WO1996018640A9 - - Google Patents

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WO1996018640A9
WO1996018640A9 WO9618640A9 WO 1996018640 A9 WO1996018640 A9 WO 1996018640A9 WO 9618640 A9 WO9618640 A9 WO 9618640A9
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nucleic acid
antisense
dna
formula
bond
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  • the present invention relates to an antisense nucleic acid analogue useful as a pharmaceutical. More specifically, by specifically binding to DNA or RNA and forming a complex, transmission of gene information carried by the DNA or RNA is selectively inactivated, activated or The present invention relates to an antisense molecule having a function to control.
  • a gene usually has a form of double-stranded DNA consisting of complementary base sequences, and a coding sequence (sense sequence) having a meaning as amino acid sequence information is encoded on one of the strands.
  • An oligonucleotide complementary to this sequence can specifically bind to the sense sequence, and in the case where this binding is strong, gene function is selectively inactivated by closing a target constant region. , Can be activated or controlled.
  • a compound having such a function is called an antisense molecule.
  • Antisense molecules can thus bind to DNA and inhibit the process by which messenger RNA is generated. It can also bind to RNA and selectively inhibit the process of translation of protein from messenger RNA (Harold M. Weintraub, Scientific American, 34-40 1990). Based on the above principle, an antisense molecule can be useful for elucidating and controlling biological functions, and thus represented by the formulas [10 1] to [1 08] and [1 1 1] to [1 12] Among them, B is a nucleobase, R and R ′ each represent hydrogen, an alkyl group or another substituent). A nucleic acid analogue has been synthesized (Murakami et al., Synthetic Organic Chemistry, vol. 48, No. 3, 180-193. 1990; Chapter Murakami, Cell Engineering, Vol. 13, No. 4, 259-266 1994).
  • A DNA type oligonucleotide derivative (partial structure is shown in the formula [10 1])
  • B RNA type oligonucleotide derivative (a partial structure is shown in Formula [102])
  • ⁇ represents a nucleobase
  • Ri represents hydrogen, an inorganic acid residue, an organic acid residue, an alkyl group, or an asyl group.
  • n represents a natural number.
  • B represents a nucleobase.
  • R represents an alkyl group or a phenyl group.
  • nucleic acid homologs have been synthesized including the above-mentioned compounds (for example, nucleic acid homologs described in US Pat. No. 5,034,506), but a satisfactory effect is necessarily obtained. It has not been developed to develop highly practical antisense molecules fully equipped with the following conditions 1) to 8). 1) Stability of binding to DNA or RNA (complex formation ability)
  • B represents adenine-9-yl, guanine-9-yl, hypoxanthine-9-yl, thymine-1-yl, uracil-1-yl or cytosine-1-yl
  • XY is the same or different and represents a nucleoside analogue represented by hydrogen, hydroxy, halogen or alkoxy
  • a DNA RNA or phosphodiester bond is It has been found that an antisense molecule containing at least one or more in the same strand of a modified or substituted nucleic acid homologue solves the above purpose and has properties as an excellent antisense molecule, and the present invention has been completed.
  • Examples of the halogen contained in the nucleoside represented by the partial structural formula [1] include chlorine, bromine and fluorine.
  • Examples of lower alkoxy include linear or branched ones having 1 to 8 carbon atoms. Specifically, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy or t-butoxy can be mentioned.
  • the antisense molecule of the present invention can exhibit resistance to various nucleolytic enzymes by including a nucleoside having the partial structure represented by the partial structural formula [1].
  • molecules having the nucleoside at the 5 'end or 3' end or at both ends exhibit remarkable resistance to hydroxylase.
  • those containing a plurality of, for example, 2 to 9 residues of the nucleoside analogues continuously or intermittently near both ends can be expected to exhibit stronger resistance.
  • the nucleoside included in the present invention is particularly soluble in water or the like, as compared to a component of a conventional antisense molecule (e.g., a force carbamate-type nucleoside that constitutes a force carbamate-type oligonucleotide derivative (formula [10 8])).
  • a conventional antisense molecule e.g., a force carbamate-type nucleoside that constitutes a force carbamate-type oligonucleotide derivative (formula [10 8]
  • Sex is extremely high. Therefore, the solubility in water and the like does not significantly decrease even if the nucleic acid analogue in which the D NA, R N A or phosphodiester bond is modified or substituted is contained in an unlimited amount of one or more residues.
  • nucleoside is contained in natural D NA or R N A, since the degree of freedom in the steric structure is less limited, the association is stabilized and the function as an antisense molecule is easily exerted.
  • the content of the nucleoside is suitably 1 residue or more and 80% or less of all residues constituting the molecule in one antisense nucleic acid analogue. It is preferable to have one or more residues and an equal number of consecutive nucleosides at each end of 5 'and 3', and the composition ratio is preferably 60% or less of all nucleoside residues of the contained molecule. is there.
  • the homogeneity of the steric helical structure of the side chain is In particular, binding to the recognition sequence It is expected to be superior to the opposite sex.
  • resistance to degradative enzymes is considered to be larger if the structure of the sugar moiety is different from that of the natural form, and should be appropriately selected according to the necessity.
  • the antisense molecule of the present invention is compared with the conventional antisense molecule, while keeping the conditions of 1) above, 2), 3), 4), 5), 6), 7). It has extremely excellent properties.
  • the antisense molecule of the present invention has extremely high solubility in water etc., particularly compared to conventional antisense molecules (eg, carbamate-type oligonucleotide derivatives (Formula [10 8])).
  • Antisense molecules of the present invention can be expected to be highly useful in the medical field because they have a high proportion of binding to highly water-soluble DNA or RNA.
  • the antisense nucleic acid analogue of the present invention When administered in vivo, forms a complex with the cationic ribosome, thereby blocking its transfer into cells, and is equivalent to or more than a nucleic acid such as natural DNA. Demonstrate an antisense effect.
  • Antisense molecules generally have higher activity and selectivity as the number of bases increases.
  • the number of bases contained in one molecule of the antisense nucleic acid analogue of the present invention is preferably 1 to 50, more preferably 4 to 30.
  • nucleotide sequence of the antisense nucleic acid homolog of the present invention is selected to be complementary to the nucleotide sequence of DNA or RNA to be inactivated, activated or controlled.
  • Antisense molecules of the invention are known to be substrates for aseH. This property is shown to be involved not only in gene inactivation but also in actively degrading hybrid RNAs when administered in vivo, and has high utility. As described later, since the antisense molecule of the present invention has a pharmacological action such as an inhibitory action on synthesis of a membrane receptor protein in HER-2 expressing cells, it is very useful as, for example, an anticancer agent.
  • the antisense molecule of the present invention inactivates the complementary genetic information of the antisense molecule according to the present invention, it is possible to use foreign substances that have invaded the living body, for example, hepes virus, influenza virus, human virus It can be used as a medicine to suppress the transmission of oncogenes and infections such as immunodeficiency virus (HIV, AIDS virus etc.). In addition, they can actively control animal and plant genes and be applied to techniques such as variety improvement. Furthermore, it has the potential as a DNA probe for analysis of gene function, infection check of hereditary diseases, bacteria and viruses, etc. It is also possible to find many other uses besides the above description.
  • foreign substances that have invaded the living body for example, hepes virus, influenza virus, human virus It can be used as a medicine to suppress the transmission of oncogenes and infections such as immunodeficiency virus (HIV, AIDS virus etc.). In addition, they can actively control animal and plant genes and be applied to techniques such as variety improvement. Furthermore, it has the potential
  • the nucleoside which is a main component of the antisense nucleic acid homologue of the present invention, is as low in cytotoxicity as natural nucleosides, and thus, the nucleic acid which is also naturally toxic to the antisense molecule in which the nucleoside is contained in DNA or RNA. Equally low.
  • the antisense molecule of the present invention When used as a pharmaceutical, the compound of the present invention is administered as it is or in a pharmaceutically acceptable non-toxic and inert carrier.
  • a pharmaceutically acceptable non-toxic and inert carrier As the carrier, one or more of liquid, solid or semi-solid diluent, filler, and other processing aids are used.
  • the pharmaceutical composition is preferably administered in dosage unit form.
  • the antisense molecules of the present invention can be administered orally, systemically, topically or rectally.
  • administering are administered in dosage forms suitable for these administration methods, for example, various oral agents, injections, inhalants, eye drops, ointments, suppositories and the like.
  • various oral agents for example, various oral agents, injections, inhalants, eye drops, ointments, suppositories and the like.
  • intra-tissue administration and local administration are preferred.
  • the dose varies depending on the type of disease, symptoms, age, body weight etc.
  • topical administration of lmg to lg as an antisense molecule is suitable once a day for adults.
  • the administration method and dose depend on the therapeutic purpose and the type of the antisense molecule of the present invention to be used. It should be changed as appropriate.
  • B is adenine-9-yl, guanine-9-yl or cytosine-1-yl
  • Bp is a protected adenine-9-yl, guanine-9-yl or cytosine- 1-yl or hypoxanthine-9-yl, thymine- 1-yl or uracil- 1-yl which is not protected.
  • Operation 2 The reaction of [1 2] obtained in Operation 1 with, for example, 4, 4 -dimethoxytrityl chloride in a pyridine solution, to obtain a 5'-hydroxy protected dimethytril compound [13] Can.
  • Operation 3 Among the solid phase methods, in order to use the reagent for automatic synthesis by the so-called amidite method, for example, a method such as phosphating agent and Kess Yu et al. (H. Koester et al., Nucleic Acids Res., 124539-4557 (1984) to obtain an amide reagent [14].
  • Operation 4 The dimethyloxytrityl compound [13] obtained in Operation 2 can be reacted, for example, with succinic anhydride in methylene chloride to obtain a succinic acid derivative [15].
  • Operation 5 The compound [15] obtained in Operation 4 is reacted with, for example, pentachlorophenol and dicyclohexylcarbodiimide (hereinafter abbreviated as DCC) in N, N-dimethylformamide to give an activated form [16 ] Can be obtained.
  • DCC pentachlorophenol and dicyclohexylcarbodiimide
  • Operation 6 The activated form [16] is reacted with, for example, a control depore glass carrier (hereinafter abbreviated as CPG carrier) in ⁇ , ⁇ -dimethyl formamide in the presence of triethlyamine to form nucleosideated CP G Carrier [17] can be obtained.
  • CPG carrier control depore glass carrier
  • Operation 7 For example, if a commercially available amidite reagent for DNA synthesis and amidite reagent [14] are appropriately combined and a CPG carrier [17] is used, the DNA synthesizer (eg, Perkin Elmer One) can be used to Antisense DNA homolog [18] containing the nucleoside derivative of the invention can be easily synthesized.
  • the DNA synthesizer eg, Perkin Elmer One
  • the homotype nucleoside prepared by the method of Oki et al. (W095 / 15964) is tetraisopropyldisiloxylated at the 3, 5, -position of formula [19] as usual, and then [20] is synthesized, [20 Trifluoromesylation of the 2'-position of] can give cyclonucleoside [21].
  • sodium halide for example, sodium bromide in DMF
  • the 2-bromide [22] can be obtained.
  • 2, -Bromide [22] as usual, It can be processed to give 2, -ha alpha genohomonucleoside [23].
  • RNA type or phosphorothioate-linked antisense nucleic acid analogue can be synthesized by the method according to the above.
  • the compounds according to the invention can be used therapeutically as free phosphoric acid, but can also be used in the form of pharmaceutically acceptable salts according to known methods.
  • salts sodium salts and potassium salts can be mentioned.
  • the alkali metal salt of the compound having free phosphoric acid according to the present invention can be obtained preferably by adding sodium hydroxide or potassium hydroxide or the like in an alcoholic solvent.
  • the compounds according to the present invention can be obtained by conventional separation and purification means, for example, extraction, concentration, neutralization, It can be isolated and purified by means of filtration, recrystallization, column chromatography, reverse phase chromatography and the like.
  • Solvates (including hydrates) of the compounds according to the present invention or salts thereof are also included in the present invention. Solvates can generally be obtained by removing excess solvent from the corresponding solvent or a suitable mixed solvent containing the corresponding solvent.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail by the following Examples, Reference Examples, Test Examples and Comparative Examples, but the present invention is not limited thereto.
  • nucleotide analogues represented by the formula [11] obtained by the method of Ohki et al. (WO 95/15964) 2.60 g of adenine base (hereinafter referred to as homotypic adenosine) suspended in 30 ml of pyridine After stirring and adding 8.43 g of benzyl chloride under ice-cooling, the mixture was allowed to react at room temperature for 1 hour. The above reaction mixture was added to a mixture of 100 ml of chloroform, 70 g of ice and 5.48 g of sodium hydrogen carbonate, and the mixture was separated to separate an organic layer. The remaining aqueous layer is further extracted twice with 50 ml of methylene chloride, and the organic layer is After combining and drying sodium sulfate, the solvent was distilled off under reduced pressure.
  • adenine base hereinafter referred to as homotypic adenosine
  • the precipitated salt was filtered off with a glass filter, and the filtrate was concentrated under reduced pressure to dryness.
  • the residue was partitioned between ethyl acetate and saturated sodium hydrogen carbonate solution each saturated with nitrogen gas, and the obtained organic layer was dried over sodium sulfate and concentrated under reduced pressure to dryness.
  • the residue was triturated with n-hexane saturated with nitrogen gas to obtain 174 mg of a compound [14] (wherein Bp is for N 6 -benzyladenine-9-yl) as a white powder.
  • the molecular weight was measured by high-speed atomic impact mass spectrometry (hereinafter abbreviated as FAB method). The amount was 871.
  • This compound [14] was used below as a reagent for an automatic DNA synthesizer (Perkin-Elmer One).
  • CPG carrier [17] (where Bp is N 6 -benzyladenine-9-yl) corresponding to nucleic acid base l ⁇ M was loaded on a column for automatic DNA synthesizer. 170 mg of the amidite reagent [14] was dissolved in 3.4 ml of anhydrous acetonitrile.
  • amidite reagent [N 6 -benzyl-5,0- (4,4, -dimethyxitylytyl) -2,2-deoxyadenosine 3'-0- (2-cyanoethyl N, N-diisopropylphosphoamida It) (Perkin-Elmer One) 500 mg was also dissolved in 5 ml of anhydrous acetonitrile, mounted on an automatic DNA synthesizer (Perkin-Elmer One) with these two reagents, and subjected to automatic synthesis according to the program of the synthesizer.
  • the sample solution (conc. Ammonia solution) in the collection vial was treated at 55 ° C. for 18 hours, and then concentrated under reduced pressure to dryness.
  • the residue was dissolved in 5 ml of 50 m acetic acid-triethyl ammonium buffer (PH 7.0) (hereinafter abbreviated as TEM buffer), and preparative reverse-phase chromatography (Preparative RP-18 (55-105 m), 125 Onk, Preparation and purification were carried out using a stream, 1 mm x 100 mm column Waters Co., Ltd.).
  • acetonitrile in 50 mM TEAA buffer.
  • an orange-colored fraction was collected when acetic acid was added, the solvent was distilled off under reduced pressure, 5 ml of 80% acetic acid was added, and the mixture was left at room temperature for 15 minutes. After confirming by TLC that the protective group had been removed, the solvent was evaporated under reduced pressure. Liquid separation extraction was performed with ethyl acetate and water, and the aqueous layer was evaporated to dryness to obtain 80 OD (260 nm) of the desired product.
  • CPG carrier [17] (wherein Bp is thymine-1-yl) corresponding to nucleic acid base 2 ⁇ M was loaded on a column for an automatic DNA synthesizer.
  • Amidite reagent [14] (provided that Bp is thymine-yl), a commercially available amidite reagent for DNA synthesis, [N 6 -benzoyl-5,-0- (4,4,-dimethyxitylytyl] )-2,-deoxyadenosine 3,-0-(2-cyanoethyl ⁇ , ⁇ -diisopropylphosphoramide ⁇ )], [ ⁇ 2 -isopyryl-5,-0-(4,4,-dimethyxitylytyl) -2, -deoxyguanosine 3'- 0- (2-cyanoethyl ⁇ , ⁇ -diisopropylphosphoramide ⁇ )], [ ⁇ 4 -benzyl -5,-0- (4,4,-dimethyxitylytyl) -2, -Deoxycytidine 3, 0-(2-cyanoethyl ⁇ , ⁇ -diisopropylphosphoramidite)]
  • hA 25 3 'to 5' phosphodiester linkage
  • Example 4 DNA-type antisense molecules containing nucleosides which are X 2 H, Y 2 H in the formula [1]: Of the homologs, those of which the base is adenine-9-yl, A is deoxyadenosine Each represents. Unless otherwise stated, the internucleoside linkage is 3 'to 5' Stel bond is shown. The following composition of hA 2 A 23 ).
  • the linkage between nucleosides is 3
  • the ' ⁇ 5' phosphodiester bond is shown, hereinafter referred to as hAwA) synthesis.
  • the linkage between nucleosides indicates a phosphodiester linkage of 3, ⁇ 5, below, which is hereinafter referred to as “hA 18 A?” Synthesis.
  • EXAMPLE 8 A DNA-type antisense molecule comprising a nucleoside that is X 2 H, Y 2 H in Formula [1]:
  • Example 1 Toxicity test on mouse fibroblasts
  • a complex with a cationic ribosome was also used in the test.
  • the components of the cationic ribosome are 3-0- (2-jetyl aminothiol) power lvamoyl-1,2- 0-dioleyl glycerol (see W094 / 19314 published) and egg yolk phosphatidylethanolamine (Nippon Yushi (stock)
  • the mixing ratio of 3: 1 was used.
  • Each test substance was added to the culture medium at a nucleoside concentration of 10, 3, 1 or 0.1 zM, respectively.
  • the mixing ratio between the cationic liposome and the antisense oligonucleotide was 2: 1.
  • the NIH3T3-HER2 cells were seeded at a density of 10 5 cells / well in a Koning's 24-well plate, and then cultured overnight at 37 ° C.-5% CO 2 in Dulbecco's modified Eagle medium. .
  • each test substance was added to the supernatant and cultured overnight.
  • the results are shown in Table 1. Table 1. Cytotoxicity test
  • NT unexamined phosphorothioate type oligonucleotide alone has relatively high cytotoxicity, and its toxicity is further enhanced when complexed with dynamic ribosome, while the antisense molecule of the present invention is not effective. Even when added alone, it did not show toxicity up to a concentration of ⁇ ⁇ ⁇ , and even when administered as a complex with a cationic ribosome, up to a concentration of 1 ⁇ M.
  • Example 2 HER-2 synthesis inhibitory activity and toxicity test of 5 ′ t CCGGTCCCAATGGAGGGGA At 3 ′ synthesized in Example 2
  • the inhibitory effect on the protein synthesis in cells of 5, t CCGGTCCCAATGGAGGGGAAt3 'obtained in Example 2 was observed.
  • This nucleotide sequence shows an antisense sequence near the 5, cap region of the HER-2 gene, which is one of the EGF receptor family and considered to be involved in malignancy of breast cancer (Woolrich et al., Science, 230 1132-1139 1985).
  • a DNA of the same sequence and a phosphorothioate type oligonucleotide of the sequence of 5 'CGG TCCCAATGGAGGGGAAT 3' were used as a control.
  • the DNA was prepared by the method described in Test Example 1 according to a conventional method using a DNA synthesizer (Applied Biosystems Inc. Model 380 B) and phosphorothioate type oligonucleotides.
  • the NIH3T3-HER2 cells shown in Test Example 1 were used as the cells.
  • a complex with a cationic ribosome was administered to the cells.
  • the cationic ribosome used and the method of addition were the same as in Test Example 1.
  • Each test substance was added to the culture medium to give a nucleoside concentration of 1, 0.1 or 0.01 iM, respectively.
  • NIH3T3-HER2 cells were seeded at a density of 10 s cells / well in a Corning 24-well plate, and then cultured overnight in Dulbecco's modified Eagle's medium at 37 ° C.-5% CO 2 .
  • the medium was changed to a medium containing 0.2% calf serum, and after 7 to 8 hours after the first addition of the test substance, the calf serum concentration was increased to 5% overnight.
  • the medium was changed to a medium containing 0.2% calf serum not containing methionine, and then each test substance was added a second time. The added concentration is the same as the first time. After 7 to 8 hours, 35 S methionine (Amersham, 3TBq / olol) was added to culture the calf serum concentration to 5% overnight.
  • test substance in which the cell morphology was determined under a microscope the test substance in which the cells were significantly atrophy was cytotoxic (indicated by + in the table), and the test substance in which the cell morphology was not changed was not cytotoxic (in FIG. Was represented.
  • HER-2 protein was immunoprecipitated by an ordinary method using an anti-human HER-2 antibody (Nichirei).
  • the antisense molecule of the present invention showed a synthetic inhibitory activity of ⁇ -2 protein equal to or higher than DNA at the same concentration as DNA.
  • Phosphorothioate type oligonucleotides have no effect at 0.01 to 0.1%, and cytotoxicity is expressed at a concentration of 1.0 ⁇ M at which the antisense molecule of the present invention or DN ⁇ exhibits effective activity, and the activity can be measured. won.
  • the sense sequence near the 5 'cap region of the HER-2 gene of the compound and DNA of the present invention does not show synthetic inhibitory activity of HER-2 protein.
  • Resistant compounds of the present invention hA 25 against nucleolytic enzymes of Test Example 3 present compound (Nuclease SI) was the test substance. As controls opening one Le, using natural DN A oligomer dA 25 to have the same sequence.
  • the 5 't CCGGTCCCAATGGAGGG GAAt3' of the substance of the present invention synthesized in Example 2 was used as a test substance.
  • a natural D NA Aori mer having the same sequence was used as a control substance.
  • the 5 't CGGTCC C CAATGGAGGG GAAt3' of the substance of the present invention synthesized in Example 2 was used as a test substance.
  • As a control natural DNA oligomers having the same sequence were used.
  • test substance was prepared in 100 mM ammonium succinate buffer solution (pH 8.9) and 1 mM EDTA solution to a final concentration of 4 OD (260 nm), and calf spleen phosphodiesterase (Boehringer) was adjusted to a concentration of 0.2 U / ml. Was added (final volume 0.1 ml). Incubate at 37 ° C. for 1 hour, sample the reaction solution, and use the Lichro-spher RP-18 (4 thighs; LD. X 125%) column as a high-performance liquid chromatograph (hereinafter abbreviated as “HP LC”) as the test substance. The degree of degradation was calculated by measuring the degree of reduction of the peak of The results are shown in Table 5. Table 5. Resistance of the substance of the present invention to calf spleen phosphodiesterase
  • Control 27.6 While the control is degraded by nearly 30%, the degree of degradation of the substance of the present invention is 1% or less, showing strong resistance to calf spleen phosphodiesterase.
  • Test Example 6 Substrate Specificity of RNase H for the Substance of the Present Invention
  • the substance of the present invention formed a hybrid with natural RNA, was recognized as a substrate for RNase H, and it was examined whether or not the RNA was degraded.
  • RNA forming a Haipuriddo poly (U) (S 2 o , w 6.6, Yamasa Shoyu Co.) was used.
  • hAisA 96.6 hAi 8 A7, hAi 4 Ai And hA 2 A 23 were shown to be RNase H substrates to the same extent as dA 25 used as a control.
  • Test Example 7 Binding stability to I3NA or RNA
  • the absorbance increases rapidly before and after a certain temperature as the temperature is raised. This is due to a decrease in the so-called hypochromicity of the nucleic acid, but by measuring the temperature (hereinafter referred to as Tm) when the absorbance has increased to half the absorbance difference before and after this, the nucleic acid and Know the complex formation ability of
  • HA25 as the test substance, hA 2 2 A3, i8A7, hAi4Ai i, the hAi oAi 5, hA 6 Ai 9 and hA 2 A 23, was used dA 25 as a control.
  • Poly (U) (S 20 , w 6.6, Yamasa Syoyu Co., Ltd.) was used as the RNA for forming the hybrid.
  • test substance or control is complexed with Poly (U), and the test substance concentration is 100 / M, 0.15 M sodium chloride, 10 mM sodium phosphate (pH 7.0), heating rate
  • test substance concentration is 100 / M, 0.15 M sodium chloride, 10 mM sodium phosphate (pH 7.0), heating rate
  • the Tm values were determined under the conditions of 0.5 ° C./min. Table 7. Tm values of the substances of the present invention to Poly (U)
  • the substance of the present invention had a good ability to form a complex with natural RNA.
  • the Tm value tended to decrease as the content of nucleoside homologs increased, but it was also shown that the content up to 40% (hAioA 15 ) is comparable to natural DNA. From these results, the antisense molecule of the present invention has low toxicity, and can be used as a medicine for suppressing infection with herpes virus, influenza virus, human immunodeficiency virus and the like and the function of oncogenes.

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