title: Variable Heavy Chain Domain Derived from a Cell-Penetrating Anti-DNA Monoclonal Antibody for the Intracellular Delivery of Biomolecules.
abstract: Targeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.
authors.affiliation: a Department of Biomedical Sciences, Graduate School of Medicine, and Department of Microbiology , School of Medicine, Ajou University , Suwon , Republic of Korea.
authors.forename: Sun-Woo
authors.initials: SW
authors.lastname: Im
authors.affiliation: a Department of Biomedical Sciences, Graduate School of Medicine, and Department of Microbiology , School of Medicine, Ajou University , Suwon , Republic of Korea.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Pravinsagar
authors.affiliation: a Department of Biomedical Sciences, Graduate School of Medicine, and Department of Microbiology , School of Medicine, Ajou University , Suwon , Republic of Korea.
authors.forename: Sae-Ran
authors.initials: SR
authors.lastname: Im
authors.affiliation: a Department of Biomedical Sciences, Graduate School of Medicine, and Department of Microbiology , School of Medicine, Ajou University , Suwon , Republic of Korea.
authors.forename: Young-Ju
authors.initials: YJ
authors.lastname: Jang
mesh_terms: D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D059447:Cell Cycle Checkpoints / Q000235:genetics; D049109:Cell Proliferation; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002522:Chlorocebus aethiops; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019169:Jurkat Cells; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000974:Antibodies, Antinuclear; D001324:Autoantigens; D057846:Cell-Penetrating Peptides; D018796:Immunoconjugates; C489390:NASP protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies
keywords: Anti-DNA antibodies; cell-penetrating antibody; intracellular delivery vehicle; variable heavy chain domain