Abstract: Vectors and a method for the identification of affector RNA molecules, such as ribozymes, external guide sequences, anti-sense RNA, and triple helix-forming RNA, that inhibit expression of target RNA molecules are disclosed. The method identifies functional affector RNA molecules by screening or selecting for those RNA molecules that inhibit expression of a fusion transcript, which includes the sequence of an RNA molecule of interest, from a library of potential affector RNA molecules. The vectors include a reporter gene encoding the fusion transcript including the RNA molecule of interest and RNA encoding the reporter protein. The vectors also include a second reporter gene encoding a second reporter protein. Expression of the second reporter protein can be used both to detect transformation or transfection of the vector into cells and as a control for effects on the expression of the first reporter protein that are not due to inhibition of expression of the RNA molecule of interest.

Abstract: Methods and compositions are described for the directed delivery of ribozymes or other compounds to specific cells which express the heme receptor on their surface using heme-bearing microparticles. Such microparticles are useful in the directed delivery and accumulation of drugs designed to treat hepatic diseases such as viral hepatitis or hepatoma.

Abstract: External guide sequence (EGS) molecules for eukaryotic RNAse P are engineered to target efficient and specific cleavage of target RNA. Engineered RNA molecules are designed and synthesized which contain specific nucleotide sequences which enable an external guide sequence for RNAse P to preferentially bind to and promote RNAse P-mediated cleavage of target RNA molecules. Short External Guide Sequence (SEGS) molecules have been constructed that, when hybridized to a target molecule, provide a minimal structure recognized as a substrate by RNAse P. The SEGS/target structure is comprised of a structures similar to the A stem and the T stem of a tRNA, the natural substrate of RNAse P. The SEGS makes up only half of these stem structures. The other half of the stem structures is provided by the target molecule. By allowing the target molecule to form more of the RNAse P substrate structure, the disclosed SEGS molecules can be significantly smaller than previous EGS molecules.

Abstract: Regulatable RNA molecules such as regulatable ribozymes, nucleic acids encoding such regulatable ribozymes, and methods of making and using such regulatable ribozymes are disclosed. Regulatable ribozymes comprise a ligand-binding RNA sequence and a ribozyme sequence capable of cleaving a separate targeted RNA sequence, wherein upon binding of the ligand to the ligand-binding RNA sequence, the activity of the ribozyme sequence against the targeted RNA sequence is altered. The ligand may be either an inorganic or an organic molecule and may be a co-drug which can be administered to specifically regulate the ribozyme activity. Regulatable RNA molecules other than ribozymes are also disclosed, such as regulatable mRNA molecules which comprise a ligand-binding RNA sequence separate from the coding sequence, wherein upon binding of a ligand to the ligand-binding RNA sequence, translation of the regulatable mRNA is altered.

Abstract: Hepatitis delta is used as a vector for inhibition of viral infection and to express proteins in vivo in a cell-specific manner. The scope of delta's use as a vector is broadened in the present invention in several important ways. For example, a delta RNA genome capable of self-replication is enlarged to carry additional information, either coding for messenger RNA for a protein, or for a targeted ribozyme, which can be delivered to liver cells using delta's normally infectious properties, or to other cell types using chimeric delta viral agents carrying altered surface proteins. In another embodiment, the delta vector is made self-limiting, so that its role in delivering targeted information is separated from its viral property of unlimited infectious replication. Targeting is achieved through the use of sequences flanking the delta sequences that have affinity for sites on RNA to be cleaved.

Abstract: Hepatitis delta is used as a vector for inhibition of viral infection and to express proteins in vivo in a cell-specific manner. The scope of delta's use as a vector is broadened in the present invention in several important ways. For example, a delta RNA genome capable of self-replication is enlarged to carry additional information, either coding for messenger RNA for a protein, or for a targeted ribozyme, which can be delivered to liver cells using delta's normally infectious properties, or to other cell types using chimeric delta viral agents carrying altered surface proteins. In another embodiment, the delta vector is made self-limiting, so that its role in delivering targeted information is separated from its viral property of unlimited infectious replication. Targeting is achieved through the use of sequences flanking the delta sequences that have affinity for sites on RNA to be cleaved.

Abstract: Regulatable RNA molecules such as regulatable ribozymes, nucleic acids encoding such regulatable ribozymes, and methods of making and using such regulatable ribozymes are disclosed. Regulatable ribozymes comprise a ligand-binding RNA sequence and a ribozyme sequence capable of cleaving a separate targeted RNA sequence, wherein upon binding of the ligand to the ligand-binding RNA sequence, the activity of the ribozyme sequence against the targeted RNA sequence is altered. The ligand may be either an inorganic or an organic molecule and may be a co-drug which can be administered to specifically regulate the ribozyme activity. Regulatable RNA molecules other than ribozymes are also disclosed, such as regulatable mRNA molecules which comprise a ligand-binding RNA sequence separate from the coding sequence, wherein upon binding of a ligand to the ligand-binding RNA sequence, translation of the regulatable mRNA is altered.

Abstract: Modified external guide sequence (EGS) molecules that mediate cleavage of specific target RNAs have been constructed. The modified molecules are external guide sequence molecules for RNAse P which are designed to specifically bind to and promote RNAse P-mediated cleavage of target RNA molecules and to have enhanced nuclease resistance. Specific regions are modified to achieve enhanced stability while maintaining RNAse P activity. Modified external guide sequence molecules suitable for use in the treatment of hepatitis B viral infections have been constructed.

Abstract: A system is described for the use of a ribozyme as a diagnostic tool for detecting the presence of a nucleic acid, protein, or other molecule, in which the formation of an active ribozyme and cleavage of an assayable marker is dependent on the presence or absence of the specific target molecule. The essential component is a ribozyme specifically but reversibly binding a selected target in combination with a labelled co-target, preferably immobilized on a support structure. When both the target and co-target are bound, the ribozyme cleaves the label from the co-target, which is then quantifiable. Since the ribozyme is reversibly bound by target and co-target, it can reassociate with additional co-target, cleaving more label, thereby amplifying the reaction signal.

Abstract: Ribozymes, sequences cleaving RNA, derived from sequences present in the hepatitis delta virus, have been engineered for greater specificity without increasing size. The specific ribozyme sequences are useful as reagents for cleaving RNA for experimental studies as well as antiviral therapies. Examples demonstrating the targeting of these sequences against HIV and Crohn's disease are described in detail. The sequences are also useful as diagnostics for the detection of hepatitis delta virus in tissue and fluid samples, as in blood banking, as well as in isolation and characterization of new viroids having ribozyme activity, using an RNA-specific hybridization method. Based on analysis of the two domain structure of the hepatitis delta virus, it is possible to construct a vector for expression of non-hepatitis delta virus proteins in mammalian cells.

Abstract: Hepatitis delta is used as a vector for inhibition of viral infection and to express proteins in vivo in a cell-specific manner. The scope of delta's use as a vector is broadened in the present invention in several important ways. For example, a delta RNA genome capable of self-replication is enlarged to carry additional information, either coding for messenger RNA for a protein, or for a targeted ribozyme, which can be delivered to liver cells using delta's normally infectious properties, or to other cell types using chimeric delta viral agents carrying altered surface proteins. In another embodiment, the delta vector is made self-limiting, so that its role in delivering targeted information is separated from its viral property of unlimited infectious replication. Targeting is achieved through the use of sequences flanking the delta sequences that have affinity for sites on RNA to be cleaved.