Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.

Ribozymes are RNA molecules that catalyse the cleavage and/or ligation of other RNA molecules. This capacity of ribozymes has therapeutic application for the inactivation of deleterious genes and the repair of mutated genes involved in many disease states. Keywords: ribozyme; catalytic RNA; antisense; gene therapy

The hammerhead ribozyme is one of the smallest of known catalytic RNAs. Its structure and mechanism have been examined using a broad range of approaches. Recently, crystal structures of the hammerhead have been reported. Within the crystal the hammerhead exists in a Y-shaped configuration in which helices I and II form the adjacent upper arms, while helix III forms the lower leg of the Y. Based on these findings, hammerheads in which helix I and II are constrained to remain adjacent and roughly parallel are expected to be catalytically active. We have examined this possibility with two novel hammerhead configurations, circular and circular/hairpin. These circular and circular/hairpin hammerhead ribozymes possess activity comparable to, and in some cases even greater than, non-constrained hammerheads. Since these novel ribozymes are constrained into a closed, and active conformation, they offer advantages for structure/function studies. Additionally, these novel ribozymes will be adventitious for antisense mediated gene inhibition, since they possess increased activity and a reduced requirement for magnesium ion, and are expected to be more resistant to nuclease degradation.

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA, Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict, Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA, The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention.

Circular hammerhead ribozymes were synthesized from linear oligoribonucleotides using T4 RNA ligase. Some of the precursors could not be efficiently circularized under standard conditions. For these molecules, the use of a DNA template allowed their efficient circularization. The template was designed to prevent the precursor from folding into an unsuitable structure. The template allowed circular ribozymes as small as 15 nucleotides in length to be efficiently synthesized at concentrations as high as 50 microM in the ligation reaction. The circular products retained their biological activity.

The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro, The reaction produces linear products, and as of yet, closed circular products have not been possible, Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells, Here, we describe an in vitro procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.

We have previously shown that circular I/II-format hammerhead ribozymes have faster catalytic rates and a reduced divalent metal-ion requirement.(1,2) The enhanced-rate and reduced metal-ion dependence are an asset for in vivo use of hammerhead ribozymes as antisense therapeutics. However, circular hammerheads suffer two disadvantages. Their Km for substrate is increased relative to the wild-type hammerhead, and as Tm-format hammerheads they place greater constraints on the sequences that can be targeted, relative to I/III-format hammerheads. Here we show that these disadvantages can be eliminated in a novel hammerhead configuration, the lariat. The lariat is a I/III-format hammerhead that was constructed by combining chemical and enzymatic methods. The lariat hammerhead ribozyme possesses the same reduced magnesium-ion requirement and enhanced activity of our circular hammerheads;; Additionally, the lariat possesses no natural 5'- or 3'-termini and is therefore expected to be of greater resistance to nuclease degradation.