Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-06-21
2004-01-27
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S025300, C514S04400A
Reexamination Certificate
active
06683166
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel modified oligonucleotides, the construction thereof, and their use in oligonucleotide-based therapies. More specifically, the invention is to novel oligonucleotides having modified internucleoside linkages which are resistant to nucleases, having enhanced ability to penetrate cells, and which are capable of binding target oligonucleotide sequences in vitro and in vivo. The modified oligonucleotides of the invention are particularly useful in oligonucleotide-based therapies utilizing the modified oligonucleotides to interrupt protein synthesis or otherwise inactivate messenger RNA or double stranded DNA.
BACKGROUND OF THE INVENTION
The application of oligonucleotides and oligonucleotide analogs (oligomers) for therapeutic uses represents a relatively new development in drug design and discovery. Several fundamental therapeutic approaches that utilize oligomers have been proposed.
One approach is based largely on interfering with gene expression through oligomer binding to a complementary RNA sequence. This application is known as “antisense” therapy because the oligomer base sequence is identical to the antisense strand of the gene that gave rise to the RNA (Uhlmann, E., et al.,
Chem Reviews
(1990) 90:543-584; and Stein, C. A., et al.,
Cancer Res
(1988) 48:2659-2668). Another approach, referred to herein as “triple helix” therapy utilizes oligomers that bind to duplex DNA as detailed below. Binding to a target DNA is sequence specific but involves different base pairing binding. Both antisense and triple helix therapies exert therapeutic effects via binding to nucleic acid sequences that are responsible for disease conditions. Such sequences are found in the genome of pathogenic organisms including bacteria, protozoa, yeasts, parasites, fungi or viruses or may be endogenous sequences (oncogenes). By modulating the expression of a gene important for establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
Another therapeutic approach that is based on the use of oligomers includes generation of “aptamers” and is disclosed and claimed in commonly owned application nos. 745,215, 659,980 and 658,849. This approach utilizes oligomers that specifically bind to proteins thereby interfering with their function. The use of oligomers that mimic the structure of certain RNA molecules that are bound by intracellular proteins has also been adduced as a therapeutic approach as described in international application no. PCT/US91/01822.
Antisense oligonucleotides are synthetic oligonucleotides which bind complementary nucleic acids (i.e. sense strand sequences) via hydrogen bonding, thereby inhibiting translation of these sequences. Therapeutic intervention at the nucleic acid level using antisense oligonucleotides offers a number of advantages. For example, gene expression can be inhibited using antisense or triple helix oligomers. Inhibition of gene expression is more efficient than inhibition of the protein encoded by the gene since transcription of a single DNA sequence gives rise to multiple copies of mRNA which, in turn, are translated into many protein molecules.
Antisense and triple helix therapies for diseases whose etiology is characterized by, or associated with, specific DNA or RNA sequences, are particularly useful. The oligomer employed as the therapeutic agent can be directly administered or generated in situ and is one that is complementary to a DNA or RNA needed for the progress of the disease. The oligomer specifically binds to this target nucleic acid sequence, thus disturbing its ordinary function.
An oligomer having a base sequence complementary to that of an mRNA which encodes a protein necessary for the progress of the disease, is particularly useful. By hybridizing specifically to this mRNA, the synthesis of the protein will be interrupted. However, it is also possible to bind double-stranded DNA using an appropriate oligomer capable of effecting the formation of a specific triple helix by inserting the administered oligomer into the major groove of the double-helical DNA. The resulting triple helix structure can then interfere with transcription of the target gene (Young, S. L. et al.,
Proc Natl Acad Sci
(1991) 88:10023-10026).
An important feature of therapeutic oligomers is the design of the backbone of the administered oligomer. Specifically, the backbone should contain internucleoside linkages that are stable in vivo and should be structured such that the oligomer is resistant to endogenous nucleases, such as nucleases that attack the phosphodiester linkage. At the same time, the oligomer must also retain its ability to hybridize to the target DNA or RNA. (Agarwal, K. L. et al.,
Nucleic Acids Res
(1979) 6:3009; Agarwal, S. et al.,
Proc Natl Acad Sci USA
(1988) 85:7079.) In order to ensure these properties, a number of modified oligonucleotides have been constructed which contain alternate internucleoside linkages. Several of these oligonucleotides are described in Uhlmann, E. and Peyman, A.,
Chemical Reviews
(1990) 90:543-584. Among these are methylphosphonates (wherein one of the phosphorus-linked oxygens has been replaced by methyl); phosphorothioates (wherein sulphur replaces one of these oxygens) and various amidates (wherein NH
2
or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen). These substitutions confer enhanced stability, for the most part, but suffer from the drawback that they result in a chiral phosphorus in the linkage, thus leading to the formation of 2
n
diastereomers where n is the number of modified diester linkages in the oligomer. The presence of these multiple diastereomers considerably weakens the capability of the modified oligonucleotide to hybridize to target sequences. Some of these substitutions also retain the ability to support a negative charge and the presence of charged groups decreases the ability of the compounds to penetrate cell membranes. There are numerous other disadvantages associated with these modified linkages, depending on the precise nature of the linkage.
It has also been suggested to use carbonate diesters. However, these are highly unstable, and the carbonate diester link does not maintain a tetrahedral configuration exhibited by the phosphorus in the phosphodiester. Similarly, carbamate linkages, while achiral, confer trigonal symmetry and it has been shown that poly dT having this linkage does not hybridize strongly with poly dA (Coull, J. M., et al.,
Tet Lett
(1987) 28:745; Stirchak, E. P., et al.,
J Org Chem
(1987) 52:4202.
WO 91/15500, published Oct. 17, 1991, teaches various oligonucleotide analogs in which one or more of the internucleotide linkages are replaced by a sulfur based linkage typically sulfamate diesters which are isosteric and isoelectric with the phosphodiester.
WO 89/12060, published Dec. 14, 1989, similarly discloses linkages containing sulfides, sulfoxides, and sulfones.
WO 86/05518, published Sep. 25, 1986, suggests a variant of stereoregular polymeric 3′,5′linkages.
U.S. Pat. No. 5,079,151 to Lampson et al., discloses a msDNA molecule of branched RNA linked to a single strand DNA via a 2′,5′ phosphodiester linkage.
Commonly owned, pending U.S. patent application Ser. No. 690,786, filed Apr. 24, 1991, the entirety of which is incorporated by notice, describes modified linkages of the formula —Y′CX′
2
Y′— wherein Y′ is independently O or S and wherein each X′ is a stabilizing substituent and independently chosen. Amide-containing linkages disclosed in commonly owned, pending U.S. patent application attorney docket number 24610-20044, filed May 28, 1992, S. Swaminathan, et al inventors, the entirety of which application is incorporated herein by reference, are also suitable for incorporation into oligomers containing one or more of the linkages disclosed herein.
Modifications of oligomers that enhance their affinity for target molecules will generally impro
Jones Robert J.
Matteucci Mark
Pudlo Jeff
Swaminathan Sundaramoorthi
ISIS Pharmaceuticals Inc.
Riley Jezia
Woodcock & Washburn LLP
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