Oligoribonucleotide compound, process for preparation and method of inactivation

The present invention relates to an oligoribonucleotide compound having a high specific ribonuclear activity, to a process for preparing this compound, as well as to a method of inactivating the target RNA in a cell by using this compound.

A number of RNA molecules existing in nature are known, such as spotted avocado virus (ASBV), satellite RNAs from the tobacco ring spot virus (STObRV) and transient striated lucerne (sLTSV) virus. a self-catalyzed cleavage. Such cleavages appear as an essential and unique part of the life cycle of these or other RNA types.

All autocatalytic RNA cleavage reactions require the presence of bivalent metal ions and a neutral or alkaline pH and these result in RNAs with 5'-hydroxy and 2 ', 3'-cyclic phosphate terminal groups (as known). from the specialized literature). The reactions are catalyzed by RNAs themselves, probably as a result of the conformation that brings reactive groups in the immediate vicinity. The positions of the self-catalytic cleavage of naturally occurring RNAs are located within the most conserved regions of the secondary RNA structure (similarly known in the literature).

Experiments on satellite RNAs from the tobacco ring spot virus (sTobRV) have led to the designation of new endoribonucleases (hereinafter referred to as "ribozymes"), these being enzymes contained in RNA, which catalyze catalytic cleavage. of target RNA molecules.

The term ribozymes, as specified in the specification, refers to molecules comprising entirely of RNA or its derivatives.

Ribozymes, of the present invention, are different from endoribonuclear RNA that is normally found in Tetrahymena Thermophila (known as IVS, or L-19 IVS RNA) and has been described extensively by Thomas Cech et al. Endoribonuclease Cech has a byte of base pairs in an active site that hybridizes to a target RNA sequence, followed by cleavage of the target RNA, with a requirement for guanosine or free guanosine derivatives. The cleavage fragments contain 5-phosphate and 3'-hydroxyl terminal groups. The limited number of nucleotides available for hybridization to an RNA substrate limits the efficacy and efficiency of Cech endoribonuclease as well as oligonucleotides generally containing less than twelve nucleotides that hybridize to a small extent to the target sequences. It also appears that the Cech endoribonuclease active site is required to be conserved with a number of nucleotides for the efficiency of endoribonuclease activity. This restricts the number of permutations of the active site sequences that can be directed towards hybridization to the target sequences, thus restricting the series of target RNA sequences cleavable by the Cech endoribonuclease. Endoribonuclease Cech also modifies the RNA by adding a free nucleotide guanosine to the 5 'end position of the cleaved RNA.

The oligoribonucleotide compound of the present invention has general formula I: ³' ^(X> n A. ^{X (X)}' ⁵' \

I TAKE

G

<img file="RO114469B1_D0001.tif" />

X. ^x (X)_b(I) wherein: each X represents a ribonucleotide which may be the same or different; where each (X)_n and (X)_n. represents an oligoribonucleotide (a) capable of hybridization with a target RNA cleavage sequence and (b) defined by a predetermined sequence, which does not naturally covalently bind to AAAGC sequences and, respectively,

RO 114469 Blivu, XCUGA-, such a target RNA sequence is not present in the compound; wherein each of n and n 'represents an integer defining the number of ribonucleotides in the oligonucleotide provided that the sum of n + n's is sufficient to allow the compound to interact stably with the target RNA sequence through base pairing; wherein each * represents the base pairing between the ribonucleotides located on each side thereof; in each continuous line represents a chemical bond that creates covalent bonds between the ribonucleotides located on any part thereof; wherein a represents an integer defining a number of the ribonucleotides provided that it can be O or 1 and if it is O, then A at position 5 'relative to (X)_ofis connected to G in position 3 'relative to (X)_of; wherein each of m and m 'represents an integer greater than or equal to 1; wherein each of the dotted lines independently represents either a chemical bond making covalent bonds between the ribonucleotides disposed on each side thereof, or the absence of such chemical bond; and in which (X)_brepresents an oligoribonucleotide, which may be present or absent, provided that b represents an integer greater than or equal to 2 if (X)_b its present.

The oligoribonucleotide compound of the present invention may also have general formula II;

<td> 3 &lt;^X) ~ NT^THAT\</td><td>x— (X)_n· ⁵</td>

<td>A</td><td>c</td>

<td>A</td><td> \</td>

<td>r \</td><td>^U\</td>

<td>G 1</td><td>^G\ 1 1</td>

<td>C 1</td><td>^g -4 i *</td>

<td>X</td><td></td>

<td>1 X I</td><td>X |</td>

<td>(X)_m * 'lm</td><td>(*&gt; M ·</td>

<td>X</td><td>X</td>

<td>(X)_b</td><td>(II)</td>

<td colspan="2">wherein: each X represents a ribbon-</td>

<td>cleotide that can</td><td>be the same or different</td>

<td>acidifying; in which each</td><td>(Xk, and (X)_n., repre</td>

target an oligrabonucleotide (a) capable of hybridization with a target RNA cleavage sequence and (b) defined by a predetermined sequence, which sequence cannot naturally form a covalent bond to the CAAAGC and XC-UG-A- sequences, respectively such a target RNA sequence is not present in the compound; wherein each of n and n 'represents an integer defining the number of ribonucleotides in the oligonucleotide provided that the sum of n + n' is sufficient to allow the compound to interact stably with the target RNA sequence through base pairing; wherein each * represents the base pairing between the ribonucleotides located on each side thereof; wherein each continuous line represents a chemical bond that creates covalent bonds between the ribonucleotides located on any part thereof; wherein a represents an integer defining a number of the ribonucleotides provided that it can be O or 1 and if it is O, then A in position 5 'relative to (X)_ofis connected to G in position 3 'relative to (X)_of; wherein each of m and m 'represents an integer greater than or equal to 1; wherein each of the dotted lines independently represents either a chemical bond, which makes covalent bonds between the ribonucleotides disposed of any part thereof, or the absence of such a chemical bond; and where [X]_torepresents an oligoribonucleotide, which may be present or absent, provided that, b represents an integer greater than or equal to 2 if (X)_b its present.

Also, the oligoribonucleotide compound of the present invention may also have general formula III:

<td> 3 &lt;^X&gt; N-^THAT\ A I</td><td>x- \ c \</td><td>- (X)_n·</td>

<td>1 A.</td><td></td><td>\ G.</td>

<td>G 1</td><td>A.</td><td>\ μ</td>

<td> 1</td><td> /</td><td rowspan="2"></td>

<td>c</td><td>G</td>

<td> 1</td><td> 1</td><td>X</td>

<td>X</td><td></td><td></td>

<td>1 X and</td><td>X</td><td></td>

<td>(X) ^ * \ 'm</td><td> &lt;&gt;%</td><td></td>

<td>it X</td><td>X</td><td></td>

&lt;^x&gt; b (III)

Wherein each X represents a ribonucleotide which may be the same or different; wherein each (X)_n_₇ and (X)_n. . represents an oligoribonucleotide (a) capable of hybridization with a target RNA cleavage sequence and (b) defined by a predetermined sequence which sequence cannot naturally form a covalent bond to the CAAAGC and XCUgA- sequences, respectively; such a target RNA sequence is not present in the compound; wherein each of n and n 'represents an integer, which defines the number of ribonucleotides in the oligonucleotide provided that the sum of n + n's is sufficient to allow the compound to interact stably with the target RNA sequence through base pairing; wherein each * represents the base pairing between the ribonucleotides located on each side thereof; in each continuous line represents a chemical bond that creates covalent bonds between the ribonucleotides located on any part thereof; wherein each of m and m 'represents an integer, which is greater than or equal to 1; wherein each of the dotted lines independently represents either a chemical bond making covalent bonds between the ribonucleotides disposed on each side thereof, or the absence of such chemical bond; and in which (X)_fa represents an oligoribonucleotide, which may be present or absent, provided that b represents an integer greater than or equal to 2, if (X)_fa its present.

The sum n + n 'is greater than or equal to 14 and each n and n' is greater than 6. The target RNA cleavage sequence is a viral sequence.

The process for obtaining the oligoribonucleotide compound according to the invention comprises the following steps: a] binding to a transfer vector comprising DNA, RNA or a combination thereof of a nucleotide sequence corresponding to the respective compound; b] transcription of the nucleotide sequence of step (a) with an RNA polymerase; and c) recovery of the compound.

The method of inactivating the target RNA in a cell, according to the present invention, comprises contacting the target RNA in the cell with the compound shown above, the compound being able to interact by base pairing with the target RNA sequence under such conditions that the compound interacts stable by pairing bases with target RNA it is cleaved.

Target RNA is a transcription of a gene that is endogenous to the cell or exogenous to the cell. The cell is prokaryotic or eukaryotic and is a plant or animal cell. The plant cell is a component of a plant and the compound is formed inside the cell or outside the cell.

By contrast, the ribozymes of the present invention efficiently hybridize to a wide variety of target RNA sequences, and do not alter the cleaved RNA target.

The ribozymes of the present invention contain a hybridization portion that is complementary to the nucleotide sequence, with at least a portion of the RNA target and a catalytic area, which is adapted to cleave the RNA target. The hybridization zone contains 9 or more nucleotides.

Preferably, the ribozymes of the present invention have a hybridization region comprising one or more arms consisting of a single RNA strand and having a complementary sequence at least part of the target RNA, said arm or arms being associated with an area. catalytic capable of cleaving said target, and where the hybridization region comprises a single RNA arm, said arm contains at least 9 nucleotides, and where the hybridization region comprises two or more RNA arms, the total number of nucleotides in the respective arms is greater than 9 nucleotides.

Ribozymes of the present invention can be prepared by methods well known in the art of RNA molecule synthesis. In particular, the ribozymes of this invention may be prepared from an appropriate RNA sequence (DNA which by transcription results in a ribbon

And which can be synthesized, according to well known methods, in the technique of DNA synthesis) chemically bound to RNA polymerase, such as, for example, a promoter for T7 RNA polymerase or SP6 RNA polymerase. A DNA sequence, corresponding to a ribozyme, of the present invention, can be ligated from a DNA transfer vector, such as plasmid DNA or bacteriophage DNA. Where the transfer vector contains an RNA polymerase promoter bound to the DNA corresponding to a ribozyme, the ribozyme may conveniently be obtained by incubation with an RNA polymerase. Ribozymes can therefore be obtained, in vitro, by incubating RNA polymerase with a DNA polymerase promoter bound to the DNA corresponding to a ribozyme, in the presence of ribonucleotides. In vivo, prokaryotic or eukaryotic cells (including mammalian cells and plant cells) may be transfected with a suitable transfer vector containing genetic material corresponding to a ribozyme in accordance with the present invention, linked to an RNA polymerase promoter such that the ribozyme is transcribed into the host cell. The transfer vectors can be: bacterial plasmids or RNA or viral DNA. Nucleotide sequences corresponding to ribozymes are generally placed under the control of strong promoters such as, for example, impure, delayed SV40, early SV40, metallothionein, or λ promoter. Ribozymes can be directly transcribed in vivo, from a transfer vector, or by choice can be transcribed as part of a larger RNA molecule. For example, DNA corresponding to the ribozyme sequence can be linked to the 3 'end of a carrier gene such as, for example, after a stop-signal translation and larger RNA molecules can help stabilize the ribozyme molecules against nuclease digestion within cells. At translation, carrier genes can cause a protein to grow, whose presence can be directly analyzed, for example, by the enzymatic reaction. The carrier gene may, for example, encode an enzyme.

Additionally, the invention provides a DNA transfer vector, which contains a DNA sequence corresponding to a promoter-linked ribozyme to prevent transcription of the ribozyme.

In a preferred method, to produce a ribozyme, two synthetic oligonucleotides of the complementary sequence are prepared by standard processes (which are known in the literature), and hybridize together. One of the oligonucleotides encodes a desired ribozyme. The respective ends of the hybridized oligonucleotides correspond to different enzymatic restrictive sites, either EcoR1, at one end, and Pst1, at the other end. After cleavage with the appropriate restrictive enzymes (EcoR1 and Pst1 in the example above), the double stranded DNA fragment can be cloned into a transfer vector. Where the plasmid vector contains an RNA polymerase promoter, derived from the DNA sequence corresponding to a ribozyme of the present invention, the RNA transcribed corresponding to a ribozyme may be convenient either in vitro or in vivo. Where the ribozyme contains two halves retained together by complementary nucleotide base pairing, each half of the ribozyme can be obtained, according to the above methods, and the halves incubated together form the ribozyme.

Preferred ribozymes of the present invention cleave the target RNA, which contains the sequence X ° UY where X ° is any ribonucleotide, U is uracil and Y is adenine, cytosine or uracil. X ° U forms part of a base pair of the flanking area, and Y is not paired with the base. Preferably, but by no means exclusive, X ° is guanidine and X ° UY is GUC or GUA. Any RNA molecule containing these sequences can be cleaved with the ribozymes of the present invention. 0 given the sequence of a transcribed RNA containing the X ° UY sequence being determined, the arms of the ribozyme sequence can be synthesized so as to complement them, and thus produce hybridization, the RNA in the target sequence flanking the X ° UY sequence. When hybridizing the arms of the ribozyme, the target RNA sequence is flanked

When X ° UY, the catalytic region of the ribozyme cleaves the target RNA within the X ° UY sequence. RNA cleavage is facilitated in the presence of magnesium or other bivalent cations, at pH about 8.0.

Thus, the preferential ribozymes of the present invention may be designed to cleave any RNA whose sequence is known. the high frequency of ribozyme-cleaved residues in RNA (1:64 for GUC in an RNA, with coincidence and equal frequency of base distribution) means that a potential number of ribozyme cleavage sites can be predicted in any target RNA. given.

According to another aspect of the present invention, there is provided a method for inactivating a target RNA sequence that includes the reaction of said RNA target with a ribozyme of the present invention.

In vivo, that is, within a cell or cells of an organism, a transfer vector, such as a vector plasmid or viral RNA, encoding one or more ribozymes, can be transferred into cells for example (a phenomenon that is known from the literature). Once inside the cell, the transfer vector can replicate, and be transcribed by cellular polymerases to produce ribozyme RNAs which then inactivates a desired target RNA.

Upon choice, a transfer vector containing one or more ribozyme sequences can be transferred into cells or introduced into cells by means of technical micromanipulation, such as microinjection, so that the transfer vector or part thereof integrates. in the genome of the host cell. Transcription of the integrated genetic material results in the development of ribozymes, which act by inactivating a specific RNA target.

Ribozimers of the present invention have extensive therapeutic and biological applications. For example, viruses that cause disease in humans and animals can be inactivated by administering to a subject infected with a virus, a ribozyme, according to the present invention, adapted to hybridize and cleave the transcribed RNA of a virus. Such ribozymes can be hybridized by parenteral administration or other means of administration. To a subject infected with a disease-causing virus, a non-virulent virus such as vaccine or adenovirus, which has been prepared so as to contain DNA corresponding to an RNA promoter-linked ribozyme, may be selected. , that ribozyme is transcribed into the host animal's cells, transfected with the prepared virus, to cleave and / or inactivate the transcribed target RNA of the virus causing the disease. Ribozymes of the present invention have application, in particular, to viral diseases caused by, for example, the Herpes simplex virus (HSV) or the AIDS virus (HIV).

Ribozymes of the present invention are also of particular importance in the inactivation of transcribed RNA in bacteria and other prokaryotic cells, plants and animals. In bacteria, the transcribed RNA, for example, bacteriophage, which causes bacterial cell death, can be inactivated by transfecting a cell with a DNA transfer vector, which is capable of producing a ribozyme, according to the present invention, and which inactivates DNA. beech. In another embodiment, the ribozyme itself may be added and absorbed by the bacterial cell to perform RNA cleavage.

RNA transcribed into plants can be inactivated using ribozymes encoded by a vector like Ti plasmid from Agrobacterium tumefaciens. When such vectors are transfected into a plant cell, the ribozymes are produced by the action of RNA polymerase and can lead to cleavage of a specific target RNA sequence. Similarly plant viruses whose RNA sequences are known, or the transcribed RNA of plant genes, can be inactivated using ribozymes.

Endogenous genes transcribed in plants, animals or other cell types can be inactivated using the ribozymes of the present invention. Similarly, undesirable or characteristic phenotypes may be mutated

RO 114469 Bl dulate. It may be possible, for example, by using the ribozymes of the present invention to remove pips from fruit or to treat human hereditary diseases caused by the production of a pernicious protein, or the overproduction of a particular protein.

The following are examples of embodiments of the invention in connection with FIG. 1 ... 17, which represents:

FIG. 1, presents the self-cleavage sites of wild-type RNA and of mutation RNAs and a representation of an electrophoretic profile of the RNA self-catalytic cleavage products:

(a). Includes conserved structures associated with cleavage sites of natural RNA in ASBV, triton satellite transcribed DNA, and satellite RNAs of sTobRV, LTSV, Solanum nodiflorum, virus, tobacco, and underground clover virus. The nucleotide sequences that are conserved between these structures are represented, while others are represented by X. The base pairing is represented by and the RNA cleavage site is indicated by an arrow.

(b). It represents conserved nucleotide sequences associated with sTobRV RNA (+) chain cleavage. The cleavage site is indicated by an arrow.

(c). An in vitro mutant of sTobRV containing an insertion of eight nucleotides (shown as a circle) together with a flanking duplication of three nucleotides (UGU with residues 7 and 9] is shown (d). Haelll sub-cloned fragments of type wild-type sTobRV and mutant D-51 in vitro were each transcribed in both (+) and (-) orientations and fractionated by radiolabeled transcription and polyacrylamide gel electrophoresis. The positions of the unpaired bases 159 and 170 transcribed from the wild type (WT) and mutant (D-51) sequences are indicated by an arrow and the quantities of cleaved products are highlighted.

FIG. 2, represents the nucleotide sequence of a ribozyme and the splitting products of the ribozyme separated by gel electrophoresis:

(a). The nucleotides inserted into the D-51 mutant (Fig. 1c) contain a BamHI restriction endonuclease site. BamHI was used to cleave the mutant DNA and the two sequences were subcloned and transcribed separately in vitro, the transcribed RNA schematically represented with the potentially unmatched bases between the indicated RNAs, with the fragment containing the cleavage site shown with an arrow is designated, as SARN, fragments containing the ribozyme is designated as Rz-RNA.

(b). (³²P) -Rz-RNA (101 bases) was incubated alone (column 1), and with unlabeled SRNA (column 2).³²P) -S-RNA was incubated alone (column 3), and with unmarked or labeled Rz-RNAs, with ³²P (columns 4 and 5, respectively).

FIG. 3, schematically represents a model of ribozyme, according to one of the objects of the present invention. Zone A represents the cleavage sequence within the target RNA. Zone B represents the catalytic zone, and zone C represents the arms of the ribozyme.

FIG. 4, shows the scheme of the ribozymes targeted against the CAT (chloramphenicol acetyl transferase) gene. Ribozymes, termed RzCAT-1,2 and 3, were targeted to three sites of the CAT gene, from a base 835 transcribed in vitro. The relative locations of the cleavage sites on the transcript are schematically represented with the bases numbered on the flanks (a). The three ribozyme sequences are represented (from (b) to (d)) with their target sequences. The amino acid sequences of the CAT gene are numbered and the predicted sites for RNA cleavage are indicated by the arrow. RzCAT-1 and 3 contain 24 base sequences derived from the (+) chain of sTobRV (region B, fig. 3), whereas RzCAT-2 contains a single AU change in this area.

FIG. 5, shows the CAT RNA cleavage result with RzCAT-1 ribozymes at 3.

(A). RNAs [³²P] -CAT were fractionated on gel after incubation, only (-) or with one of the three ribozymes,

RO 114469 Bl

RzCAT-1 at 3 (columns 1,2 and 3 respectively). The location of the entire transcript is indicated by the arrow.

(B). 5 'terminal base analysis. The 3 'fragments produced by CAT mRNA ribozyme cleavage were [5'-³²P] kinases, purified on gel, subjected to complete digestion of nucleases, and the terminal residues released were fractionated by electrophoresis on polyacrylamide gel, at pH = 3.5. The 5 'terminal nucleotides, determined by reference to markers (column M), where A, U and G are from fragments produced by RzCAT, from 1 to 3 ( columns 1, 2 and 3, respectively).

FIG. 6, the catalytic activity curve of the RzCAT-1 ribozyme on CAT RNA is represented. The quantities of cleavage products of nucleotide 139 were quantitatively established and plotted. Further representation shows accumulation of base fragment 139 over time after polyacrylamide gel electrophoresis.

FIG. 7, shows the relative cleavage rate of CAT RNA under different temperature conditions. The RNA substrate is represented by a solid line. In each case, the splitting product is represented by a dotted line.

FIG. 8, represents three ribozymes (correspond to RzCAT-2) having flank arms or sequences of different sizes.

FIG. 9, shows the scheme for obtaining a ribozyme comprising catalytic antisense RNA, containing in each RzCAT cleavage domain, from 1 to 3.

FIG. 10, shows ribozyme hybridization to target sequences containing GUA (10a) and GUU (10b) motifs in CAT mRNA.

FIG. 11, shows the sites for autocatalytic cleavage of RNA from Citrus exocortis virus (CEV) RNA and its complement.

FIG. 12, represents the RzCEV25x (+) ribozyme hybridized to the target CEV RNA (a), and an image of gel electrophoresis of (+) CEV RNA and (-) CEV complementary RNA, incubated with RzCEV25x (+) (b, columns 1 and respectively 2. The cleavage product is indicated by an arrow).

FIG. 13 shows RzCAT ribozyme hybridizing to its target sequence (a) and RzSCMoV ribozyme (b). The catalytic domain of each ribozyme is encircled. There are also marked differences between the catalytic zones of RzSCMoV, compared to RzCAT-2.

FIG. 14, shows RzCEV2 ribozyme hybridizing to a target sequence of Citrus exocortis virus (CEV) RNA. The cleavage site corresponds to nucleotide-336 in the CEV RNA sequence. Alteration to the nucleotide sequence in the catalytic domain of sTobRV is circumscribed (a). Fig. 14 (b) shows an electrophoretic image of a control RNA (chain (-) of CEV) RNA, column 7 and chain (+) of CEV RNA column 8, after incubation with RzCEV2.

FIG. 15 shows RzCAT2 ribozyme (a) compared to RzCAT-2B ribozyme (b). The catalytic domains are encircled. Changes in the catalytic domain of RzCAT-2B compared to RzCAT-2 are also circumscribed.

FIG. 16 is a schematic of plasmid pJ35SN.

FIG. 17, is a graphical presentation of an average of four experiments on the inhibition of CAT expression in plants (tobacco protoplasts).

In the following embodiments, reactions and manipulations involving DNA, such as ligation, reduction of enzyme digestion, bacterial transformation, DNA sequencing, etc., have been conducted using standard techniques, such as those described in the speciation literature (e.g., Maniatis et al. .). RNA manipulations were also conducted according to standard techniques as described by Uhlenbeck.

Example 1. Self-catalytic cleavage of mutation sTobRV RNA

A consensus of the domains associated with cleavage of naturally occurring RNA sites in ASBV, triton-transcribed DNA, and satellite RNAs of sTobRV, LTSV, velvety tobacco virus (VMoV), Solanum nodiflorum (SNMV) and viruses underground clover paint (SCMoV) is as shown in fig. 1a. The nucleo sequences are presented

EN 114469 Bl tides, which are conserved between these structures, while non-conserved sequences are represented by X. An additional U is positioned after the residue. ¹In the LTSV (+) chain.

The domain associated with the self-catalyzed cleavage of the (+) chain of sTobRV has been studied to highlight the enzymatic activity of the substrate in this domain. First, cloned sTobRV cDNAs were subjected to mutagenesis using an oligonucleotide insertion-binding linker (BamH1).

Construction of a vector for sTobRV expression in vitro

A 160 Taq 1-Spe 1 fragment of sTobRV cDNA was isolated from pSP653 (according to known methods in the literature) and ligated to the phosphatase-treated Acc 1Spe 1 residue, pGEM 4 to reform the Acc 1 site. A resulting clone was linearized with Acc 1 treated with phosphatase and a 359 bp Taq 1 fragment, from sTobRV cDNA was linearized. The resulting clones were protected by the presence of a circularly permuted 520 bp sTobRV cDNA sequence, containing excessive terminal residues, from 277 to 81 (pTTS). The sTobRV sequence is flanked by promoters for T7 and SP6 RNA polymerases, and transcription, results in an increase of (+) or (-) targeting RNA containing two sites for self-cleavage.

In vitro mutagenesis

The plasmid pTTS (50 µg) was linearized with BamH1, treated with S1 nuclease, and cultured to remove a single BamH1 site. The resulting compound, pTTSB, was treated with 2 x 1 O⁴ units of DNase 1, in 20 mM Tris-HCl, pH = 7 and 0.15 mM MgCl₂, for a period of 10 minutes and at a temperature of 37 ° C. The resulting linear DNAs were oriented and / or blocked at the end, using T₄ DNA polymerase and were purified by 0.7% gel electrophoresis of LGT agarose and extraction. BamHI kinase linker sequences (CGGATCCG) were ligated to linearize the plasmid overnight at room temperature in the presence of 5% polyethylene glycol. Then, the reactions were assimilated by BamHI, and the linear plasmid DNAs were repurified by 0.7% gel electrophoresis of LGT agarose (this was found to be necessary to remove the last traces of the circular plasmid together with the unbound linkers). Plasmids were recirculated using T₄ DNA ligase and transformed into E.coli DH-1. Colonies (larger than 1000) were scraped from agar plates, grown in liquid culture until saturation, and mixed populations of plasmid DNAs were prepared. The mixture of sTobRV cDNA insertions was excised by restriction of the assimilation enzyme to flanking EcoR1 and Pst1 sites, purified by 1% gel electrophoresis of LGT agarose and sub-cloned at the EcoR1Pst1 end, phosphate-treated pGEM-4. The resultants were again combined, raised in liquid culture and plasmid DNA was prepared. Plasmid DNAs were treated with BamHI, to cleave only those plasmids containing a BamHI linker sequence and the linear forms were again purified by electrophoresis in two rows on 0.7% agarose AGT gel, recirculated with T₄DNA ligase and transformation into E.coli DH-1. The individual transformants were protected for the approximate position of the BamHI linker inserted in the sTobRV sequence, by the restriction of the assimilation enzyme, sub-cloned to M13 mp 19 and sequenced by the dideoxynucleotide terminal chain technique.

A plurality of sTobRV mutants resulted and nucleotide sequence analysis showed that each mutant contained a BamHI linker sequence (CGGATCCG] inserted together with flanking or deletion of sTobRV sequences. Mutants were transcribed in vitro and RNAs were assayed. In terms of the ability to undergo cleavage, a 52-nucleotide sequence containing both substrate and cleavage portions of sTobRV RNA was identified from this experiment. This nucleotide sequence 52, shown in FIG. 1b, contained the conserved sequence domain required for self-cleavage of other RNAs (fig. 1a). One mutant, designated D-51, contained an eight-nucleotide sequence

B 114469 BamH1 linker block inserted between three duplicate sTobRV nucleotides numbered 7 to 9. This mutant underwent auto-catalytic cleavage of RNA.

The base pairs 97 and 108 of the Haelll fragments containing the wild-type 52-nucleotide cleavage sequence and the D-51 RNAs (as shown in Figs. 1b and 1c) were excised from the sequenced plasmid clones. Fragments were ligated to the Smal site of pGEM4 and protected to obtain both insertion orientations. Plasmids were linearized using EcoR1 and the (+) and (-) strands of RNAs with base fragments 159 and 170 were transcribed using 200 units / ml of T7 RNA polymerase at pH = 7.5, in 50 mM Tris- HCl, 10 mM NaCl, 6 mM MgCI₂, 2mM spermidine, 1000 units / ml RNazine, 500 pM ATP, CTP and GTP with 200 pM (a³²P) UTP. The RNAs were fractionated by electrophoresis on 10% polyacrylamide, 7 molar urea, 25% formamide gel and autoradiographed.

As shown in FIG. 1 d, cleavage (-) of the transcribed RNA was not observed. This was to be expected since the (-) chain does not contain a self-catalytic cleavage site. In the (+) chains of both the wild type and of the D-51 sequences, the cleavage took place, and the cleavage of the D-51 RNA was somewhat less efficient than that of the wild type (fig. 1d). This experiment indicates that the region with a single-stranded loop, on the right-hand side of the 52-nucleotide sequence, at the self-catalytic cleavage of RNA, is not essential.

Separation of enzymatic and substrate activities

Using the restriction BamH1 endonuclease site inserted into D-51, HaelllBamH1 and BamH1 -Haelll flank fragments were obtained and each was sub-cloned into an E. coli palsmid suitable for in vitro transcription. This resulted in the removal of the single-stranded loop moved from the self-cleavage domain, splitting the region into two RNA segments (Fig. 2a). The smaller Haelll-BamH1 fragment contained nucleosides from 321 to 9, including the current cleavage site, and was named the S fragment. The BamH1 -Haelll fragment containing nucleotides from 7 to 48 of sTobRV was termed a ribozyme or Rz fragment. E.coli plasmids used for in vivo transcription were pGEM4 and pGEM3 (as known in the literature). These expression plasmids contain:

(a), an origin of replication;

(b). selectable gene for drug resistance (Amp^r);

(c). a multiple cloning site flanked by RNA polymerase promoters that can be used to obtain in vitro transcription.

T7 DNA polymerase treated, Kpn1-digested RzpGEM3 and Xbal-digested S-pGEM4 were transcribed using SP6 RNA polymerase under the same conditions as described above. As shown in FIG. 2, both S. and Rz-RNAs did not show significant degradation when incubated alone (Fig. 2b, columns 1 and 3) under conditions suitable for high-efficiency self-cleavage (50 ° C, 20 mM MgCl₂, pH = 8.0). The labeled Rz-RNA also appears unaltered after incubation with S-RNA (Fig. 2b, columns 2 and 5). However, when S-RNA was mixed with Rz-RNA, efficient cleavage of S-RNA occurred (Fig. 2b, columns 4 and 5) resulting in two fragments. The product dimensions were coordinated with the S-RNA stranding (84 bases) at the normal site between nucleotides # 359 and # 1, to give fragments adjacent to positions 5 'and 3' of nucleotides 67 and 17, respectively. This shows that SARN acts as a substrate for ribonucleotide cleavage with Rz-RNA, which acts catalytically.

A model of a ribozyme based on the catalytic region of sTobRV RNA is shown in FIG. 3. Ribozyme has two arms or flanking sequences, of single-stranded RNA, presented at C, hybridizing to complementary sequences on an RNA substrate, and the RNA is cleaved. Each flanking sequence presented at C contains 8 ribonucleotides. Number of ri

EN 114469 The block of bonucleotides contained in region C is not critical. However, sufficient nucleotides must be present to allow ribozyme to hybridize to an RNA target. It turns out that four nucleotides in each C region need to be present, at least, for hybridization.

The catalytic region B contains sequences that are very good in the areas where the spinning occurs naturally (spontaneously) (see fig. 1a). From the comparison of the cleavage domains of the known sequences, it appears that the base pair portion of II is not important, as is the presence of a loop associated with one of its ends.

The cleavage site within the RNA target is represented at A (in Fig. 3) as GUC. Based on our experiments (not shown), and others in the specialized field, on cleavage sites in naturally occurring RNAs, it was found that the GUA, GUC, CUC, AUC and UUC sequences also act as cleavage sites, within RNA.

Example 2. Demonstration of the design, synthesis and activity of ribosomes, with a new and high endoribonuclear activity

As an illustration of this invention, three ribozymes have been designed, which are targeted against the transcript of a commonly used indicator gene, derived from bacteria, Tn9 Chloramphenicol Acetyl Transferase (CAT), which can develop resistance to bacteria, plants and animals and can be easily tested. These ribozymes, designated Rz-CAT from 1 to 3, correspond to the potential cleavage of GUC sites in CAT RNA at positions 139-140, 494-495 and 662-663, respectively. The sequences of these ribozymes are shown in FIG. 4. In each case, the flank sequences that hybridize the target CAT RNA contained 8 nucleotides. The catalytic zone was chosen so as to correspond to that of sTobRV RNA, shown in FIG. 3.

The CAT gene that was obtained from pCM4 was subcloned as a BamH1 fragment into pGEM-32. This plasmid was linearized with Hind III and transcripts of the CAT gene were obtained using T7 RNA polymerase with 220 μΜ (a³²P) UTP. Ribozyme sequences were synthesized as oligodezoxynucleotides, Rz CAT1,2 and 3. These were kinase, ligated with an EcoRI-Pstl portion of pGEM4 treated with phosphatase and incubated with the Klenow fragment of DNA polymerase 1, before bacterial transformation. The linearized EcoRI plasmids were transcribed with T7 RNA polymerase to produce ribozymic RNAs. Ribozymes were incubated with CAT transcript, in mM Tris-HCl, pH = 8.0, 20 mM MgCl₂, at 50 ° C, for 60 min, and the products were fractionated by gel electrophoresis containing 5% polyacrylamide, 7 M urea, 25% formamide before autoradiography.

When the CAT 840-nucleotide transcript was incubated with any of the three ribozymes, efficient, high sequence specificity cleavage occurred (Fig. 5), resulting in two RNA fragments from each reaction. The fragment sizes were compatible with the sites. predicted for cleavage (base fragments 139 and 696, 494 and 341, 662 and 173 were the products of catalytic cleavage 5 'and 3' of RzCAT-1,2 and 3, respectively). The conditions required for catalytic cleavage with ribozymes were similar to those observed for cleavage reactions, which occur naturally (as known in the literature), with more efficient cleavage occurring, at high pHs, temperature and concentrations. bivalent cation (no data are presented). When present in molar excess, the three ribozymes catalyze the almost complete cleavage of CAT RNA substrate, after 60 min, in 50 mM Tris-HCl, pH = 8.0, 20 mM MgCl₂, the temperature of 50 ° C. under similar conditions, with 0.1 pM substrate and 3 μΜ ribozymes, T_1/2 of the CAT mRNA substrate were 3.5 and 2.5 min, in the presence of RzCAT1,2 and, respectively, 3. The ribozyme sequences were inactive compared to the complement of the RNA substrate (chain (+)), and in

EN 114469 Bl oligodeoxyribonucleotide (data not shown). The 3 'cleavage terminal fragments of each ribozyme-catalyzed reaction were isolated and the 5' kinase kinase position was isolated.³²P (50 mM Tris-HCl, pH = 9, 10 mM MgCI₂, 10 mM DTT, with uCi γ³²-Ρ ATP and 5 units T4 polynucleotide kinase, for 30 min, at 37 ° C). By efficient kinase of the fragments, it is indicated that they possess 5 'terminal clusters, similar to those resulting from cleavage, which occurs naturally.

The nucleotides of the fragments obtained by cleavage of the CAT sequences by RzCAT were determined from 1 to 3. Briefly, the radiolabelled fragments were purified on a 5% polyacrylamide gel and subjected to digestion, with an equal volume of 500 units / ml RNase T1, 25 units / ml RNase T2 and 0.125 mg / ml RNase, in 50 mM ammonium acetate pH = 4.5, for 120 min, at 37 ° C. The products were fractionated on a 20% polyacrylamide gel containing 25 mM sodium citrate, pH = 3.5 and 7 molar urea. Fig. 5b shows that the cleavage of the CAT sequences by RzCAT-1 to 3 occurs precisely in front of nucleotides A, U and G. respectively.

The terminal sequences of the CAT gene fragments were determined directly using the technique of partial enzymatic assimilation (as known in the literature), using specific splitting on a partially, nucleotide basis. The fragment sequence confirms that the cleavage occurs at the expected locations within CAT RNA (not shown).

Enzyme catalysis

To demonstrate that ribozymes cause cleavage of the CAT mRNA substrate in a catalytic manner, each was incubated with a molar excess of substrate, under conditions that would favor both cleavage and product dissociation.

Fig. 6 shows the results of an experiment, where, after 75 min, at 50 ° C, pH = 8.0, in 20 mM MgCl₂, 10 pmol of RzCAT-1 catalyzed the specific cleavage of a truncated substrate of 163 pmol CAT mRNA (173 bases) to give 5 'and 3' fragments of bases 139 and 34, respectively. On average, each ribozyme participated in more than ten cleavage reactions. After 75 min, a non-specific RNA cleavage ratio was observed at 50 ° C due to extreme conditions, but 70% of the remaining intact RNAs (163 mol) accumulated as the base fragment 139. Similar results were obtained for RzCAT-2 and 3 (no data are presented), and thus it turns out that each acts as an RNA enzyme.

Example 3. Effect of temperature on ribozyme activity

The effect of temperature on the speed of ribozyme activity in vitro was examined.

Reactions were monitored for RzCAT-1, 2 and 3, respectively, substrates at 37 ° C and 50 ° C.

In this experiment, the reactions for each ribozyme were performed in duplicate, using the reaction conditions of Example 2. One sample was incubated at 37 ° C, the other at 50 ° C. The samples at the reaction time, up to 90 min and the reaction progression were analyzed by denatured polyacrylamide gel electrophoresis. Fig. 7 shows the reaction progress for each of the ribozymes, from 1 to 3, at 37 ° C and 50 ° C. The reaction rate of each ribozyme increases with increasing reaction temperature.

Reaction time required for 50% cleavage (t_1/2) from CAT RNA is presented in table 1:

RO 114469 Bl

Table 1

<td>Temperature, ° C</td><td>RzCAT-1</td><td>RzCAT-2</td><td>RzCAT-3 _{/ P}(Minutes)</td>

<td> 50</td><td> 3,5</td><td> 3,5</td><td> 2,5</td>

<td> 37</td><td> 55,0</td><td> 70,0</td><td> 65,0</td>

As shown in Table 1, the reaction rate of ribozymes at 37 ° C is approximately 20 times lower than the reaction rate at 50 ° C.

Example 4. Effect of long variation - 15 thousand ribozyme arms (or flank sequence) on the catalytic activity of ribozyme

The arms or flanking sequences of a ribozyme hybridize the ribozyme to 20 RNA targets, followed by RNA cleavage. In this experiment, the effect on the cleavage rate of a target sequence, obtained by altering the complementarity dimensions 25 and, therefore, the length of the mating bases, of the arms of the ribozyme, to the target sequence was investigated.

Ribozymes were produced with 4, 8 and 12 complementary bases of sequence 3, one target RzCAT-2, on each arm (Fig. 8a). Ribozymes were prepared according to the methods of Example 2. The activity of ribozyme was determined by incubating the RNA ribozyme with CAT RNA, as described above.

The ribozyme having a complementation of 4 bases, on each arm, does not cleave the RNA substrate. The ribozyme having a complementation of 8 bases on 40 each arm split the CAT substrate, as well as the ribozyme having a complementation of 12 bases. Ribozymes with a complement of 12 bases have more efficiently cleaved the RNA target, as can be judged by the in vitro reaction rate, than ribozymes with a smaller number of complement bases. Therefore, it appears necessary to have a complementarity greater than four bases, increasing the size of the ribozyme hybridization zone 50 as their reaction rate increases.

In a second experiment, the efficiency of the reaction of a ribozyme having complementarity (a) to the full length of the target transcribed CAT RNA and multiple catalytic domains (b) was investigated.

Four GUC target sites, in CAT RNA sequences, were chosen. The catalytic domains of ribozyme against these sites were "inserted" in a complete anti-sense (-] sequence for the CAT transcript and catalytic activity was tested.

The four sites chosen were the three designated by RzCAT-1, 2 and 3, described above and an additional site, which can be described as follows:

Our CAT Site # 192

His His Ala Val C ys Asp Gly

5' 3’

CAU CAU GCC GUC UGU GAU GGC wherein: “192 refers to amino acid 192 of the CAT polypeptide and refers to the cleavage site.

Oligodeoxyribonucleotides containing catalytic domains of the ribozyme and rotations of each of these cleavage sites were used for M13 mutagenesis experiments to produce a sequence containing the full complement of the CAT sequence, but with the four catalytic domains of the ribozyme itself. M13 mutagenesis was performed by binding of oligonucleotides containing ribozyme insertions to single-stranded DNAs, containing uracil, followed by complementary DNA synthesis, containing the insertion. Complementary DNAs were recovered as a result of cloning into a suitable E. coli species (as known in the speciation literature).

RO 114469 Bl

The resulting double-stranded CADN was cloned into an in vitro expression vector to produce RNA ribozyme, using the T7 transcription system. Ribozyme activity was determined by incubating ribozyme RNA with SAT transcript followed by gel electrophoresis of the reaction mixture, after glyoxal treatment to denature nucleic acids.

Autolithic cleavage occurred at all expected sites on the CAT transcript. Accordingly, the flanking sequences of the arms or ribozyme may extend along the entire length of the RNA transcript, which must be cleaved.

Fig. 9 shows schematically a catalytic anti-sense RNA containing each of the four ribozymes. Catalytic antisense RNA contains about 9OO bases.

Under the above conditions, the ribozyme and the target sequences form highly molecular complexes probably through extensive base pairing. Strongly denaturing treatment, such as glyoxal treatment, during electrophoresis is required to resolve the reaction products.

Example 5. Target sequences for ribozyme cleavage

The GUA motif in the mRNA was tested to see if a ribozyme would affect RNA cleavage at this sequence.

A specific site from CAT mRNA was selected, including the GUA motif (fig. 1Oa) and a suitable and tested ribozyme sequence was prepared, in terms of activity. The ribozyme contained the arms of 8 ribonucleotides.

□ the synthetic ligonucleotides corresponding to the ribozyme of FIG. 10, were prepared according to example 2 and the double-stranded cDNA was cloned into an in vitro expression vector from E.coli, in order to produce ribozyme RNA using the T7 polymerase transcription system. Ribozyme activity was determined by incubation of ribozyme RNA with CAT mRNA, followed by gel electrophoresis of the reaction mixture as described.

Ribozyme cleaves at the GUA target site (not shown). Accordingly, the GUA motif in RNA is a substrate for the ribozymes of the present invention. This is not completely unexpected, since a naturally occurring site from the satellite RNA of the transient striated virus of the lucerne requires the recognition of a GUA site.

Similarly, a GUU motif from the CAT RNA target sequence was tested with a suitable ribozyme (see Fig. 10b) and cleavage was performed.

Example 6. Splitting of the viral RNA ribozyme

The viroid RNA, in the form of viroid RNA from Citrus exocortis, was cleaved using a ribozyme of the present invention, in the target RNA of Citrus exocortis (CEV) two target GUC sites were chosen. A site in the complementary catenary sequence was also chosen. Ribozymes were prepared against all of these sites and their activity was tested. Ribozymes were named CEV9x (+), CEV9x (-) and CEV25x (+). Fig. 11 presents the three cleavage sites in CEV RNA for each of these ribozymes.

Ribozymes were prepared according to the previous methods. The ribozyme RzCEV25x (+) is shown in Fig. 12. This ribozyme cleaves the GUC motif at nucleotide 116a CEV RNA.

Fig. 12b shows the cleavage of CEV RNA with RzCEV9x (+) ribozyme. With the ribozyme RzCEV9x [-) no cleavage was observed.

This experiment indicates that ribozymes are active against target RNA sequences from various sources. This is to be expected, as all RNAs are made up of basic ribonucleotide bricks containing adenine, guanine, cytosine and uracil, regardless of their origin, from animals, plants or microbes.

Example 7. Examples of ribozymes having variable catalytic domains

A ribozyme targeted against a CAT-2 site was prepared using the RNA catalytic domain sequence

RO 114469 Satellite underground clover virus (SCMoV) satellite. 0 complementarity of twelve bases of the flank sequence of the ribozyme arm was incorporated into the RzSCMoV ribozyme project. The RzCAT-2 and RzSCMoV ribozymes are shown in FIG. 13a and 13b respectively. The loop region of RzSCMoV contains 5 nucleotides containing the AAAUC sequence. This is in contrast to the loop region of RzCAT-2, which contains 4 nucleotides comprising the AGAG sequence. In addition, RzSCMoV contains a C, in the catalytic area instead of U * in RzCAT-2. The different sequences from RzSCMoV compared to RzCAT-2 are marked.

RzSCMoV was produced according to example 2. RzSCMoV was found to be active, resulting in two cleavage products, as expected.

In another experiment, the target site of Citrus exocortis viroid (CEV) at nucleotide -336 in its complementary RNA was cleaved using a ribozyme (RzCEV-2] having the sequence shown in Fig. 14a. The loop region designated by the letter "L" in Fig. 14 comprises six nucleotides having the sequence 3'-CCTATA-5 ', which is distinct from the loop region of sTobRV which comprises four nucleotides with the sequence 3'-AGAG-5'. This ribozyme cleaves the complementary CEV target of RNA at position -336, as shown in the electrophoretic profile of FIG. 14b.

This experiment indicates that the number of nucleotides and nucleotide sequences in the loop area is not important in ribozyme activity. In these experiments, ribozyme was produced according to the methods described above in the invention.

In another experiment, the effect of base pairing in the catalytic domain (trunk region) was investigated.

A modified ribozyme containing four additional base pairs was prepared and tested. In FIG. 15a, the RzCAT-2 ribozyme sequence hybridized to CAT target RNA is presented. The test ribozyme is shown in FIG. 15b, with the additional base pairs tested closed. The ribozyme assay has activity comparable to that of RzCAT-2. This indicates that the base pairing area of ​​the catalytic domain of the ribozyme can be of variable length without affecting the catalytic activity.

It has been observed (data not shown) that the stable in vivo form of sTobRV RNA transcripts expressed in transgenic plants is circular at the beginning, probably due to ligation of the 5 'and 3' terminal groups. Therefore, the use of two autolytic cleavage sites flanking a sequence of interest in an RNA transcribed in vivo is likely to result in a circularized product that may have greater stability than the linear transcript. This seems to design a new method for in vivo stabilization of ribozyme sequences. This operation is called circularization.

Example 8. In vivo activity of ribozymes In this example, the in vivo activity of ribozymes in plant cells is investigated.

Experimental protocol

Plasmids containing anti-CAT (CAT = chloramphenicol acetyl transferase) or anti-CAT / ribozyme gene constructs (see below) were introduced into tobacco protoplasts, in the same amount and proportion as each other, together with another plasmid, which contained a functional CAT construction gene. CAT activities were measured and compared with the baseline level of gene activity.

Materials and methods [a], Electroporation and CAT tests

They were executed as described in the literature. Briefly, Nicotiana plumbaginifolia T5 line protoplasts were prepared from a two-day suspension after subculture, suspended in 10 mM HEPES, pH = 7.2, 150 mM NaCl, 0.2 M mannitol, and adjusted to a density of 3 x 10⁶/ Ml. Electroporation was performed using a single pulse of 50 ms at 250 V. Protoplasts were diluted 10 times and cultured for 20 h at room temperature.

RO 114469 The temperature is 26 ° C and in the dark. These were cleaved by sonication and extracts were obtained. Normalized extracts for protein content were tested for CAT activity in vitro using¹⁴C-chloramphenicol and acetyl CoA. The reaction products were separated by thin layer chromatography and autoradiography. The degree of reaction was calculated by obtaining radioactive derivatives of the product in the model.¹⁴C-chloramphenicol.

(B). Construction genes

Construction genes were introduced into 0.1 ml protoplast suspensions as plasmid DNAs, which were purified from bacteria, by extraction and centrifugation at two-cycle density gradient equilibrium (CsCI equilibrium). These were resuspended in 10 mM Tris / 1 mM EDTA /, pH = 7.5 for use.

Active CAT construction genes were born on the designated plasmid pCAT7 +. It derived by fusion of a CAT gene sequence (from plasmid pCM4, as known in the literature) into plasmid pJ35SN (derived from p35SN, also known in the literature), so that the construction genes have former:

5 '3' inside the plasmids with the following names:

pJ35SN = This plasmid vector, the sketch of which is shown in FIG. 16, contains a 355 CaMV promoter and a 3 'adenylation nopalin synthetase signal from the plant, which can be represented as follows:

5' 3'

<td>35</td><td>SN0</td>

pCAT7- = This contains the CAT gene sequence inserted into pJ35 SN, so that the transcription will result in CAT antisense RNA, which can be represented as follows:

5' 3'

<td>35</td><td>'How</td><td>NOS</td>

pCAT19- = This contains the CAT gene with four ribozyme catalytic domains included in it (see example 4 and Fig. 9), inserted into pJ35SN, so that the transcription will result in the production of antisense CAT RNA, which can be represented as follows. :

5' 3’

<td>35</td><td>CAT *</td><td>NOS</td>

<td>35</td><td>HOW</td><td>NOS</td>

35S refers to the CaMV 35S (cauliflower mosaic virus) promoter, NOS to the polyadenylation nopalin synthetase signal, T / C to the transcript.

Along with 0.2 µg of pCAT7 +, various over-construction genes were added as described below. The construction genes found the catalytic domain insertions

Results

The following table shows the relative activities of CAT in cells, after 20 h, after electroporation. The activity is expressed as a percentage conversion of the chloramphenicol substrate into a one-hour test.

RO 114469 Bl

<td rowspan="2">Treatment</td><td colspan="4">pg of electroporated plasmid</td><td rowspan="2">conversion %</td>

<td>pCAT7 +</td><td>pJ35SN</td><td>pCAT7-</td><td>pCAT19-</td>

<td>1A</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 0</td>

<td>1B</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 0</td>

<td>2A</td><td> 0,2</td><td> 18</td><td> -</td><td> -</td><td> 21</td>

<td>2B</td><td> 0,2</td><td> 18</td><td> -</td><td> -</td><td> 46</td>

<td>3A</td><td> 0,2</td><td> 9</td><td> 9</td><td> -</td><td> 28</td>

<td>3B</td><td> 0,2</td><td> 9</td><td> 9</td><td> -</td><td> 32</td>

<td>4A</td><td> 0,2</td><td> -</td><td> 18</td><td> -</td><td> 26</td>

<td>4B</td><td> 0,2</td><td> -</td><td> 18</td><td> -</td><td> 19</td>

<td>5A</td><td> 0,2</td><td> 9</td><td> -</td><td> 9</td><td> 19</td>

<td>5B</td><td> 0,2</td><td> -</td><td> -</td><td> 9</td><td> 22</td>

<td>6A</td><td> 0,2</td><td> -</td><td> -</td><td> 18</td><td> 14</td>

<td>6B</td><td> 0,2</td><td> -</td><td> -</td><td> 18</td><td> 16</td>

(for each treatment A "and" B, they are duplicates).

The following conclusions can be drawn from 25 of these results:

(a). The introduction of the CAT gene of construction leads to an insignificant CAT activity - to compare 2A, B with 1A, B. there is a variation between duplicates. 30 From the trends observed in the other samples (see "b" and "c" below) it is likely that 2A has an abnormally low activity.

(b). The simultaneous introduction of a 35 antisense construction gene leads to a decrease in activity level - to compare 3A, B and 4A, B. The size of the decrease is directly related to the level of the antisense gene added as a plasmid - by 4 compared to 3A, B and 4A, B.

(c). The concomitant introduction of the combined antisense / ribozyme gene construct leads to a decrease in gene activity - compare 5A, B and 45 6A, B. In addition, the decrease is more marked than for the corresponding levels of the antisense construction gene - compare 5A, B with 3A, B and 6A, B with 4A, B. 50

The average results for four in vivo experiments are shown in FIG. 17. In this figure, "control" means treatment 2. Antisense "means treatment 4. Catalytic" means treatment 6 and "foundation" represents treatment 1.

Catalytic ribozyme inhibits CAT activity 47%, compared to 34% for antisense ribozyme.

The introduction of ribozyme-bearing genes into plant cells inhibits the activity of the genes against which they were targeted. In addition, the inhibition is higher for the corresponding antisense RNA molecules.

These results indicate that ribozymes will be active in cells, animals, plants or microbes against a range of target RNA molecules.

The mechanism of action of ribozymes in this example is unclear. For example, antisense ribozyme can irreversibly hybridize to an RNA target and catalyze the cleavage of the phosphodiester linkage at one or more target sites selected along

RO 114469 Blade of RNA target. Alternatively, cellular enzymes can disassemble antisense RNA from its target sequence so that the target RNA is cleaved into two or more fragments. 5

Example 9. In vivo ribozyme activity In animal cells, the activity of ribozymes in inactivating an RNA target in mammalian cells is demonstrated in this example.

Materials and methods

Active building genes encoding ribozymes were transfected into a cell line from widely accessible monkey kidneys. In this method, 15 x 3 O were contacted^B/ ml COS1 cells suspended in saline buffer with 10% fetal calf serum, with different construction genes. In order to perform the electroporation of the DNA from the cells, an electrical discharge was applied. The transfected cells were incubated at 37 ° C for 48 h in culture medium and before testing for CAT and luciferase activity. 25

Construction CAT genes appeared in the plasmid designated pTK CAT (as known in the literature). This plasmid is derived by introducing a CAT gene sequence into the pSV2 plasmid so that it is under the control of the herpes simplex virus thymidinquinase promoter.

The coding construction gene appeared in plasmid pSV232A (known in the literature) containing the luciferase gene fused with the early SV40 promoter. DNA encoding ribozymes was ligated into the Xbal site at the 3 'end of the luciferase gene, according to standard methods, which are known in the literature (Maniatis et al.).

The following constructs were prepared using the known standard techniques (Maniatis et al.):

PFC58 = This plasmid vector contains DNA encoding the RzCAT1 ribozyme fused to the 3 'end of the luciferase gene in a non-functional orientation.

It can be described as follows:

<img file="RO114469B1_D0002.tif" />

where: 232A refers to the 35 pSV232A sequence, early SV40 refers to the early SV40 promoter and small T is DNA, encoding the small T intervention sequence of SV4D. This construct results in the production of a molecule encoding 40 luciferase and RzCAT-1 ribozyme, the latter being in such an orientation that it is not expected to be catalytic.

pFC4 = This plasmid is the same as pFC58, except that RzCAT-1 45 is replaced by RzCAT-3.

pFC1-6 = This plasmid is the same as pFC58, except that RzCAT-1 is replaced by RzCAT-3, in the sense of orientation (5'-3 '). 50 pFC2D = This plasmid is the same as pFC1-6 except that, RzCAT-3 is replaced by RzCAT-2 flanking sequences of eight nucleotides.

pFC12 = This plasmid is the same as pFC2D, except that the RzCAT-2 ribozyme contains flanking sequences of twelve nucleotides.

pFC5D = This plasmid contains the CAT gene with four ribozyme catalytic domains included in it (see example 4 and Fig. 9) for orientation (5'-3'J, which gives rise to an inactive ribozyme on the transcript. This plasmid can be described as follows:

<td>SV40 early</td><td>CAT gene containing ribozyme domains</td><td>Poli A⁺</td>

♦

Non-catalytic RNA

RO 114469 Bl pFC54 = This plasmid is the same as pFC5O, except that the CAT gene and the ribozyme domains are in the antisense orientation (3'-5 ').

pFC64 = This plasmid splits the SV40 promoter and the polyadenylation signals with pFC5O and contains the wild-type CAT gene with non-inserted ribozyme domains. This gene is in an antisense orientation and thus does not produce any protein or CAT.

pFC65 = This plasmid is the same as pFC64, except that the wild-type CAT gene is in the (5'-3 ') orientation direction and thus produces CAT protein.

tests

The activity of luciferase was tested according to the methods known in the specialized literature. Briefly, the COS cells were lysed at 48 h after transfection and the cell lysate was incubated with luciferin, the substrate of lysifferase, and the liminiscence detected using a scintillation counter. 25

CAT activity was also measured using COS cell lysates (cell lysates were divided into two, and each portion tested for luciferase or CAT activity), according to the method known in the literature.

In in vivo assays, pFC58 and pFC4 did not carry out CAT activity in transfected cells. This activity was designated as CAT activity 100% and 0% CAT suppression. CAT activity in cells transfected with other plasmids was measured relative to pFC58. The percentage of CAT suppression was measured as:

(. test - x 100 ^Howz normalized control of luciferase production. HOW_test-CAT tests the result for test constructions. HOW_controller| -CAT tests for construct control (pFC4 and pFC58).

Lucifer production is an internal control for electroporation and gives a measure of ribozyme production within each individually electroporated tissue culture plate.

Results

Experiment (s) pg electroporated plasmid / 1.5 x 1CT cells

<td>Treatment</td><td>pTKCAT</td><td>pFC58</td><td>pFC20</td><td>pFC1-6</td><td>pFC12</td><td>Suppression% CAT</td>

<td> 1</td><td> 5</td><td></td><td> 2</td><td></td><td></td><td> 56</td>

<td> 2</td><td> 5</td><td></td><td> 1</td><td> 1</td><td></td><td> 53</td>

<td> 3</td><td> 5</td><td></td><td></td><td></td><td> 2</td><td> 40</td>

<td> 4</td><td> 5</td><td> 2</td><td> -</td><td> -</td><td></td><td> 0</td>

All the experimental treatments were conducted in duplicate and an average value was given.

Experiment (s)

<td></td><td colspan="3">pg electroporated plasmid / '</td><td colspan="3">', 5 x 1CP cells</td>

<td>Treatment</td><td>pTKCAT</td><td>pFC1-6</td><td>pFC20</td><td>pFC12</td><td>pFC4</td><td>Suppression% CAT</td>

<td> 5</td><td> 5</td><td> 2</td><td></td><td></td><td></td><td> 75</td>

<td> 6</td><td> 5</td><td> -</td><td> 2</td><td></td><td> -</td><td> 75</td>

<td> 7</td><td> 5</td><td></td><td> 4</td><td></td><td></td><td> 62</td>

<td> 8</td><td> 5</td><td> 1</td><td> 1</td><td></td><td></td><td> 70</td>

<td> 9</td><td> 5</td><td></td><td></td><td> 4</td><td></td><td> 51</td>

<td> 10</td><td> 5</td><td></td><td> -</td><td> -</td><td> 2</td><td> 0</td>

RO 114469 Bl

The treatments from 5 to 10 were performed in duplicate, and an average value was given.

Experiment [iii] pg electroporated plasmid / 1.5 x 1CP cells

<td>Treatment</td><td>pTKCAT</td><td>pFC1-6</td><td>pFC4</td><td>Suppression% CAT</td>

<td> 11</td><td> 5</td><td> 2</td><td></td><td> 66</td>

<td> 12</td><td> 5</td><td></td><td> 2</td><td> 0</td>

The treatments were performed in duplicate and an average value was given. Experiment [iv] pg electroporated plasmid

<td>Treatment</td><td>pTKCAT</td><td>pFC50</td><td>pFC54</td><td>pFC64</td><td>pFC65</td><td>Suppression% CAT</td>

<td> 13</td><td> 5</td><td> 2</td><td></td><td></td><td></td><td> 0</td>

<td> 14</td><td> 5</td><td></td><td> 2</td><td> -</td><td></td><td> 26</td>

<td> 15</td><td> 5</td><td></td><td></td><td> 2</td><td></td><td> 2</td>

<td> 16</td><td> 0</td><td></td><td></td><td></td><td> 2</td><td>N / A</td>

Each of the treatments from 13 to 16 20 was performed in four copies.

The CAT sense construct (treatment 16) produced high levels of CAT activity. Therefore,% suppression is not applicable (NA). 25

A number of experiments were conducted replacing the TK promoter of pTKCAT with the human metallothionein promoter. When this construct was cotransfected into C0S1 cells with plasmids 30 encoding one or more ribozymes, a marked decrease in CAT activity was observed.

The above results clearly demonstrate the inactivation of in vivo activity of 35 ribozymes in human cells.

While the efficacy of in vivo ribozymes is thought to be caused by one or more catalytic regions, which are capable of cleavage of an RNA target, the presence of such regions in "Antisense" RNA ribozymes may not actually lead. , in vivo cleavage if the entire RNA / RNA-antisense molecule does not collapse. However, regardless of whether the molecule collapses or not, the above examples demonstrate the efficacy of ribozyme in inactivating the RNA target. Thus, the invention is applicable to all ribozymes having a catalytic zone capable of producing cleavage and a hybridization zone, regardless of where cleavage in the RNA target actually occurs. The hybridization zone can be so large that it causes the RNA / ribozyme combination to stand together and prevent cleavage of the RNA target into separate components, even if the catalytic area itself is capable of cleavage.