AU722973B2 - Grb3-3 gene, variants and uses thereof - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFCATION FOR A STANDARD PATENT

(ORIGINAL)

.00.

0*0.0: Name of Applicant: Actual Inventors: Address for Service: Rhone-Poulenc Rorer S.A.

Fabien SCHWEIGHOFFER AND Bruno TOCQUE DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000 Grb3-3 gene, variants and uses thereof Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us: Q:\OPER\PDB\67247-94.279 6/10/98 GRB-3 ANTI- SENSE POLYNUCLEOTIDES The present invention relates to anti-sense polynucleotides complementary to the gene designatedI Grb3-3, and their u_ es and to an isolated polypeptide encoded by Grb3-3.

Various genes, called oncogenes and suppressor genes, are involved in the control of cell division. Among them, the ras genes and their products generally designated p2l proteins, play a key role in the control of cell proliferation in all eukaryotic o rganisms where they have been searched out. In varticullar, it has been shown that certain snecific -modifications of these proteins cause them to lose tCheir normal control and lead them to become oncogenic.

Thus, a large number of human tumours have been associated with the presence of modified ras genes.

:Likewise, an o-,erexpression of these p21 proteins can *lead to a deregulation of cell1 pro i-Earation. An-r understanding of the exract role of these p 2 1 proteins in cells, of their mode of operation and theiLr characteristics therfore constitut.es a major stakefr the understanding and the therapeuti1c approach to carcinogenesis.

-Various factors involved in the ras-dependent signalling pathway have been identifid onths are tChe Grb2- gene which. encodes a protein of 23-25 kfla having a SF13--SH2-SH3 strcture (Lowenstein et al., Cell '(1592) 431; Matuoka et al. PINAS 89 (1992) 9015).

The poroduct of the Grb2 gene 'appears to interact wih the tyrosine phoophorylated POtciz's via its SH2 domain, and with a factor fov- exchanre 0-4 GDP of the SOS class via its S93 domain (Egan et Nature 36.3 (1l,93) 45) it would thur. be one of the components of the transformant activity- of the product 6fE the, ras gene. The present invention deriveA from the demonstration of the cloning and characterizatiofl of an isoform of the Grb2 gene, designated Gtb3-3, possessing a. deletion in the 3H2 domain. This gone is expressed in adult tissues: the correspcndina wMRA is present in the fomof a singlie band of 1.5 kb, and is translated into a 19 kDa proteln. Because of Its daletion in the SH2 domdin, the pr--duct of the Grb3-3 gene is no longer capable of 4-tractrig with the tyrosine phosphoryltd proteins (phos~phoz-Vlat.d EGF reeptor), but it retains the capacity i.nteract with the pr~nline-~rich domains of the SOS ur eis Because oi its deletion, the product oi the Grb3-3 gene is thum. ca-paIble of preventing the cel.lular effects of the product of the Grb2 gene. The transfer in vivo of antisense variants of this gene therefore makes it possible to interfere with the processes of proliferation, differentiation a nd/or cell death., The invention therefore provides an antisense polynucleotide comprising the complementiary strand of the nucleotide sequence of SEQ ID NO:l, which antisense polynucleotide inhibits the expression of Grb2 and Grb3- 3.

The invention also provides an antisense IZI- AL/ ncletie comprising part of the complementary strand of the nucleotide sequence of SEQ ID NO:1, which antisense polynucleotide specifically inhibits the expression of Grb3-3.

In particular such polynucleotides may comprise a sequence complementary to the sequence joining the Nterminal SH3 domain and the residual SH2 domain of Grb3- 3.

Such antisense polynucleotidesmay be antisense RNAs.

The antisense sequences of-the invention may be expressed in a target cell in order to make it possible to control the transcription of cellular mRNAs. Such sequences can for example be transcribed, in the target cell, into RNAs complementary to the cellular mRNAs Grb2 or Grb3-3 and thus block their translation into protein, according to the technique described in patent EP 140 308. Such sequences may consist of all or part of the nucleic sequence SEQ ID NO. 1, transcribed in the reverse orientation.

The invention also provides an isolated polypeptide having the amino acid sequence encoded by the polynucleotide of SEQ ID NO:1.

As indicated above-, Grb2 .isA protein which is at least bifunctional, and which is anchored via its o SH2 domain to specific sequences phospborylated at the tyrosine, and via its two SH3 domains, to the exchange factors of the SOS family. Grb3-3 having lost its capacity to associate with proteins phosphorylated at the tyrosine can therefore only form a complex with the SOS proteins. Grb3-3 can therefore prevent the recruitment of the Grb2-SOS complex by the receptors of the self-phosphorylated growth factors or by associated ge proteins which are also phosphorylated at the tyorsine 2uch as SEC or IRS!. Grb3-3 being capable of blocking this recruitment 1 it is capable of blocking mytogenic pathways and of inducing cell death. The Aipplicant has indeed demonstrated that the arb3-3 protein was expressed during certain physiological Processes suchi as for example the maturation-of the thymuo in rats.

The Applicant has also shown that Grb3-3 is capable of inducing cell death by apoptosin of various cell types.

it was possible to detect these conkpletely advantageous properties by injecting recombinant prorein into the 3T3 fibrobla sts and (ii) by by transferring the sequence encoding Grb3-3 into the 371T3 cells (Example Grb3-.3 is therefore capable of inducing the cellular death of viable cells such as immortalized, cancer or embryonic cells. As shown in the examples, Grb2; is capable of preventing the effects of Grb3-3.

Moreover, a search f or the 9:tpressvion of Grb3I- 3 caPrri ed out during the infection,.of lymphocytic .cells by the HIV virus- made it possible-to show that the -massive viral production observed 7 days after the infection is correlated with an overexp~ession of the *Grb3 -3 mRNA by the inf ected cells (Example 5) This, experiment shows that eliminating or blocking the cellular effects of Grb3-3 can also) make it poosible to maintain alive cells infected especially with HIV, and thus allow the T4 lymphocytes to continue to play a ~~~role of immune defence.

The nuclaic saglaance. aCC=Tdi% to t-he 4nvention can be used as SUCh, fOr sxam~1 ate injection into man or animals, to ind.ce a protection or to treat cancers. in particular,. they can. be injected in the form of naked DN4A accordia.g to the -ehique described i~n application WO 90/11092. They can also be administered in complaxed far~i, for example with DEE-dexctran (Pagano et al., AT. Virol. 1 (1967) 891), with nuclear protains (Kaneda et al., Science 243 (1989) 375), with lipids (Fslgna= st al., PMkB 84 (198V) 7413), in the to=m of liposomes (Fraley at al., J. Bicd. Chem. 253 (1980) 10411) and the like.

Preferably, the nucleic segiLnces according to the invention form part of a ivector. The uise of such veacr indeed makas it possible to improve the administr-ation ot the nuclaic acid into th@ calls to be treated, and also to increame its atability in th~e maid cellswh-:6 cels, hca makes it possible to obtain a durable therapeutic effect. Furthermere, it is possible to introduce several nucleic acid sequences into the same vector, which also increases thle efficacy of the treatment.

The vector used may be of diverse origin, as' long as it is capable of transforming animal cells, preferably human tumour cellIs. In a preferred embodiment of the invention, a viral vector is used which can be chosen from eg. adenoviruses, retroviruses adeno-asoociated viruses (AAV), herpes virus,cytomegalovi4rus (CMV) or vaccinia virus_ Vectors derived from adenoviruses, -ratroviruses or AAVs incorporating heterologous; nucleic acid sequences have been described in the literature [Ak-l. at al." Nature Geneti-Ca 3 (1993) 224; Strateord-Perricaudet et al.,.

Human Gene Therapy 1 (1990) 241; .P 185 573, Levrero et al., Gene 101 (1991) 1.95; Le Gal la Salle et al.,, Science 259 (1.993) 988; Roemer and Friedmann, Eur. J.

Biochem. 208 (1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., New Biol. 2 (1990) 739; Mivanohara et al., New Biol. 4 (1992) 238; W091/18088J.

0 The present invention therefore also relates to any recombinant virus comprising, inserted into its genome, an anti-sense polynu( 1eotide as defined before.

Advantageously, the recombinant virus according to the invention is a defective virus. The tarm "defective virus" designates a virus in-a-pable of replicating in the target cell. Generally, the genome of the defective viruses used within the framework of the present invention is therefore devoid of at least the sequences necessary for the replication of the said virus in the infected cell. These regions can either be removed (completely or partially), or rendered nonfunctional, or substituted by other sequences and especially by the nucleic acid of the invention.

Preferably, the defective virus nevertheless conserves 10 the sequences of its genome which are necessary for the encapsulation of the viral particles.

It is particularly advantageous to use the ~nucleic sequences of the invention in a form incorporated in an adenovirus, an AAV or a defective 15 recombinant retrovirus.

As regards adenoviruses, various serotypes exist whose structure and properties vary somewhat, but which are not pathogenic for man, and especially nonimmunosuppressed individuals. Moreover, these viruses do not integrate into the genome of the cells which they infect, and can incorporate large fragments of exogenous DNA. Among the various serotypes, the use of the type 2 or 5 adenoviruses (Ad2 or Ad5) is preferred within the framework of the present invention. In the case of the Ad5 adenoviruses, the sequences necessary for the replication are the E1A and E1B regions.

The defective recombinant viruses of the invention can be prepared by homologous recombination between a detective virus and a PI&SMId carrying, inta= alia. the nucleotide sequence ag defined above (Levrero et al., Gene 101 (1991) 195; Graham, EMO a.

3 (12) (1984) 2917).- The homologous recombination is produced after co-transfection of the said viruses and plasmid into an appropriate cell line. The cell line used should preferably be transformable by the said elements, and (ii) contain sequences capable of Complementing the part of the genome of the defective virus, preferably in integrated for= so as to avoid the risks of recombination. An example of a line which can be U;Sed for the preparation of defective recombinant adenoviruses, therm may be mentioned the human embryonic kidney line 293 (Grahe~m etal., J. Gen.

-Viral. 36 (1977) 59) which contains especially, integrated into its genome, the left part of the genome of an Ad ade-novirus (1 )As exCample of a line which can be used for the preparation of detactive recombinant retr-oviruses, there may be mentioned the .6200. CPJP line (Danos and Mulligan, PHAS 85 (1988) 6460).

Then the viruses which have multiplied are recove red and purified according to conventional molecular biology tachnq.,ez.

The present invention also provides a pharmaceutical composi3tionl containimng at least one vector, eg recombinant virus or an antisense polynucleotide as defined above in association with a pharmaceutically accaptable vehicle.

The pharmaceuti.cal compositions of the 9 invention can be formulated for eg.topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration* Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected, optionally directly into the tumour to be treated. These may be in particular isotonic, sterile, saline solutions 10 (egponosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, 15 permit the constitution of injectable solutions-.

The doses of nucleic acids (sequence or vector) used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, of the nucleic acid to be expressed, or alternatively of the desired duration of treatment. Generally, as regards the recombinant viruses according to the invention, the latter are formulated and administered in the form of doses of between 104 and 1014 pfu/ml, and preferably 106 to 1010 pfu/ml. The term pfu ("plaque forming unit") corresponds to the infectivity of a virus solution, and is determined by infecting an appropriate cell culture

I

and measuring, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

Such pharmaceutical compositions can be used in man, for the treatment and/or prevention of cancer.

In particular the products of the invention are capable of modulating the activity of ras proteins, they make it possible to intervene in the cancer development process, and in particular, they can inhibit the activity of oncogenes whose transformant activity depends on a p21-functional GAP interaction. Numerous cancers have indeed been associated with the presence :of oncogenic ras proteins. Among the cancers most often 1 containing mutated ras genes, there may be mentioned especially adenocarcinomas of the pancreas, of which 90 have a Ki-ras oncogene mutated on the twelfth codon (Almoguera et coll., Cell 53 (1988) 549), adenocarcinomas of the colon and cancers of the thyroid 20 (50 or carcinomas of the lung and myeloid leukaemias (30 Bos, J.L. Cancer Res. 49 (1989) 4682) More generally, the compp.itions according to the invention can be used for treating any type of pathology in which an abnormal cell proliferation is observed, by inducing apoptosis, as well as any pathology characterized by a cell death by apoptosis (AIDS, Huntington's chorea, Parkinson), by 5s R blocking the effects of Grb3-3.

The present invention will be more fully described with the aid of the following examples.

Legend to the Figures Figure 1 schematic representation of the structural domains of Grb2 and Grb3-3.

Figure 2 study of the binding of Grb3-3 to the EGFreceptor (Figure 2a) and to proline-rich peptides (Figure 2b).

Figure 3 effect of Grb3-3 on the transactivation, by ras, of an RRE derived from the polyoma virus enhancer.

Figure 4 demonstration of Grb3-3-induced cell death S. on 3T3 fibroblasts.

Figure 5 demonstration of the expression of Grb3-3 in cells infected with the HIV virus.

General molecular biology techniques The methods conventionally used in molecular 2* biology, such as preparative extractions of plasmid 20 DNA, centrifugation of plasmid DNA in caesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, extraction of proteins with phenol or phenolchloroform, DNA precipitation in saline medium with ethanol or isopropanol, transformation in Escherichia coli, and the like are well known to persons skilled in the art and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory 12 Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley Sons, New York, 1987].

The pBR322 and pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories).

For the ligations, the DNA fragments can be separated according to their size by agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol/chloroform mixture, precipitated with o* ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the supplier.

15 The filling of the protruding 5' ends can be carried out by the Klenow fragment of DNA polymerase I of E. coli (Biolabs) according to the specifications of Sthe supplier. The destruction of the protruding 3' ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5' ends is carried out by a controlled treatment with S1 nuclease.

The site-directed mutagenesis in vitro with synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al.

[Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

13 The enzymatic amplification of DNA fragments by the so-called PCR technique [Polymerase-catalyzed Chain Reaction, Saiki R.K. et a.l, Science 230 (1985) 1350-1354; Mullis K.B. et Faloona Meth. Enzym.

155 (1987) 335-350] can be carried out using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the specifications of the manufacturer.

The verification of the nucleotide sequences can be carried out by the method developed by Sanger et 10 al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] 9* using the kit distributed by Amersham.

EXAMPLES

1. Isolation of the Grb3-3 gene The Grb3-3 gene was isolated by screening a 15 human DNA library by means of a probe derived from the sequence of the Grb2 gene.

500,000 lambda gtll recombinant phages carrying DNA fragments derived from a human placenta library (Clontech) were screened by means of a probe derived from the sequence of the Grb2 gene. The probe used corresponds to the first 8 amino acids of the Grb2 protein, and has the following sequence: ATGGAAGCCATCGCCAAATATGAC (SEQ ID No. 2) positive clones were thus identified. The insert of these 10 clones was isolated in the form of EcoRI fragments, cloned into the plasmid M13mpl8 and sequenced. Among these 10 clones, 9 carried inserts identical to the Grb2 sequence. Only one of them carried an insert of a size smaller than the Grb2 gene, because of a deletion in the SH2 domain (Figure 1).

Analysis of the remaining sequence revealed a perfect identity with the corresponding regions of Grb2, including in the non-coding 5' and 3' regions. The open reading frame of this clone encodes a protein of 177 amino acids (SEQ ID No. containing 2 SH3 domains bordering an incomplete SH2 domain (Figure The

C.

amino acids deleted in the SH2 domain (residues 60 to

C*

10 100 of the Grb2 protein) correspond to the residues involved in the binding of Grb2 to the peptides containing phosphorylated tyrosines.

2. Binding activity of the Grb3-3 protein As indicated above, the Grb2 protein is the 15 mediator of the interaction between the phosphorylated growth factor receptors and the SOS factors. This example demonstrates that the Grb3-3 protein is incapable of interacting with the phosphorylated

EGF

receptor but that it conserves its capacity to interact with a proline-rich peptide derived from the sequence of the human SOS1 factor.

The binding capacity of Grb3-3 was studied using biotinylated Glutathione-S-transferase

(GST)

fusion proteins. This type of fusion permits a rapid and efficient purification of the recombinant products.

For that, the sequences of the invention were expressed in the E. coli TG1 strain in the form of fusion proteins with GST according to the technique described by Smith and Johnson [Gene 67 (1988) 31]. Briefly, the Grb2 and Grb3-3 genes were first modified by introducing a BamHI site on either side of the start and stop codons. For that, the open reading frames of these genes were amplified by PCR by means of the following oligonucleotides: Oligonucleotide I (5')(SEQ ID No. 3):

GAATTCGGATCCATGGAAGCCATCGCCAAATATGACTTC

Oligonucleotide II (SEQ ID No. 4): 10 GAATTCGGATCCTTAGACGTTCCGGTTCACGGGGGTGAC The underlined part corresponds to the BamHI site created, followed or preceded by the start and stop codons.

The genes thus amplified were then cloned in 15 the form of BamHI fragments into the vector pGEX 2T (Pharmacia) linearized by the same enzyme, in 3' and in frame in a cDNA encoding GST. The vectors thus obtained were then used to transform the E. coli TG1 strain. The cells thus transformed were precultured overnight at 37 0 C, diluted 1/10 in LB medium, supplemented with IPTG in order to induce the expression (2 hours, 25 0 C) and then cultured for about 21 hours at 25 0 C. The cells were then lysed, and the fusion proteins produced affinity-purified on an agarose-GSH column. For that, the bacterial lysate is incubated in the presence of the gel (prepared and equilibrated with lysis buffer) for 15 minutes at 4 0 C. After 3 washes with Tris-HCl buffer pH 7.4, the proteins are eluted in the presence of a tris-HCl buffer pH 7.7 containing an excess of GST. The supernatent is harvested and centrifuged.

The same procedure was used to prepare a mutant of Grb2 in which the glycine 203 is replaced by an arginin (Grb2G203R) and a Grb3-3 mutant in which the glycine 162 is replaced by an arginin (Grb3-3G162R).

The Grb2G203R mutant has been described as no longer having any activity in a test of reinitiation of DNA synthesis (Lowenstein et al., previously cited). The lr S 10 Grb3-3G162R mutant carries the same mutation in the ."e same position, and should therefore also be inactive.

These mutants were prepared by mutagenesis by PCR on the Grb2 and Grb3-3 genes using, in the oligonucleotide I described above, and in the 15 following oligonucleotide III in which the mutated codon is underlined: Oligonucleotide III (SEQ ID No.

GACGTTCCGGTTCACGGGGGTGACATAATTGCGGGGAAACATGCGGGTC

The fragments thus amplified were then eluted, reamplified by PCR by means of the oligonucleotides I and II, and then cloned into the vector pGEX 2T. The mutuants were then produced as described above.

The GST fusion proteins (GST-Grb2, GST-Grb3- 3, GST-Grb3-3G162R and GST) were then biotinylated by conventional techniques known to persons skilled in the art (cf. general molecular biology techniques as well as Mayer et al., PNAS 88 (1991) 627), and used as probes to determine the binding to the immobilized phosphorylated EGF receptor and then to a peptide derived from hSOSI 2.1. Binding to the phosphorylated EGF receptor Procedure The EGF receptor used was purified from A431 cells by immobilization on WGAsepharose according to the technique described by Duchesne et al., (Science 259 (1993) 525). 2 gg of this receptor were first stimulated by 1 pM EGF, 10 min at *i 22 0 C, and then incubated, with or without cold ATP M) in the presence of 2.5 mM MnC12 in HNTG buffer mM Hepes, 150 mM NaCI, 0.1 Triton, 10 glycerol, pH=7.5) at 4 0 C for 2 min. The phosphorylation of the receptor is then stoped by adding a degradation buffer.

15 The samples are then deposited on a 4-20% SDS-PAGE gel and then transferred onto polyvinylgdene difluoride membranes (PVDF). The blots were then incubated in the presence of various biotinylated GST fusions (2 gg/ml) and then revealed by means of alkaline-phosphatase coupled streptavidin (Promega). The EGF receptors were also subjected to an immunoblotting in the presence of anti-phosphotyrosine antibodies (anti-PY) in order to verify that the receptors have indeed been phosphorylated.

Results: The results obtained are presented in Figure 2a. They show, as expected, that the Grb2 protein interacts with the EGF receptor in phosphorylated form alone. They also show that the Grb3-3 protein does not bind the EGF receptor, regardless of its degree of phosphorylation.

2.2. Binding to a peptide derived from hSOS1 Procedure the following two proline-rich peptides were synthesized: hSOS1 Peptide GTPEVPVPPPVPPRRRPESA This peptide corresponds to residues 1143 to 1162 of the hSOS1 protein (Li et al., Nature 363 (1993) 83) responsible for the interaction between Grb2 and hSOS1 (SEQ ID No.

6).

3BP1 Peptide PPPLPPLV This peptide is derived from the 3BP1 protein, which is known to efficiently bind 6the SH3 domain of Abl and Src (Cicchetti et al., 0** Science 257 (1992) 803) (SEQ ID No. 7).

15 Each of these peptides (1 Ml, 10 mg/ml) was immobilized on nitrocellulose membrane. The membranes were then incubated in a blocking buffer (20 mM Tris pH=7.6, 150 mM NaC1, 0.1 Tween, 3 bovine albumin).

The membranes were then incubated overnight at 4 0 C in the presence of the various biotinylated GST fusions (4 ug/ml) and then revealed by means of alkaline phosphatase-coupled streptavidin (Promega).

Results The results obtained are presented in Figure 2b. They show that Grb3-3, like Grb2, is capable of binding the hSOS1 peptide. They also show that this interaction is specific since no binding is observed with the 3BP1 peptide. Moreover, the results also show that the Grb3-3G162R mutant is no longer 19 capable of binding the hSOS1 peptide, which confirms the importance of this residue and the functional role of this interaction.

3. Activity of the Grb3-3 protein This example demonstrates that, in spite of its deletion in the SH2 domain, the Grb3-3 protein has a functional effect.

The activity of the Grb3-3 protein was 0* studied by determining its capacity to cooperate with 10 ras for the transactivation of a promoter possessing ras response elements (RRE) and governing the expression of a reporter gene.

The procedure used has been described for Sexample in Schweighoffer et al., Science 256 (1992) 15 825. Briefly, the promoter used is a synthetic promoter composed of the murine promoter of the thymidine kinase gene and 4 repeated PEA1 elements derived from the polyoma enhancer (Wasylyk et al., EMBO J. 7 (1988) 2475) Py-TK promoter. This promoter directs the expression of the reporter gene, in this case of the bacterial gene for chloramphenicol acetyl transferase (CAT) Py-TK-CAT vector. The vectors for expressing the tested genes were constructed by inserting the said genes, in the form of BamHI fragments, into the BglII site of the plasmid pSV2. This site makes it possible to place the genes under the control of the early promoter.

ER22 cells which are 40 confluent were transfected with 0.5 pg of the vector Py-TK-CAT alone (Py) or in the presence of of the expression vector carrying, under the control of the early SV40 promoter, the gene Grb2, 2 pg, Grb3-3, 2 gg, Grb2(G203R) 2 pg, Grb3-3(G162R) 2pg, or Grb3-3, 2 pg Grb2, 2 pg. In each case, the total quantity of DNA was adjusted to pg with an expression vector without insert. The transfection was carried out in the presence of lipospermine (Transfectam, IBF-Sepracor). The cells were maintained for 48 hours in culture in a DMEM Smedium supplemented with 0.5 foetal calf serum. The e CAT activity (transactivation of the RRE) was then ~determined as described by Wasylyk et al. (PNAS o* S (1988) 7952).

so w5 The results obtained are presented in Figure 3. They show clearly that the expression of the Grb3-3 protein prevents the effects of the activation of a growth factor receptor. They also show that Grb2 in excess prevents the effects of Grb3-3 on the response to the growth factor.

4. Grb3-3 induces cellular apoptosis This example demonstrates the direct involvement of Grb3-3 in cellular apoptosis. This property offers particularly advantageous applications for the treatment of pathologies resulting from a cellular proliferation (cancers, restenosis, and the like).

The induction of cellular apoptosis by Grb3-3 was demonstrated by injecting recombinant protein into 3T3 fibroblasts and (ii) by by transferring the Grb3-3 encoding sequence into the 3T3 cells.

Injection of the recombinant protein The recombinant Grb3-3 protein was prepared in the form of fusion protein with GST according to the procedure described in Example 2. The fusion protein was then treated with thrombine (0.25 Sigma) in order to separate the GST part, and then purified by ion-exchange chromatography on a monoQ column. The fractions containing the recombinant protein were then concentrated by means of Microsep microconcentrators (Filtron) in a 20 mM phosphate buffer (pH 7) containing 100 mM NaCl. The purified protein thus obtained was 15 injected (1 to 3 mg/ml) into cultured 3T3 cells by means of an automatic Eppendorf microinjector. The *5 cells were then incubated at 34 0 C and photographed at regular intervals in order to follow the morphological transformations. The results obtained show that 5 hours after the injection of Grb3-3, most of the cells were dead whereas the injection under the same conditions of Grb2 or of the Grb3-3 mutant (G162R) had no effect on the viability of the cells.

(ii) Transfer of the sequence encoding the recombinant protein A plasmid was constructed comprising the sequence SEQ ID No. 1 encoding the Grb3-3 protein under the control of the early promotyer of the SV40 virus.

The 3T3 fibroblasts which are 40 confluent were transfected in the presence of lipospermine (Transfectam, IBF-Sepracor) with 0.5 or 2 tg of this expression plasmid. 48 hours after the transfection, 50 of the cells were in suspension in the medium, and the remaining cells, adhering to the wall, exhibited very substantial morphological changes (Figure 4).

SAnalysis by agarose gel electrophoresis showed, moreover, that the cells had an oligo-nucleosomal

DNA

S 10 fragmentation pattern characteristic of dead cells (Figure In contrast, the cells transfected under the same conditions with a Grb2, Grb3-3 (G162R) or Grb2 (G203R) expression plasmid retain a normal morphology, are always viable and show no DNA fragmentation. As shown in Figure 4, the co-expression of Grb2 makes it possible to prevent the effects of Grb3-3.

These results therefore clearly show that Grb3-3 constitutes a killer gene capable of inducing cellular apoptosis. As indicated above, this property offers particularly advantageous applications for the treatment of pathologies resulting from a cellular proliferation such as especially cancers, restenosis and the like.

Demonstration of the expression of Grb3-3 in lymphocytes infected by the HIV virus This example shows that, during the cycle for infection of the T lymphocytes by the HIV virus, the relative proportion of the Grb2 and Grb3-3 mRNAs is modified, and that the Grb3-3 messenger is overexpressed at the time of massive viral production and cell death.

Peripheral blood lymphocytes were infected with the HIV-1 virus at two dilutions (1/10 and 1/100) for 1, 4 or 7 days. The mRNAs from the cells were then analysed by inverse-PCR by means of oligonucleotides specific for Grb2 and Grb3-3 in order to determine the relative proportion of the Grb2 and Grb3-3 messengers.

The Grb3-3-specific oligonucleotides used are the following: Oligonucleotide IV ATCGTTTCCAAACGGATGTGGTTT

(SEQ

*0 ID No. 8) Oiigonucleotide V ATAGAAATGAAACCACATCCGTTT

(SEQ

ID No. 9) The results obtained are presented in Figure 5. They show clearly that 7 days after the infection with the HIV virus, the Grb3-3 mRNA is overexpressed.

As shown by assaying the p24 protein and the virus reverse transcriptase, day 7 also corresponds to the period during which a massive viral production is observed.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the RA1- exclusion of any other element or integer or group of elements or integers.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: RHONE-POULENC RORER S.A.

STREET: 20, avenue Raymond ARON CITY: ANTONY COUNTRY: FRANCE POSTAL CODE: 92165 (ii) TITLE OF INVENTION: Grb3-3 gene, its 10 variants and their uses.

(iii) NUMBER OF SEQUENCES: 9 (iv) COMPUTER READABLE FORM 0 MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible 15 OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE SYSTEM: PatentIn Release Version #1.25 (EPO) INFORMATION FOR SEQ ID NO.: 1: SEQUENCE CHARACTERISTICS: LENGTH: 933 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 37. .567 OTHER INFORMATION: /product= "Grb3-3"1 (xi) SEQUENCE DESCRIPTION: SEQ ID No. 1: *GAATTCGGC-G CTGCTGAGCA CTGAGCAGGG CTCAGA ATG GAA GCC ATC GCC AAA 54 *Met Giu Ala Ile Ala Lys 1 TAT GAC TTC- AAA GCT ACT GCA GAC GAO GAC CTG AGO TTC AAA AGG GG 102 .Tyr Asp Phe Lys Ala Thr Ala Asp Asp Asp Leu Ser Phe Lys Arg Giy 10 15 GAC ATO OTC AAG OTT TTG AAC GAA GA.A TGT GAT CAG AAC TOG TAC AAG 1 Asp Ile Leu Lys Val Leu As n Glu Glu Cys Asp Gin Asn Trp Tyr Lys 30 OCA GAG OTT AAT GGA AA GAC GGC TTC ATT CCC AAG AAC TAO ATA GAA 198 *Ala Glu Letu Asn Gly Lys Asp Oly Phe Ile Pro Lys Asn Tyr Ile Giu ATG A.AA met Lys CGA GAT Ara Asp CCA CAT CCG TTT GGA AAC GAT GTC CAG CAC TTC AAG GTG CTC Pro His Pro Phe Gly Asn Asp Val. Gin His Phe Lys Val Leu 60 65 GGA GCC GGG AAG TAC TTC CTC TGG GTG GTG AAG TTC AAT TCT Gly Ala Gly Lys Tyr Phe Leu Trp Val Val. Lys Phe Asn Ser 80

S.

S

S

S.

S.

TTG AAT GAG CTG GTG GAT TAT CAC AGA TCT ACA TCT GTC TCC AGA AAC Leu ASn Giu Leu Val Asp Tyr His Arg Ser Thr Ser Val. Ser Arg Asn 90 95 100 CAG CAG ATA TTC CTC CGG GAC ATA GAA CAG C-TG CCA CAG CAG CCG ACA Gin Gin Ile Phe Leu Arg Asp Ile Glu GIn Val Pro Gin Gin Pro Thr 105 110 115 TAC CTC CAG CCC CTC TTT GAC TTT GAT CCC CAG GAG CAT CGA GAG CTC Tyr Val. Gin Ala Leu Phe Asp Phe Asp Pro Gin Clu Asp Gly Ciu Leu 120 125 130 GCC TTC CCC CGG GGA CAT TTT ATC CAT CTC ATG CAT AAC TCA CAC CCC Gly Phe Arg Arg Gly Asp Phe Ile His Val Met Asp Asn Ser Asp Pro 35 140 145 150 AAC TGG TGG AAA GGA GCT TGC CAC CCC CAG ACC CCC ATG TTT CCC CC Asn Trp Trp Lys Gly Ala Cys His Gly Cln lhr Gly Met Phe Pro Arg 155 1 60 165 AAT TAT CTC ACC CCC CTG AAC CCC AAC CTC TAGAGTCAA GMAGCA-ATTA Asn Tyr, Val Thr Pro Val Asr. Arg ASn Val.

170 175 246 294 342 390 438 486 534 584 644 704 764 824 884 933

-TTAACAAA

GACACCAGC

TGGTTGGAAC

TAAATTAAGA

CTTTTTTTTC

ACTCCTAGCT

GTGAAAA-ATG

TGTCAGGGAG

TTTAGGGGGT

AGAGT'-TTTA

TTCCTTTTTT

GACGCCAATA

TAAAACACAT ACAAAAGAAT TAAACCCACA AGCTGCCTCT TGCAGA.ACAC CTCCCGOTC ACCCTCTCAC CCTCTCACTT CGGAGGCC CTTGCATTTA AAAATGCCAA AACTTACCTA TTACAAATTT TCACTCCTGC TCCTCTTTCC CCTCCTTTGT CTCTTCTCTC CATCAGTGCA TGACGTTTAA GGCCACGTAT AAAAACAAGA AACCAAAAAA CCCGAATTC INFORMATION FOR SEQ ID NO.: 2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRMAflEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: OLIGONUCLEOTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2: ATGGAAGCCA TCGCCAAATA TGAC 24 INFORMATION FOR SEQ ID NO.: 3: SEQUENCE

CHARACTERISTICS:

LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear i (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL

SOURCE:

10 ORGANISM: OLIGONUCLEOTIDE

I

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 3: GAATTCGGAT CCATGGAAGC CATCGCCAAA TATGACTTC 39 INFORMATION FOR SEQ ID NO.: 4: SEQUENCE

CHARACTERISTICS:

15 LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL

SOURCE:

ORGANISM: OLIGONUCLEOTIDE

II

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 4: GAATTCGGAT CCTTAGACGT TCCGGTTCAC GGGGGTGAC 39 INFORMATION FOR SEO ID NO.: SEQUENCE

CHARACTERISTICS:

LENGTH: 49 base pairs TYPE: nucleic acid STRANDEDNESS: single 28 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: OLIGONUCLEOTIDE

III

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: GACGTTCCGG TTCACGGGGG TGACATAATT GCGGGGAAAC ATGCGGGTC 49 INFORMATION FOR SEO ID NO.: 6: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids 10 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: Protein .9 (vi) ORIGINAL SOURCE: ORGANISM: hSOS1 Peptide (residues 15 1143 to 1162) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gly Thr Pro Glu Val Pro Val Pro Pro Pro Val Pro Pro Arg Arg Arg 1 5 10 Pro Glu Ser Ala INFORMATION FOR SEQ ID NO.: 7: SEQUENCE

CHARACTERISTICS:

LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: ORGANISM: Peptide 3BP1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 7: Pro Pro Pro Leu Pro Pro Leu Val 1 INFORMATION FOR SEQ ID NO.: 8 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs *i TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: OLIGONUCLEOTIDE

IV

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 8: ATCGTTTCCA AACGGATGTG GTTT 24

**S

INFORMATION FOR SEO ID NO.: 9: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL

SOURCE:

ORGANISM: OLIGONUCLEOTIDE

V

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 9: ATAGAAATGA AACCACATCC GTTT 2

Claims (9)

1. An antisense polynucleotide comprising the complementary strand of the nucleotide sequence of SEQ ID NO:1, which antisense polynucleotide inhibits the expression of Grb2 and Grb3-3.

2. An antisense polynucleotide comprising part of the complementary strand of the nucleotide sequence of SEQ ID NO:1, which antisense polynucleotide specifically inhibits the expression of Grb3-3.

3. An antisense polynucleotide according to claim 2 comprising a sequence complementary to the sequence joining the N-terminal SH3 domain and the residual SH2 domain of Grb3-3.

4. An antisense polynucleotide according to claim 1, 2 or 3 which is an antisense RNA. Vector comprising an antisense polynucleotide according to any one.of claims 1 to 4.

6. Vector according to claim 5, which is a viral 0 vector.

7. Vector according to claim 6, which is derived from an adenovirus, retrovirus, AAV, HSV virus, CMV or vaccinia virus.

8. Vector according to claim 6 or 7, which is 25 defective for replication.

9. Pharmaceutical composition comprising an antisense polynucleotide according to any one of claims 1 to 4 or one or more vectors according to any one of claims 5 to 8 in association with a-pharmaceutically acceptable vehicle. Pharmaceutical composition comprising one or more antisense polynucleotides according to any one of claims 1 to 4, in a form complexed with DEAE-dextran, nuclear protein or lipid, in crude form or incorporated ,4-s 35 into liposomes. -31-

11. An isolated polypeptide having the amino acid sequence encoded by the polynucleotide of SEQ ID NO:1. DATED this ninth day of June 2000. Rhone-Poulenc Rorer S.A. by DAVIES COLLISION CAVE Patent Attorneys for the Applicant