In recent years, a nova1 class of pharmaceuticals has been developed which bind in a predictable way to certain nucleic acid target sequences aiming at selective inhibition of expression of ...disease‐causing genes. The chemical structure of these so‐called antisense oligonucleotide compounds must be altered relative to their natural models to render them stable under in vivo conditions and to allow their penetration to the site of action inside cells. In this article, the principle of antisense oligonucleotide function, the structure of antisense oligonucleotide analogues, the different strategies for improvement of their biological potency, and selected reports on successful in vivo studies and first clinical investigations using these antisense oligonucleotides are discussed.
Single ultraviolet (u.v.) irradiation of mammalian cells in culture evokes the transcriptional activation of various proto-oncogenes, among them members of the fos/jun family which are known to play ...an important role in cell proliferation and differentiation. u.v. exposure of mammalian skin results in growth arrest and cell death followed by hyperproliferation of epidermal cells. To obtain information in vivo about a possible relationship between u.v.-induced proto-oncogene expression and cellular alterations, we have analysed the expression of c-fos, fosB, c-jun, junB, bcl-2 and bax in rat epidermis after single and chronic u.v. irradiation. We present data demonstrating that the transcripts of these genes are constitutively expressed in the epidermis and that expression is differentially modulated by u.v. exposure. Single u.v. irradiation causes a rapid and sustained increase in c-jun, junB and c-fos mRNA and a decline in bcl-2 transcripts, whereas expression of bax remained unchanged. c-Fos and c-Jun immunoreactivity was localized throughout the epidermal cell layers 1.5 h after single irradiation, but restricted to basal cells at 48 h suggesting an involvement in both u.v.-induced apoptosis and hyperproliferation. 48 h after chronic exposure a significantly higher induction and a totally different pattern of epidermal proto-oncogene expression was detectable which may be associated with malignancy. Superfusion of rat skin with c-fos antisense oligodeoxynucleotides inhibited the increase in c-Fos immunolabeled epidermal cells 1.5 h after single u.v. irradiation demonstrating that antisense oligodeoxynucleotides are capable of penetrating mammalian skin and modulating the u.v. response in vivo. However, suppression of the early c-Fos activation did not significantly affect the formation of sunburn cells in the u.v.-exposed epidermis. Thus, c-Fos does not seem to play a major role in u.v.-induced apoptosis or other members of the fos/jun family may compensate for a loss in c-Fos.
Vive la différence! Replacement of the base‐linker amide functionality in peptide nucleic acids (PNAs) by an isostructural C−C double bond (shown schematically) leads to a dramatic change in DNA ...affinity, preferred strand orientation, and triplex‐forming properties, and thus highlights the importance of electrostatic properties over geometric properties of the amide functionality in PNA/DNA recognition.
Unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG)-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists for Toll-like receptor 9. We here describe a new class of ...CpG ODNs, the so-called P-Class, which combines preferred properties of known CpG ODN classes. This P-Class contains two palindromic sequences, enabling it to form concatamers, multimeric units, where each molecule is bound via Watson-Crick basepairing to a second and a third palindrome. The type I interferon-inducing potency and efficacy of the double-palindromic P-Class ODN is substantially higher than that of previously described C-Class ODNs, and they stimulate superior cytokine production upon in vivo application. The multimeric structures of the P-Class can be resolved to monomers and dimers by formulation in low-salt buffer, retaining the strong and potent immune effects. Taken together, we have discovered a novel class of CpG ODNs, the P-Class, with promising superior activity for disease application.
The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1–4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5–8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal ...part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐(thymin‐1‐yl)acetylglycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their Tm values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.
Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of ...these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3‘-end (PPS−C16). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3‘- and 5‘-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS−C16 ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS−C16 ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS−C16 ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.
The synthesis of bile acid–oligodeoxynucleotide conjugates via the 3-OH group of the bile acids is described. When used in vivo in rats, covalent conjugation of an oligodeoxynucleotide via a linker ...to cholic acid resulted in an increased biliary excretion of bile acid–oligodeoxynucleotide conjugates compared to unconjugated oligodeoxynucleotides.
The synthesis of bile acid–oligodeoxynucleotide conjugates via the 3-OH group of the bile acids is described. When used in vivo in rats, covalent conjugation of an oligodeoxynucleotide via a linker to cholic acid resulted in an increased biliary excretion of bile acid–oligodeoxynucleotide conjugates compared to unconjugated oligodeoxynucleotides.
The automated on‐line synthesis of DNA‐3′‐PNA (PNA=Polyamide Nucleic Acids) chimeras 1 – 3 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the aminoterminal part of ...the PNA either via a N‐(2‐mercaptoethyl)‐ (X=S), a N‐(2‐hydroxyethyl)‐ (X=O), or a N‐(2‐aminoethyl)‐ (X=NH) N‐(thymin‐1‐yl)acetylglycine unit. Furthermore, the DNA‐3′‐PNA chimera 4 without a nucleobase at the linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their Tm values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on duplex stabilization was determined to be in the order O>S≈NH, rendering the phosphodiester bridge the most favored linkage at the DNA/PNA junction. The observed strong duplex‐destabilizing effects, when base mismatches or non‐basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest that the base at the linking unit contributes significantly to duplex stabilization.
Polyamide-linked nucleic acid analogs (PNAs) are DNA mimics in which the deoxyribose phosphate backbone is replaced by uncharged N-(2-aminoethyl)glycine units. PNA is the only example of a biopolymer ...whose backbone structure differs significantly from that of natural nucleic acids which nevertheless follows Watson--Crick base-pairing rules with complementary strands. Initially, PNAs were investigated as potential antisense compounds. They block both translation and transcription. More recently, their improved affinity to complementary DNA has suggested that PNAs may have further value as diagnostic tools for detecting genetic mutations. PNAs have not, however, been found to serve as substrates for nucleic acid processing enzymes, in particular, for polymerases. The search has been discouraged in part by crystallographic and biochemical studies that suggested that polymerases bind to the negative charges of the phosphate backbone of the nucleic acid substrates via highly conserved amino acid residues. Here, we show for the first time that PNAs can at least serve as primers for certain DNA polymerases and reverse transcriptases, even though no phosphate residues are present in the primer to interact with the polymerase.
The anti-herpes simplex virus type 1 (anti-HSV-1) efficacy of a series of oligonucleotides was determined as a function of their chemical structure. All oligonucleotides consisted of the same ...sequence directed against the translation initiation codon of the HSV-1 immediate early gene. The oligonucleotides were modified with phosphorothioate linkage patterns according to various protection strategies against nucleolytic degradation. We show that nuclease resistance is the dominant factor that determines the antiviral efficacy in this series. A minimal protection strategy, consisting of end-capping and pyrimidine protection, has proven to be particularly useful, because it not only yields nuclease-resistant oligonucleotides but also minimizes non-sequence-specific effects.
