Paul S. Miller

Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

XPB, a subunit of TFIIH, is a target of the natural product triptolide

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.

Molecular Basis for Zinc Potentiation at Strychnine-sensitive Glycine Receptors

The divalent cation Zn2+ is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn2+. Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn2+ potentiation site on GlyRs and account for the differential sensitivity of GlyR α1 and GlyR α2 to Zn2+ potentiation. In addition, juxtaposed to this Zn2+ site, which is located externally on the N-terminal domain of the α subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn2+ potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn2+ binds to a site on the extracellular outer face of the GlyR α subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.

Cell Cycle Modulation of Gene Targeting by a Triple Helix-forming Oligonucleotide

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targetingin vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in ∼20–30% of target sequences. 75–80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.

Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process.

Elutriator‐purified human monocytes were cultured in a serum‐free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate.

Chapter 30. Oligonucleotide Inhibitors of Gene Expression in Living Cells: New Opportunities in Drug Design

Publisher Summary This chapter describes some of the oligomers and experiments in which they have been used to control gene expression at the messenger RNA (mRNA) level, particularly in living cells. It appears that this concept could be exploited to develop clinically useful drugs to control the growth of viruses or malignant cells. Present day drugs that bind or intercalate in the minor groove of DNA have their limited ability to recognize specific nucleic sequences. In that way, they cannot take full advantage of the sequence information available in DNA, and thus are unable to inhibit expression of specific genes. The preparation, introduction, and expression of anti-sense RNAs require rather sophisticated molecular biology techniques. They can be prepared in the laboratory and then introduced into cells by microinjection or expressed in cells after transfection with plasmids carrying an anti-sense gene. The control of gene expression can, in theory, be achieved by single-stranded nucleic acids, that can specifically read complementary mRNA sequences via Watson–Crick base-pairing interactions. There exists a simpler approach that involves the use of short anti-sense oligonucleotides or oligonucleotide analogs prepared by chemical synthesis. There are three types of oligonucleotides that are oligodeoxyribonucleotides, oligomers having modified sugar–phosphate backbones, and oligomers derivatized with specific functional groups. Little is known about the stability, distribution, acute and/or chronic toxicity, or immunogenicity of compounds in vivo belonging to the anti-sense oligonucleotides or oligonucleotide analogs class. The promising results obtained in cell culture studies suggest that anti-sense oligonucleotide analogs offer a unique approach for the rational design of drug.

Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen)

Purpose: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the bcl-2 mRNA, is currently being evaluated in several Phase II and Phase III clinical trials. However, the mechanism of action of this molecule appears to depend on a combination of antisense plus nonantisense events. Indeed, the very idea that bcl-2 is a critical target is, at least in part, an extrapolation from experiments in which intracellular bcl-2 protein concentrations have been dramatically increased, yielding chemoresistant cells. Experimental Design: In this work, we down-regulated the expression of bcl-2 protein by 80–90% by two different antisense RNA strategies (antisense RNA and small interfering RNA) in DU145 prostate cancer cells. Results: Even after down-regulation of bcl-2 protein expression by either one of these strategies, the cellular phenotype induced by subsequent G3139 treatment (inhibition of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mock-infected or wild-type DU145 cells in which bcl-2 protein expression had not been down-regulated previously. Conclusions: These results strongly suggest that bcl-2 expression in DU145 cells is not strongly associated with the prolife phenotype and that the mechanism by which G3139 produces its cytostatic effects in this cell line is bcl-2 independent.

Triplex formation by oligonucleotides containing novel deoxycytidine derivatives

Homopurine sequences of duplex DNA are binding sites for triplex-forming oligodeoxyribopyrimidines. The interactions of synthetic duplex DNA targets with an oligodeoxyribopyrimidine containing N4-(6-amino-2-pyridinyl)deoxycytidine (1), a nucleoside designed to interact with a single C-G base pair interruption of the purine target tract, was studied by UV melting, circular dichroism spectroscopy and dimethylsulfate alkylation experiments. Nucleoside 1 supports stable triplex formation at pH 7.0 with formation of a 1-Y-Z triad, where Y-Z is a base pair in the homopurine tract of the target. Selective interaction was observed when Y-Z was C-G, although A-T and, to a lesser extent, T-A and G-C base pairs were also recognized. The circular dichroism spectra of the triplex having a 1-C-G triad were similar to those of a triplex having a C(+)-G-C triad, suggesting that the overall structures of the two triplexes are quite similar. Removal of the 6-amino group from 1 essentially eliminated triplex formation. Reaction of a triplex having the 1-C-G triad with dimethylsulfate resulted in a 50% reduction of methylation of the G residue of this triad. In contrast, the G of a similar triplex containing a U-C-G triad was not protected from methylation by dimethylsulfate. These results are consistent with a binding mode in which the 6-amino-2-pyridinyl group of 1 spans the major groove of the target duplex at the 1-C-G binding site and forms a hydrogen bond with the O6 of G. An additional stabilizing hydrogen bond could form between the N4 of the imino tautomer of 1 and the N4 amino group of C.

Proton NMR and Optical Spectroscopic Studies on the DNA Triplex Formed by d-A-(G-A)7-G and d-C-(T-C)7-T

Triplex and duplex formation of two deoxyribohexadecamers d-A-(G-A)-G (a) and d-C-(T-C)-T (b) have been studied by UV, CD, fluorescence, and proton NMR spectroscopy. Optical studies of a and b at dilute concentrations (microM range) yielded results similar to those seen for polymers of the same sequence, indicating that these hexadecamers have properties similar to the polymers in regard to triplex formation. The CD spectra of concentrated NMR samples (mM range) are similar to those observed at optical concentrations at both low and high pH, making possible a correlation between CD and NMR studies. In NMR spectra, two imido NH-N hydrogen bonded resonance envelopes at 12.6 and 13.7 ppm indicate that only the duplex conformation is present at pH greater than 7.7. Four new NH-N hydrogen-bonded resonance envelopes at 12.7, 13.5, 14.2, and 14.9 ppm are observed under acidic conditions (pH 5.6) and the two original NH-N resonances gradually disappear as the pH is lowered. Assignment of these four peaks to Watson-Crick G.C. Hoogsteen T.A Watson-Crick A.T, and Hoogsteen C+.G hydrogen-bonded imidos, respectively, confirm the formation of triple-stranded DNA NMR results also show that triplex is more stable than duplex at the same salt condition and that triplex melts to single strands directly without going through a duplex intermediate. However, in the melting studies, a structural change within the triple-stranded complex is evident at temperatures significantly below the major helix-to-coil transition. These studies demonstrate the feasibility of using NMR spectroscopy and oligonucleotide model compounds a and b for the study of DNA triplex formation.

Initiation of DNA Interstrand Cross-link Repair in Mammalian Cells

Interstrand cross‐links (ICLs) are among the most cytotoxic DNA lesions to cells because they prevent the two DNA strands from separating, thereby precluding replication and transcription. Even though chemotherapeutic cross‐linking agents are well established in clinical use, and numerous repair proteins have been implicated in the initial events of mammalian ICL repair, the precise mechanistic details of these events remain to be elucidated. This review will summarize our current understanding of how ICL repair is initiated with an emphasis on the context (replicating, transcribed or quiescent DNA) in which the ICL is recognized, and how the chemical and physical properties of ICLs influence repair. Although most studies have focused on replication‐dependent repair because of the relation to highly replicative tumor cells, replication‐independent ICL repair is likely to be important in the circumvention of cross‐link cytotoxicity in nondividing, terminally differentiated cells that may be challenged with exogenous or endogenous sources of ICLs. Consequently, the ICL repair pathway that should be considered “dominant” appears to depend on the cell type and the DNA context in which the ICL is encountered. The ability to define and inhibit distinct pathways of ICL repair in different cell cycle phases may help in developing methods that increase cytotoxicity to cancer cells while reducing side‐effects in nondividing normal cells. This may also lead to a better understanding of pathways that protect against malignancy and aging. Environ. Mol. Mutagen., 2010.