James McSwiggen

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector‐based model was used in which the siRNA and the HBV vector were codelivered via high‐volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA‐treated groups compared with the control siRNA‐treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation. (HEPATOLOGY 2005.)

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2′-O-Me nucleotides, 1-2 other 2′-modified nucleotides at positions U4 and U7, and a 3′-3′-linked nucleotide “cap.” Eight 2′-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2′-NH substitutions at U4 and U7, or 2′-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only 0.1 min. The addition of a 3′-3′-linked nucleotide “cap” (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

Optimizing the Cell Efficacy of Synthetic Ribozymes SITE SELECTION AND CHEMICAL MODIFICATIONS OF RIBOZYMES TARGETING THE PROTO-ONCOGENE c-myb

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5′-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2′-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

Chapter 30. Catalytic RNA (Ribozymes) as Drugs

Publisher Summary The predominant activity found in the naturally occurring catalytic RNA— that is, ribozymes —is the ability to splice or cleave the RNA molecules in a sequence-specific manner. Sequence specificity results from the base-pairing of ribozyme sequences with nucleotides near the cleavage site of the target RNA. Ribozymes function in an intramolecular (cis) reaction to splice or cleave their own RNA sequence, and they can also function in Trans to cleave another RNA molecule. Because of their sequence specificity, ribozymes show promise as therapeutic agents to down-regulate a given RNA species in the background of cellular RNAs. Specifically, the messenger RNA (mRNA) coding for a protein associated with a disease state may be selectively cleaved. This cleavage event renders the mRNA untranslatable and attenuates the proteins expression. To function as therapeutic agents, ribozymes must be deliverable to the target organs and cells and must be able to destroy the target RNA in a time that is short relative to the natural half-life of that RNA. Delivery of ribozymes to the target cells can be accomplished either through exogenous drug delivery approaches or through gene therapy approaches, with each approach, raising different issues for ribozyme delivery, stability, activity, and potential toxicity. Unmodified RNA is unstable in biological sera. Therefore, a significant challenge, in using ribozymes as drugs, is to modify them chemically to increase their stability, while retaining their catalytic activity. A number of structural modifications have been applied to oligonucleotides in general to enhance the nuclease resistance. Improvements in the chemical synthesis of RNA have expanded the ability to modify ribozymes. This chapter outlines the progress in utilizing ribozymes as human therapeutic agents.