Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-02-03
2001-07-17
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S005000, C435S006120, C436S501000, C514S04400A, C536S025300
Reexamination Certificate
active
06262241
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to materials and methods for detecting and modulating the activity of RNA. The invention generally relates to the field of “antisense” compounds, compounds which are capable of specific hybridization with a nucleotide sequence of an RNA. In accordance with preferred embodiments, this invention is directed to the design, synthesis and application of oligonucleotides and to methods for achieving therapeutic treatment of disease, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins and cleaving RNA in site specific fashions.
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as “antisense” agents. The oligonucleotides or oligonucleotide analogs complementary to a specific, target, messenger RNA, mRNA sequence are used. A number of workers have reported such attempts. Pertinent reviews include C. A. Stein & J. S. Cohen,
Cancer Research,
vol. 48, pp. 2659-2668 (1988); J. Walder,
Genes & Development,
vol. 2, pp. 502-504 (1988); C. J. Marcus-Sekura,
Anal. Biochemistry,
vol. 172, 289-295 (1988); G. Zon,
Journal of Protein Chemistry,
vol. 6, pp-131-145 (1987); G. Zon,
Pharmaceutical Research,
vol. 5, pp. 539-549 (1988); A. R. Van der Krol, J. N. Mol, & A. R. Stuitje,
BioTechniques,
vol. 6, pp. 958-973 (1988) and D. S. Loose-Mitchell,
TIPS,
vol. 9, pp. 45-47 (1988). Each of the foregoing provide background concerning general antisense theory and prior techniques.
Thus, antisense methodology has been directed to the complementary hybridization of relatively short oligonucleotides to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs which are designed to bind in a specific fashion to—which are specifically hybridizable with—a specific mRNA by hybridization. Such analogs are intended to inhibit the activity of the selected mRNA—to interfere with translation reactions by which proteins coded by the mRNA are produced—by any of a number of mechanisms. The inhibition of the formation of the specific proteins which are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits.
A number of chemical modifications have been introduced into antisense oligonucleotides to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA, and to improve their pharmacokinetic properties. At present, however, no generalized antisense oligonucleotide therapeutic or diagnostic scheme has been found. The most serious deficiency of prior efforts has been the complete lack of a termination event once appropriate hybridization takes place or the presence of only a termination event that is so inefficient that a useful potency cannot be achieved due to the inability of oligonucleotides to be taken into cells at effective concentrations. The activity of the antisense oligonucleotides presently available has not been sufficient for effective therapeutic, research reagent, or diagnostic use in any practical sense. Accordingly, there has been and continues to be a long-felt need for oligonucleotides and oligonucleotide analogs which are capable of effective therapeutic and diagnostic antisense use.
This long-felt need has not been satisfied by prior work in the field of antisense oligonucleotide therapy and diagnostics. Others have failed to provide materials which are, at once, therapeutically or diagnostically effective at reasonable rates of application.
Initially, only two mechanisms or terminating events have been thought to be operating in the antisense approach to therapeutics. These are the hybridization arrest mechanism and the cleavage of hybridized RNA by the cellular enzyme, ribonuclease H (RNase H). It is likely that additional “natural” events may be involved in the disruption of targeted RNA, however.
These naturally occurring events are discussed by Cohen in
Oligonucleotides: Antisense Inhibitors of Gene Expression,
CRC Press, Inc., Boca Raton, Fla. (1989). The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides; P. S. Miller & P.O.P. Ts'O,
Anti-Cancer Drug Design,
2:117-128 (1987), and &agr;-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second “natural” type of terminating event is the activation of RNase H by the heteroduplex formed between the DNA type oligonucleotide and the targeted RNA with subsequent cleavage of target RNA by the enzyme. The oligonucleotide or oligonucleotide analog, which must be of the deoxyribo type, hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of antisense agents which are thought to operate by this type of antisense terminating event. R. Y. Walder and J. A. Walder, in
Proceedings of the National Academy of Sciences of the U.S.A.,
Vol. 85, pp.5011-5015 (1988) and C. A. Stein, C. Subasinghe, K. Shenozuka, and J. Cohen, in
Nucleic Acids Research,
Vol 16, pp.3209-3221 (1988) describe the role the RNase H plays in the antisense approach.
To increase the potency via the “natural” termination events the most often used oligonucleotide modification is modification at the phosphorus atoms. One oligonucleotide analog that has been developed in an effort to secure hybridization arrests is a methyl phosphonate oligonucleotide. Such analogs of oligonucleotides, analogs in the sense that the ordinary structure of the oligonucleotide has been modified into one or more methylphosphonate-substituted structures, have been extensively reported on. A number of authors including K. L. Agarwal & F. Riftina,
Nucleic Acids Research,
vol. 6, pp. 3009-3024 (1979); P. S. Miller, J. Yano, E. Yano, C. Carroll, C. Jayaraman, K. & P.O.P. Ts'o,
Biochemistry,
vol. 18, pp. 5134-5143 (1979); K. Jayaraman, K. McParland & P.O.P. Ts'o,
Proceedings of the National Academy of Sciences of the U.S.A.,
vol. 78, pp. 1537-1541 (1981); P. S. Miller, K. B. McPa
Acevedo Oscar Leobardo
Cook Phillip Dan
Ecker David J.
Guinosso Charles John
Kawasaki Andrew
ISIS Pharmaceuticals Inc.
Marschel Ardin H.
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
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