Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides.

A 21-mer oligodeoxynucleotide complementary to the polyadenylation signal for human hepatitis B virus (HBV) was complexed to a soluble DNA-carrier system that is targetable to hepatocytes via asialoglycoprotein receptors present on those cells. A cell line, HepG2 (2.2.15) that possesses asialoglycoprotein receptors and is permanently transfected with hepatitis B virus (ayw subtype) was exposed to complexed antisense DNA or controls. In the presence of complexed antisense DNA, the concentration of hepatitis B surface antigen in medium was 80% lower than controls after 24 h. Furthermore, during the next 6 days, there was no significant increase in surface antigen concentration in the presence of complexed antisense DNA. The inhibition could be effectively blocked by competition with an excess of free asialoglycoprotein. Total protein synthesis remained unchanged by exposure to complexed antisense sequences under identical conditions. In addition, HBV DNA in the medium and cell layers after 24-h exposure to complexed antisense sequences was 80% lower than in controls. The data indicate that antisense oligonucleotides complexed by a soluble DNA-carrier system can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of hepatitis B viral gene expression and replication.     

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5′ and 3′ HNA sequences to conventional phosphorothioate oligonucleotides and to a 2′-O-methoxyethyl (2′-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2′-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.     

Inhibition of expression of a mouse alpha-globin gene by plasmids that include antisense oligonucleotides.

Plasmid-borne DNAs, corresponding to 68-base oligodeoxynucleotides, synthesized in the antisense or sense configuration and based on the nucleotide sequences of various regions of the mouse alpha-globin mRNA, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from E. coli (Ecogpt) into mouse erythroleukemia (MEL) cells by protoplast fusion. Specific inhibition of the synthesis of alpha-globin was observed only in the cells transformed with the plasmids with antisense 68-mers that corresponded to the cap site as well as the site of initiation of translation of alpha-globin mRNA (Oligo-A); Other plasmids with antisense 68-mers that corresponded to the regions of the exon/intron junctions, the individual exons, or the 3' untranslated region were ineffective. This antisense RNA efficiently reduced the production of alpha-globin to 9-18% of the endogenous level after induction with hexylmethylene-bis-acetoamide (HMBA). Moreover, most of the antisense transformants did not show any decrease in the expression of the c-myc gene at the early phases of differentiation of MEL cells. Thus, we propose a hypothesis that the early decline in levels of c-myc mRNA may be independent of and uncoupled from the program of globin synthesis during the differentiation of MEL cells.     

Recovery of cell cycle delay following targeted gene repair by oligonucleotides

We have previously shown that activation of the homologous recombinational repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long-term impact of this stalling on the growth of cells that have enabled gene repair events. Using a mutated eGFP gene as an episomal reporter, we show that corrected (eGFP-positive) cells contain only a few active replication templates 2 weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By 6 weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP-negative) population. These results indicate that the initial stalling is transient and eGFP-positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.     

Enhancement of ribosomal frameshifting by oligonucleotides targeted to the HIV gag-pol region.

The pol gene of all retroviruses is expressed as a gag-pol fusion protein which is proteolytically processed to produce all viral enzymes. In the human immunodeficiency virus (HIV), the gag and pol genes overlap by 241 nucleotides with pol in the -1 phase with respect to gag. The gag-pol fusion is produced via a -1 ribosomal frameshifting event that brings the overlapping, out-of-phase gag and pol genes into translational phase. Frameshifting occurs at a so called 'shift site' 8-10 nucleotides upstream of a hairpin loop which may play a role in the regulation of frameshifting. We have fused this region of HIV-1 to the 5' end of the firefly luciferase reporter gene in order to quantitatively measure ribosomal frameshifting both in cells and by in vitro translation. A series of 2'-O-methyl oligonucleotides was designed to specifically bind the sequences which flank the gag-pol hairpin. Ribosomal frameshifting is enhanced up to 6 fold by those oligonucleotides which bind the area just 3 to the stem. Oligonucleotides which bind 5' to the stem have no effect on frameshift efficiency. In addition, we have constructed a series of fusion genes which mimic the effect of the bound oligonucleotides with intramolecular hairpins. The results suggest that increasing RNA secondary structure downstream of the shift site increases the frequency of ribosomal frameshifting, and that this effect can be mimicked by antisense oligonucleotides.     

Inhibition of human immunodeficiency virus type 1 Rev-Rev-response element complex formation by complementary oligonucleotides.

Complementary 18-mer oligodeoxynucleotides (oligonucleotides) specifically inhibited the formation of human immunodeficiency virus Rev-Rev-response element (RRE) complexes. Inhibition of Rev-RRE binding required blockage of G-7819 to G-7820 in band shift assays. Structural studies revealed both local and distal effects. RRE structure was also disrupted by oligonucleotides targeted to other minor stems, by altering RNA renaturation conditions, or by reducing Rev concentrations--indicating a dynamic RRE structure and involvement of a minor RRE stem in the maturation of initial Rev-RRE complexes. Thus, complementary oligonucleotides alter RRE structure and may prove useful for the design of therapeutic anti-RRE oligonucleotides.     

Control of gene expression by oligonucleotides covalently linked to intercalating agents

Oligodeoxynucleotides covalently linked to intercalating agents selectively recognize the complementary sequence of the oligonucleotide. The intercalating agent provides an additional binding energy which stabilizes the complex. These substances can be used in vitro to block mRNA translation. In cell cultures they are able to inhibit the cytopathic effect of viruses, such as influenza virus and the oncogenic virus SV40. They kill trypanosomes in culture as a result of protein synthesis inhibition. A reactive group can be attached to an oligodeoxynucleotide in order to achieve site-directed modifications of the target sequence. Metal complexes of EDTA, phenanthroline or porphyrins induce cleavage reactions of the phosphodiester backbone in both DNA and RNA. Photoactive groups can be used to modify bases in the complementary sequence. The double helix can be recognized and modified by oligonucleotides that bind to the major groove, forming a local triple helix. These site-directed modifications may inhibit biological processes. The oligonucleotide can be made resistant to nuclease digestion by substituting the synthetic alpha-anomers of nucleosides to the natural beta-nucleosides. These results provide the basis for the design of gene-specific inhibitors that can be used as tools in molecular and cellular biology. They also suggest new approaches for the rational development of selective anti-viral, anti-parasitic, and anti-tumoral agents.     

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera–TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.     

Inhibition of CD4 expression by antisense oligonucleotides in PMA-treated lymphocytes

To decrease CD4 expression on T helper (Th) lymphocyte surface, antisense oligonucleotides (AS-ODNs), delivered by the cationic liposome DOTAP, were assayed in vitro on rat spleen lymphocytes. Four 21-mer ODNs (AS-CD4-1, AS-CD4-2, AS-CD4-3, and AS-CD4-4) directed against the translation start region of the cd4 gene were designed. AS-CD4-1 was phosphorothioate (PS)-modified in each base, and the other three were PS-modified at both ends and in the internal pyrimidine residues. Four ODN controls (fully PS-modified ODN-A and partially modified ODN-B, ODN-C, and ODN-D) were also assayed. CD4 resynthesis was stimulated by treatment with phorbol 12-myristate 13-acetate (PMA) at the same time as the incubations with the ODN. After 24 hours of treatment, CD4 expression was measured by immunofluorescence staining and flow cytometry. CD4 reexpression in rat PMA-treated lymphocytes was counteracted by 40% by means of AS-CD4-2 and AS-CD4-4 treatments. On the other hand, AS-CD4-3 produced only 20% inhibition, similar to that produced by ODN-B, and AS-CD4-1 did not have any significant effect compared with control ODNs. Both AS-CD4-2 and AS-CD4-4 decreased CD4 mRNA, as determined by RT-PCR, and in addition, they did not affect the expression of other surface lymphocyte molecules. Inhibition of surface CD4 expression remained at least 72 hours. The addition of both AS-ODNs did not further increase the effect obtained separately by each AS-ODN. Treatment of rat PMA-lymphocytes with two concentrations of AS-CD4-2 and AS-CD4-4 added 24 hours apart did not further decrease CD4 expression. In summary, AS-CD4-2 and AS-CD4-4 could constitute a good strategy to inhibit CD4 expression on Th lymphocytes and modulate their function.     

Luminescence quenching of Ru-labeled oligonucleotides by targeted complementary strands.

The yield of hole injection into guanines of different oligonucleotide duplexes by a photooxidizing tethered Ru(II) complex is examined by measuring the luminescence quenching of the excited complex. This yield is investigated as a function of the anchoring site of the complex (on a thymine nucleobase in the middle of the sequence or on the 5' terminal phosphate) and the number and position of the guanine bases as compared with the site of attachment of the Ru(II) compound. In contrast to other studies, the tethered complex, [Ru(tap)(2)(dip)](2+), is a non-intercalating compound and has been shown previously to produce an irreversible photocrosslinking between the two strands as the ultimate step of hole injection. The study of luminescence quenching of the anchored complex by emission intensity and lifetime measurements for the different duplexes indicates that a direct contact between the complex and the guanine nucleobase is needed for the electron transfer to take place. Moreover, for none of the sequences a clear contribution of a static quenching is evidenced independently of the two types of attachment of the [Ru(tap)(2)(dip)](2+) complex to the oligonucleotide. A comparison of the fastest hole-injection process by electron transfer to the excited anchored [Ru(tap)(2)(dip)](2+), with the rate of the photo-electron transfer between the same complex free in solution and guanosine-5'-monophosphate, indicates that the hole injection by the anchored complex is slower by a factor of 10 at least. A bad overlap between donor and acceptor orbitals is probably the cause of this slow rate, which could be attributed to some steric hindrance induced by the complex linker.     

Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure.

Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem. We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor. The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex. However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA. In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene. Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA. Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance.