title: Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown.
abstract: Knockdown of genes by RNA interference (RNAi) in vitro requires methods of transfection or transduction, both of which have limited impact in vivo. As a virus-free approach, we chemically coupled cell surface receptors internalizing antibodies to the short interfering RNA (siRNA) carrier peptide protamine using the bispecific cross-linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone to carry siRNA. This complex can efficiently find, bind and internalize into receptor-positive cells in vitro and in vivo, which can be checked by flow cytometry, fluorescence microscopy and western blotting. This method obtains results similar to those of siRNA targeting molecules engineered by genetic fusions between receptor-binding and siRNA carrier units, with the advantage of using readily available purified proteins without the need for engineering, expression and purification of respective constructs. The procedure for coupling the complex takes ∼ 2 d, and the functional assays take ∼ 2 weeks.
authors.affiliation: Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Bäumer
authors.affiliation: Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany.
authors.forename: Neele
authors.initials: N
authors.lastname: Appel
authors.affiliation: Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Terheyden
authors.affiliation: University Cancer Center (UCC), Medical Systems Biology, Medical Faculty, Technische Universität (TU) Dresden, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, Muenster, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Rossig
authors.affiliation: Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany.|Department of Medicine IV, Hematology and Oncology, University Hospital Halle, Halle, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Müller-Tidow
authors.affiliation: Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany.
authors.forename: Wolfgang E
authors.initials: WE
authors.lastname: Berdel
authors.affiliation: Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Muenster, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bäumer
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism*; D066246:ErbB Receptors / Q000276:immunology; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D011479:Protamines / Q000737:chemistry / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000906:Antibodies; D004337:Drug Carriers; D008301:Maleimides; D011479:Protamines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C071675:sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; D066246:ErbB Receptors