| 2'-deoxy-2'-fluoro modified rna -> Monitor Keywords |
|
|
  2'-deoxy-2'-fluoro modified rnaUSPTO Application #: 20060036087Title: 2'-deoxy-2'-fluoro modified rna Abstract: The present invention refers to an RNA molecule with catalytic activity comprising at least one modified nucleoside, wherein the hydroxy group at the 2′-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups, a process for the preparation of modified RNA molecules and the use of modified RNA molecules as therapeutic agents and biocatalysts. (end of abstract) Agent: Alison J. Baldwin Mcdonnell Boehnen Hulbert & Berghoff - Chicago, IL, US Inventors: Fritz Eckstein, Wolfgang Pieken, Fritz Benseler, David B. Olsen, David M. Williams, Olaf Heindenreich USPTO Applicaton #: 20060036087 - Class: 536023100 (USPTO) Related Patent Categories: Organic Compounds -- Part Of The Class 532-570 Series, Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component, Carbohydrates Or Derivatives, Nitrogen Containing, Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20060036087. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] Certain naturally occuring ribonucleic acids (RNAs) are subject to self-cleavage. The first reported example is the cleavage of the ribosomal RNA precursor of the protozoan Tetrahymena (for a review see Cech, Ann. Rev. Biochem. 59 (1990), 543-568) which requires guanosine as cofactor. A number of examples of RNA cleavage have been subsequently discovered in viroid, virusoid and satellite RNAs (for reviews see Sheldon et al. in Nucleic Acids and Molecular Biology (1990) Vol. 4, pg. 227-242, ed. F. Eckstein and D. M. J. Lilley, Springer Verlag Berlin Heidelberg; Symons, TIBS 14 (1989), 445-450). These cleavages involve site-specific breakage of a phosphodiester bond in the presence of a divalent cation such as Mg.sup.2+, generating a 5'-hydroxyl and a 2',3',-cyclic phosphodiester terminus. Sequence analysis around the site of self-cleavage of several of such RNAs has led to the identification of a common structural feature essential for cleavage which was named a "hammerhead" structure (Hutchins et al., Nucleic Acids Res. 14 (1986) 3627-3640). This structure consists of three helices and 13 conserved nucleotides (framed in below scheme) which form a three dimensional structure amenable to cleavage at one particular position. The self-catalyzed cleavage is normally an intramolecular process, i.e. a single RNA molecule contains all the functions necessary for cleavage. However, Uhlenbeck (Nature 328 (1987), 596-600) has demonstrated that this hammerhead structure does not have to be embodied in one strand but can be made up of two strands. These two strands combine to form the hammerhead structure which leads to phosphodiester bond cleavage (indicated by an arrow) in one of the strands (strand S) whereas the other (strand E) remains unaltered and can participate in many cleavage reactions. This strand meets the definitions of an enzyme and is called a ribozyme. Whereas the framed sequences (below scheme) are conserved the others may vary provided that the structure of base paired and the single stranded regions remains intact. TABLE-US-00001 [0002] The cleavage reaction after the trinucleotide GUC has been studied in detail (Ruffner et al., Gene 82 (1989), 31-41; Fedor and Uhlenbeck, Proc. Natl. Acad. Sci. USA 87 (1990), 1668-1672). Ribozymes with new specificities have also been constructed (Haseloff and Gerlach, Nature 334 (1988), 585-591) indicating that cleavage can for example also take place after the sequences GUA, GUU, CUC, AUC and uuC. [0003] Further examples for RNA enzymes are the hairpin RNA (Hampel et al., Nucleic Acids Res. 18 (1990), 299-304), as well as RNA containing proteins such as the telomerase (Greider and Blackburn, Nature 337 (1989), 331-337) and the RNase P (Baer vet al., in Nucleic Acids and Molecular Biology (1988), Vol. 0.3, pp. 231-250, ed. F. Eckstein and D. M. J. Lilley, Springer Verlag, Berlin/Heidelberg). [0004] Ribozymes are potentially of interest for use as therapeutic agents (for review see Rossi and Sarver, TIBTECH 8 (1990), 179-183). A possible strategy would be to destroy an RNA necessary for the expression of both foreign genes such as viral genes and particular endogenous genes. This requires the construction of a RNA molecule which is able to form a hammerhead or a hairpin structure with the target RNA and to cleave this at a predetermined position. A first application to the inhibition of the HIV-1 virus by this strategy has been reported (Sarver et al., Science 247 (1990), 1222-1224). Other examples of the action of targeted hammerhead ribozymes in vivo are Cammeron and Jennings (Proc. Natl. Acad. Sci. USA 86 (1986), 9139-9143) and in vitro Cotten et al. (Mol. Cell. Biol. 9 (1989), 4479-4487). [0005] Further, other useful catalytic properties of ribozymes are known, e.g. dephosphorylase and nucleotidyl transferase activities (see Patent Application WO88/04300). Therein RNA enzymes are disclosed which are capable of dephosphorylating oligonucleotide substrates with high sequence specifity, which distinguishes them from known protein enzymes. RNA molecules also can act as RNA polymerases, differing from protein enzymes in that they use an internal rather than an external template. Thus, various heteropolymers can be constructed by variant RNA enzyme forms. This enables the formation for example of messenger RNA molecules for particular proteins or peptides. Furthermore, Herschlag and Cech (Nature 344, (1990), 405-409) describe an RNA enzyme with DNase acitivity. [0006] To be useful as a therapeutic agent the RNA enzyme has to be introduced into target cells. There are a priori two methods for delivery of the ribozyme into the target cells: [0007] (a) exogenous delivery of a preformed synthetic RNA; [0008] (b) endogenous transcription of a ribozyme-coding gene located on a plasmid. [0009] A great disadvantage of method (a) resides in the very low stability of RNA molecules under physiological conditions due to their fast degradation by a variety of ribonuclease enzymes present in the living cell. The disadvantages of method (b) result from the great difficulties of specifically and stably inserting a ribozyme-coding gene into the cells of higher organisms. Furthermore, the problem of degradation also occurs with in vivo synthesized RNA molecules. [0010] Therefore the problem underlying the present invention was to provide RNA molecules comprising both catalytic activities and enhanced stability against chemical and enzymatical degradation, which can be employed as therapeutical agents or as biocatalysts in biochemical or biotechnological processes. [0011] It was however known from a recent paper by Perreault et al. (Nature 344 (1990), 565-567) that certain modifications of the RNA enzyme, e.g. the incorporation of 2'-deoxyribonucleotides at a few positions of a ribozyme lead to a great impairment of the catalytic activity. [0012] It was now surprisingly found that certain chemical modifications at the 2'-position of the ribose sugar which enhance the stability of an RNA molecule do not considerably affect and/or abolish the catalytic properties of ribozymes. [0013] Therefore it is an object of the present invention to provide an RNA molecule with catalytic activity comprising at least one modified nucleoside, wherein the hydroxy group at the 2'-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups. [0014] The catalytic activity of an RNA molecule according to the present invention comprises advantageously at least one of the group consisting of nucleotidyl transferase, dephosphorylase, deoxyribonuclease and sequence specific endoribonuclease activities. Preferably the catalytic activity comprises a sequence specific endoribonuclease activity. More preferably the RNA is a hammerhead ribozyme as described above. Especially preferred is that the ribozyme can combine with another RNA strand to form a hammerhead structure consisting of two strands, wherein the modified RNA strand is the E strand as described above. [0015] Although a hammerhead ribozyme is especially preferred, other RNA enzymes are encompassed also by the present invention, e.g. the Tetrahymena ribozyme (Cech, Ann. Rev. Biochem. 59 (1990), 543-568) in naturally occuring form or a shortened form thereof (Zang et al., Biochemistry 27 (1988), 8924-8931), and especially the Hairpin RNA (Hampel et al., Nucleic Acids Res. 18 (1990) 299-304) or RNA containing proteins such as the RNase P (Baer et al., in Nucleic Acids & Molecular Biology (1988), Vol. 3, pp 231-250, ed. F. Eckstein and D. M. J. Lilley, Springer Verlag Heidelberg), the telomerase (Greider and Blackburn, Nature 337 (1989), 331-337). [0016] The incorporation of a modifier group at the 2'-position of the ribose sugar appears also to be particularly useful for RNA with new functions either derived at by a procedure that depends on alternate cycles of selection (Tuerk and Gold, Science 249 (1990), 505-510; Ellington and Szostak, Nature 346 (1990), 818-822) or any other method. [0017] The modifier group replacing the hydroxy group at the 2'-position of the ribose sugar is selected from halo, sulfhydryl, azido, amino, monosubstituted amino, and disubstituted amino groups. The halo group can be a fluoro, chloro, bromo or iodo group, wherein the fluoro group is preferred. The substituents of the substituted amino group are preferably C.sub.1-C.sub.3 alkyl and or hydroxyalkyl groups. Most preferably the modifier group is a halo or an unsubstituted amino group. [0018] The incorporation of a modifier group at the 2'-position of the ribose sugar significantly increases the RNA stability against enzymatic cleavage. It was confirmed that 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-aminouridine incorporated at specific positions of a ribozyme prevented cleavage at these positions by RNase A (see FIG. 3+4). This enzyme cleaves at the 3'-position of pyrimidine nucleosides and requires the presence of the 2'-hydroxyl group (Uchida and Egami (1971), in The Enzymes Vol. IV, 3rd ed. (Ed. P. D. Boyer), Academic Press, pp. 205-250). Furthermore, results obtained with polynucleotides show that the presence of the 2'-amino function also slows down degradation by unspecific nucleases such as snake venom phosphodiesterase (Hobbs et al., Biochemistry 12 (1973), 5138-5145). The presence of a 2'-halogroup also inhibits nucleases such as DNase I (Hobbs et al., Biochemistry 11 (1972), 4336-4344). Results with polynucleotides also show that the presence of a halogen at the 2'-position of a nucleotide protects against the action of human serum nucleases (Black et al., Virology 48 (1972) 537-545). Thus, protection by incorporation of a modified ribose sugar according to the present invention will be rather general and not be restricted to RNases which depend on the presence of the 2'-hydroxyl group. [0019] In a ribonucleic acid the ribose sugar is linked to a nucleotide base via a N-glycosidic bond. The nucleotide base, which is attached to the modified ribose sugar in an RNA molecule of the present invention is selected from the group consisting of bases naturally occuring in RNA and substituted bases. Preferably the modified ribose is attached to adenine, guanine, cytosine and/or uracil, which are the natural bases in RNA. The modified ribose, however, can also be attached to substituted bases, preferably selected from the group consisting of xanthine, hypoxanthine, 2,6-diamino purine, 2-hydroxy-6-mercaptopurine and purine bases substituted at the 6-position with sulfur or pyrimidine bases substituted at the 5-position with halo or C.sub.1-C.sub.5 alkyl groups, especially bromo or methyl groups. Most preferably the nucleotide base attached to the modified ribose sugar is uracil. [0020] The modified nucleosides which are incorporated into a RNA molecule are either previously described compounds or compounds which can be prepared in analogy to known compounds. The mostly preferred fluoro and amino analogs of ribonucleosides have been described previously, 2'-deoxy-2'-fluorocytidine (Doerr & Fox, J. Org. Chem. 32 (1967), 1462; Mengel & Guschlbauer, Ang. Chem. 90 (1978), 557-558); 2'-deoxy-2'-fluoroadenosine (Ikehara & Miki, Chem. Pharm. Bull. 26 (1978), 2449-2453), 2'-deoxy-2'-fluorouridine (Codington et al., J. Org. Chem. 29 (1964), 558-564), 2'-deoxy-2'-aminouridine (Verheyden et al., J. Org. Chem. 36 (1971), 250) and 2'-deoxy-2-aminocytidine (Verheyden et al. (1971) supra). For the synthesis of some of these compounds more recent synthetic procedures can be employed. The 2'-deoxy-2'-fluorocytidine can be prepared from 2'-deoxy-2'-fluorouridine by the method of Sung (J. Org. Chem. 47 (1982), 3623-3628). The same method can be used for the transformation of 2'-deoxy-2'-azidouridine to 2'-deoxy-2'-azidocytidine (Verheyden et al. (1971), supra). The latter can be reduced to 2'-deoxy-2'-aminocytidine by the method of Mungall et al. (J. Org. Chem. 40 (1975), 1659). [0021] The synthesis of the 2'-deoxy-2'-fluoronucleoside 5'-triphosphates can be carried out either according to Ludwig (Acta Biochim. et Biophys. Acad. Sci. Hung. 16 (1981), 131-133) or Ludwig and Eckstein (J. Org. Chem. 54 (1989), 631-635). The 2'-deoxy-2'-aminouridine and -cytidine 5'-triphosphates can be prepared as described for the diphosphates by Hobbs et al. (Biochemistry 12 (1973), 5138-5145) with the modification that pyrophosphate is employed instead of phosphate. The 2'-deoxy-2'-fluoronucleoside 31-phosphoramidites for automated oligonucleotide synthesis can b prepared by the method of Sinha et al. (Nucleic Acids Res. 12 (1984), 4539-4557). For the synthesis of the corresponding 2'-amino derivatives, the amino group can be protected by trifluoroacetylation according to Imazawa and Eckstein (J. Org. Chem. 44 (1979), 2039-2041). [0022] An RNA according to the present invention comprises at least one modified nucleoside, wherein the hydroxy group at the 2'-position of ribose is replaced by a modifier group. A preferred embodiment of the present invention is an RNA or guanosine or cytidine or uridine) contain modified sugars, while the remaining three nucleosides contain unmodified sugars. More preferably the modified nucleoside is pyrimidine nucleoside, i.e. cytidine or uridine or a substituted derivative thereof. Most preferably the modified sugar is 2'-fluoro ribose or 2'-amino ribose. Examples for this embodiment are the hammerhead ribozymes E2 and E3, which were derived from a hammerhead ribozyme E1 described by Fedor and Uhlenbeck (Proc. Natl. Acad. Sci. USA 87 (1990), 1668-1672). In E2 all uridine residues are replaced by 2'-deoxy-2'-fluorouridine and in E3 all uridine residues are replaced by 2'-deoxy-2'-aminouridine residues. The ribozymes E2 and E3 show a ribonuclease activity which is comparable to that of the unmodified RNA molecule E1. [0023] In a further preferred embodiment of the present invention all nucleosides of two different kinds contain modified sugars, while the remaining two nucleosides contain unmodified sugars. More preferably all pyrimidine nucleosides, i.e. cytidine and uridine (including substituted pyrimidine bases) contain modified sugars, most preferably 2'-fluoro or 21-amino ribose derivatives. [0024] Still a further embodiment of the present invention is an RNA molecule comprising a modification pattern (i.e. which nucleosides are modified and which are unmodified) which is designated as a so-called "selective modification pattern". An RNA comprising selective modification pattern is a molecule wherein nucleosides at specifically selected locations can be modified while nucleosides at other specifically selected locations can be unmodified. For instance, nucleotides which are known to be hypersensitive sites for ribonucleases (e.g. due to the secondary structure of the RNA molecule) should be modified to achieve an extended life time of the RNA molecule. An example for a ribonuclease-hypersensitive site is provided at position 21 of ribozyme E1. As shown in FIG. 3 the RNA molecule is cleaved at this position by RNase A with very high intensity. [0025] Still a further embodiment of the present invention is a RNA molecule additionally comprising at least one modified internucleotidic phosphodiester linkage. Examples for suitable modified phosphodiester linkages are methyl phosphonate groups or phosphorothioate groups, the latter being especially preferred. Preferably at least the 5'-terminal phosphodiester linkage and/or the 31-terminal phosphodiester linkage of the RNA molecule is modified. More preferably the 5'-terminal phosphodiester linkage and the last three 3'-terminal phosphodiester linkages are modified. [0026] It was found, that the presence of modified internucleotidic linkages alone was not sufficient to provide increased stability against degradation. However, the combined presence of 2'-modified ribose sugars together with modified internucleotidic linkages showed an additive stability enhancing effect. A more than fiftyfold increase in stability confered by both modifications outweighs the decreased efficiency in cleavage compared to a unmodified ribozyme. Continue reading... Full patent description for 2'-deoxy-2'-fluoro modified rna Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this 2'-deoxy-2'-fluoro modified rna patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like 2'-deoxy-2'-fluoro modified rna or other areas of interest. ### Previous Patent Application: Purification of a triple helix formation with an immobilized oligonucleotide Next Patent Application: Natural promoters for gene expression in c1 metabolizing bacteria Industry Class: Organic compounds -- part of the class 532-570 series ### FreshPatents.com Support Thank you for viewing the 2'-deoxy-2'-fluoro modified rna patent info. IP-related news and info Results in 1.63279 seconds Other interesting Feshpatents.com categories: Electronics: Semiconductor , Audio , Illumination , Connectors , Crypto , |
||
