WO1996004294A1

WO1996004294A1 - Nucleic acid to initiate the activity of ribonuclease h or reverse transcriptase

Info

Publication number: WO1996004294A1
Application number: PCT/EP1995/003059
Authority: WO
Inventors: Michael STÜRZL; Matthias GÖTTE; Hermann Heumann; Wolfgang Brysch
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1994-08-01
Filing date: 1995-07-31
Publication date: 1996-02-15
Also published as: EP0775153A1; DE4427219A1

Abstract

The invention pertains to a nucleic acid with a sequence binding to an RNA, where the sequence (a) initiates the activity of an Rnase H and is a DNA sequence, and/or (b) initiates the activity of a reverse transcriptase at a site different from the primer binding site and is an RNA sequence. The invention also pertains to a method for producing such a nucleic acid and to its use.

Description

Nucleic acid to initiate the act of RNas e H b zw. reve r ser transcript ase.

description

The invention relates to a nucleic acid for the initiation of the activity of RNase H or reverse transcriptase, a method for producing such a nucleic acid and the use thereof.

It is known that retroviruses are responsible for many diseases of humans, animals and possibly also plants. The acquired immunodeficiency syndrome (AIDS) should be mentioned in particular as an example.

Retroviruses are therefore studied intensively. They have an RNA genome that is translated into a DNA molecule. As such, they integrate into the DNA of infected cells. The integrated DNA acts as a template for the synthesis of viral mRNA. RNA is translated into DNA by a retroviral enzyme called reverse transcriptase (RT). This enzyme requires a primer that binds to a specific location on the viral RNA, the primer binding site (PBS). One such primer is the cellular tRNA ^Lvβ3 . This is packaged (selected) in the viral capsid during the maturation of the virus particles. This ensures the initiation of the translation of RNA into DNA. The reverse transcriptase synthesizes a DNA strand complementary to the RNA on the primer and simultaneously degrades the transcribed RNA strand with its RNase H activity. A DNA double strand is then formed by the DNA-dependent polymerase activity of the reverse transcriptase.

Many attempts have been made to inhibit the replication of retroviruses. However, none of these attempts have so far brought the desired success.

The present invention is therefore based on the object of providing a means with which the replication of retroviruses can be inhibited.

According to the invention this is achieved by the subject matter of the claims. The present invention is based on the idea of using the activity of an RNase H or a reverse transcriptase to inhibit retroviral replication.

An RNase H is an enzyme of the cell as well as the virus, especially the retrovirus. In the latter case, RNase H is associated with reverse transcriptase. The term "RNase H" used in the claims includes such an above enzyme. An RNase H has the activity of degrading the RNA in an RNA / DNA hybrid.

This is used according to the invention by providing a nucleic acid which has a DNA sequence which is complementary to an RNA to be degraded. The RNA can be of any type and of any origin. It is preferably cellular or viral RNA, particularly preferably retroviral and very particularly preferably an RNA comprising the primer binding site or parts thereof. Cellular tRNA ^Lvs3 is also particularly preferred and that part of this tRNA which binds to the primer binding site is very particularly preferred. The above DNA sequence is intended to degrade the complementary RNA through the activity of an RNase H. For example, the former is degraded with a DNA sequence that is complementary to the primer binding site.

A reverse transcriptase is a retroviral enzyme that translates RNA into DNA. For this, the reverse transcriptase requires the binding of an RNA primer to the primer binding site. One such RNA primer is the cellular tRNA ^Lvs3 . Recent investigations indicate that an RNA / RNA or RNA / DNA complex is essential for the initiation of the activity of a reverse transcriptase, the nucleotide sequence being of little importance.

This is used according to the invention by providing a nucleic acid with an RNA sequence which binds to a site different from the primer binding site. The RNA sequence preferably comprises 18 nucleotides. Furthermore, a conserved site in the retroviral RNA is preferred as the site to be bound. The RNA sequence is intended to redirect the natural initiation site of the reverse transcription to one or more artificial sites. The RNA / DNA hybrids initiated there are subjected to the RNase H associated with the reverse transcriptase. An uncontrolled arises

Degradation of the retroviral RNA, which leads to the synthesis of incomplete virus genomes and ultimately to the inhibition of viral replication.

The above nucleic acids preferably have structural elements which allow colocalization of the nucleic acids with the RNA to be bound, that is to say packaging both into the retroviral capsid. Such structural elements can be cellular tRNA molecules that are selected specifically for the virus. HIV selects tRNA ^Lys \ tRNA ^Lys2 , tRNA ^{,, e} and tRNA ^Lvs3 . These tRNA molecules and parts thereof, for example the anticodon-stem-loop structure of the tRNA ^Lvs3 or a part thereof, are seen as preferred structural elements. In a particularly preferred form, the nucleic acids which initiate the activity of an RNase are present as DNA analogs of the cellular tRNA ^Lys3 , ie as tDNA ^Lvs3 or modulated tDNA- ^Lvs3 . The latter does not bind to the primer binding site, but, depending on the modulation, to other retroviral and / or cellular sequences. However, nucleic acids which initiate the activity of a reverse transcriptase at artificial sites are particularly preferably present as modulated tRNA ^Lys3 analogs. Furthermore, nucleic acids are preferred which are chimera from parts of tDNA ^Lys3 or modulated tDNA ^Lys3 and of tRNA ^Lyβ3 or modulated tRNA ^Lys3 .

The above nucleic acids can also have chemically modified deoxy or ribonucleotides. This increases the intracellular stability of the nucleic acids, in particular if the modifications are present at the 3 'end of the nucleic acids. Modifications of deoxy or ribonucleotides are known. As an example, reference is made to FIGS. 1 and 2. 1 shows the chemical structure of an oligodeoxyribonucleotide portion, FIG. 2 shows the chemical structure of an oligoribonucleotide portion. The structures shown in FIG. 1 and FIG. 2 are understood as sections from longer oligonucleotide chains.

In FIG. 1, the letter B denotes an organic base, such as adenosine (A), guanosine (G), cytidine (C), thymidine (T) or uridine (U), which is attached to the N9 (A, G) or N1 ( C, T, U) position is coupled to the deoxyribose. The oligodeoxy ribonucleotide portions can e.g. be chemically modified as follows:

1. All R 'positions of an oligodeoxyribonucleotide portion are substituted

2. The R ¹ positions of an oligodeoxyribonucleotide portion are differently substituted

5 'BpBpBp- (Bp-) nB-pBpBp- ^B 3'

I I I I 1 1 1

Ria Ria R a Rlb Ria Ria Ri

B = desocyribonukeotide dA, dC, dG, dt or DU depending on the sequence p = internucleotide phosphate n = an oligodeoxyribonucleotide section with a length between 1 and 120 bases

3. The R ¹ positions of an oligodeoxyribonucleotide portion are alternately substituted

5 * Bp- (B- _p -Bp) nBpB 3 '

I I I I

_R 1a Rlb Ria Rlb

B = deoxyribonucleotide dA, dC, dG, dT or dU depending on the sequence p = internucleotide phosphate n = an oligodeoxyribonucleotide section with a length between 1 and 120 bases

in Figure 2, the letter B denotes an organic base such as adenosine (A), guanosine (G), cytidine (C) or uridine (U), which is at the N9 (A, G) or N1 (C, U) position the ribose is coupled. The oligoribonucleotide components can be chemically modified, for example, as follows: 4. All R ¹ positions of an oligoribonucleotide portion are substituted

5. The R ¹ positions of an oligoribonucleotide portion are substituted differently

5 "B- _p -B- _p -Bp- (Bp-) _n BpBpBpB ₃ -

1 IIIIIIR ^1a Ria Ria Rlb Ria Ria ia

B = ribonucleotide dA, dC, dG or dU depending on the sequence p = internucleotide phosphate n = an oligoribonucleotide segment with a length between 1 and 120 bases

6. The R ¹ positions of an oligoribonucleotide portion are alternately substituted 5 ^' Bp- (BpBp) _n -B- _p -B

I I I i

_R 1a Rlb _R 1a _R ^1b

B = ribonucleotide dA, dC, dG or dU depending on the sequence p = internucleotide phosphate n = an oligoribonucleotide section with a length between 2 and 120

Bases

The R ⁴ position of the compounds described in 4.1 - 4.6, 5.1 - 5.6 or 6.1 - 6.6 are substituted

7.1 R ⁴ = O

7.2 R ⁴ = F

7.3 R ⁴ = CH ₃

The nucleic acid fraction of the nucleic acids described in the present invention can consist of a fraction as described under 1.1-1.6, 2.1-2.6, 3.1-3.6, 4.1-4.6, 5.1-5.6, 6.1-6.6 or 7.1-7.3, whose base sequence is complementary to a partial sequence of the viral or cellular target RNA. At the 3 'and / or 5' end of this complementary sequence, one or more nucleic acid segments can be found, as under 1.1-1.6, 2.1-2.6, 3.1-3.6, 4.1-4.6, 5.1-5.6, 6.1-6.6, 7.1-7.3 described, which, due to their respective base sequence, form a secondary and tertiary structure which specifically bind to viral proteins, for example reverse transcriptase, nucleocapsid

Proteins, or cellular proteins binds. Said nucleic acid segments can also contain base modifications, as shown for example in FIG. 3.

In addition to the nucleic acid proportions, the nucleic acids described in the present invention can also be other molecules, e.g. Contain coupled peptides, proteins, phospholipids, steroids, which specifically bind to viral or cellular proteins instead of or in addition to a nucleic acid secondary or tertiary structure. The nucleic acids are preferably with a substance which inhibits virus multiplication, particularly preferably ribozyme or 2 ',

5'-coupled oligoadenylates.

According to the invention, a method for producing the above nucleic acids is also provided. Such a process advantageously comprises the following process steps:

(a) binding an activated β-cyanoethyl nucleotide to a support,

(b) cleavage of the 5'-dimethoxytrityl protective group of the nucleotide from (a),

(c) addition of a further activated β-cyanoethyl nucleotide, (d) oxidation or, if necessary, modification of the phosphite group of the sugar-phosphate backbone,

(e) binding the still free 5'-hydroxyl groups with a protective group, and

(f) Carrying out a further synthesis cycle or cleaving the nucleotide chain from the carrier. The above chemical modifications are inserted in step (d). This can be done in the usual way.

Nucleic acids according to the invention can be introduced into a person to be treated by conventional methods. For example, T-lymphocytes can be isolated from an HIV-infected person and the nucleic acids according to the invention can be introduced into them by known measures, such as electroporation.

It is also possible to produce, ie to express, nucleic acids according to the invention, especially if they consist of ribonucleotides, such as modulated tRNA ^Lys3 (see FIG. 4), only in the person to be treated. For this purpose, it is advantageous to insert the DNA coding for such a nucleic acid into an expression vector such as pNEOtp-muNTSI (cf. Hemann et al., DNA and Cell Biology, (1994) and the expression plasmid obtained, as indicated above, in It goes without saying that the above DNA can comprise all sequences which can influence the expression of a nucleic acid according to the invention. For example, reference is made to FIG. 5, in which a DNA is indicated which is suitable for tRNA ^Ly " ³ (a) or modulated tRNAL ^Ly83 (b) The bold sequence relates to (modulated) tRNA ^Lyβ3 , while the weakly printed sequence relates to regulatory regions such as the termination sequence of polymerase III (oligo No. 9). The DNA of FIG. 5 is inserted into the expression vector pNEOtp-muNTSI digested with Sall, whereby an expression plasmid suitable for introduction into a person to be treated is obtained will.

Nucleic acids according to the invention are distinguished in that they are targeted

Hydrolyze RNA, particularly cellular or viral, very particularly retroviral RNA. For this purpose, the activity of cellular as well as viral RNase H, in particular associated with reverse transcriptase, is used. Also the activity reverse transcriptase used as such. Nucleic acids according to the invention are therefore ideally suited for inhibiting the replication of retroviruses, in particular of HIV viruses. The present invention thus opens up the possibility of diagnosing and treating infections or diseases which are caused by the above viruses.

Brief description of the drawing:

1 shows the chemical structure of an oligodeoxyribonucleotide portion of a nucleic acid according to the invention,

2 shows the chemical structure of an oligoribonucleotide portion of a nucleic acid according to the invention, FIG. 3 shows the structure of tRNA ^Ly83 and modified bases thereof,

Fig. 4 shows the structure of tRNA ^Ly " ³ and modulated tRNA ^Ly * ³ , Fig. 5 shows the DNA for tRNA ^{Ly, 3} (a) and modulated tRNA ^{Ly, 3} (b)

6 shows chemically treated tDNA ^Lyβ3 under native and denaturing conditions, FIG. 7 shows the titration of the tDNA ^Lyβ3 and tRNA ^Lys3 with HIV-1-RT,

Figure 8 shows the RNA region of HIV-1 and the primer binding site

9 shows the time-dependent hydrolysis of RNA in the presence of NCp7 or without NCp7.

The invention is illustrated by the following examples.

Example 1: Synthesis of tDNA ^Lys3 and investigation of its structure

1. Synthesis of tDNA ^Lva3 The DNA analogue of tRNA ^Lys3 (see FIG. 3) was as described above ben, chemically synthesized. An automatic synthesizer from Applied Biosystems was used for this. After the synthesis, the tDNA ^{Lys3 was} purified by gel electrophoresis. The sequence of the tDNA ^Lys3 is identical to that of the tRNA ^Lys3 . However, it has no modified bases. The uridine ribonucleosides are also replaced by corresponding thymidine deoxynucleotides.

2. Radioactive labeling of the tDNA ^Ly * ³

For the structural ^{examinations,} the tDNA ys3 was radioactively marked on the 3'termin or alternatively 5'terminus. The 3'terminal labeling was carried out enzymatically using "transferase" and [σ- ³² P] dd ATP. The 5 'labeling was also carried out enzymatically using polynucleotide kinase and [- ³² P] ATP.

3. Identification of single-stranded thymidines and adenosines of the tDNA ^Ly * ³ by chemical modification. To detect single-stranded thymidines, the tDNA ^Ly * ³ radiolabelled at the 3 'term was incubated under native conditions at 37 ° C with OsO ₄ (bipyridine) ₂ . Single-stranded adenosines were detected by incubating the tDNA ^Ly83 with diethyl pyrocarbonate (DEPC). Corresponding control reactions were carried out at 95 ° C. All thymidines and all adenosines are chemically modified under these conditions, since the tDNA ^{Lys3 is} unfolded and is therefore completely single-stranded. A subsequent pyrimidine treatment leads to strand breaks on the chemically modified nucleotides of the tDNA ^ys3 . The reaction products were analyzed by gel electrophoresis.

In Fig. 6, lane (1) of the OsO ₄ and lane (3) of the DEPC reaction show the tDNA ^Lys3 under native conditions and lanes (2) and (4) show the tDNA ^Ly83 under denaturing conditions. A comparison of the reactions between native and denaturing agents shows that thymidines and adenosines of tDNA ^Lys3 exist which are not modified and are therefore paired with bases. The tDNA ^Lys3 structure determined in this way is a tRNA ^Lys3 structure analogous.

Example 2: Comparison of the interaction of the natural replication primer tRNA ^' ^Ly * ³ and chemically synthesized tDNA ^Ly * ³ with the reverse transcriptase (RT) of HIV-1

1. Preparation of the tRNA ^Ly ' ³

For all experiments comparing the structural and functional ^{properties of} the tRNA ^Lyβ3 and tDNA ^Lys3 , purified tRNA ^Lys3 isolated from ^{rabbit liver was} used (Raba et al., (1979) Eur.J.Biochem. 97, 305-318). This tRNA ^Lys3 is ^{identical in} sequence to the human tRNA ^Lys3 .

2. Preparation of the HIV-1 RT. The expression plasmid E. coli was used to synthesize recombinant HIV-1 RT

M15 :: pDMI.1 :: pRT6H-PR (Le Grice, S.F.J. and Grüninger-Leitch, F. (1990) Eur.J.Biochem. 187, 307-314). The protein processing was carried out as described there.

3. Radioactive end labeling of the tDNA ^Lys3 and tRNA ^Lyt3

The 5 'end of the natural tRNA ^Lys3 was firstly dephosphorylated with alkaline phosphatase and then, as described above in Example 1, with l - ³² P] ATP radiolabeled.

4. Titration of the tDNA ^Ly " ³ and tRNA ^Ly * ³ with HIV-1-RT

100 ng tRNA ^Lys3 or tDNA ^y * ³ were incubated in separate reaction batches with different RT amounts at 37 ° C. The batches were analyzed by gel electrophoresis under native conditions. It was found that both nucleic acids are completely complexed by the protein in a five-fold molar RT excess. The RT binds both nucleic acids highly cooperatively, since at an insignificantly lower RT excess (fourfold), both the tDNA ^Lys3 and the tRNA ^{Lys3 are} completely uncomplexed.

7 shows the gel electrophoretic analysis of the two band-shift experiments. Lane C is the control lane without HIV-1 RT, the other lanes show the titration of the nucleic acid with HIV-1 RT in the molar ratios 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7 and 1: 8. The positions of the tRNA / RT or tDNA / RT complexes and the uncomplexed nucleic acids are marked in the figure.

This analysis shows that the natural tRNA ^Lys3 and the chemically synthesized tDNA Lys3 ^have a comparable affinity for HIV-1-RT.

Example 3: Substrate specificity of the RT associated RNaseH domain

1. Synthesis of the RNA template

The RNA template was synthesized enzymatically by run-off transcription using the T7 RNA polymerase. The RNA sequence is shown in Fig. 8.

2. Radioactive labeling of the RNA template

The radioactive labeling at the 5 'terminus of the RNA template was carried out as described in Example 1 above. A 3 'terminal labeling was carried out with [ ³² P] pCp and T4-RNA polymerase.

3. Hybridization of the RNA template with the primer tRNA ¹ * ³ or tDNA ^Lys3

The primer and template were prehybridized in a molar ratio of 1.2: 1. The components were first heat denatured, then 10 min. incubated at 55 ° C and finally cooled to 37 ° C. The prehybridized substrates, 10ng template / tRNA and 10ng template / tDNA, were then washed with a triple molar RT excess for 1 min. at 37 ° C beer. This was followed by a gel electrophoretic analysis.

It was shown that the RNA / RNA substrate remains intact, while the RNA / DNA substrate is hydrolyzed by the RT-associated RNaseH activity.

Example 4: Catalytic hydrolysis of the primer binding site by HIV-1-RT, nucleocapsid protein (NCp7) and tDNA ^Ly * ³ under native conditions

1. NCp7 synthesis

Chemically synthesized NCp7 was used (De Rocquigny et al., (1992) Proc.Natl.Acad, Sci. USA 89, 6472-6476).

2. Kinetic analysis of RNA hydrolysis

55 pmol RNA template were incubated with 5.5 pmol tDNA ^Lyβ3 and 15 pmol RT at 37 ° C. In separate reaction batches, 20 pmol of NCp7 was additionally incubated. The reactions were stopped after various times and analyzed by gel electrophoresis.

FIG. 9 shows that in the presence of NCp7 the 10-fold molar excess of RNA is completely hydrolyzed. The RNA hydrolysis is accelerated 20-fold compared to the control reaction without NCp7. It also shows that HIV-I-RT, NCp7 and tDNA ^{Lys3 are} the components of a system for the targeted catalytic degradation of the primer binding site. Since the above reaction conditions correspond to native conditions, the result obtained is of the greatest importance for the in vivo application of the present invention.

3. Further analysis with an HIV-1 wild-type RNA template The above experiment, described under 2., was carried out analogously with a HIV-1 wild-type RNA template performed (length approx. 320 nucleotides). This RNA template is characterized by the natural structure of the viral RNA genome. In this experiment, too, in which the natural situation could be largely simulated, it was shown that the PBS is degraded in the presence of NCp7 or alternatively by prehydbridization. Thus structured RNA and DNA molecules are unfolded by NCp7, specifically hybridized and used as a substrate of the RNase H associated with RT. This result implies that the natural replication ^primer tRNA ^Lys3 packaged in the virus capsid can also be the target of DNA-induced hydrolysis. In this reaction, the tRNA ^{Lys3 acts} as an RNA template for the RT / RNase H-catalyzed RNA

Hydrolysis. Analogously, a ribozyme can also be used for tRNA ^Lys3 hydrolysis. The colocalization of the rection ^partners in the capsid (nucleic acid ^according to the invention, tRNA ^Lys3 , RT and NCp7) means that only the selected tRNA ^" ^Lys3 and not the cytoplasmic tRNA is hydrolyzed.

Claims

claims

1. Nucleic acid with a sequence binding to an RNA, part of the nucleic acid

(a) initiates the activity of an RNase H and is a DNA sequence, and / - or

(b) the activity of a reverse transcriptase is initiated at a site other than the primer binding site and is an RNA sequence.

2. Nucleic acid according to claim 1, characterized in that the sequence binds to a viral and / or cellular RNA.

3. Nucleic acid according to claim 2, characterized in that the cellular RNA is the tRNA ^Lys3 .

4. Nucleic acid according to one of claims 1 to 3, characterized in that the nucleic acid has structural elements which allow its colocalization with the RNA to be bound.

5. Nucleic acid according to claim 4, characterized in that the structural elements are analogous to those which also allow a colocalization of tRNA and viral RNA.

6. Nucleic acid according to claim 4 or 5, characterized in that the structural elements bind to a reverse transcriptase and / or a nucleocapsid protein.

7. Nucleic acid according to one of claims 4 to 6, characterized in that that the structural elements come from the tRNA ^Lys3 .

8. Nucleic acid according to one of claims 4 to 7, characterized in that the nucleic acid is tDNA ^Lys3 modulated tDNA ^Lys3 .

9. Nucleic acid according to one of claims 4 to 7, characterized in that the nucleic acid is modulated tRNA ^ys3 .

10. Nucleic acid according to one of claims 4 to 7, characterized in that the nucleic acid is a chimera of parts of tDNA ^Lys3 or modulated tDNA ^Lys3 and of tRNA ^Lys3 or modulated tRNA ^Lys3 .

1 1. Nucleic acid according to one of Claims 1 to 10, characterized in that the nucleic acid is coupled to a substance which inhibits virus multiplication.

12. Nucleic acid according to claim 1 1, characterized in that the substance is a ribozyme.

13. Nucleic acid according to one of claims 4 to 6, which has structural elements analogous to those of the cellular tRNA ^Lys3 .

14. Nucleic acid according to claim 13, characterized in that the genann¬ th structural elements bind to a reverse transcriptase and / or a nucleocapsid protein.

15. Nucleic acid according to claim 14, characterized in that said reverse transcriptase or the nucleocapsid protein stems from the HIV virus.

1 6. Nucleic acid according to one of claims 1 3 to 1 5, characterized gekennzeich¬ net that the binding of the nucleic acid, the binding of cellular tRNA ^' competitively inhibits ^ys3 .

17. Substances which have structural features which bind to a reverse transcriptase and / or a nucleocapsid protein, it being possible for the substances to be compounds of amino acids and / or nucleotides and / or further organic compounds.

18. Substances according to claim 17, characterized in that the bound reverse transcriptase or the nucleocapsid protein stems from the HI virus.

19. Substances according to claim 17 or 18, characterized in that the binding of these substances competitively inhibits the binding of cellular tRNA ^Lys3 to the reverse transcriptase or a nucleocapsid protein.

20. A method for producing the nucleic acid according to claim 1, comprising the following method steps:

(a) binding an activated β-cyanoethyl nucleotide to a support,

(b) cleavage of the δ'-dimethoxytrityl protective group of the nucleotide from (a),

(c) addition of a further activated β-cyanoethyl nucleotide,

(d) oxidation or, if necessary, modification of the phosphite group of the sugar-phosphate backbone,

(e) binding the still free 5'-hydroxyl groups with a protective group, and

(f) carrying out a further synthesis cycle or cleaving the nucleotide chain from the carrier.

21. Use of the nucleic acid according to one of claims 1 to 13 as a medicament or part of a medicament preparation for hydrolysis of RNA.

22. Use according to claim 21, wherein the RNA is viral or cellular RNA.

23. Use according to claim 21 or 22, wherein the RNA hydrolysis serves to inhibit viral replication.

24. Use according to claim 23, wherein the replication of retroviruses, in particular HI viruses, is inhibited.

25. Use of the substances according to one of claims 13 to 19 as a medicament or part of a medicament preparation for the competitive blocking of the binding site for tRNA ^Lys3 on a reverse transcriptase or a nucleocapsid protein.

26. Use according to claim 25, wherein the reverse transcriptase or the nucleocapsid protein are derived from the HI virus.

27. Use according to claim 26, wherein the replication of the HI virus is inhibited.