Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-04-22
2002-09-03
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C436S501000, C514S04400A, C536S022100, C536S023100
Reexamination Certificate
active
06444806
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with oligonucleotide-carbohydrate conjugates which are resistant to nuclease degradation and can be used as inhibitors of gene expression. More particularly, the invention pertains to such conjugates which are preferably formed using carbohydrates (e.g., sucrose) coupled via crosslinkers (e.g., 1,5-bis(succinimidooxycarbonyloxy)pentane) to the 3′-ends of oligonucleotides. The invention also relates to a method of inhibiting gene expression wherein a conjugate is transported into a cell where it can react with a macromolecule (e.g., mRNA and double-stranded DNA) to form a complex, and thus inhibit gene expression. Thus, these conjugates can be used as oligonucleotide drugs useful in treating diseases caused by the expression of specific genes.
2. Description of the Prior Art
Recent studies have indicated that oligonucleotides have potential therapeutic value. Oligonucleotides may act as antisense inhibitors of gene expression in a number of ways. Single-stranded oligodeoxynucleotides may arrest translation by forming heteroduplexes with mRNA, or may arrest transcription by forming triple-strand helices with duplex DNA. Catalytic ribonucleotides (ribozymes) may inhibit gene expression by cleaving complementary mRNAs. Furthermore, double-stranded oligonucleotides may bind to protein sites that recognize specific sequences of bases, thereby inhibiting polymerases. (Ghosh et al.,
Progress in Nucleic Acid Research and Molecular Biology,
42:79-126, 1992).
Two factors may limit the use of oligonucleotides as therapeutic agents. First, unmodified oligonucleotides are particularly susceptible to degradation by ubiquitous nucleases. Many of these nucleases exhibit 3′ to 5′ exonuclease activity and require a hydroxyl group at the 3′-end of the DNA molecule for activity. Second, the therapeutic action of oligonucleotides is limited by their low ability to enter the target cell.
Many approaches have been taken to limit the nuclease susceptibility of oligonucleotides. One approach has been to modify the phosphorous atom of the phosphodiester linkage to produce phosphorothioates, methylphosphorates, and phosphoramidates. (Uhlamann et al.,
Chem. Rev.,
90:543-584,1990; Goodchild,
Bioconjugate Chem.,
1:165-186, 1990). Although this approach has given significant resistance to nuclease degradation, the ability of the oligonucleotide to support RNase-mediated degradation of RNA is compromised. (Dagel et al.,
Antisense Res. Develop.,
1:11-20, 1991). RNase H is a nuclease which degrades only RNA that is hybridized to DNA and is commonly used to gauge the ability of an oligodeoxynucleotide to form a stable duplex with RNA. However, phosphorothioate oligonucleotides incorporating 2′-deoxy-2′-fluoroadenosine, -guanosine, -uridine, and -cytidine did enhance duplex stability as measured by melting temperature (T
m
) without compromising base-pair specificity. (Kawasaki et al.,
J. Med. Chem.,
36:831-841, 1993).
Terminal phosphorothioate and methane phosphorate modifications were shown to protect an oligonucleotide from exonucleases such as snake venom phosphodiesterase and spleen phosphodiesterase. (Stein et al.,
Nucleic Acids Research,
16:3209-3221, 1988; Agrawal et al.,
Tetrahedron Lett.,
27:5575-5578, 1986). Additionally, terminal methane phosphonate modifications were useful in protecting oligodeoxynucleotides from nuclease digestion in fetal calf serum. (Tidd et al.,
Br. J. Cancer,
60:343, 1989).
Modification of pyrimidines at their 5- and/or 6-positions resulted in enhanced nuclease stability of deoxyoligonucleotides in fetal calf serum and did not inhibit RNase H-mediated degradation of RNA. (Sanghvi et al.,
Nucleic Acids Research,
21:3197-3203, 1993). Also, deoxyoligonucleotides modified at the 3′- and/or 5′-terminal phosphate with phosphoroamidate linkages blocked exonucleolytic degradation in human serum, fetal calf serum, and cell media; furthermore, these terminal modifications maintained high binding affinity of the oligonucleotide for the complementary DNA sequence as shown by thermal denaturation measurements. (Shaw et al.,
Nucleic Acids Research,
19:747-750, 1991).
The inclusion of an 8 base-pair sequence encoding a hairpin loop on the 3′ end effectively stabilized a deoxyoligonucleotide against degradation by phosphodiesterase-1 and in fetal calf serum. Thermal denaturation experiments showed that the T
m
of this oligonucleotide hybridized to its complementary mRNA was unaffected by the presence of the secondary structure modification. (Kahn et al.,
Nucleic Acids Research,
21:2957-2958, 1993).
An oligonucleotide containing internucleotide phosphorothioates and 3′ alkylamine exhibited in vitro stability to nucleases in fetal calf serum and in cell culture. Additionally, treatment of HeLa cells with this oligonucleotide inhibited expression of the target gene 15 days following initial oligonucleotide treatment. Confocal microscopy revealed that an oligonucleotide with both modifications exhibited greater accumulation and release from cytoplasmic vesicles compared to an oligonucleotide containing only the 3′ alkylamine addition. Increased intracellular accumulation and distribution of oligonucleotides are factors known to affect their biological availability. (Tam et al.,
Nucleic Acids Research,
22:977-986, 1994).
Although the studies described above demonstrate that specific modifications of oligonucleotides result in enhanced resistance to nucleases, these investigations have not linked oligonucleotide modifications to improved methods of cellular uptake. Unfortunately, little is known about the interaction of oligonucleotides with cell membranes.
Letsinger et al.,
Proc. Natl. Acad. Sci. USA,
86:6553-6556, 1989, linked a cholesteryl group to the 3′-terminal internucleotide phosphorus of phosphodiester and phosphorothioate oligonucleotides in order to increase oligonucleotide solubility in cell membranes. These compounds significantly inhibited replication of human immunodeficiency virus (HIV) in tissue culture.
The presence of polylysine at the 3′-end of oligonucleotides complementary to the HIV-1 splice donor site resulted in a significant reduction in the production of viral structural proteins and virus tissue in infected cultures. (Stevenson et al.,
J. Gen. Virol.,
70:2673-2682, 1989). The authors postulated that the alteration in lipid solubility imparted by an attached polylysine moiety affects both the ability of an oligonucleotide to cross the cell membrane and its intracellular distribution.
Recently, gene-transfer methods have been developed that exploit natural receptor-mediated endocytosis pathways for the delivery of DNA into cells. One such class of receptors are the lectins, which specifically recognize carbohydrate residues. Ligands for cell receptors such as asialorosomucoid (Wu et al.,
J. Biol. Chem.,
262:4429-4432, 1987), albumin-bound insulin (Huckett et al.,
Biochem. Pharmacol.,
40:253-263, 1990), transferrin (Wagner et al.,
Proc. Natl. Acad. Sci. USA,
87:3410-3414, 1990), and viral proteins (Cotten et al.,
Methods Enzymol.,
217:618-644, 1992) have been successfully used to import DNA molecules into cells. In each case, the ligand was conjugated to DNA-binding protein and incubated with DNA. Resultant ligand-coated DNA was able to bind to receptors on the cell surface and was subsequently internalized.
Drawbacks of this approach include the difficulty of controlling the protein conjugation chemistry (Plank et al.,
Bioconjugate Chem.,
3:533-539, 1992), and limited gene delivery due to accumulation of DNA complexes in intracellular vesicles. Zatloukal et al.,
Ann. N.Y. Acad. Sci.,
660:136-153, 1992). Cotten et al.,
Proc. Natl. Acad. Sci. USA,
87:4033-4037,1990, have attempted to overcome these problems by replacing large protein ligands with smaller compounds that mimic ligand-binding to a receptor. These researchers constructed an artificial ligand for th
Nozawa Iwao
Veerapanani Dange
Hisamitsu Pharmaceutical Co. Inc.
Hovey & Williams, LLP
Marschel Ardin H.
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