title: Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate.
abstract: Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVβ-gal, resulted in enhanced β-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of β-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
authors.affiliation: Centre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Uppal Road, Hyderabad 500007, Andhra Pradesh, India.
authors.forename: Srinath
authors.initials: S
authors.lastname: Govindarajan
authors.forename: Jeyarajan
authors.initials: J
authors.lastname: Sivakumar
authors.forename: Prathyusha
authors.initials: P
authors.lastname: Garimidi
authors.forename: Nandini
authors.initials: N
authors.lastname: Rangaraj
authors.forename: Jerald M
authors.initials: JM
authors.lastname: Kumar
authors.forename: Nalam M
authors.initials: NM
authors.lastname: Rao
authors.forename: Vijaya
authors.initials: V
authors.lastname: Gopal
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D006801:Humans; D008024:Ligands; D051379:Mice; D016513:Mice, SCID; D058990:Molecular Targeted Therapy / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D011479:Protamines; D055550:Protein Stability / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D044967:Serum / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D014162:Transfection
chemical_list: D001672:Biocompatible Materials; D057846:Cell-Penetrating Peptides; D004247:DNA; D003850:Deoxyribonuclease I; D008024:Ligands; D011479:Protamines; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D013925:Thromboplastin; C508053:ERBB2 protein, human