EP0707493A1 - Co-localisation d'une molecule cible et de ses inhibiteurs - Google Patents

Co-localisation d'une molecule cible et de ses inhibiteurs

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Publication number
EP0707493A1
EP0707493A1 EP95904785A EP95904785A EP0707493A1 EP 0707493 A1 EP0707493 A1 EP 0707493A1 EP 95904785 A EP95904785 A EP 95904785A EP 95904785 A EP95904785 A EP 95904785A EP 0707493 A1 EP0707493 A1 EP 0707493A1
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Prior art keywords
ribozyme
hiv
rna
trna
target molecule
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German (de)
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EP0707493A4 (fr
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John J. Rossi
Garry P. Larson
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City of Hope
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City of Hope
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Priority claimed from US08/185,827 external-priority patent/US5827935A/en
Application filed by City of Hope filed Critical City of Hope
Publication of EP0707493A1 publication Critical patent/EP0707493A1/fr
Publication of EP0707493A4 publication Critical patent/EP0707493A4/fr
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead

Definitions

  • This invention relates to mechanisms for bringing two or more molecules together in a living cell. More particularly, the invention relates to mechanisms for bringing together with a cell a target molecule and an inhibitor therefore in a manner effective to increase the concentration of the inhibitor with respect to the target. For example, the invention relates to mechanisms for increasing the cellular concentration of a ribozyme with respect to a target mRNA molecule to be cleaved by the ribozyme.
  • One embodiment of this invention relates to chimeric tRNA LYS ribozyme molecules which compete effectively with tRNA LYS for binding to HIV-1 reverse transcriptase. These chimeric molecules provide a co-localization mechanism for delivering inhibitors of HIV-l and reverse transcriptase to the virion particle itself.
  • RNA is unusual in its ability both to store information in its nucleotide seguence and to function as an enzymatic catalyst of specific reactions (1,2) .
  • This combination of attributes has created opportunities for engineering RNA enzymes (ribozymes) which can be used to cleave and functionally inactivate targeted RNAs.
  • ribozymes Some of the attributes of ribozymes which make them attractive candidates for therapeutic agents are their ability to site-specifically cleave targeted RNAs, cleave multiple substrates, and their ability to be engineered for improved cleavage specificity and enhanced catalytic turnover (3,4).
  • catalytic motifs which have been successfully modified and/or adapted for use in ribozy e applications.
  • RNAse P the hammerhead and hairpin motifs
  • self- cleaving domain of the hepatitis delta virus (5,3,6,7).
  • Each of these engineered ribozymes only requires a divalent metal cation for activity (usually MG**) which participates in the chemistry of the cleavage reaction (8,9,10).
  • ribozymes can be tailor made to cleave viral transcripts, thereby leaving cellular transcripts untouched. Because of this, HIV is a prime target for ribozyme inactivation. This concept was successfully tested by intracellularly expressing a hammerhead ribozyme targeted to a gag cleavage site, which resulted in up to a 40 fold reduction in viral p24 antigen production in HeLa CD4+ cells challenged with HIV (11,12).
  • ribozyme cleavage sites there are hundreds of potential ribozyme cleavage sites along the length of the viral geno ic and subgenomic RNAs. Since the virus mutates rapidly, and can become resistant to most drugs developed to inhibit a single viral target (13) , ribozymes have become an important alternative for anti-viral therapeutic agents since multiple ribozymes targeted to a number of different sites can be simultaneously delivered to cells for inhibition of HIV (14) . There are two times in the viral life-cycle when ribozymes could be effective against HIV infection.
  • RNA The first immediately following infection prior to proviral DNA formation, when all or part of the viral genome is still in the form of RNA, and the second following the establishment of integrated provirus from which spliced and a full length viral transcript ⁇ are produced (15,16).
  • An important consideration is the observation that HIV can infect quiescent T-lymphocytes, wherein proviral DNA synthesis is initiated but is incompletely reverse transcribed (17) . If a ribozyme is present in the infected cell cytoplasm, it theoretically could protect cells from permanent infection by cleaving the RNA at this early step, before the T-cell becomes activated.
  • Yamada, et al. (18) have demonstrated a 50-100 fold reduction in HIV proviral DNA formation in cells expressing a hairpin ribozyme targeted to a site in the 5' leader sequence.
  • RNAs very little is known about the mechanisms regulating the pathway of movement from transcription through translation, and in the case of HIV, from transcription through packaging.
  • nuclear transcripts are processed and migrate along specific tracks, which predicts non uniform distributions of specific nuclear transcripts (31) .
  • RNAs can be specifically localized within the cytoplasm as well (32) .
  • RNAs From the prospective of ribozyme therapeutic applications, capitalizing upon the localization properties of RNAs could facilitate intracellular functioning of ribozymes by allowing them to co-localize with their target RNAs.
  • Sullenger and Cech (1993) (33) (incorporated herein by reference) have directly tested this idea by utilizing the dimerization and packaging signal of a Moloney murine leukemia virus genomic RNA to co-localize a hammerhead ribozyme with its target, the lac Z gene carried by another recombinant Moloney viral vector. They found that up to 90% inhibition of infective virus production could be achieved as a result of co-packaging the ribozyme and the lacZ target containing viral RNAs.
  • RNA binding proteins such as HIV-1 encoded NCp7 and cellular hnRNP Al can facilitate ribozyme catalytic turnover .in vitro.
  • the ribozyme-target co-localization strategy described in Serial No. 08/185,827 involves utilizing the tRNA LYS3 primer for reverse transcriptase (RT) as a vehicle for co-localizing a ribozyme with HIV genomic RNA, and potentially into the virion itself.
  • the strategy is based upon the well established interactions of HIV RT with cellular tRNA LYS3 , which is the primer tRNA used by all the mammalian lentiviruses.
  • This tRNA is selectively bound by RT, and in the presence of the nucleocapsid protein NCpl5 (or NCp7) , unwinds the aminoacyl stem of the tRNA, allowing it to base pair with the viral PBS (38).
  • the tRNA-ribozyme is expressed as a Pol III transcript when transfected into 293 cells, and the ribozyme moiety is not processed from the transcript, although the 5' precursor segment of the tRNA-ribozyme is processed normally. By including the CCA in the transcripts, which is normally added post- transcriptionally to the tRNA, these molecules are not subject to the normal 3' processing events. (3) The tRNA-ribozyme is exported to the cytoplasm, making it available for binding with RT. (4) When the tRNA ribozyme is transiently transfected into 293 cells, there are equivalent levels of tRNA-ribozyme transcript to endogenous tRNA LYS3 . (5) Co-transfeetion of the tRNA- ribozyme gene with pNL4-3 DNA into 293 cells resulted in a 4 to 12 fold reduction in infectious virus production relative to control constructs. See Figure 5.
  • Co-localization means the positioning of two or more molecules within a living cell, one of which is a target and the other an inhibitor of the target that the concentration of the inhibitor with respect to the target is increased within the cell and function or expression of the target is constrained or inhibited.
  • Co-localization may be accomplished by covalent linkage (cis-ribozyme) or via co-targeting the viral capsid.
  • a specific embodiment of co-localization pursuant to this invention entails the positioning within a living mammalian cell of a ribozyme adjacent a virion particle to cleave virion RNA.
  • This invention provides co-localization mechanisms and living cells in which an inhibitor and a target are co-localized by such mechanisms.
  • An important object of the invention is to provide novel intracellular immunogens for vaccines against viral infections.
  • One preferred embodiment of this invention provides novel chimeric tRNA LYS -ribozyme molecules that compete effectively with tRNA LYS for HIV-1 reverse transcriptase binding sites.
  • the chimeric human tRNA LYS -ribozymes inhibit reverse HIV transcription by delivering inhibitors such as ribozymes of HIV-1 reverse transcriptase directly to the virion particle and render it non-functional.
  • the chimeric molecules of this invention thus serve as highly specific non-toxic therapeutic agents.
  • chimeric molecules also reveal a novel, site specific RNA cleaving activity of HIV-1.
  • Figure 1 shows the structure of one chimeric ribozyme.
  • This tRNA LYS -ribozyme construct has been cloned into a Blue Script transcription vector using Sacll and Xhol restriction sites. Following linearization at the Sacll site the chimeric RNA can be transcribed in vitro using bacteriphase T-7 RNA polymerase. There is also a Mae I restriction site in between the tRNA and ribozyme moieties, allowing the tRNA to be transcribed independently of the ribozyme.
  • FIG. 1 This gel shift experiment shows binding of the chimeric tRNA LYS -ribozyme to HIV-1 reverse transcriptase.
  • the eight lanes of the gel from left to right are:
  • tRNA LYS in vitro transcript which has extra bases at both the 5' and 3' ends.
  • the extra 5' bases are from the Blue Script poly linker between the T-7 promoter and the Xhol site.
  • tRNA LYS -ribozyme iri vitro transcript which has the same extra 5' bases as tRNA LYS , but terminates at Sacll site at the end of the ribozyme moiety.
  • tRNA LYS -ribozyme transcript incubated with HIV-1 reverse transcriptase.
  • This Figure 2 shows that the chimeric tRNA- ⁇ ribozyme specifically binds to HIV-1 reverse transcriptase by a shift in radioactivity when HIV-1 reverse transcriptase is present.
  • Cold tRNA LYS -ribozyme competes with tRNA LYS for binding to HIV-l reverse transcriptase as indicated by the reduced radioactive shift in lane 8.
  • FIG. 3 This experiment demonstrates cleavage of a 162 nucleotide, radioactively labelled HIV-l RNA containing the primer binding site plus sequences upstream of this and including the AUC cleavage signal for the ribozyme.
  • the cleavage products are 101 and 61 bases. The extent of cleavage increases with increasing temperature.
  • FIG. 4 Demonstration of the novel RNAse activity of HIV-l reverse transcriptase when tRNA LYS -ribozyme and HIV-l primer binding site transcripts are incubated together in the presence of HIV-l reverse transcriptase.
  • the tRNA LYS -ribozyme is radioactively labelled, and the HIV-l RNA is non-radioactive.
  • the cleavage products result in the tRNA moiety being separated from the ribozyme moiety.
  • This result also demonstrates that the chimeric tRNA LYS -ribozyme cannot serve as a primer for HIV-l reverse transcriptase.
  • the lanes are, left to right: tRNA LYS -ribozyme alone, tRNA LYS -ribozyme plus HIV-l reverse transcriptase, no deoxyribonucleoside triphosphates; tRNA LYS -ribozyme plus HIV-l reverse transcriptase plus deoxyribonucleoside triphosphates; last two lanes same as lane 3 except lane 4 has AMV reverse transcriptase and lane 5 has MLV reverse transcriptase. The black dots mark the HIV-l reverse transcriptase cleavage products. Unlabelled HIV-l primer binding site containing 162 nucleotide transcript was present in each lane. None of the reverse transcriptases can utilize the tRNA LYS -ribozyme as a primer since it has 12 nucleotides at the 3' end which cannot base pair with the HIV-l primer binding site RNA.
  • Figure 5 Illustrates A: RT binding to tRNA LYS3 - ribozyme.
  • B Primer extension analyses demonstrating nuclear localization of chimeric transcript. The primer for the tRNA-ribozyme is in the ribozyme moiety, and the primer for tRNA LYS3 is at the 3 ' end of the tRNA.
  • C Results of infectious virus assays carried out with supernatents from 293 cells transfected with tRNA- ribozyme or control construct (ribozyme minus tRNA in same vector) and co-transfected with pNL4-3. Three independent experiments are presented.
  • Figure 6 illustrates the tRNA Lys3 -ribozyme which is the starting molecule.
  • the asterisks indicate sites which UV crosslink to HIV RT or are protected from RNAse digestion in the presence of RT.
  • a deliberately created mismatch in the ribozyme pairing arm is indicated with a boxed in nucleotide pair. This was done to eliminate a stretch of 4T's in the ribozyme gene which could serve as a Pol III termination site.
  • the authentic termination site (5 U's or T's in DAN) is underlined.
  • the T loop- stem and aminoacyl acceptor stem which pair with the HIV primer binding site are overlain with a heavy line.
  • Figure 7 is a schematic representation of nef and 3 , UTR region to be included in ribozyme and GH reporter systems.
  • the delineating sequences are the extremities of the DNA amplified by PCR. These sequences are from the pNL4-3 proviral clone and encompass the region of nucleotides 9389 through 9704.
  • Figure 8 represents a construct containing anti- HIV-1 ribozyme expressed in context of dimerization domain and RRE to facilitate co-localization with HIV full-length genomic RNAs.
  • the invention provides various co-localization mechanisms. These mechanisms include, among others, (i) utilization of specific RNA trafficking pathways to both the target and the inhibitor, (ii) utilization of protein interaction with inhibitor and target molecules, e.g., HIV-l RT (see Sullenger and Cech (33)), (iii) use of cellular proteins which subcellularly compartmentalize the inhibitor to the target or a specific target site; (iv) use of cis-acting sequence substituents on ribozyme transcripts to direct the ribozyme to a specific subcellular trafficking pattern or site; (v) ribozymes which include any molecule or moiety that specifies a distinct intracellular trafficking pattern and target localization site.
  • inhibitor and target molecules e.g., HIV-l RT (see Sullenger and Cech (33)
  • cellular proteins which subcellularly compartmentalize the inhibitor to the target or a specific target site
  • cis-acting sequence substituents on ribozyme transcripts to direct the
  • 08/185,827 describes somewhat different co- localization strategy with the tRNA LYS3 -ribozyme chimeras (see Progress Report section) , which are bound by HIV reverse transcriptase allowing alignment of the ribozyme during packaging of the virus.
  • This approach has been successful and has led to a reduction of infective viral titer as a consequence of co-expressing chimeric tRNA- ribozy es with HIV proviral DNA.
  • One of the goals of this invention is to develop genetic variants of tRNA LYS3 which maintain the sequence and structural features required for interaction with a ribozyme for cleavage, yet are dissimilar enough from cellular tRNA LYS3 so as not to interfere with normal cellular metabolism.
  • the use of these variants will also be coupled with enhanced intracellular expression systems.
  • the identification of molecules which can still interact with the primer binding site of HIV (which means leaving at least the 3' segment of the amino-acyl acceptor stem intact) , thereby allowing alignment of a ribozyme (appended to the 3' end) with a cleavage site adjacent to the viral primer binding site is contemplated.
  • tRNA LYS3 - ribozyme chimeric gene Since high levels of expression of the tRNA LYS3 - ribozyme chimeric gene during transient transfection were observed, it is reasonable that inserting multiple, tandem copies of the tRNA ribozyme chimeric genes in a vector such as adeno associated virus (AAV) can also lead to high level expression.
  • AAV adeno associated virus
  • a potential strategy for increasing the intracellular levels of the chimeric ribozyme transcript is to express them from heterologous promoters. For those variants which lack the A or B boxes, this will be a necessity. For variants which have maintained these elements, site directed changes which eliminate the promoter function will allow testing of these constructs using heterologous promoters.
  • Several candidate promoters have been developed for ribozyme expression.
  • the human U6 snRNA gene has a Pol III promoter element which is 5' of the coding sequence (Parry, et al. (39)). Transcription terminates after a string of 5 Uracil residues, resulting in a RNA with well defined ends.
  • this promoter can be used to transcript ribozyme containing RNAs which localize to the cytoplasm.
  • a potential advantage of this promoter is that transcription can be engineered to initiate at the +1 sequence of the tRNA molecule, thus eliminating any need for processing a 5' leader, and allowing the synthesis of a very defined transcript.
  • b- The 3 ' untranslated region fUTR) as an RNA trafficking signal-model for ribozvme-target co- localization.
  • RNAs may "track" along specific paths following transcription and transport to the cytoplasm (reviewed in 31) .
  • messenger RNAs which localize to specific regions of the cytoplasm as well.
  • the most well studied localized RNAs are the oocyte and early embryo mRNAs of Drosophila and Xenopus (32) .
  • Other mRNAs such as actin have been shown to localize to cytoskeletal components (40, 41, 42) .
  • the signal for localization for many of the mRNAs which have been studied resides in the 3 ' untranslated region (32,42).
  • Actin isoforms contain very few differences in amino acid coding sequences, but the 3' UTR's are isoform specific, and evolutionarily conserved within a given isoform family, suggesting an important functional role (43) .
  • the ⁇ -actin and ⁇ -actin UTR's may be used to test their potential for co-localizing ribozyme and target mRNA's intracellularly.
  • a similar approach involves using the HIV-l 3 ' UTR, which is present in all HIV transcripts.
  • the basic strategy is to incorporate the 3' UTR of interest onto a reporter construct as well as to incorporate the same UTR onto a ribozyme transcript.
  • the 293 and HeLa cell lines were used for the studies.
  • the reporter construct to be used is depicted below and contains the human growth hormone (GH) gene driven by the SIV-1 LTR promoter. This system produces a readily quantifiable (using a radioimmunoassay) secreted protein.
  • the linear range of response of GH expression to plasmid concentration in the 293 cell line was established. The expression of this construct is not dependent upon TAT expression, although a 10 fold stimulation of expression in the presence of SIV TAT was observed. If the results look promising in the 293 cell line, confirmation testing in HeLa cells will be carried out.
  • the 3 ' UTRs will be appended to both the growth hormone and ribozyme expression cassettes. To do this, the human 3-actin or ⁇ -actin 3' UTRs will be isolated from human genomic DNA or m
  • UTRs are: beta actin oligo 5'
  • Ribozyme constructs may be made in the adeno associated virus vector backbone. The constructs will be encapsidated in collaboration with Saswati Chatterjee's laboratory, and transduced into three A293 or HeLa cell lines. Stable lines will be selected from G418, and levels of ribozyme expression will be monitored via primer extension and northern gel analyses.
  • a non-cleaving mutant control For each ribozyme, a non-cleaving mutant control will be used.
  • the controls for 3' UTR effects will utilize comparison of the efficiency of reporter gene inhibition as a function of having the ⁇ - versus ⁇ -actin 3' UTRs, which localize to different intracellular compartments, appended to the reporter and ribozyme transcripts.
  • Several ribozyme targets in the SIV leader region have been established which will be tested in conjunction with the UTRs. These ribozymes have been tested for substrate interaction using an in vitro gel shift assay, and identified by this process sites in the SIV LTR which are most accessible to binding. In each case where binding was shown to be efficient, good cleavage activity by the ribozyme was observed.
  • the first set of sequences appended to the GH reporter construct included the last 20 bases of the pNL4-3 proviralnef coding sequence and extended to the 3 ' terminus of the LTR. Much of this region is included in all of the viral messenger and full length genomic transcripts. This sequence contains the poly A additional signal and putative transcriptional termination region (45) , but most importantly lacks cis- acting regulatory signals such as the RRE, INS and CRS. This region was isolated using PCR primers and appended to both the GH reporter gene construct and the ribozyme transcriptional units as described above.
  • control constructs included the AAV poly A and termination signals, which were appended to the ribozyme and GH reporter constructs as well as mutant, non- cleaving ribozymes. Again, efficacy was measured by inhibition of growth hormone secretion in transient transfection assays of the GH construct into stable cell lines expressing the ribozyme constructs as described above.
  • c Co-localization of anti-HIV-l ribozymes with full length viral transcripts via the dimerization domain and the viral RRE.
  • the third strategy for co-localizing ribozyme and target RNAs will capitalize upon the unique RNA-RNA interaction of the dimerization domain of HIV (which is facilitated by the NCp7 nucleocapsid protein) (46-49) in combination with the RRE (to force cytoplasmic translocation of the ribozyme containing transcripts) .
  • the rationale for these studies is that ribozyme containing RNAs which harbor the signals required for packaging can be co-localized with unspliced viral mRNAs and genomic RNAs via interactions of the dimerization domains.
  • the most probable targets for ribozyme interactions will be full-length viral RNAs, destined for encapsidation or translation into viral structural proteins.
  • chimeric molecules have been tested in cell free assays for their ability to bind to HIV-l reverse transcriptase and their inhibitory activity on HIV-l reverse transcriptase polymerization activity.
  • the ribozyme moiety targets the cleavage of HIV-l viral RNA at a known hammerhead cleavage site immediately upstream of the primer binding site for initiation of reverse transcription in the HIV-l viral RNA.
  • the site chosen for initial study, and reported here is an AUC in which cleavage is immediately after the C. This site is absolutely conserved in all HIV-l isolates sequenced to date.
  • the chimeric RNAs, which are specifically bound by HIV-l reverse transcriptase, should be carried into newly formed HIV-l virions during viral assembly.
  • the chimeric primers effectively block HIV-l revere transcription, making them a novel, highly target specific, and unique anti-HIV-l therapeutic agent.
  • the tRNA LYS portion contains within its mature coding sequence the elements required for transcription by human RNA polymerase III, thereby making it feasible to insert the gene, rather than the RNA, into human cells.
  • This activity is of unknown function in the viral replication cycle, but may play an important role in the use of chimeric RNAs by freeing the ribozyme moiety from the tRNA moiety such that it can cleave one or both of the viral RNAs encapsidated in the HIV-l virion.
  • HIV and other lentiviral RNAs co- equalized with a ribozyme provide intracellular and therapeutic agents and vaccines for mammalian lentiviral infections. Such therapeutic agents and vaccines are administered in known manner by viral mediated delivery, e.g., AAV or retroviral deliveries.
  • viral mediated delivery e.g., AAV or retroviral deliveries.
  • the idea of chimeric tRNA LYS -ribozyme molecules which effectively compete with tRNA LYS for binding to HIV-l reverse transcriptase is novel. It provides a possible mechanism for specifically delivering inhibitors of HIV-l reverse transcriptase to the virion particle itself. Such inhibitory agents will render these viral particles non-functional, and thus serve as highly specific, non- toxic therapeutic agents.
  • RNAse cleavage activity associated with HIV-l reverse transcriptase. This activity was only shown to cleave HIV-l RNA, not the primer. This activity cleaves twice in the primer binding site, and only substrates paired with tRNA LYS .
  • RNA attached at the 3' end of the tRNA LYS need not be a ribozyme, but any extra RNA which can base air with the HIV-l target upstream of the primer binding site. If a ribozyme is joined to the tRNA, other cleavage sites such as CUC, or CUA which are on the HIV-l sequence just to the 3 ' side (downstream) of the AUC site, can be targeted. It is not necessary to make an entire tRNA LYS -ribozyme fusion because it is now known that the last 18 nucleotides of tRNA LYS fused to the ribozyme are also bound by HIV-l reverse transcriptase. Genetic variants of tRNA LYS which compete better than tRNA LYS for binding to HIV-l transcriptase are included in the invention.
  • the ribozyme fusions to tRNA LYS allow specific targeting of the ribozyme to HIV-l virion. Since all retroviruses use cellular tRNAs for priming, this invention provides a general strategy for inhibiting other retroviruses as well.
  • Existing ribozyme technology makes use of specific base pairing between ribozyme and target, but this is accomplished by diffusion of the ribozyme until it finds a target RNA.
  • This invention uses well known retroviral packaging pathways to specifically carry the ribozyme into the virion, and get it bound to the correct site on the viral RNA for cleavage.

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Abstract

Cette invention concerne des mécanismes permettant de co-localiser dans une cellule vivante une molécule cible et un inhibiteur de cette même molécule cible, ainsi que de nouvelles molécules de ribozyme ARNtLYS chimères qui sont en forte concurrence avec l'ARNtLYS pour les sites de liaison de la transcriptase inverse du VIH-1. Ces ribosymes d'ARNtLYS humaines chimères inhibent la tanscription inverse du VIH-1 en apportant directement à la particule du virion des inhibiteurs tels que des ribozymes de la transcriptase inverse du VIH-1 et la rendent non fonctionnelle. Les molécules chimères de cette invention servent par conséquent d'agents et de vaccins thérapeutiques non toxiques hautement spécifiques utilisables contre les infections virales, y compris lentivirales. Ces molécules chimères ont également permis de découvrir une nouvelle activité de clivage de l'ARN à spécificité de site du VIH-1.
EP95904785A 1994-01-24 1994-12-02 Co-localisation d'une molecule cible et de ses inhibiteurs Withdrawn EP0707493A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/185,827 US5827935A (en) 1992-05-27 1992-05-27 Chimeric tRNAlys -ribozyme molecules
US185827 1994-01-24
PCT/US1994/013798 WO1995019788A1 (fr) 1994-01-24 1994-12-02 Co-localisation d'une molecule cible et de ses inhibiteurs

Publications (2)

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EP0707493A1 true EP0707493A1 (fr) 1996-04-24
EP0707493A4 EP0707493A4 (fr) 1999-03-31

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EP (1) EP0707493A4 (fr)
AU (1) AU692208B2 (fr)
CA (1) CA2157015A1 (fr)
WO (1) WO1995019788A1 (fr)

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Publication number Priority date Publication date Assignee Title
US7919583B2 (en) 2005-08-08 2011-04-05 Discovery Genomics, Inc. Integration-site directed vector systems
WO2010008562A2 (fr) 2008-07-16 2010-01-21 Recombinetics Procédés et matériaux pour produire des animaux transgéniques

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387775A1 (fr) * 1989-03-16 1990-09-19 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Unité génétique pour inhiber la fonction d'ARN
WO1993023569A1 (fr) * 1992-05-11 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif d'inhibition de la replication virale
WO1993024133A1 (fr) * 1992-05-27 1993-12-09 City Of Hope MOLECULES CHIMERIQUES DE RIBOZYMES D'ARNt?LYS¿

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387775A1 (fr) * 1989-03-16 1990-09-19 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Unité génétique pour inhiber la fonction d'ARN
WO1993023569A1 (fr) * 1992-05-11 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif d'inhibition de la replication virale
WO1993024133A1 (fr) * 1992-05-27 1993-12-09 City Of Hope MOLECULES CHIMERIQUES DE RIBOZYMES D'ARNt?LYS¿

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HERSCHLAG D ET AL: "AN RNA CHAPERONE ACTIVITY OF NON-SPECIFIC RNA BINDING PROTEINS IN HAMMERHEAD RIBOZYME CATALYSIS" EMBO JOURNAL, vol. 13, no. 12, 15 June 1994, pages 2913-2924, XP000567895 *
ROSSI J J ET AL: "RIBOZYMES AS ANTI-HIV-1 THERAPEUTIC AGENTS: PRINCIPLES, APPLICATIONS, AND PROBLEMS" AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 8, no. 2, February 1992, pages 183-189, XP002026934 *
See also references of WO9519788A1 *
SULLENGER B A ET AL: "TETHERING RIBOZYMES TO A RETROVIRAL PACKAGING SIGNAL FOR DESTRUCTION OF VIRAL RNA" SCIENCE, vol. 262, 3 December 1993, pages 1566-1569, XP000567869 *

Also Published As

Publication number Publication date
EP0707493A4 (fr) 1999-03-31
WO1995019788A1 (fr) 1995-07-27
AU1333595A (en) 1995-08-08
AU692208B2 (en) 1998-06-04
CA2157015A1 (fr) 1995-07-27

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