AU7905198A - 3'-monophosphorylating oligonucleotides - Google Patents

Description

Translation from Chinese of PCT/CN98/00102 3'-Monophosphorylated Oligonucleotides 5 Technical area This invention involves modified oligonucleotides, in particular 3'-OH monophosphorylated oligonucleotides, 10 which can be used as therapeutic agents with increased stability, as well as reduced toxicity and side effects. Technical background 15 Oligonucleotides, including anti-sense and anti-gene oligonucleotides (Triplex Forming Oligonucleotides) are used as inhibitors of gene expression. These represent a new kind of therapeutic approach developed in recent years (Wagner, RW, Nature, 1994, 372, 333-335; Crook, ST, 20 Annu. Rev. Pharmacol. Toxical., 1992, 32, 329-376; Helene, C, Eur. J. Cancer, 1994, 30A, 1721-1726; Crooke, ST, Antisense Nucleic Acids Drug Dev., 1996, 6, 141-147). Their mechanisms of action include: inhibition of viral gene replication, transcription and translation; 25 inhibition of the transcription and translation of harmful genes within humans; and the degradation of targeted RNA through the action of in vivo RNase H. These oligonucleotides belong to a class of therapeutic agents exhibiting high specificity with low toxicity and 30 side effects. Examples of antisense oligonucleotides that are currently undergoing clinical trials are the anti-HIV agent GEM91 (Phase II), the anti-inflammatory agent ISIS2302 (Phase II), the anti-cancer agents 2 ISIS3521 (Phase I) and ISIS5132 (Phase I), the anti-CMV retinitis agent ISIS2922 used in AIDS patients (Phase III) , LR-3001 used for the treatment of chronic marrow leukaemia (Phase I), etc (Genetic Engineering News, 1996, 5 16, 29-34). Oligonucleotides are easily degraded by in vivo enzymes after entering the human body and before they can penetrate target organs and cells. Therefore they do not 10 reach their expected therapeutic potential (Hoke, G.D., et al, Nucleic Acids Res., 1991, 19, 5734-5748). Hence, it is clearly critical to be able to increase the stability of oligonucleotides, especially their stability to enzymatic degradation. In this way, not only will 15 their therapeutic efficacy be increased and the required dose reduced, but the cost of treatment and the extent of side effects will also be reduced somewhat. So as to increase the stability of oligonucleotides and 20 to extend their half-life in the human body, a series of approaches involving chemical and structural modifications have been investigated, as reported in a number of publications. For example: a phosphorothioate diester bond replaces a phosphate diester bond (WO 25 9115500, 1991); and two oligonucleotides are ligated via their 3' and 3' or 5' and 5' ends (EP 464638, 1992), etc. At present, most anti-sense oligonucleotides use chemical modifications to increase their stability, of which the phosphorothioate modification process is regarded as 30 being one of the most ideal. However, such materials are not natural products and exhibit a high degree of "foreignness" within human cells. Moreover, the cost of synthesis of these modified oligonucleotides is higher 3 than that of unmodified oligonucleotides, and they are less chemically stable than their natural phosphate diester cousins. The phosphorothioate-modified oligonucleotides do not exhibit any specificity in their 5 activity, and they can bind with some important intra cellular proteins, thereby influencing the ability of cells to transmit signals, as well as carry out their other biological functions. These oligonucleotides exhibit strong immunogenicity, being able to stimulate 10 the body's immune system. Also, the in vivo metabolites of phosphorothioate diester analogues can cause toxic side effects in the human body (Stein, CA, Trends in Biotechnology, 1996, 14, 147-149). This invention is therefore focussed on developing a better method of 15 modifying oligonucleotides. Aim of the invention The aim of this invention is to produce 3' 20 monophosphorylated oligonucleotides, as examples of oligonucleotides with increased stability, and decreased toxicity and side effects. Details of the invention 25 This invention produces 3'-monophosphorylated oligonucleotides with the structural formula 5'd (NNN... ... NNN) p3' or oligo (dN) -3' P, where N=A, G, C, T; p3' or 3'P = the 3' monophosphate group. 3'-Monophosphorylated 30 oligonucleotides were synthesised on a 3'-phosphate solid phase column (3'-phosphate CPG, produced by Glen Research Company; full name is 2-[2-(4,4' dimethoxytrityloxy)ethylsulphomyl]ethyl-succinoyl long 4 chain alkylamino-CPG) using a 391EP DNA synthesiser (ABI Company), followed by deprotection in concentrated ammonia solution. 5 The stability of these 3'-monophosphorylated oligonucleotides to enzymolysis was tested. Snake venom phosphodiesterase was used to mimic a 3' -- 5' exonuclease, and DNase I was used to mimic human endonuclease, in an in vitro comparison of the stability 10 to 3' -+ 5' exonuclease and endonuclease of unmodified oligonucleotides, 3'-monophosphorylated oligonucleotides, phosphorothioate-modified oligonucleotides and 3'-partly phosphorothioate-modified oligonucleotides. Further stability studies were carried out in serum and within 15 cells. The results showed that 3'-phosphorylated oligonucleotides were resistant to snake venom phosphodiesterase, and that their stability in serum and in cells was clearly greater than that of 3'-unmodified or 3'-partly modified phosphorothioate oligonucleotides. 20 Yet their stability was only slighter greater than the phosphorothioate-modified oligonucleotides. Moreover, 3'-phosphorylation did not affect the speed with which oligonucleotides could enter cells, which is faster than that of phosphorothioate-modified oligonucleotides. 25 These results also indicate that 3' -+ 5' exonuclease is a major enzyme present in serum and cells, and that it requires a 3'-OH substrate. After the 3'-OH group in an oligonucleotide is phosphorylated, the oligonucleotide can no longer act as a substrate for the 3' -+ 5' 30 exonuclease. Therefore, such an oligonucleotide can resist the action of 3' -+ 5' exonuclease, its retention time in human serum is prolonged, and so it can be more 5 effectively transported into target cells. Due to the increased stability of 3'-monophosphorylated oligonucleotides in cells, they can more efficiently bind to the complementary sequence of target genes. 5 3'-Monophosphorylated oligonucleotides can only be digested by 3' -+ 5' exonuclease after the 3'-phosphate has been cleaved by phosphomonoesterase. The high stability of 3'-monophosphorylated oligonucleotides in 10 serum and cells indicates low phosphomonoesterase activity in these sites. In addition, as DNA replication requires a primer with a 3'-OH end, as soon as 3'-monophosphorylated 15 oligonucleotides bind to DNA, they inhibit viral DNA replication and RNA reverse transcription. Most of the anti-sense oligonucleotides in current use are modified phosphorothioate oligonucleotides with a 3'-OH end, and they do not inhibit viral DNA replication or RNA reverse 20 transcription. Moreover, 3'-monophosphorylated oligonucleotides do not affect normal base pair matching, therefore it can be guaranteed that such modified oligonucleotides will correctly and specifically bind to target DNA or RNA. 25 In summary, 3'-monophosphorylated oligonucleotides, designed according to the specific sequences in the DNA or RNA of harmful genes such as viral or tumour genes, can selectively bind with target DNA and RNA, inhibit 30 viral DNA replication, transcription and translation, and can also inhibit the transcription and translation of harmful genes in the human body. They can also utilise in vivo RNase H to cause digestion of target RNA with 6 which they are bound. Therefore, due to the above mechanisms and advantages, 3'-monophosphorylated oligonucleotides have even more biological functions. Moreover, studies on anti-sense oligonucleotides designed 5 against the hepatitis B virus showed that 3' monophosphorylated oligonucleotides are more than twice as capable of inhibiting the expression of hepatitis B viral genes compared with unmodified oligonucleotides. 10 Advantage of the present invention 3'-Monophosphorylated oligonucleotides as described in this invention have the advantage over phosphorothiolated oligonucleotides of being more stable and more readily 15 taken up by cells. Furthermore, as the phosphate group is a natural component of nucleic acids, use of that group as a modifying agent does not introduce a foreign element into the body, and its degradation metabolites do not cause any toxic side effects. Thus, this system is 20 safer than other chemical modification methods, and from an overall perspective, it is superior to the commonly used phosphorothiolated oligonucleotides. 3' Monophosphorylated oligonucleotides can selectively bind target DNA and RNA, as well as inhibit viral DNA 25 replication, transcription and translation. Hence, 3' monophosphorylated oligonucleotides have the potential to become useful drugs in the clinic. List of figures 30 Fig 1: Resistance of the four oligonucleotides to digestion by snake venom phosphodiesterase 7 Fig 2: Resistance of the four oligonucleotides to digestion by DNase I Fig 3: Stability of the four oligonucleotides in 40% 5 human serum Fig 4: Stability of the four oligonucleotides in HeLa cells 10 Fig 5: Concentration of the four oligonucleotides in HeLa cell cytoplasm Fig 6: Concentration of the four oligonucleotides in HeLa cell nuclei 15 Examples Example 1:Synthesis of 3'-monophosphorylated oligonucleotides and other oligonucleotides 20 The following four oligonucleotides were synthesised on a 391 EP DNA synthesiser (ABI Company). The sequences of these oligonucleotides and their 3'-modifications are listed below: 25 1) 3' OH: 5' d (ATAGGGGCAT) 3' 2) 3' P: 5' d(ATAGGGGCAT)P 3' 3) SP: 5' d(A.T.A.G.G.G.G.C.A.G.A) 3' 4) 3SP: 5' d(TTGAGGATGGAGCCCTGGA.C.C.A) 3' 30 . indicates the phosphorothiolate diester linkage The first oligonucleotide has a 3'-OH group; The second oligonucleotide has a 3'-monophosphate group; 8 The third oligonucleotide contains all phosphorothiolate diester linkages and has a 3'-OH group; The fourth oligonucleotide contains three phosphorothiolate diester linkages, with the rest being 5 phosphate diester linkages, and has a 3'-OH group. Sequence 2 (the 3'-monophosphorylated oligonucleotide) was synthesised on a 0.2pmole 3'-phosphate solid phase column (3'-phosphate CPG, product of Glen Research 10 Company, full name is 2-[2-(4,4' dimethoxytrityloxy)ethylsulphomyl]ethyl-succinoyl long chain alkylamino-CPG) using a 391EP DNA synthesiser (ABI Company) on a 0.2ptmole cycle. Sequence 1 (the 3'-OH oligonucleotide) was synthesised on a 0.2pmole dT solid 15 phase column (Glen Research Company), using an analogous synthesis cycle. Sequences three and four were synthesised on a dA solid phase column (Glen Research Company), using an analogous synthesis cycle, except that where a phosphorothiolate modification was required, a 20 thiolating agent was used instead of an oxidising agent (see Iyer, RT, et al. J. Org. Chem. 1990, 55, 4693-4699). After synthesis was complete, the oligonucleotide carrier was recovered, and was treated with concentrated ammonia solution at 55'C for 15 hours to remove protecting 25 groups. The resulting solution was removed and vacuum dried, and the residue redissolved in 200pl 50% formamide. The product was purified by 7 mol/L urea-20% PAGE, the band cut under UV, and dialysed with double distilled H 2 0 to remove salts, and the concentrate stored 30 at -20 0 C. The amount of product was determined at A 260

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9 Example 2:Stability of the four oligonucleotides towards snake venom phosphodiesterase The four oligonucleotides described in example 1 (3' OH, 5 3' P, SP, 3SP) were separately 5-32p labelled by T4 polynucleotidekinase (T4 PNK). To 50pmol of oligonucleotide were added 50pCi [y- 32 P] -ATP, 2 units of T4 PNK, 1pLl of lOx buffer, and double-distilled

H

2 0 to 10pl. The mixture was incubated at 370C for 1 hour and 10 the labelled oligonucleotides purified as in example 1 using PAGE. Unlabelled oligonucleotide was added to give a final concentration of 5pnol/L, snake venom phosphodiesterase was added to 100pU/ml, the mixture buffered to pH 8.0 using 10mmol/L Tris-HCl, 50mmol/L 15 MgCl 2 and 0.1% bovine serum albumin (BSA), and then incubated at 370C. Samples (5pl) were removed at 0, 0.5, 1, 1.5, 2, 4, 8, 12 and 24 hours, an equal volume of loading buffer (98% formamide, 10mmol/L EDTA, 0.025% Xylene Cyanol FF, 0.025% Bromophenol Blue) was added, the 20 solution mixed and analysed on 7mol/L urea-20% PAGE. The results showed that the resistance of 3' P, SP and 3SP to snake venom phosphodiesterase is somewhat similar, and they are all much more resistant than 3' OH. The differences are significant and are shown in Figure 1. 25 Example 3: Stability of the four oligonucleotides to DNase I (an endonuclease) As for example 2, to each of the 3P-labelled 30 oligonucleotides was added unlabelled oligonucleotide to a final concentration of 5pmol/L. DNase was added to 100U/ml, the mixture buffered to pH 8.0 using 10mmol/L Tris-HCl, 5mmol/L MgCl 2 and 0.1% BSA, and then incubated 10 at 37 0 C. Samples (5ptl) were removed at 0, 0.5, 1, 1.5, 2, 4, 8, 12 and 24 hours, an equal volume of loading buffer was added, the solution mixed and analysed on 7mol/L urea-20% PAGE. The results showed that the 5 stability of 3' P to DNase I digestion is slightly lower than that of SP, but is much higher than that of 3' OH and 3SP. Furthermore, 3SP is particularly sensitive to the action of DNase I. See Figure 2. 10 Example 4:Stability of the four oligonucleotides in 40% human serum As for example 2, to each of the 32 P-labelled oligonucleotides was added unlabelled oligonucleotide to 15 a final concentration of 5pmol/L. Human serum was added to 40% and the mixture incubated at 37 0 C. Samples (5pil) were removed at 0, 0.5, 1, 1.5, 2, 4, 8, 12 and 24 hours, an equal volume of loading buffer was added, the solution mixed and analysed on 7mol/L urea-20% PAGE. The results 20 showed that 3' P is most stable, with around 50% remaining after incubation for 24 hours. This is clearly superior to the other three oligonucleotides, with the 3' OH being the least stable in this system. See Figure 3. 25 Example 5:Stability of the four oligonucleotides inside cells HeLa cells (2x10 5 ) were inoculated onto 35mm culture dishes, 1.5ml of cell culture medium (DMBM containing 10% 30 FCS) was added, and the cells were grown at 37 0 C under 5%

CO

2 . After the cells had grown to 40%-60% confluence, the culture medium was discarded, and 200pl of 5'- 32 P labelled 3' OH, 3' P, SP or 3SP transfection mixture 11 (containing 4pl Lipofectin and 0.2pmol/L of the respective oligonucleotide) was added. After incubating for 5 hours, the transfection mixture was discarded, 2ml of DMBM culture medium containing 10% FCS was added, and 5 the cells were again incubated at 370C under 5% C02. The cells were digested after 5, 12, 24, 36, 48 and 72 hours, centrifuged, and the pellet washed with an acid solution to remove oligonucleotides from the cell surface (see Gao WY, et al. J. Bio. Chem. 1990, 265, 20172-20178; 10 Lappalainen K. et al. Biochim. Biophys. Acta, 1994, 1196, 201-208). The cells were then lysed for 10 minutes at room temperature by adding lml TES solution (20mmol/L Tris-HCl pH 8.0, 10mmol/L EDTA, 1% SDS). The mixture was extracted twice with an organic phase 15 (phenol: chloroform: isoamyl alcohol 50:48:2), two volumes of absolute ethanol were added to precipitate the DNA, and the precipitate dissolved in 10pl of distilled water. The DNA was analysed on 7mol/L urea-15% PAGE, autoradiographed, and black-density scanned. The amount 20 of full-length oligonucleotides remaining within cells after various culture times was determined as a proportion of the amount of full-length oligonucleotide remaining after being cultured for 5 hours, with the latter being taken as 100%. The results showed that in 25 HeLa cells, 3' P was markedly more stable than 3SP and 3' OH, and also SP was relatively stable. See Figure 4. Example 6.Distribution of the four oligonucleotides within cells 30 HeLa cells (5x10 4 ) were inoculated in 12 well plates, 0.5ml of cell culture medium (DMBM containing 10% FCS) was added, and the cells were grown at 37 0 C under 5% C02.

12 After the cells had grown to 40%-60% confluence, the culture medium was discarded, and 100pl of 5'- 32 p labelled 3' OH, 3' P, SP or 3SP transfection mixture (containing 4pil Lipofectin and 0.2pmol/L of the 5 respective oligonucleotide) was added. Samples were set up in triplicate. After 5 hours of incubation, 400pl of DMBM culture medium containing 10% FCS was added, and the cells were again incubated at 37'C under 5% CO 2 . After 0, 2, 4, 8, 18, 24 and 30 hours, the culture medium was 10 discarded, the cells were washed once with 0.5ml PBS, digested with 100pl of trypsin for 6 minutes, and then 100pl of DMBM containing 10% FCS was added. Cells were counted three times so as to obtain an average count, and then centrifuged at 5000 rpm. As for example 5, the 15 pellet was washed with an acid solution to remove oligonucleotides from the cell surface. The cytoplasm and nuclei were separated (see Weintraub H, et al. Cell. 1983, 32, 1191-1203) and the radioactivity contained in the cytoplasm and nuclei was measured. The amount of 3' 20 OH, 3' P, SP and 3SP contained in the cytoplasm and nuclei at different times was calculated, and averaged over the triplicate samples. The concentrations of 3' OH, 3' P, SP and 3SP in cytoplasm and nuclei are shown in Figures 5 and 6. Under the experimental conditions, 3' 25 OH and 3' P entered the cell nucleus more quickly than SP and 3SP, with SP entering relatively slowly, and 3SP being the slowest of all. Whereas 3SP is a relatively long oligonucleotide, 3' OH and 3' P are of the same length and SP has an extra nucleic acid. As can be seen 30 from the results, the 3'-monophosphorylated modification does not affect the speed with which the oligonucleotide enters the cell.

Claims (2)

1. A class of 3'-monophosphorylated oligonucleotides, 5 characterised by the structure: 5'd(NNN... ... NNN) p3' or oligo(dN)-3'P, where N=A, G, C, T;

2. The uses of the 3'-monophosphorylated oligonucleotides 10 described in Claim 1, whereby these oligonucleotides bind target DNA and RNA, inhibit the replication, transcription and translation of viral and harmful genes, and can be used as therapeutic agents.