title: An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.
abstract: Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing.
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Konrad
authors.initials: K
authors.lastname: Bienk
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Michael Lykke
authors.initials: ML
authors.lastname: Hvam
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Malgorzata Maria
authors.initials: MM
authors.lastname: Pakula
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Frederik
authors.initials: F
authors.lastname: Dagnæs-Hansen
authors.affiliation: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.affiliation: Novozymes Biopharma, Novozymes A/S, Brudelysvej 32, 2880 Bagsværd, Denmark.
authors.forename: Birgitte Mølholm
authors.initials: BM
authors.lastname: Malle
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kragh-Hansen
authors.affiliation: Albumedix Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom.
authors.forename: Jason
authors.initials: J
authors.lastname: Cameron
authors.affiliation: Novozymes Biopharma, Novozymes A/S, Brudelysvej 32, 2880 Bagsværd, Denmark.
authors.forename: Jens Thostrup
authors.initials: JT
authors.lastname: Bukrinski
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. Electronic address: kenh@inano.au.dk.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Howard
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D005167:Factor VII / Q000235:genetics; D005260:Female; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D012709:Serum Albumin / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005167:Factor VII; D002784:Cholesterol
keywords: Albumin; Biodistribution; Drug delivery; Factor VII; Half-life extension; siRNA