Cleavage of intracellular hepatitis C RNA in the virus core protein coding region by deoxyribozymes

Abstract

Hepatitis C virus (HCV) infection represents an important global health problem. Current antiviral therapeutics for HCV have proven inadequate in stemming the disease process. A novel therapeutic strategy involves the use of deoxyribozymes, also known as DNA enzymes or DNAzymes. These catalytic DNA molecules, designed to target and cleave specific RNA sequences, have shown promise in in vitro experimental models for various diseases and may serve as an alternative or adjunct to current HCV drug therapy. We designed and tested several deoxyribozymes that can bind and cleave highly conserved RNA sequences encoding the HCV core protein in in vitro systems. One of these deoxyribozymes reduced the level of our HCV RNA target by 32% and 48% after 24 h of cell exposure when tested in human hepatoma and epithelial cell lines, respectively. As this deoxyribozyme showed significant cleavage activity against HCV core protein target RNA in human cells, it may have potential as a therapeutic candidate for clinical trial in HCV infected patients.

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes

“…The Dz Dz858-15-15 [72] decreased the level of viral RNA by 30 --50% depending on cell type. Both mRNA and protein expression from the cloned core gene were reduced substantially when the gene was co-transfected with anti-core Dz CDz [19].…”

“…Other Dzs also were constructed to bind conserved sequences of the core protein regions of the viral RNA [19,72]. The Dz Dz858-15-15 [72] decreased the level of viral RNA by 30 --50% depending on cell type.…”

“…ENV [55][56][57] TAT, TAT-REV [58][59][60][61] TAR [62] GAG, NEF [63] HIV-1 Integrase [64] Vpr [65] Hepatitis B X [66,67] S, C [68,69] DR1 and PA regions of pre-genomic RNA [70] Hepatitis C Core [19,71,72] NS3 [73,74] NS5B [19] IRES [17] Influenza A PB2 [75,76] M1 [77] Influenza B BM2 [21] Respiratory syncytial virus (RSV) N [15,78] Severe acute respiratory syndrome coronavirus (SARS-CoV) 5'-Untranslated region of a highly conserved fragment of SARS genome [79] Epstein-Barr virus LMP1 [80][81][82][83][84][85] Human papilloma virus type 16 E6/E7 [48] Japanese encephalitis virus 3'-Non-coding regions of RNA genome [16] Bacteria Ampicillin-resistant bacteria TEM1, TEM3 b-Lactamase [14] Methicillin-resistant Staphylococcus aureus blaR1 [86] MecR1 [87] Mycobacterium tuberculosis…”

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

“…Target RNA for in vivo deoxyribozyme-catalyzed cleavage References Egr1 zinc finger transcription factor [139 -142] Epstein-Barr virus latent membrane protein [143] Hepatitis B virus X protein [144] Hepatitis B virus HBs and HBe antigens [145] Hepatitis C virus core protein [146] HIV-1 Gag [147 -149] HIV-1 Tat protein [150,151] HIV-1 5'-untranslated region [152] Human telomerase reverse transcriptase [153] Influenza virus A [154] Isocitrate lyase from M. tuberculosis [155] c-Jun leucine zipper transcription factor [156,157] b-Lactamase [158,159] 12-Lipoxygenase [160] c-Myc proto-oncogene [161 -163] Ornithine decarboxylase [164] Pencillin-binding protein [165] PML/RARa fusion gene of acute promyelocytic leukemia [166] Respiratory syncytial virus (RSV) nucleocapsid protein [167] SARS associated coronavirus 5'-untranslated region [168] Survivin [169] TGF-b1 [170] Twist helix-loop-helix transcription factor [171] Vascular endothelial growth factor receptor 2 (VEGFR) [172] This tabulation is representative, not comprehensive. Additional examples and reviews are cited in ref.…”

Activity of Core‐Modified 10–23 DNAzymes against HCV

“…For the particular case of HCV, as far as we know, only one report dealing with a DNAzyme carrying four 2'-O-methylnucleotides consecutively located on the distal ends of the annealing arms is known. [20] On the other hand, due to the sensitive structure-activity relationship the catalytic core presents, there are only a few examples of chemical modifications in the 10-23 DNAzyme catalytic core, using for example 2'-O-methylnucleosides, [18] phosphorothioates, [21] amino-acid-like bases, [22] or abasic phosphoramidites. [23] In this sense, the 2'-C-methyl-2'-deoxynucleosides [24] constitutes an interesting nucleoside class, because they show differential preferred sugar conformations depending on the absolute configuration at the 2'-carbon.…”