Cellular delivery of hammerhead ribozymes conjugated to a transferrin receptor antibody.
Chemically-modified hammerhead ribozymes are sequence-specific RNA enzymes that can cleave target mRNA. These molecules have potential application as biological tools to understand gene expression and as therapeutic agents for the selective down-regulation of genes implicated in disease. However, as a result of their polyanionic character and relatively large molecular weights, ribozyme delivery to target cells is relatively inefficient. Using nuclease resistant 2'-O-methyl-modified ribozymes targeting the c-erbB1 oncogene, we have evaluated the potential use of human monoclonal transferrin-receptor antibody (TRA)-ribozyme conjugates for the improved delivery of ribozymes to A431 tumour cells. A 37-mer ribozyme derivatized with a free thiol-group at the 5'-end and bearing an internal [32P]-radiolabel was conjugated to either TRA or a non-specific IgG antibody using the heterobifunctional crosslinker, succinimidyl 4-(maleimido methyl)cyclohexane-1-carboxylate (SMCC). Up to six molecules of the ribozyme could be conjugated to one molecule of antibody. Cellular uptake studies in cultured human epidermoid A431 carcinoma cells showed that approximately a three-fold increase in cellular association could be obtained with the TRA-ribozyme conjugate compared to the free ribozyme. Cellular association of the conjugate was temperature-dependent and was inhibited by competition with excess free transferrin receptor antibody implying that conjugate uptake was consistent with the transferrin receptor-mediated endocytosis pathway. Treatment of cells with monensin further enhanced TRA-ribozyme conjugate cell association indicating that ribozyme delivery of conjugates may be further improved by strategies that modulate vesicular trafficking in cells.
Hudson
A J
AJ
Pharmaceutical Sciences Research Institute, Department of Pharmaceutical and Biological Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
Normand
N
N
Ackroyd
J
J
Akhtar
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000493:pharmacokinetics*; D001483:Base Sequence; D004795:Enzyme Stability; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000493:pharmacokinetics*; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000493:pharmacokinetics*; D011990:Receptors, Transferrin / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D007476:Ionophores; D016337:RNA, Catalytic; D011990:Receptors, Transferrin; D008985:Monensin