Methods of using oligomers containing modified pyrimidines
First Claim
1. A method to evaluate a candidate antisense oligomer for its ability to inhibit gene expression, which method comprises:
- microinjecting varying amounts of said candidate antisense oligomer into a host cell along with (a) a target vector for the expression of a gene containing a target sequence for said candidate antisense oligomer, and (b) a control vector for the expression of a control gene encoding a detectable protein, wherein said control gene does not contain said target sequence; and
measuring expression of the target gene and the control gene;
wherein increasing inhibition of the target gene expression, but not of the control gene expression, as the amount of said candidate antisense oligomer increases, indicates the ability of said candidate antisense oligomer to inhibit gene expression.
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Abstract
Novel oligomers are disclosed which have enhanced ability with respect to forming duplexes or triplexes compared with oligomers containing only conventional bases. The oligomers contain the bases 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine or related analogs. The oligomers of the invention are capable of (i) forming triplexes with various target sequences such as virus or oncogene sequences by coupling into the major groove of a target DNA duplex at physiological pH or (ii) forming duplexes by binding to single-stranded DNA or to RNA encoded by target genes. The oligomers of the invention can be incorporated into pharmaceutically acceptable carriers and can be constructed to have any desired sequence, provided the sequence normally includes one or more bases that is replaced with the analogs of the invention. compositions of the invention can be used as pharmaceutical agents to treat various diseases such as those caused by viruses and can be used for diagnostic purposes in order to detect viruses or disease conditions.
544 Citations
15 Claims
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1. A method to evaluate a candidate antisense oligomer for its ability to inhibit gene expression, which method comprises:
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microinjecting varying amounts of said candidate antisense oligomer into a host cell along with (a) a target vector for the expression of a gene containing a target sequence for said candidate antisense oligomer, and (b) a control vector for the expression of a control gene encoding a detectable protein, wherein said control gene does not contain said target sequence; and measuring expression of the target gene and the control gene; wherein increasing inhibition of the target gene expression, but not of the control gene expression, as the amount of said candidate antisense oligomer increases, indicates the ability of said candidate antisense oligomer to inhibit gene expression. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of inhibiting the expression of at least one selected protein in a cell, wherein the protein is translated from RNA, the method comprising the steps of:
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introducing into the cell an oligomer comprising at least 8 nucleomonomers wherein at least one of the nucleomonomers comprises a base of formula (1) or (2) ##STR12## wherein each X is independently O or S;
R2 is cyano, or R2 is C2-12 1-alkynyl optionally substituted with halogen or an alkynyl group, or R2 is C2-12 1-alkenyl optionally substituted with halogen or an alkynyl group, or R2 is a C2-12 heteroaromatic group containing 5-6 ring atoms in which one to three of the ring atoms independently is nitrogen, oxygen or sulfur, wherein the heteroaromatic group is optionally substituted on a ring carbon atom by oxygen, halogen or C1-4 alkyl or substituted on a ring nitrogen by C1-4 alkyl, or R2 is --C.tbd.C--Z wherein Z is hydrogen or C1-10 alkyl or C1-10 haloalkyl with 1 to 6 halogen atoms, or Z is a (1-alkynyl)-heteroaromatic group optionally substituted on a ring carbon by oxygen or C1-4 alkyl or optionally substituted on a ring nitrogen by C1-4 alkyl;Pr is (H)2 or a protecting group, with the proviso that when at least one of the nucleomonomers of the oligomer comprises deoxyuridine 5-substituted by vinyl, 1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl, 1-octenyl, 1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-octynyl, then the remainder of the nucleomonomers comprising the oligomer are not solely comprised of phosphodiester linked 2'"'"'-deoxyadenosine, 2'"'"'-deoxyguanosine, 2'"'"'-deoxycytidine, thymidine or a combination thereof; and permitting the oligomer to form a duplex with the RNA whereby expression of the protein is inhibited. - View Dependent Claims (12, 13, 14)
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15. A method of introducing an oligomer into cells, comprising:
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mixing an oligomer comprising at least 8 nucleomdnomers wherein at least one of the nucleomonomers comprises a base of formula (1) or (2) ##STR13## wherein each X is independently O or S;
R2 is cyano, or R2 is C2-12 1-alkynyl optionally substituted with halogen or an alkynyl group, or R2 is C2-12 1-alkenyl optionally substituted with halogen or an alkynyl group, or R2 is a C2-12 heteroaromatic group containing 5-6 ring atoms in which one to three of the ring atoms independently is nitrogen, oxygen or sulfur, wherein the heteroaromatic group is optionally substituted on a ring carbon atom by oxygen, halogen or C1-4 alkyl or substituted on a ring nitrogen by C1-4 alkyl, or R2 is --C.tbd.C--Z wherein Z is hydrogen or C1-10 alkyl or C1-10 haloakyl with 1 to 6 halogen atoms, or Z is a (1-alkynyl)-heteroaromatic group olptionally substituted on a ring carbon by oxygen or C1-4 alkyl or optionally substituted on a ring nitrogen by C1-4 alkyl;Pr is (H)2 or a protecting group, with the proviso that when at least one of the nucleomonomers of the oligomer comprises deoxyuridine 5-substituted by vinyl, 1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl, 1-octenyl, 1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-octynyl, then the remainder of the nucleomonomers comprising the oligomer are not solely comprised of phosphodiester linked 2'"'"'-deoxyadenosine, 2'"'"'-deoxyguanosine, 2'"'"'-deoxycytidine, thymidine or a combination thereof, with a permeation enhancing agent to form a complex; and incubating the complex with the cells under conditions where the complex is introduced into the cells.
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Specification