AU669487B2

AU669487B2 - Ribosomal structure derived from the genomic RNA structure of the delta hepatitus virus

Info

Publication number: AU669487B2
Application number: AU25583/92A
Authority: AU
Inventors: Marta Blumenfeld; Gilbert Thill; Marc Vasseur
Original assignee: Genset SA
Current assignee: Merck Biodevelopment SAS
Priority date: 1991-09-03
Filing date: 1992-09-03
Publication date: 1996-06-13
Anticipated expiration: 2012-09-03
Also published as: FR2680797A1; ATE212057T1; EP0602157A1; AU2558392A; FR2680797B1; DE69232351T2; DE69232351D1; WO1993005157A1; JPH06510196A; EP0602157B1

Abstract

The present invention concerns a ribosomal structure derived from genomic RNA of the delta hepatitus virus consisting in one or more fragments of said genomic RNA of the delta hepatitus virus, capable of having a catalytic trans cleavage activity on another molecule containing a given RNA nucleic acid sequence.

Description

DPI DAYE 05/04/93 AOJP DATE 10/06/93 APPLN. ID 25583/92 PCT NUMBER PCT/FR92/00840 AU9225583 IJLVA14i NU 1I nflNx11~jvlltmI I, REVETS (PCT) (51) Classification internationale des brevets 5 (11) Numniro de publication internationale: WVO 93/05157 C12N 15/51, A61K 31/70 Al (3 aed ulcto nentoae 8mr 93(80.3 C12N 9/00 (3 aed ulcto nentoae 8mr 93(80.3 (21) Numno de la demnande internationale: PCT/FR92/00840 (74) Mandataire: WARCOIN, Jacques; Cabinet Regimbeau, 26, avenue K16ber, F-751 16 Paris (FR).

(22) Date de d~p6t international: 3 septembre 1992 (03.09.92) (81) Etats disign~s: AU, CA, JP, KR, US, brevet europ~en (AT, Donnev relatives i la priorit6: BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, 9 1/10872 3 septembre 199' (03.09.9 1) FR NL, SE).

(71) Diposant (pour tous les Etats dc~signbs sauf US): GENSET Pubie [FR/FR]; 1, passage Etienne-Delaunay, F-75011I Paris Avec rapport de recherchze internationale.

(FR).

(72) Inventeurs; et Inventeurs/D~posants (US settlemnent) :BLUMENFELD, Marta [FR/FR]; 75, rue Bobillot, F-75013 Paris e" TRILL, Gilbert [FR/FR]; 24, rue Victor-Hugo, F-922707 Bois-Colombes VASSEUR, Marc [FR/FR]; 25, 6 8 7 boulevard Arago, F-75013 Paris (FR).

(54) Title: RIBOSOMAL STRUCTURE DERIVED FROM THE GENOMIC RNA STRUCTURE OF THE DELTA HEPA- TITUS VIRUS (54)Titre: STRUCTURE RIBOZYMIQUE DERIVEE DE L'ARN GENOMIQUE DU VIRUS DE L'HEPATITE DELTA (57) Abstract The present invention concerns a ribosomal structure derived from genomic RNA of the U-Jelta hepatitus virus consisting in one or more fragments of said genomic RNA of the delta hepatitus virus, capable of having a catalytic trans cleavage activity on another molecule containing a given RNA nucleic acid sequence.

(57) Abr~gi La pr~sente invention concerne une structure ribozymique d6rivee de Y'ARN gi~nomnique du virus de l'h~patite delta consistant dans un ou plusieurs fragments dudit ARN g~nomique du virus de l'h~patite delta, et capable d'exercer une activit6 catalytique de coupure en trans sur une autre moI~cule comportant une sequence sp6cifique d'acide nucl~ique dWARN.

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WO 93/05157 PCT/FR92/00840 RIBOSOMAL STRUCTURE DERIVED FROM THE GENOMIC RNA STRUC

T

URE OF THE DELTA HEPATITUS VIRUS The present invention relates to a novel ribozyme structure which is capable of trans cleavage of a DNA or RNA target molecule.

In detail, this novel ribozyme structure is an RNA molecule which is derived from the genomic RNA of the human hepatitis delta virus.

The genomic RNA of the human hepatitis delta virus shows autocatalytic activities. Using RNA fragments of a reduced size which preserve an autocatalytic cleavage activity and which are derived from genomic viral RNA, a catalytic portion and a substrate portion have successfully been separated according to the invention in a distinctive manner as will be explained hereinafter.

Consequently, a cleavage can be obtained in a binary reaction by mixing the ribozyme structure which has a catalytic activity with a heterologous RNA or DNA substrate. This characterization of a fragment which has a catalytic cleavage activity allows these ribozyme structures of the genomic RNA of the hepatitis delta virus to be used as a "human" type ribozyme structure for the cleavage of target RNA' in vitro or in vivo for experimental, therapeutic or prophylactic purposes.

i 25 Ribozymes are RNA molecules which are naturally capable of exercising catalytic activities, in particular RNAse activities. Using such natural ribozymes, it has been attempted to establish artificial ribozyme structures which are capable of performing enzymatic cleavages on specific target RNAs.

Such ribozymes thus provide therapeutic tools which can be used within approaches of the "antisense" oligonucleotide type, especially for specifically degrading pathogenic type target RNAs.

Ribozymal activities seem to be widespread, and catalytic RNAs have been described for bacteria, protozoans, plants and .tlimal systems. The first ribozymal activities were revealed, first of all, in systems for pnrsp~ 1 -2 splicing class I introns, and then in RNase P. More recently, it was found that the genomes of a large number of RNA viruses, plants or even mammals also had ribozymal activities which appeared during the viral multiplication cycle. Finally, certain RNAs transcribed from the satellite DNA regions in tritons also show a natural ribozymal activity which cleaves concatemer transcripts.

Analysis of the structure of genomic ribozymes of certain plant viruses has revealed common reactional sequences and secondary consensus structures which have allowed ribozymes to be constructed which can act as tailor-made cleavage agents for specific RNA sequences.

Analysis of the regions involved in these cleavages has brought to light a common structure termed "hammerhead" 15 or which is required and sufficient for bringing about the nuclease reaction.

A second class of ribozymes which are also derived from a plant virus shows a catalytic "hairpin" i structure, or structure.

j 20 These different ribozyme types function in vitro !I but the efficiencies for in vivo cleavage in animal or human cells are still relatively ineffective. This relative inefficiency is probably at least partly due to the fact that all these ribozymes are derived from plant viruses and not from animal or even human viruses.

The hepatitis delta virus is a human RNA virus which is systematically associated with the hepatitis B virus. Both the genom and the antigenom RNA of this virus have an autocatalytic cleavage activity. The sequence of i 30 the RNA fragment which has a very short length and presents ribozymal activity bears no resemblance whatsoever to the and type catalytic motifs which are known already. The hepatitis delta virus replicates in human hepatocytes and its ribozymal activity functions perfectly in those cells. Its ribozymal activity has been selected naturally to function in these human liver cells and this model provides a perfect example of what could be an active ribozyme in humans.

e i The genomic RNA of the hepatitis delta virus, 3 which has a length of 1700 nucleotides, contains, indeed, a region which is capable of performing an autocatalytic cleavage. This autocatalytic cleavage can be performed by a subfragment which is 89 nucleotides long, whose secondary structure can be represented schematically in the form shown in Figure 1.

This 89 nucleotide-long structure contains a "pseudoknot" type configuration. The aut:catalytic cleavage is performed in the phosphodiester bond shown by the arrow in Figure 1.

Using this ribozymal fragment which has an autocatalytic activity, there have been provided, according to the present invention, ribozyme structures with a catalytic trans o1 activity which can be exerted on a second RNA target molecule during a binary reaction.

This invention provides novel ribozymal molecules capable of effective cleavage of DNA or RNA target molecules, in particular pathologic ones, in human, animal or even plant cells, for therapeutic or prophylactic purposes.

This concerns novel ribozymal trans activity. The molecule which is responsible for this trans cleavage has a structure and a sequence which do not correspond to any other ribozymal, trans-acting structure which has previously been described.

Using techniques for the chemical synthesis of RNA, RNA subfragments which correspond to different regions of the molecule, shown in Figure 1, were prepared.

These various fragments were subsequently tested in reconstruction experiments to distinguish the substrate fragments from the catalytic fragments.

The present invention therefore relates to a ribozyme structure derived from Sgenomic RNA of the hepatitis delta virus consisting of one or more fragments of said genomic RNA of the hepatitis delta virus, capable of having a catalytic trans cleavage activity on another molecule containing a specific RNA nucleic acid sequence.

i 25 Disclosure of the Invention According to this invention there is provided a ribozyme structure derived from genomic RNA of the hepatitis delta virus consisting of one or more fragments of said genomic RNA of the hepatitis delta virus, capable of having a catalytic trans cleavage activity on another molecule containing a specific RNA nucleic acid sequence, wherein said ribozyme structure is composed of one or two fragment(s) from the sequence of the 74 nucleotides which contain the nucleotides from 16 to 89 shown in Figure 2.

By "other molecule" there is to be understood according to the present application that a molecule other than genomic RNA of the hepatitis delta virus is cleaved.

[N:\LIBuu]00730:KEH wood 4 4 In a particularly suitable embodiment, the ribozyme structure is composed of one or two fragment(s) from the sequence of the 74 nucleotides which contain the nucleotides from 16 to 89 shown in Figure 2.

The preferred ribozymal structures amongst the structures tested, i.e. the most effective ribozymal structures in terms of cleavage activity, contain a single fragment.

In particular, the ribozymal structure according to the invention can consist of the 74 nucleotide fragment containing the nucleotides from 16 to 89, or this same fragment 16-89 in which only part of the nucleotides to 77 have been deleted. When all of the sequence 50-77 has been deleted, the fragment loses its catalytic cleavage activity.

The ribozymal structure according to the invention will preferably contain a sequence of at least 12 nucleotides.

According to the invention, this nucleic acid sequence of this other molecule corresponds to a genomic RNA fragment of the hepatitis delta virus, which is a substrate of this catalytic trans cleavage activity.

In particular, it will be possible for this nucleic acid sequence of this other molecule, which is a substrate of the catalytic activity, to contain the sequence 5'XGGCC3' where X C or U, this ribozymal structure being capable of having a nuclease or ribonuclease activity on the phosphodiester bond between X and G.

H 30 In an embodiment, it will be possible for this nucleic acid sequence of this other molecule to contain the 8 nucleotide fragment, which covers the nucleotides to 12, which are shown in Figure 2.

The catalytic cleavage activity takes place in a suitable fashion in the presence of divalent metal ions, especially magnesium.

In an embodiment of the invention, the ribozymal structure according to the invention is inserted into F RA 7 this other molecule and is capable of having an r 5 autocatalytic cleavage activity on this other molecule into which it is inserted.

As has been shown, the ribozymal structure according to the invention will have a ribonuclease activity, especially an endoribonuclease activity, on another molecule which contains a polyribonucleotide of a specific sequence.

The ribozymal structure according to the invention can be used for therapeutic or prophylactic purposes in prokaryotic or eukaryotic, animal or plant cells, it being possible, thus, for this other molecule to be heterologous to animal or plant cells.

It will be possible to use the viral particle of hepatitis delta as an encapsidation system, or any other vector for intracellular delivery of ribozymal structures according to the invention, be this in bacteria or in eukaryotic, animal or plant cells.

Other advantages and characteristics of the present invention will become apparent in the light of the detailed description which follows. This description refers to Figures 1 to 4.

Figure 1 shows the potential secondary "pseudoknot" like structure of an autocatalytic fragment of the genomic hepatitis delta virus RNA.

Figure 2 shows the potential structure of the binary trans cleavage complex by a ribozyme derived from the genomic strand of the hepatitis delta virus.

Figure 3 shows the autoradiograph of a polyacrylamide gel on which the products from an incubation of a ribozyme structure according to the invention with its substrate were run, after various periods of time.

Figure 4 shows an analysis of the products of an autocatalytic cleavage run with a 71 nucleotide-long ribozyme structure with a deletion in the self-paired structure 50-77 on 10% polyacrylamide/7 M urea.

Example 1 SPRA Amongst the combinations tested, it has been Sfound, as has been shown, that it is possible to perform C .j L I 6 a trans cleavage using a fragment composed of, for example, 13 nucleotides (1 to 13) of Figure 2 as substrate and a molecule composed either of two fragments containing the nucleotides from 16 to 62 and 63 to 89, respectively, or of a single, 74 nucleotide-long fragment containing the nucleotides from 16 to 89, as ribozymal structure.

This 74 nucleotide ribozyme was obtained by in vitro transcription of a bicatenary DNA matrix by means of RNA polymerase T7. This matrix has been obtained by chemical synthesis of the DNA sequences, hybridizing them, and subsequently amplifying the product obtained by means of a PCR reaction. Using suitable primers, this PCR reaction allows the transcription promotion sequences which enable the polymerase T7 to function to be adjoined.

Figure 2 illustrates the 74 nucleotide ribozyme structure and the structure of its 13 nucleotide substrate.

This 74 nucleotide-long ribozyme is capable of cleaving the substrate rapidly. If the ribozyme is incubated in the presence of the substrate over variable periods of time from 0 to 60 minutes in a buffer containing 10 mM MgCl 2 50 mM Tris-pH 8.0 and 2 mM spermidine, it can be found (Figure 3) that a cleavage takes place. Figure 3 shows the autoradiograph of a polyacrylamide gel on which the products of incubations of the ribozyme with its substrate after various periods of time are run. The substrate was labeled at the 5' with gamma-ATP 32 P by means of T4-polynucleotide kinase and purified on polyacrylamide gel before being used. It can be seen that a large portion of this substrate is cleaved during the first minutes of incubation, which cleavage is shown by a labeled, 5 nucleotide-long band which appears rapidly under conditions under which the ribozyme functions, that is to say in the presence of magnesium.

The controls carried out in the presence of EDT are negative, which shows that an enzymatic reaction which is S dependent on the presence of magnesium (or other divalent P L c Y Ah 7 ions) is observed, which is a characteristic of ribozymes.

Example 2 The experimental results which follow show that the presence of the paired secondary structure, which is situated between nucleotides 50 and 77, is partly not indispensable for the catalytic activity of this ribozyme.

Starting from the structure of an 89 nucleotidelong ribozyme as the one described in Figure 1, this is followed by experiments in which certain regions are deleted, in particular deletions of the hairpin structure between nucleotides 50 and 77. Indeed, this hairpin is an extremely stable secondary structure which is found again in all potential configurations calculated by energy minimalization programs which are applied to the structural analysis of this ribozyme, and it is probably effectively present in the active structure.

To find out if this paired secondary structure played a catalytic role or else a structural role, nucleotides 53 to 61 and 66 to 74 were deleted. These deletions were performed by synthesizing a DNA fragment with the desired sequence and then amplifying this fragment by means of PCR as.described above in the case of the 74 nucleotide ribozyme. After amplification using the suitable primers and transcription by means of T7 RNA polymerase, a 71 nucleotide-long ribozyme structure which preserves its autocatalytic activity and functions at 37°C and at 600C is obtained (Figure 4).

The ribozyme was labeled at the 5' with gamma-ATP 32 P using T4 polynucleotide kinase and purified on polyacrylamide gel before use. It is denatured at for 1 minute in the presence of 1 mM EDTA and then cooled rapidly to 0°C on ice (snap-cool). The reaction medium is then modified by adding 40 mM Tris pH 8.0 and 10 mM MgCl 2 and subsequently incubated at 370C (2 to 7) or at 0 C (8 to 13). After various periods of time, aliquot ~~RA samples are taken: 1 minute (2 2 minutes (3 9), 5 minutes (4 to 10), 10 minutes (5 11), 20 minutes 4 8 (6 12) and 60 minutes (7 13). In the control the reaction was stopped after period t 0 minute before adding 10 mM MgC 2 The native ribozyme (89 nucleotides long) cleaves its RNA with tl/2 of 15 seconds, while the deleted ribozyme has a tl/2 of 3 minutes. The deletion therefore modifies the general structure of the ribozyme, which modifies the reaction kinetics but does not directly affect the catalytic site.

It is therefore possible to reduce the size of the functional ribozyme structure of Figure 2 by partially reducing the length of this loop 50-77.

r fi I_ -9- KEYS TO FIGURE 3: T1 Substrate only, incubated in the reaction medium for T2 Substrate incubated in the reaction medium in the presence of ribozyme HAV and in the presence of mM EDTA C Cleavage kinetics substrate incubated in the presence of ribozyme HAV M Size marker indicating the position of the expected cleaved band _L C-

Claims

1. A ribozyme structure derived from genomic RNA of the hepatitis delta virus consisting of one or more fragments of said genomic RNA of the hepatitis delta virus, capable of having a catalytic trans cleavage activity on another molecu'e containing a specific RNA nucleic acid sequence, wherein said ribozyme structure is composed of one or two fragment(s) from the sequence of the 74 nucleotides which contain the nucleotides from 16 to 89 shown in Figure 2.

2. A ribozyme structure as claimed in claim 1, which consists of the 74 nucleotide a:agment containing the nucleotides from 16 to 89, which are shown in Figure 2.

3. A ribozyme structure as claimed in claim 1, which consists of a fragment obtained from the 74 nucleotide fragment composed of nucleotides 16 to 89 in Figure 2, in which fragment part of the nucleotides 50 to 77 have been deleted.

4. A ribozyme structure as claimed in any one of claims 1 to 3, which contains a sequence of at least 12 nucleotides.

A ribozyme structure as claimed in any one of claims 1 to 4, wherein this nucleic acid sequence of this other molecule corresponds to a genomic RNA fragment of the hepatitis delta virus, which is a substrate of this catalytic trans cleavage activity.

6. A ribozyme struc.tre as claimed in claim 5, which is capable of having a S 20 catalytic trans cleavage activity on the phosphodiester bond between X and G where SX C or U of a nucleic acid sequence or RNA sequence, respectively, which contains S the sequence 5'XGGCC3'.

7. A ribozyme structure as claimed in claim 5 or claim 6, which is capable of having a catalytic cleavage activity on a nucleic acid sequence of this other molecule which contains the 8 nucleotide fragment which covers the nucleotides 5 to 12, which are shown in Figure 2.

8. A ribozyme structure as claimed in any one of claims 1 to 7, which is inserted into this other molecule and is capable of having an autocatalytic cleavage activity on this other molecule into which it is inserted.

9. A ribozyme structure as claimed in any one of claims 1 to 8, which is capable of having a ribonuclease, in particular endoribonuclease, activity on another molecule containing a polyribonucleotide. A ribozyme structure as claimed in any one of claims 1 to 9, which can be used for carrying out the a therapeutic or prophylactic treatment method in prokaryotic or eucaryotic, animal or plant cells. /VT: 0 [N:\LIBuu]00730:KEH I r 1 B 11 11, A ribozyme structure derived from genomic RNA of the hepatitis delta virus, said structure substantially as hereinbefore described with reference to any one of the Examples. Dated 16 April, 1996 Genset Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:\LIBuu]00730;KEH ,i