OLIGONUCLEOTIDES WITH RNA CLEAVAGE ACTIVITY

Abstract: The present invention relates to oligonucleotides with RNA cleavage activity called ribozymes having sequence (I) in which ----X and Y---- are target specific RNA recognition sequences, A, C, G and U are deoxynucleotides and at least one a and/or g is a 2'-substituted adenosine or guanosine derivative respectively, P is either a) A-U or b) C-G and, W is either i) a nucleotide loop sequence or ii) dial bridges connected with phosphodiester or substituted neutral phosphotriester derivative linkages.

Description: Oligonucleotides with RNA Cleavaσe Activity The present invention relates to oligonucleotides w th RNA cleavage activity. RNA is known to have endoribonuclease activity Catalytic RNA molecules called ribozymes have been used to bind to target RNA molecules and catalyse their cleavage, thus blocking tne activity of the target RNA. The so-called hammerhead ribozymes have been widely studied. The recognition site ana catalytic site of these oligoπbonucleotides are well characterised and πcozymes containing a recognition sequence specific for any αesired target RNA which contains a specified triplet can oe constructed. These compounds can therefore ne considered as potential therapeutic agents with possibly higher biological activity than the simple antisense oligonucleotides 111 . The structure of a typical hammerhead πbozyme is snown m Figure 1. A G C and U are all πbonucleotides . Experiments show that externally supplied ribozymes produce only a transient effect. The catalytic effect is destroyed once the ribozyme nas been degraded. The accessibility of the target site also limits ribozyme cleavage activity. Protein oounα at the cleavage site has been shown to block ribozyme activity. Further, ribozyme catalytic efficiency has oesn shown to depend on whether the ribozyme expression occurs in the same cell compartment as that occupied by the target DNA. The hammernead ribozyme has been shown to have no effect in prokaryot c cells wnen the ribozyme and target RNA were generated from different genes, whereas it can function at a 1:1 riDozyme : target ratio if co-localization m the same cell is maintained. Studies' 11 have shown that a high (1000:1) ribozyme: suns rate ratio is needed for inhibition m vivo in eukaryotic cells. This suggests that the catalytic potential of the ribozyme is not being achieved. Eckstein et al introduced 2' -ammo or 2' -F substituents into the pyπmidine positions of the hammerhead ribozyme (21 m The cleavage positions of the r oozymes in cellular extracts were not determined but the all-pyr idme substitutions together with phosphorothiolate substitutions at the 3' terminus gave compounds with a markedly increased stability. The influence of these chemical modifications on the catalytic activity of the riDoz mes was negligible. Ξproat et al {i found that ribozymes containing 2'-0-Allyl substituents m all but six positions of the catalytic core are resistant to nuclease attack to some extent ana retain their catalytic activity. Cedergreen et al { S ) found that mixeα DNA / RNA oligo ers with 4-7 noopositions are active in cleaving substrate RNA. These oligomers are three orders of magnitude more stable than the all-RNA ribozymes in incubation w th RNase A and yeast extract. Recent results have suggested that the nbonucleot de backoone is not a strong requirement for catalytic cleavage of mRNA 141 . When the 2' -OH groups m the ham ernead structure were systematically replaced with H residues it was observed that the presence of only four 2 ' -OH groups at defined positions m the catalytic core (the core being nucleot des 3 to 15.1 shown m Figure 1) were necessary to snow catalytic activity. Referring to Figure 1, these positions are G 5 , G j , A 9 and A 15 1 If the A\ A 1 * and one nucleotide in helix III (possioly C 1S l) were also left unmodified, catalytic activity was found to reach 1/lOth of the all-2'-0H RNA ribozyme. If the suDstituents were 2'-0-Allyl groups the essential 2' -OH groups needed for catalytic activity were found to be ϋ\ G' , A6 , G a , G 12 , and A 15 L . Those snown m bold are positions identical to the DNA analogue. The kcat/ for this ribozyme was only 5 times lower than for the all 2' -OH ribozyme. Other modifications of the 2' -OH groups in the catalytic core of the hammerhead ribozyme have been reported. Williams et al"" found only a 15-fold, reduction m catalytic activity if the 2' -OH groups at G 5 and G 8 are replaced with 2' -NH- and tne 2' -OH group a t G 12 is replaced with a 2'-F group. Olsen et ai (91 noted only a small decrease m catalytic activity if the 2' -OH group at one of the following adenosmes : A s , A 9 or A 15 - 1 , A 14 , A 13 is re D laced by 2 '-F. Replacement of more 2'-0H-s, however, s not allowed with this substituent. Pyle ec al (6) studied the Tetrahymena ribozyme and showed that tne essen t ial -OH groups are important for stabilising base-backbone tertiary interactions by hydrogen bonding. It is not clear, however, whether this is the case, or whether the 2' -OH groups are important for the direct binding of the catalytically important metal ion. Perreault et al π suggest t ha t t he 2 ' - OH groups of A 9 and G 3 are directly involved m Mg 2 ' Dindmg. In a recen t model McLaughlin et al α > proposed a role for the 2' -OH as of G s or G 8 . They interact with H 2 0 molecules bound in the first co-ordination sphere of the Mg 2* cofactor. In an effor t t o determine the minimal sequence requirements for ribozyme ac t ivity Jennings et al (11) observed that the catalytic ac t ivi t y of a hammerhead ribozyme in which the loop of nucleo t ides 1 0.1 to 11.1 (see Figure 1) is replaced by four deoxyuridines decreases only by a factor 3 when compared with tne original s t ructure. This modified structure has been termed the "mmiribozyme structure" . F or exogenous application of preformed synthetic ribozymes several impor t ant problems can be identified. It is desirable to: 1 . increase stability against RNase degradation 2 . increase stability against exonuclease degradation and 3. facilitate cellular uptake. The present invention provides in a first aspect oligonucleotides having the following sequence 5 ' X a Y a C U A g g a A C - G g U P w in which X and Y are target specific RNA recognition sequences A is deoxyadenosine C is deoxycytidine G is deoxvguanosine U is deoxyuridine a is 2' substituted deoxyadenosine according to formula la below, or a 2' substituted arabinofuranosyl adenosine according to formula Ic.below, or adenosine g is 2' substituted deoxvguanosine according to formula lb below or a 2' substituted arabinofuranosyl guanosine according to formula Id below, or σuanosine aαemns la lb adenme HO ne HO R Id in which R is selected from -CF j H ; -COOH ; -CONH, ; -NHCORi ; - SH ; and combinations thereof; R_ being -H, -C(0)OH, or -CH 2 -C(0)OH and wherein at least one of the groups a or g of the oligonucleotide is other than adenosine or guanosine respectively; and in which P is either a) A-U or b) C-G W is either i) nucleotide loop sequence Ha if P is A-U; 3' 5' -G-G-A-G-C-C-C-C- 11a or nucleotide IOOD seσuence lib if P is C-G 3' -C-C-G-U-U-A-G-G- Ilb or ii) diol bridges III connected with phosphodiester or substituted neutral phosphotriester derivative linkages m(Z) III in which each diol bridge (Z) is of formula IV [ 0 - (CH 2 ) n - 0 ] - IV n is 1-10 and m diol bridges are connected together, m being 2-10. There is no requirement that the length of the diol be the same in each diol bridge; n may be' independently selected for each bridge from l to 10. Preferably n is 3 in each bridge and there are four bridges; m is 4. X and Y are target specific recognition sequences made up of any deoxyribonucleosides N depending on the target RNA sequence. X and Y may be of the same or different length. There is no need for the molecule to be symmetrical. Each may be 4 to 25 nucleotides long, preferably 6 to 20 nucleotides. If X and Y are too short the oligonucleotide looses its specificity. X or Y may include stabilising modifications. For example, two or three natural 3' -5' phosphodiester linkages at at least the 3' end of X may be modified in an attempt to protect the oligonucleotide from attack by 3' -exonucieases. Particularly, 3' -5' phosphodiester linkage may be replaced by phosphorothiat-e linkages such as thiophosphodiester linkages. The 2'-R substituent is a non-nucleophylic group which is isoteric and isopolar with the replaced -OH group. The substituent has both H-bond donor and acceptor abilities. A 2' -COOH substituent, which can chelate withMg 2* , is advantageous because it is known to be important for Mg J* binding. The modifications according to the invention which involve the 2'-R substitution of nucleotides in the catalytic/cleavage region of the oligonucleotide can provide a desirable increase in stability against degradation and can increase catalytic activity due to improved Mg 2* binding. Replacement of the -G-G-A-G-C-C-C-C- loop sequence or the -C-C-G-U-U-A-G-G- loop sequence with aliphatic diol oπdges simplifies the large scale synthesis of these oligonucleotides and eliminates unwanted mtermolecular interactions. Oligonucleotides according to the invention with diol bridges can ne made by machine more easily and more cheaply than conventional ribozymes. The modifications according to the invention wnich involve substituting dials in the loop region of the oligonucleotide can provide a desirable facilitation of cellular uptake. The oligonucleotides of the present invention may be used as intermediates for further modification to improve their ease of up-take by the cell (in comparison to unmodified oligonucleotides of the invention and known ribozymes) , for example by the attachment of carrier molecules. The emoodiments navmg diol bridges are considered to be particularly useful as intermediates for this purpose. The oligonucleotides of the present invention are potential antagonists of a wide range of therapeutic targets whicn involve over-expression of products. By binding to specific targets on mRNA and cleaving the mRNA they can stop translation and hence "switch off" a specific gene. In cases where expression of a proαuct (e.g. an enzyme or a protein) by a gene is causative of an illness or disfunction this could lead to a cure. Alternatively a putative gene as a source of a problem phenomenon could be "switched off" selectively ana the tnerapeutic effect observed. The oligonucleotides of the present invention are potential anticancer and antiviral agents and could also be used as ant -inflammatory and anti-ulcer drugs. The stability of the oligonucleotides of the present invention suggests that they could be used in nano olar amounts. Th s offers a significant improvement over known ribozymes which have to be used in relatively large amounts to compensate for tneir mtracellular degradation by nucleases . The oligonucleotides of the invention may achieve true catalytic activity: i.e. they will not be destroyed in the cleavage reaction. The invention can also provide novel intermediates which are 2' substitutednucleosides needed to construct the oligonucleotides of the present invention. Such reactive intermediates may be protected where appropriate by a removable protecting group. The invention can also provide a method which comprises the synthesis of an oligonucleotides according to the invention from building blocks including deoxynucleotides and 2' substituted nucleosides . Fiσures Figure 1 shows the nucleotide sequence of a hammerhead ribozyme. Figure 2 shows an example of therapeutic target interaction between the myc oncogene mRNA and an oligonucleotide of the invention of the type described in Example 1. Figure 3 shows a synthetic scheme for producing 2' - CF 2 H substituted nucleosides. Figure 4 shows a synthetic scheme for producing 2 ' - COOH substituted nucleosides. Figure 5 shows a synthetic scheme for producing the nucleoside 9- (2' -C-difluoromethyl - β - D arabino - furanosyl) - 6 Ethoxy purine. Examples The following are given as examples of the present invention. Example l An oligonucleotide having the following sequence 3 ' N.N. N.N N N N N a N N N N N N N N _. C U *. g g a A C - G g U A - U W in which N represents any deoxyribonucleotide recognition sequence specific for the target RNA N.N represents a thiophosphodiester linkage replacing a natural 3' -5' phosphodiester linkage between two nucleotides A is deoxyadenosine C is deoxycytidine G is deoxyguanosine U is deoxyuridine a is 2' substituted deoxyadenosine according to formula la below g is 2 ' substituted deoxyguanosine according to formula lb below e la lb in which R is selected from -CF j H ; -COOH ; -CONH 2 ; -NHCOR l ; - SH ; and combinations thereof; R. being -H, -C(0)OH or -CH 2 -C(0)0H and W is nucleotide loop sequence Ila. 3' 5' -G-G-A-G-C-C-C-C- Ha Example 2 An oligonucleotide having the following sequence 3' 5' N.N. .N N N N N a N N N N N N N N a C U A g g a A C - G g U C - G W in which N represents any deoxyribonucleotide recognition sequence specific for the target RNA N.N represents a thioohosphodiester linkage replacing a natural 3' -5' phosphodiester linkage between two nucleotides A is deoxyadenosine C is deoxycytidine G is deoxyguanosine U is deoxyuridine a is 2' substituted deoxyadenosine according to formula la below g is 2' substituted deoxyguanosine according to formula lb below guanme la in which R is selected from -CFjH ; -COOH ; -CONH, ; -NHCOR,. ; - SH ; and combinations thereof; ?_._ being -K, -C(0)OH or -CH 2 -C(0)OH and W is nucleotide loop sequence lib. 3' 5' -C-C-G-U-U-A-G-G- lib Example 3 An oligonucleotide having the following sequence 3' 5' N.N.N.N N N N a N N N N N N N N C U g a A C - G g U A - U -O- (CH 2 ) 3 -0- -O- (CH 2 ) : -0- -O- (CH 2 )--0- -O- ;CH,),-O- in which N, N.N, A, C, G, U, a and g are as defined for Example 1 and the diol bridges are connected with p hosphodiester or substituted neutral ohosohotriester derivative linkages. Example 4 An oligonucleotide having the following sequence m which N, N.N, A, C, G, U, a and g are as defined for Example 1 and the diol bridges are connected with phosphodiester or substituted neutral phosphotπester derivative linkages. Example 5 A therapeutic target interaction between mRNA and an oligonucleotide of the invention is shown in Figure 2. The -myc oncogene mRNA secondary structure is shown from nucleotide l to nucleotide 900. Translation starts at nucleotide 421 and the triplet cleavage site s positions 433 to 435 (GUU) . Example 6 A scnemat c synthesis for 2' -CF 2 H substituted nucleosides s shown in Figure 3. 3 s a purine or pyπmidine heterocycie, .e. adenine, cytosme, guanme or uracil which may be protected where appropriate. Compound 7 wherein 3 is 6 - Ethoxypurme is 9 - (2'- C difluoromethyl D - arabinofuranosyl) - 6 - Ethoxypurme Example ** A schematic synthesis for 2 ' - C00H substituted nucleosides is snown in Figure 4 3 is as given m Example 6 Compound 14 wnere 3 s Undine is 3- (2' - C - Carboxyl- Uridme D - arab o furanosyl) - Uridme Example 8 Syntnesis of 2' -D fluoromethyl Substituted Nucleoside One group of novel analogs are 2 ' -difluoromethyl sunstit ted nucleoside analogs 3elow is described tne synthesis for a representative analog corresponding to Compound 7 of Example 6 The synthetic scneme is snown in Figure 5 S-Ethoxy-N9-S-D-rιoofuranosylpurιne was prepared according to literature procedures from nosme. S-Et oxy-3' -5' -0- (1, 1,3 ,3 -tetra sooropylάisiloxyl) -V9-S-D- ribofuranosylpunne (1) To a solution of 1 (1.67 g, 5.64 mmol) m pyridme (50 mi) TIPDS Cl, (1.94 ml, 6.20 mmol) was added. The mixture was stirred at rt for 3 hrs , evaporated and coevaporateα w th toluene (20 ml) . The oily residue was partitioned oetween water (50 ml) and diethylether (150 ml) . Combined organic layers were dried with sodium sulfate, filtered and evaporated to give a yellowish oil (2.98 g, 98%) FCC (5 cm x 15 cm, petrol ether/ethylacetate 2.1) yielded 2.37 g (78%) of a colorless oil . 1H-NMR: 8.45 (s, 1H) , 8.07 (s, 1H) , 6.02 (d, 1H) , 5.07 (m, 1H) , 4.64 (q, 2H) , 4.54 (d, 1H) , 4.10 ( , 3H) , 3.24 (s, 1H) , 1.50 (t, 3H) , 1. 1 0 (m, 28H) . 6-Ethoxy-3' ,5'0- (l,1.3, -tetraisopropyldisiloxyl) -N9-b-D- rιbofuran-2-ulosylPurιne (2) To a suspension of chromiu (VI) oxide (0.400g, 4 mmol) dichloromethane (12 ml) pyrid e (0.644-mi, 8 mmol) was added and the αeep rεα solution was stirred at rt for 10' . Subsequent addition of 2 (0.539 g, 1 mmol) and acetic anhydride (0.38 ml, 4 mmol) gave a brownish suspension. After stirring at rt for 1 hr the mixture was added to a supernatant of ethylacetate (80 ml) over a 3 cm layer of silica gel in a 3 cm column. After washing the column with additional 50 ml of ethylacetate the filtrate was evaporated (waterbath below 25°C!) . Subsequent coevaporation with toluene, chloroform and diethylether (30 ml each) yielded a colorless oil (0.054 g, 94%) . 3 was directly used for the synthesis of 4. ό-Ethoxy-2' -f1, 1-difluoro-l- (phenylsulfonyl)methyl} -3' , 5' -O- (1. 1,3, 3-tetraisopropyldisiloxγl) -N9- β -D-arabinofuranosylpurine(3) To a solution of 3 (0.404 g, 0.75 mmol) in THF (10 ml) and HMPA (1 mi) difluoromethylphenylsulfone (1 mmol, 0.192 g) was added and the mixture was cooled to -78°C. LDA (1 ml of a 1.5 M solution in THF, Aldrich) was added and the reaction mixture was stirred at this temperature. TLC after 1 hr indicates complete conversion. After warming to rt saturated aqueous ammonium chloride (20 ml) was added and the mixture was extracted with chloroform (100 ml) . Combined organic layers were dried with sodium sulfate, filtered and evaporated to yield 1.32 g of a brownish oil . FCC (3 cm x 15 cm, dichloromethane/methanol 97.3) afforded 4 as a yellowish foam (0.33 g, 61%) . 1 H-NMR: 8.41 (s, 1H) , 8.18 (s, 1H) , 7.84 ( , 2H) , 7.73 (m, 1H) , 7.54 ( , 2H) , 6.73 (s, 1H) , 6.29 (s, 1H) , 4.99 (d, 1H) , 4.67 (q, 2H) , 4.05 (dd, 2H) , 3.98 (m, 1H) , 1.49 (t, 3H) , 1.15 (m, 28H) . 6-Ethoxy-2' -C-difluoromethyl-N9-S-D-arabinofuranosylPurine (7) To a mixture of 4 (0.218 g, 0.3 mmol) and a 0.1 M solution of S lj in THF (1 5 ml, 1.5 mmol) HMPA (0. 60 ml, 3.45 mmol) was added. The color changed from deep blue to purple. The mixture was stirred at rt. The purple color changed gradually to greenish within 10' after which TLC indicated complete reaction 2 ml of saturated aqueous ammonium chloride were added and the resulting mixture was evaporated. The residue was partitioned between saturated sodium thiosulfate (40 ml) and ethylacetate (50 ml) . Combined organic layers were dried with sodium sulfate, filtered and evaporated to yield 0.187 g (106%) of a brownish foam. The crude 4 (homogeneous on tic) was dissolved in THF (10 ml) and TBAF (0. 1 M in THF, Aldricn, 0.6 mi, 0.66 mmol) was added. The yellowish solution was stirred at rt overnight. After evaporation the residue was purified by FCC (2 cm x 12 cm, chloroform/methanol 9:1) to give a colorless amorphous solid (70 mg, 67%) . 'H-NMR: 8.52 (s, 1H) , 8.42 (s, 1H) , 6.46 (m, 2H) , 6.23 (t, 1H, J = 50Hz) , 6.14 (d, 1H) , 5.21 (m, 1H) , 4.58 (m, 2H) , 4.36 (m, 1H) , 3.86 (m, 1H) , 3.67 (m, 2H) , 1.41 (t, 3H) . 19 F-NMR spectra of compounds 3 and 7 are in total agreement with the above structures. ό-Ethoxypurme is a synthetic svnthon for adenine; we did not convert to an adenosine derivative on the nucleoside level, because the 6-ethoxy group is a suitable base protecting group during oligonucleotide synthesis. ibliographv ( 1 ) Cotten, M. ; Trends in Biotechnology 1990, 8, 174-178. ( 2 ) Pieken, W.A., Olsen, D.B., Benseler, F., Aurup, H., and Eckstein, F. Science 1991, 253, 314-317 ( 3 ) Heidenreich, O. and Eckstein, F. J. Biol . Chem . 1992, 267, 1904-1909 ( 4 ) Paoella,G. ; Sproat,B.S.; Lammond, A. I, EMBO Journal 1992, 11, 1913-1919 (5 ) Yang, J.-H. ; Usman, N.; Chartrand, P.; Cedergreen, R.; Biochemistry 1992, 31, 5005-5009. ( 6 ) Pyle, A.M. ; Murphy, F.L.; Cech, T.R. Nature 1992, 358, 123-128. ( 7 ) Perreault, J.P.; Labuda,D.; Usman, N. ; Yang, J-H. ; Cedergreen, R. Biochemis ry 1991,30, 4020-4025 ( 8 ) Williams, D.M. ; Pieken, W.A.; Eckstein, F. ; P . N. A . Ξ . 1992, 89, 918-921. ( 9 ) Olsen,D.B. ; Benseler, F.; Aurup,H, . Pieken,W.A. and Eckstein, F. Biochemis try 1991, 30, 9735-9741 ( 10 ) Fu D.J. and McLaughlin L.W. Biochemistry 1992 31 10941-10949 (13 ) McCall,J.M. ; Hendry, P.;Jennings P.A. P . N. A . S . 1992 89 5710-5714

Claims: Claims 1. An oligonucleotide having the following sequence 3' ■ X a Y a C U A g g a A C - G g U P w in which X and Y are target specific RNA recognition sequences A is deoxyadenosine C is deoxycytidine G is deoxyguanosine U is deoxyuridine a is 2' substituted deoxyadenosine according to formula la below, or 2' substituted arabinofuranosyl adenosine according to formula Ic below or, adenosine g is 2' substituted deoxyguanosine according to formula lb below, or a 2' substituted arabinofuranosyl guanosine according to formula Id below, or guanosine nine la lb adenine ic HO Id in which R is selected from -CF 2 H ; -C0OH ; -CONH, ; -NHCOR, ; SH ; and combinations thereof; t being -H, -C(0)OH or (CH,)C(0)OH and in which and wherein at least one of the groups a or g of the oligonucleotide is other than adenosine or guanosine respectively; P is either a) A-U or b) C-G W is either i) nucleotide loop sequence Ila if P is A-U; 3' -G-G-A-G-C-C-C-C- Ila or nucleotide loop sequence lib if P is C-G; 3 ' 5 ' - C- C-G-U-U-A-G-G Ufa or i ) diol bridges III connected with pnosphodiester or substituted neutral phosphotπester derivative linkages m (Z) III wn ch each diol bridge (Z) is of formula IV - [ 0 - (CH 2 ) n - 0 ] - IV n is 1-10 ana m diol bridges are connected together, nemg 2 to 10. 2. An oligonuceotide according n claim 1 in which there s more than one appearance of a 2' substitution m the sequence. 3 An oligonucleotide according to claim 1 or 2 in which W is diol bridges III and n is 3 in each diol bridge and is 4. 4. An oligonucloetide according to any preceding claim in which X and Y, which may be the same or different length, are each 4 to 25 nucleotides long. 5. An oligonucleotide according to claim 4 in which X and Y are each 6 to 20 nucleotides long. 6. An oligonucleotide according to any preceding claim in which X or Y include stabilising modifications. 7. An oligonucleotide according to any preceding claim in which three natural 3' -5' phosphodiester- linkages at the 3' end of X are replaced by thiophosphodiester linkages. 8. An oligonucleotide according to any preceding claim, in which W is diol bridges, for use as an intermediate the preparation of a modified oligonucleotide exhibiting enhanced cellular uptake. 9. A 2' substituted nucleoside a or g as defined in claim l, or a protected derivative thereof, for use an an intermediate in the preparation of an oligonucleotdie according to claim l. 10. A method for synthesising an oligonucleotide according to claim 1 which method uses in a synthetic step an intermediate nucleoside according to claim 9.

Inventors: BENSELER FRITZ, KOTZOREK GERD, LUDWIG JANOS, BENSELER, FRITZ, KOTZOREK, GERD, LUDWIG, JANOS
Applicants: BENSELER FRITZ, KOTZOREK GERD, LUDWIG JANOS, RIBONETICS GMBH, BENSELER, FRITZ, KOTZOREK, GERD, LUDWIG, JANOS
Application year: 1993
Main classification: C12N9
IPC codes: A61K38, C07H21, C12N15