeds J.M., Graham M.J., Truong L., Cummins L.L. Quantitation of phosphorothioate oligonucleotides in human plasma. Anal. Biochem.1996; 235:36–43.

    Not in our library10.1006/abio.1996.0088

26. 26.

    Gaus H.J., Owens S.R., Cooper S., Cummins L.L. Online HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabolites. Anal. Chem.1997; 69:313–319.

    Not in our library10.1021/ac960557q

27. 27.

    Crooke S.T., Wang S., Vickers T.A., Shen W., Liang X-h. Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol.2017; 35:230–237.

    Not in our library10.1038/nbt.3779

28. 28.

    Miller C.M., Harris E.N., Tanowitz M., Donner A.J., Prakash T.P., Swayze E.E., Seth P.P. Receptor-mediated uptake of phosphorothioate antisense oligonucleotides in different cell types of the liver. Nucleic Acid Ther.2018; 28:119–127.

    Not in our library10.1089/nat.2017.0709

29. 29.

    Seth P.P., Siwkowski A., Allerson C.R., Vasquez G., Lee S., Prakash T.P., Wancewicz E.V., Witchell D., Swayze E.E. Short antisense oligonucleotides with novel 2′-4′ conformationaly restricted nucleoside analogues show improved potency without increased toxicity in animals. J. Med. Chem.2009; 52:10–13.

    Not in our library10.1021/jm801294h

30. 30.

    Gaus H., Miller C.M., Seth P.P., Harris E.N. Structural determinants for the interactions of chemically modified nucleic acids with the stabilin-2 clearance receptor. Biochemistry. 2018; 57:2061–2064.

    Not in our library10.1021/acs.biochem.8b00126

31. 31.

    Graham M.J., Crooke S.T., Lemonidis K.M., Gaus H.J., Templin M.V., Crooke R.M. Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration. Biochem. Pharmacol.2001; 62:297–306.

    Not in our library10.1016/s0006-2952(01)00669-4

32. 32.

    Graham M.J., Crooke S.T., Monteith D.K., Cooper S.R., Lemonidis K.M., Stecker K.K., Martin M.J., Crooke R.M. In vivo distribution and metabolism of a phosphorothioate oligonucleotide within rat liver after intravenous administration. J. Pharmacol. Exp. Ther.1998; 286:447–458.

    Not in our library

33. 33.

    Prakash T.P., Yu J., Kinberger G.A., Low A., Jackson M., Rigo F., Swayze E.E., Seth P.P. Evaluation of the effect of 2′-O-methyl, fluoro hexitol, bicyclo and Morpholino nucleic acid modifications on potency of GalNAc conjugated antisense oligonucleotides in mice. Bioorg. Med. Chem. Lett.2018; 28:3774–3779.

    Not in our library10.1016/j.bmcl.2018.10.011

34. 34.

    Kragh-Hansen U., Watanabe H., Nakajou K., Iwao Y., Otagiri M. Chain Length-dependent Binding of Fatty Acid Anions to Human Serum Albumin Studied by Site-directed Mutagenesis. J. Mol. Biol.2006; 363:702–712.

    Not in our library10.1016/j.jmb.2006.08.056

35. 35.

    Schmidt K., Prakash T.P., Donner A.J., Kinberger G.A., Gaus H.J., Low A., Østergaard M.E., Bell M., Swayze E.E., Seth P.P. Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor. Nucleic Acids Res.2017; 45:2294–2306.

    Not in our library10.1093/nar/gkx060

36. 36.

    Mullick A., Crooke R.M., Graham M. J. Antisense inhibition of CD36 expression and therapeutic uses thereof. 2012; WO2012149465A2.

    Not in our library

37. 37.

    Pandey S.K., Wheeler T.M., Justice S.L., Kim A., Younis H.S., Gattis D., Jauvin D., Puymirat J., Swayze E.E., Freier S.M. et al. Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1. J. Pharmacol. Exp. Ther.2015; 355:310–321.

    Not in our library10.1124/jpet.115.226969

38. 38.

    Jauvin D., Chretien J., Pandey S.K., Martineau L., Revillod L., Bassez G., Lachon A., McLeod A.R., Gourdon G., Wheeler T.M. et al. Targeting DMPK with antisense oligonucleotide improves muscle strength in myotonic dystrophy type 1 mice. Mol. Ther.–Nucleic Acids. 2017; 7:465–474.

    Not in our library10.1016/j.omtn.2017.05.007

39. 39.

    Campostrini G., Bonzanni M., Lissoni A., Bazzini C., Milanesi R., Vezzoli E., Francolini M., Baruscotti M., Bucchi A., Rivolta I. et al. The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability. Cardiovasc. Res.2017; 113:1256–1265.

    Not in our library10.1093/cvr/cvx122

40. 40.

    Song K.S., Scherer P.E., Tang Z., Okamoto T., Li S., Chafel M., Chu C., Kohtz D.S., Lisanti M.P. Expression of Caveolin-3 in skeletal, cardiac, and smooth muscle Cells: Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteinS. J. Biol. Chem.1996; 271:15160–15165.

    Not in our library10.1074/jbc.271.25.15160

Cited by papers we hold 4

• Possibilities and limitations of antisense oligonucleotide therapies for the treatment of monogenic disorders · 2024
  No notice
• Delivery of oligonucleotide‐based therapeutics: challenges and opportunities · 2021
  No notice
• The powerful world of antisense oligonucleotides: From bench to bedside · 2020
  No notice
• Therapeutic siRNA: state of the art · 2020
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.