Innovir Laboratories, Inc.

Yale University

Other

Shaji T George

Allan R Goldberg

Garry B Takle

Martina Werner

Andy Shih

Jeffrey Michael Bockman

Michael Ma

Umberto Pace

Efficient methods and compositions are provided for the targeted delivery of effective concentrations of compounds, including nucleic acid molecules and oligonucleotides such as EGSs, ribozymes and antisense, proteins, peptides, carbohydrate, and synthetic organic and inorganic molecules, or combinations thereof, to cells, especially hepatocytes. In the preferred embodiment, the compound is an negatively charged oligonucleotide which binds in a stoichiometric ratio to a water soluble, positively charged macrocycle such as a porphyrin, which targets and protects the oligonucleotide. The porphyrin protects the compound to be delivered and delivers the compound preferentially to certain cells and tissue types. In another embodiment, the porphyrin has anti-human hepatitis virus activity, when administered alone, which is significantly enhanced when in combination with an antiviral compound, especially an oligonucleotide.

Delivery of nucleic acids by porphyrins

Disclosed are a method and compositions for delivering nucleic acids to bacterial cells. The method does not require manipulation of the bacteria and is therefore particularly suited to delivery of nucleic acids to bacteria in natural environments, including inside animals bodies. The method generally involves conjugating the nucleic acid to be delivered with a cationic porphyrin and bringing the conjugate and the target bacterial cells into contact. Both the porphyrin and conjugated nucleic acid are taken up by the bacterial cells and the nucleic acid can then have a biological effect on the cells. Specifically disclosed is a method for converting drug-resistant bacterial cells to drug-sensitive cells by delivery of external guide sequences to the cells which then promote cleavage of RNA molecules involved in conferring the drug-resistant phenotype on the cells. The drug-resistant phenotype of the cells is thus converted to a drug-sensitive phenotype. The drug-sensitive cells are then susceptible to drug therapy. Also disclosed is a method and compositions for killing eukaryotic pathogens or converting drug-resistant eukaryotic cells to drug-sensitive cells. The method involves the delivery of external guide sequences, ribozymes, or vectors encoding external guide sequences or ribozymes, to eukaryotic cells. Preferred target eukaryotic cells for the disclosed method include algae, protozoa, fungi, slime mold, and cells of helminths.

Phenotypic conversion of cells mediated by external guide sequences

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.

Stabilized external guide sequences

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.

Heme-bearing microparticles for targeted delivery of drugs

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. This makes SEGS molecules especially useful as therapeutic agents since they are easier to manufacture and administer in quantity.

Short external guide sequences

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.

Regulatable RNA molecule

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.

Ribozyme compositions and expression vectors

Hepatitis delta virus compositions and expression vectors

Regulatable nucleic acid therapeutic and methods of use thereof

EGS-mediated inactivation of target RNA

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. In one embodiment, the target is a nucleic acid hybridizing to complementary sequences that form part of the ribozyme; in a second embodiment, the target is a protein or other macromolecule which is bound by interactions with a portion of the ribozyme molecule in another embodiment, a thermostable ribozyme is used, so that improperly bound ribozyme is destabilized and inactive at elevated temperatures. A method for isolating regularable ribozymes is also disclosed. The regulatable ribozymes are useful in the method for detecting the presence of a specific macromolecule, or can be used in in vitro or in vivo methods for inactivation or activation of the cleavage of target RNA molecules.

Growing community of inventors

New York, NY, United States of America

Shaji T George