US5908779A
Expired / Lapsed
US
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Targeted RNA degradation using nuclear antisense RNA
Assignee: University of Connecticut
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Filing: 1993-12-01
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Granted: 1999-06-01
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Expires: 2013-12-01
Abstract
Abstract The invention pertains to a novel method and construct for regulating gene expression through inhibition of gene expression by nuclear antisense RNA. The construct provides an efficient means for introducing, expressing and accumulating the antisense RNA in the nucleus. The antisense RNA hybridizes to the sense mRNA in the nucleus, thereby preventing both processing and cytoplasmic transport. The construct comprises a promoter, antisense sequences, and a cis- or trans-ribozyme which generates 3'-ends independently of the polyadenylation machinery and thereby inhibits the transport of the RNA molecule to the cytoplasm. The construct may also comprise a histone stem-loop structure that assists in stabilizing the transcripts against exonucleolytic degradation.
Inventors
- 👤 Gordon G. Carmichael
- 👤 David B. Batt
- 👤 Zhong Liu
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 13:55
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12N15/63; C12N15/79; C12N15/85; C12N5/00; C12N5/06; C12N5/0602; C12N5/0634; C12N5/0647; C12N2310/00; C12N2310/10; C12N2310/11; C12N2310/111; C12N2310/12; C12N2310/127; C12N2310/50; C12N2310/53; C12N2501/00; C12N2501/40; C12N2501/415; C12N2501/65
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
105 words
An isolated mammalian cell containing a DNA construct comprising: a. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; b. a DNA segment operably linked to said transcriptional promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 2
Dependent Claim
105 words
An isolated mammalian cell containing a DNA construct comprising: a. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; b. a DNA segment operably linked to said transcriptional promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 3
Dependent Claim
110 words
An isolated mammalian cell containing a vector having incorporated therein a DNA construct comprising: a. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; b. a DNA segment operably linked to said transcriptional promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 4
Dependent Claim
110 words
An isolated mammalian cell containing a vector having incorporated therein a DNA construct comprising: a. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; b. a DNA segment operably linked to said transcriptional promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 5
Independent Claim
119 words
A method of inhibiting the expression of a gene in a mammalian cell comprising introducing into a mammalian cell a DNA construct comprising: a. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; b. a DNA segment operably linked to said transcriptional promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 6
Independent Claim
119 words
A method of inhibiting the expression of a gene in a mammalian cell comprising introducing into a mammalian cell a DNA construct comprising: a. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; b. a DNA segment operably linked to said transcriptional promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 7
Independent Claim
143 words
A method of inhibiting the expression of a gene in a mammalian cell, comprising the steps of: a. introducing into a mammalian cell a DNA construct comprising: i. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; ii. a DNA segment operably linked to said transcriptional promoter; and iii. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene, whereby a transformed cell is obtained; and b. expressing the DNA segment to thereby inhibit the expression of said gene.
Claim 8
Independent Claim
143 words
A method of inhibiting the expression of a gene in a mammalian cell, comprising the steps of: a. introducing into a mammalian cell a DNA construct comprising: i. a transcriptional promoter which provides efficient expression of an operably linked DNA segment such that inhibition of expression of a gene can be achieved with a 5-fold excess of antisense over sense RNA; ii. a DNA segment operably linked to said transcriptional promoter; and iii. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene, whereby a transformed cell is obtained; and b. expressing the DNA segment to thereby inhibit the expression of said gene.
Claim 9
Dependent Claim
75 words
A DNA construct comprising: a. a human cytomegalovirus promoter or Rous sarcoma virus promoter; b. a DNA segment operably linked to said promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 10
Dependent Claim
75 words
A DNA construct comprising: a. a human cytomegalovirus promoter or Rous sarcoma virus promoter; b. a DNA segment operably linked to said promoter; and c. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 11
Dependent Claim
8 words
A cell containing the construct of claim 5.
Claim 12
Dependent Claim
10 words
A vector having incorporated therein the construct of claim 5.
Claim 13
Dependent Claim
12 words
A vector according to claim 7 wherein said vector is a plasmid.
Claim 14
Dependent Claim
13 words
A composition comprising the construct of claim 5 and a physiologically acceptable vehicle.
Claim 15
Dependent Claim
13 words
A composition comprising the vector of claim 7 and a physiologically acceptable vehicle.
Claim 16
Dependent Claim
13 words
A composition comprising the vector of claim 8 and a physiologically acceptable vehicle.
Claim 17
Independent Claim
92 words
A method of inhibiting the expression of a gene in a cell, comprising introducing into a cell a DNA construct comprising: a. a human cytomegalovirus promoter or Rous sarcoma virus promoter; b. a DNA segment operably linked to said promoter; and c. sequences encoding a cis-ribozyme operably liked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 18
Independent Claim
92 words
A method of inhibiting the expression of a gene in a cell, comprising introducing into a cell a DNA construct comprising: a. a human cytomegalovirus promoter or Rous sarcoma virus promoter; b. a DNA segment operably linked to said promoter; and c. sequences encoding a cis-ribozyme operably liked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and inhibits the expression of said gene.
Claim 19
Independent Claim
118 words
A method of inhibiting the expression of a gene in a cell, comprising the steps of: a. introducing into said cell a DNA construct comprising: i. a human cytomegalovirus promoter or a Rous sarcoma virus promoter; ii. a DNA segment operably linked to said transcriptional promoter; and iii. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and regulates the expression of said gene, whereby a transformed cell is obtained; and b. expressing the DNA segment to thereby inhibit the expression of said gene.
Claim 20
Independent Claim
118 words
A method of inhibiting the expression of a gene in a cell, comprising the steps of: a. introducing into said cell a DNA construct comprising: i. a human cytomegalovirus promoter or a Rous sarcoma virus promoter; ii. a DNA segment operably linked to said transcriptional promoter; and iii. sequences encoding a cis-ribozyme operably linked to said DNA segment, such that expression of the DNA segment produces a ribonucleotide sequence which is complementary to a ribonucleotide sequence transcribed from a gene, and said expressed ribonucleotide sequence is accumulated in the cell nucleus and regulates the expression of said gene, whereby a transformed cell is obtained; and b. expressing the DNA segment to thereby inhibit the expression of said gene.
References Cited (Backward Citations)
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