Single-stranded oligodeoxynucleotide mutational vectors
AssigneeValiGen (US), Inc.
InventorsRichard A. Metz, Bruce L. Frank, Debra M. Walther
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Abstract
The invention concerns the introduction of predetermined genetic changes in target genes of a living cell by introducing an oligodeoxynucleotide encoding the predetermined change. The oligodeoxynucleotides are effective in mammalian, avian, plant and bacterial cells. Specific end modifications that greatly increase the effectiveness of the oligodeoxynucleotides in bacteria are described. Surprisingly, unmodified oligodeoxynucleotides can be as effective in mammaliancells, including in vivo hepatocytes, as the modified nucleotides and can be as effective or more effective than chimeric oligonucleotides that consist of a mixture of deoxynucleotides and 2′-O-methyl ribonucleotides.
Claims34
A composition for making a predetermined genetic change in a targeted chromosomal gene of a mammalian or avian cell, comprising:
- (a) a single-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′ end nucleotide, having at least 25 deoxynucleotides and not more than 65 deoxynucleotides, and having a sequence comprising at least two regions each of at least 8 deoxynucleotides that are each, respectively, identical to at least two regions of the targeted chromosomal gene, which regions together are at least 24 nucleotides in length, and which regions are separated by at least one nucleotide in the sequence of the targeted chromosomal gene or in the sequence of the oligodeoxynucleotide or both such that the sequence of the oligodeoxynucleotide is not identical to the sequence of the targeted chromosomal gene;
- and
- (b) a macromolecular carrier selected from the group consisting of
- (i) an aqueous-cored lipid vesicle, wherein the aqueous core contains the single-stranded oligodeoxynucleotide,
- (ii) a lipid nanosphere, which comprises a lipophilic salt of the single-stranded oligodeoxyniucleotide, and
- (iii) a polycation having an average molecular weight of between 500 daltons and 1.3 Md wherein the polycation forms a salt with the single-stranded oligodeoxynucleotide.
A compound for making a predetermined genetic change in a targeted chromosomal gene of a mammalian or avian cell, comprising:
- a single-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′ end nucleotide, having at least 25 deoxynucleotides and not more than 65 deoxynucleotides, and having a sequence comprising at least two regions each of at least 8 deoxynucleotides that are each, respectively, identical to at least two regions of the targeted chromosomal gene, which regions together are at least 24 nucleotides in length, and which regions are separated by at least one nucleotidc in the sequence of the targeted chromosomal gene or in the sequence of the oligodeoxynucleotide or both, such that the sequence of the oligodeoxynucleotide is not identical to the sequence of the targeted chromosomal gene.
A compound for making a predetermined genetic change in a targeted gene in a bacterial cell, comprising:
- (a) a single-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′ end nucleotide, having at least 15 deoxynucleotides and not more than 35 deoxynucleotides, and having a sequence comprising at least two regions each of at least 7 deoxynucleotides that are each, respectively, identical to at least two regions of the targeted gene, which regions together are at least 14 nuclcotides in length, and which regions are separated by at least one nucleotide in the sequence of the targeted gene or in the sequence of the single-stranded oligodeoxynucleotide or both, such that the sequence of the oligodeoxynucleotide is not identical to the sequence of the targeted chromosomal gene;
- (b) a 5′ modification wherein the internucleotide linkage attached to the 5′ end nucleotide is a phosphorothioate linkage or wherein a N′-hydroxyalkyl substituted 3,3,3′,3′-tetra substituted indocarbocyanine dye is attached through a linker to the 5′ hydroxyl of the 5′ end nucleotide;
- and
- (c) a 3′ modification wherein the internucleotide linkage attached to the 3′ end nucleotide is a phosphorothioate linkage or wherein a deoxycytidine or thymidine nucleotide is 3′-3′ linked to the 3′ hydroxy of the 3′ end nucleotide or both.
A compound for making a predetermined genetic change in a targeted gene in a plant cell, comprising a single-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′ end nucleotide, having at least 21 deoxynucleotides and not more than 55 deoxynucleotides, and having a sequence comprising at least two regions each of at least 8 nucleotides that are each, respectively identical to at least two regions of the targeted gene, which regions together are at least 20 nucleotides in length, and which regions are separated by at least one nucleotide in the sequence of the targeted gene or in the sequence of the single-stranded oligodeoxynucleotide or both, such that the sequence of the oligodeoxynucleotide is not identical to the sequence of the targeted chromosomal gene.
Dependencies are inferred from the claim text.