AU674656B2 - Chimeric tRNAlys-ribozyme molecules - Google Patents
Chimeric tRNAlys-ribozyme molecules Download PDFInfo
- Publication number
- AU674656B2 AU674656B2 AU21694/92A AU2169492A AU674656B2 AU 674656 B2 AU674656 B2 AU 674656B2 AU 21694/92 A AU21694/92 A AU 21694/92A AU 2169492 A AU2169492 A AU 2169492A AU 674656 B2 AU674656 B2 AU 674656B2
- Authority
- AU
- Australia
- Prior art keywords
- trnalys
- hiv
- ribozyme
- chimeric
- reverse transcriptase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- AIDS & HIV (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Description
OPI DATE 30/12/93 AOJP DATE 10/03/94 APPN. ID 21694/92 1111 lllllll PCT NUMBER PCT/US92/04362 IIll iill Il AU9221694 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 International Publication Number: WO 93/24133 A61K 31/70, C07H 21/02 Al C12N 15/10, 15/11 (43) International Publication Date: 9 December 1993 (09.12.93) (21) International Application Number: PCT/US92/04362 (81) Designated States: AU, CA, JP, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LU, MC, NL, (22) International Filing Date: 27 May 1992 (27.05.92) SE).
(71) Applicant (for all designated States except US): CITY OF Published HOPE [US/US]; 1500 East Duarte Road, Duarte, CA With international search report.
91010-0269 (US).
(72) Inventors; and Inventors/Applicants (for US only) ROSSI, John, J. [US/ US]; 346 Cimmeron Trail, Glendora, CA 91740 (US).
LARSON, Garry, P. [US/US]; 164 East Kelby Street, Covina, CA 91723 (US).
(74) Agent: IRONS, Edward, 919 18th Street, Suite 800, Washington, DC 20006 (US).
(54)Title: CHIMERIC tRNALYS.RIBOZYME MOLECULES (57) Abstract The invention provides novel chimeric tRNALYS-ribozyme molecules that compete effectively with tRNALYs for HIV-1 reverse transcriptase binding sites. The chimeric human tRNALYS-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 the invention thus serve as highly specific non-toxic therapeutic agents. These chimeric molecules also reveal a novel, site specific RNA cleaving activity of HIV-1.
WO 93/24133 PCT/US92/04362 -1- CHIMERIC tRNA-YS-RIBOZYME MOLECULES This invention was made with government support under Grant No. AT 25959 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION This invention relates to chimeric tRNALYS ribozyme molecules which compete effectively with tRNALYS for binding to HIV-1 reverse transcriptase.
These chimeric molecules provide a mechanism for delivering inhibitors of HIV-1 transcriptase to the virion particle itself.
BACKGROUND OF THE INVENTION "It has been demonstrated that the entire tRNALYS molecule as well as various segments of the tRNA per se are capable specifically of interacting with HIV-1 transcriptase. See Barat, et al. EMBO Journal 8:3279-3285 (1989); Khan, et al. J. Bio. Chem 267:6689-6695 (1992); Weiss, et al., Gene 111:183-197 (1992). Ben-Artzi, Proc. Natl. Acad. Soc. USA 89:927-931 (1992) reports an RNAse cleavage activity associated with HIV-1 reverse transcriptase. This activity is shown to cleave only HIV-1 RNA, not the primer.
Prior to this invention there has been no report of chimeric tRNALYS-ribozyme molecules.
.SUMMARY OF THE INVENTION Accordingly, the present invention provides chimeric human tRNALYS_ ribozymes.
Chimeric tRNALYS-ribozyme molecules of the invention compete effectively with tRNALYs for HIV-1 reverse transcriptase binding sites. The /y chimeric human tRNALYS-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. Accordingly, the present invention further provides a method for blocking HIV-1 reverse transcriptase which includes the introduction of a gene which expresses a chimeric human tRNALYs ribozyme into human cells.
These chimeric molecules also reveal a novel, site specific RNA cleaving activity of HIV-1.
EXAMPLES AND DESCRIPTION OF THE FIGURES Figure 1 shows the structure of one chimeric ribozyme. This tRNALYS-ribozyme construct has been cloned into a Blue Script transcription vector using Sac11 and Xhol restriction sites. Following linearization at the Sac11 site the chimeric RNA can be transcribed in vjmt using bacteriphage T-7 RNA polymerase. There is also a Mae I
S
S restriction site in between the tRNA and ribozyme moieties, allowing the tRNA to be :"15 transcribed independently of the ribozyme.
Figure 2. This gel shift experiment shows binding of the chimeric tRNALYS_ o ribozyme to HIV-1 reveres transcriptase. The eight lanes of the gel from left to right are: 1. Molecular weight marker; 20 2. tRNALys in yjio 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. There are six extra nucleotides at the 3' derived from the nucleotides after S the CCA of the tRNA to the Mae I site which separates the tRNA from the ribozyme.
3. tRNALYS-ribozyme in vitr transcript which has the same extra 5' bases as tRNALYs, but terminates at Sac 1 site at the end of the ribozyme moiety.
4. tRNALys transcript incubated with HIV-1 reverse transcriptase.
CVT P '110 C INWORDACKIEUVENWl IJ372"IEP WO 93/24133 PCT/US92/04362 -3tRNALYS-ribozyme transcript incubated with HIV-1 reverse transcriptase.
6. tRNALYS transcript incubated with AMV reverse transcriptase.
7. tRNALYS-ribozyme incubated with AMV reverse transcriptase.
8. tRNALYS with competing, non-radioactively labelled tRNALYS-ribozyme incubated with HIV-1 reverse transcriptase.
This Figure 2 shows that the chimeric tRNALYS-ribozyme specifically binds to HIV-1 reverse transcriptase by a shift in radioactivity when HIV-1 reverse transcriptase is present. Cold tRNALYS-ribozyme competes with tRNALYS for binding to HIV-1 reverse transcriptase as indicated by the reduced radioactive shift in lane 8.
Figure 3. This experiment demonstrates cleavage of a 162 nucleotide, radioactively labelled HIV-1 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.
Figure 4. Demonstration of the novel RNAse activity of HIV-1 reverse transcriptase when tRNALYS-ribozyme and HIV-1 primer binding site transcripts are incubated together in the presence of HIV-1 reverse transcriptase. The tRNALYS-ribozyme is radioactively labelled, and the HIV-1 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 tRNALYS-ribozyme cannot serve as a primer for HIV-1 reverse transcriptase.
WO 93/24133 PCT/US92/04362 -4- The lanes are, left to right: tRNALYS-ribozyme alone, tRNALYS-ribozyme plus HIV-1 reverse transcriptase, no deoxyribonucleoside triphosphates; tRNALYS-ribozyme plus HIV-1 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-1 reverse transcriptase cleavage products. Unlabelled HIV-1 primer binding site containing 162 nucleotide transcript was present in each lane. None of the reverse transcriptases can utilize the tRNALYS-ribozyme as a primer since it has 12 nucleotides at the 3' end which cannot base pair with the HIV-1 primer binding site RNA.
GENERAL DESCRIPTION OF THE INVENTION Genetic fusions consisting of the entire mature coding sequence or 18 bases of the 3' end of human tRNALYS have been fused to hammerhead ribozyme containing RNAs with base pairing capabilities to the HIV-1 sequences immediately 5' or upstream of the primer binding site. The 3' terminal 18 nucleotides of the tRNALYS are complementary to the primer binding site.
These chimeric molecules have been tested in cell free assays for their ability to bind to HIV-1 reverse transcriptase and their inhibitory activity on HIV-1 reverse transcriptase polymerization activity. The ribozyme moiety targets the cleavage of HIV-1 viral RNA at a known hammerhead cleavage site immediately upstream of the primer binding site for initiation of reverse transcription in the HIV-1 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-1 isolates seauenced to date.
WO 93/24133 PCT/US92/04362 The chimeric RNAs, which are specifically bound by HIV-1 reverse transcriptase should be carried into newly formed HIV-1 virions during viral assembly.
The chimeric primers effectively block HIV-1 reverse transcription, making them a novel, highly target specific, and unique anti-HIV-1 therapeutic agent.
In addition, the tRNALYS 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.
Studies of the binding of the chimeric molecules to HIV-1 reverse transcriptase, revealed that the complex of chimeric tRNALYS-ribozyme, or 18 3' nucleotides of tRNALYS-ribozyme, or tRNALYS with an extra 6 nucleotides appended tc the 3' end, when base paired to the primer binding site signal of HIV-1 RNA, serves as a substrate for a novel ribonuclease activity associated with HIV-1 reverse transcriptase. This activity results in cleavage of the primer at a site very close to the 3' end of the tRNALYS molecule, CCA-3'. 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-1 virion.
GENERAL PURPOSE OR UTILITY OF THE INVENTION The idea of chimeric tRNALYS-ribozyme molecules which effectively compete with tRNALYS for binding to HIV-1 reverse transcriptase is novel. It provides a possible mechanism for specifically delivering inhibitors of HIV-1 reverse transcriptase to the virion particle itself. Such inhibitory agents will render these viral particles non-functional, and thus WO 93/24133 PCT/US92/04362 -6serve as highly specific, non-toxic therapeutic agents.
It has been demonstrated that the entire tRNALYS molecule, as well as various segments of the tRNA itself are capable of specifically interacting with HIV-1 reverse transcriptase. No one has shown that chimeric molecules such as the the ones described could specifically bind to HIV-1 reverse transcriptase. No other work has described that such molecules are inhibitory to HIV-1 reverse transcriptase polymerase activity. There is one published report of an RNAse cleavage activity associated with HIV-1 reverse transcriptase. This activity was only shown to cleave HIV-1 RNA, not the primer. This activity cleaves twice in the primer binding site, and only substrates paired with tRNALYS.
The RNA attached to the 3' end of the tRNALYS need not be a ribozyme, but any extra RNA which can base pair with the HIV-1 target upstream of the primer biU-12 site. If a ribozyme is joined to the tRNA, other cleavage sites such as CUC, or CUA which are on the HIV-1 sequence just to the 3' side (downstream) of the AUC site can be targeted. It is not necessary to make an entire tRNALYS-ribozyme fusion because it is now known that the last 18 nucleotides of tRNALYS fused to the ribozyme are also bound by HIV-1 reverse transcriptase. Genetic variants of tRNALYS which compete better than tRNALYS for binding to HIV-1 transcriptase are included in the invention.
The ribozyme fusions to tRNALYS allow specific targeting of the ribozyme to the HIV-1 virion. Since all retroviruses use cellular tRNAs for priming, this invention provides a general strategy for inhibiting other retroviruses as well. Existing ribozyme WO 93/24133 PCT/US92/04362 -7technology 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.
Claims (6)
1. Chimeric human tRNALY s ribozymes.
2. A construct including a ribozyme fused to the coding sequence of the 3' end of human tRNA LYs
3. A construct as defined by claim 2 in which said ribozyme is a hammerhead ribozyme and said coding sequence is the eighteen nucleotides of tRNALYs
4. A construct as depicted by Figure 1.
5. A method for blocking HIV-1 reverse transcriptase which includes introduction of a gene which expresses a construct as defined by claim 1 into human cells.
6. A chimeric human tRNALys-ribozyme substantially as hereinbefore described with reference to any one of figures 2 to 4. f
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US1992/004362 WO1993024133A1 (en) | 1992-05-27 | 1992-05-27 | CHIMERIC tRNALYS-RIBOZYME MOLECULES |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2169492A AU2169492A (en) | 1993-12-30 |
| AU674656B2 true AU674656B2 (en) | 1997-01-09 |
Family
ID=22231102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU21694/92A Ceased AU674656B2 (en) | 1992-05-27 | 1992-05-27 | Chimeric tRNAlys-ribozyme molecules |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU674656B2 (en) |
| DE (1) | DE69231334T2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5998193A (en) * | 1994-06-24 | 1999-12-07 | Gene Shears Pty., Ltd. | Ribozymes with optimized hybridizing arms, stems, and loops, tRNA embedded ribozymes and compositions thereof |
-
1992
- 1992-05-27 AU AU21694/92A patent/AU674656B2/en not_active Ceased
- 1992-05-27 DE DE69231334T patent/DE69231334T2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| AU2169492A (en) | 1993-12-30 |
| DE69231334D1 (en) | 2000-09-14 |
| DE69231334T2 (en) | 2001-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3529135B2 (en) | How to cut a specific RNA strand | |
| EP0402402B1 (en) | Inhibition of htlv-iii by exogenous oligonucleotides | |
| US8846893B2 (en) | Non-ionically bound constructs, and compositions and kits comprising domains | |
| Ohkawa et al. | Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus. | |
| US4806463A (en) | Inhibition of HTLV-III by exogenous oligonucleotides | |
| Huber et al. | Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase. | |
| AU708194B2 (en) | Reduction of nonspecific hybridization by using novel base-pairing schemes | |
| JP4330797B2 (en) | Ribozyme capable of inhibiting expression of CCR5 receptor | |
| KR20220111322A (en) | Antisense-type guide RNA to which a functional region for editing target RNA is added | |
| Burmeister et al. | 2-Deoxy purine, 2-O-methyl pyrimidine (dRmY) aptamers as candidate therapeutics | |
| IL106396A (en) | Pseudo-half-knot rna formation by hybridization of antisense oligonucleotides to target rna's secondary structure | |
| EP0596901B1 (en) | CHIMERIC tRNA LYS -RIBOZYME MOLECULES | |
| US5827935A (en) | Chimeric tRNAlys -ribozyme molecules | |
| AU674656B2 (en) | Chimeric tRNAlys-ribozyme molecules | |
| Tsurumi et al. | Further characterization of the interaction between the Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3'-to-5'exonucleolytic activity and stability of initiation complex at primer terminus | |
| Zamecnik | History of antisense oligonucleotides | |
| AU692208B2 (en) | Inhibitors and target molecule co-localization | |
| Malik et al. | Late sigma factor of bacteriophage T4. Formation and properties of RNA polymerase-promoter complexes. | |
| Furman et al. | Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase. | |
| Bano et al. | HIV-1 VprB and C RNA cleavage by potent 10–23 DNAzymes that also cause reversal of G2 cell cycle arrest mediated by Vpr genes | |
| Leaman et al. | Controlling gene expression with 2-5A antisense | |
| RU2770691C2 (en) | DNA APTAMERS TO HUMAN PrimPol PRIMASE AND DNA POLYMERASE | |
| Wichert et al. | Selection of a high activity ribozyme against cytostatic drug resistance-associated glypican-3 using an in vitro assay containing total tumor RNA | |
| SINGH et al. | Design of hammerhead ribozymes to distinguish single base changes in substrate RNA | |
| JP2004507244A (en) | The new array |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |