CHIMERIC tRNA<LYS>-RIBOZYME MOLECULES

Abstract: The 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 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.

Description: CHIMERIC tRNa LYS -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 tRNA LYS ; bozyme molecules which compete effectively with tRNA LYS 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 bee: demonstrated that the entire tRNA LYS 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 9:927-931 (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 tRNA LYS -ribozyme molecules. SUMMARY OF THE INVENTION 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. 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 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 iji vitro using bacteriphage 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. Figure 2. This gel shift experiment shows binding of the chimeric tRNA LYS -ribozyme to HIV-1 reveres transcriptase. The eight lanes of the gel from left to right are: 1. Molecular weight marker; 2. 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. There are six extra nucleotides at the 3' derived from the nucleotides after the CCA of the tRNA to the Mae I site which separates the tRNA from the ribozyme. 3. tRNA LYS -ribozyme jLn vitro transcript which has the same extra 5' bases as tRNA LYS , but terminates at Sacll site at the end of the ribozyme moiety. 4. tRNA LYS transcript incubated with HIV-1 reverse transcriptase. 5. tRNA LYS -ribozyme transcript incubated with HIV-1 reverse transcriptase. 6. tRNA LYS transcript incubated with AMV reverse transcriptase. 7. tRNA LYS -ribozyme incubated with AMV reverse transcriptase. 8. tRNA LYS with competing, non-radioactively labelled tRNA LYS -ribozyme incubated with HIV-1 reverse transcriptase. This Figure 2 shows that the chimeric tRNA LYS -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-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 tRNA LYS -ribozyme and HIV-1 primer binding site transcripts are incubated together in the presence of HIV-1 reverse transcriptase. The tRNA LYS -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 tRNA LYS -ribozyme cannot serve as a primer for HIV-1 reverse transcriptase. The lanes are, left to right: tRNA LYS -ribozyme alone, tRNA LYS -ribozyme plus HIV-1 reverse transcriptase, no deoxyribonucleoside triphosphates; tRNA LYS -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 tRNA LYS -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 tRNA LYS 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 tRNA LYS 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 sequenced to date. 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 tRNA LYS portion contains within its mature coding sequence the elements required for transcription by human RNA poly erase 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 tRNA LYS -ribozyme, or 18 3' nucleotides of tRNA LYS -ribozyme, or tRNA LYS with an extra 6 nucleotides appended to 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 tRNA LYS 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 tRNA LYS -ribozyme molecules which effectively compete with tRNA LYS 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 serve as highly specific, non-toxic therapeutic agents. It has been demonstrated that the entire tRNA LYS 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 tRNA Lγs . The RNA attached to the 3' end of the tRNA LYS need not be a ribozyme, but any extra RNA which can base pair with the HIV-1 target upstream of the primer biding 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 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-1 reverse transcriptase. Genetic variants of tRNA LYS which compete better than tRNA LYS for binding to HIV-1 transcriptase are included in the invention. The ribozyme fusions to tRNA LYS 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 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 A. 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: CLAIMS : 1. Chimeric human tRNA LYS -ribozymes. 2. A construct comprising 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 tRNA LYS . 4. A construct as depicted by Figure 1. 5. A method for blocking HIV-1 reverse transcriptase which comprises introduction of a gene which expresses a construct as defined by claim 1 into human cells.

Inventors: LARSON GARRY P, ROSSI JOHN J, LARSON, GARRY, P., ROSSI, JOHN, J.
Applicants: HOPE CITY, LARSON GARRY P, ROSSI JOHN J, CITY OF HOPE
Application year: 1992
Main classification: A61K31
IPC codes: A61K38, C07H21, C12N9, C12N15