# US 2016/0355833 A1 · Methods and Means for Obtaining Modified Phenotypes

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- Number: US 2016/0355833 A1
- Document ID: US20160355833A1
- Status: Published application
- Kind: A1
- Application number: 15/212,142
- Filed: 2016-07-15
- Published: 2016-12-08
- Earliest claimed: 1999-08-13
- Assignees: Commonwealth Scientific and Industrial Research Organisation
- Applicants: Commonwealth Scientific and Industrial Research Organisation
- Inventors: Peter Michael Waterhouse; Ming-Bo Wang
- Web page: https://patentgrep.com/patent/US20160355833A1

## CPC

- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 9/2405: {Glucanases}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8247: {involving modified lipid metabolism, e.g. seed oil composition}
- C12N 15/8283: {for virus resistance}
- C12Y 302/01031: Beta-glucuronidase (3.2.1.31)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell. Preferably, the unpolyadenylated, target-specific RNA is provided by transcription of a chimeric gene comprising a promoter and a DNA region encoding the target-specific RNA.

## Claims

### Claim 1

(canceled)

### Claim 2

A chimeric DNA comprising a promoter operably linked to a target specific DNA region which encodes an unpolyadenylated hairpin RNA, wherein the target specific DNA region comprises a target specific sense nucleotide sequence and a target specific antisense nucleotide sequence, wherein the target specific antisense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of a complement of a part of an RNA molecule transcribed or produced from a nucleic acid of interest in an animal cell, wherein the target specific sense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of the part of the RNA molecule transcribed or produced from the nucleic acid of interest, wherein the target specific sense nucleotide sequence and the target specific antisense nucleotide sequence are separated and linked by a spacer sequence, such that the unpolyadenylated hairpin RNA resulting from transcription of the target specific DNA region comprises the spacer sequence located between sense and antisense nucleotide sequences in the unpolyadenylated hairpin RNA, and the sense and antisense nucleotide sequences are complementary to each other so as to form the unpolyadenylated hairpin RNA.

### Claim 3 (depends on 2)

The chimeric DNA of claim 2, wherein said target specific sense nucleotide sequence corresponds to a translated region of the nucleic acid of interest.

### Claim 4 (depends on 2)

The chimeric DNA of claim 2, wherein the target specific sense nucleotide sequence corresponds to an untranslated region of the RNA molecule produced from the nucleic acid of interest.

### Claim 5 (depends on 2)

The chimeric DNA of claim 2, wherein the promoter is recognized by a eukaryotic RNA polymerase I or III and the DNA further comprises a terminator for the polymerase I or III.

### Claim 6 (depends on 2)

The chimeric DNA of claim 2, wherein the nucleic acid of interest is a gene incorporated in the genome of the animal cell.

### Claim 7 (depends on 2)

The chimeric DNA of claim 2, wherein the nucleic acid of interest is an endogenous gene of the animal cell.

### Claim 8 (depends on 2)

The chimeric DNA of claim 2, wherein the nucleic acid of interest is a viral nucleic acid.

### Claim 9 (depends on 2)

The chimeric DNA of claim 2, wherein the unpolyadenylated hairpin RNA lacks a 5′ cap structure.

### Claim 10 (depends on 2)

The chimeric DNA of claim 2, wherein the spacer sequence has a length of from 4 to 200 basepairs.

### Claim 11 (depends on 2)

The chimeric DNA of claim 2, wherein the promoter is a constitutive promoter.

### Claim 12 (depends on 2)

The chimeric DNA of claim 2, wherein the promoter is an inducible promoter.

### Claim 13 (depends on 2)

The chimeric DNA of claim 2, wherein the promoter is recognized by a single subunit RNA polymerase from a bacteriophage.

### Claim 14 (depends on 2)

The chimeric DNA of claim 2, wherein the promoter is recognized by a eukaryotic RNA polymerase III and the DNA further comprises a terminator for the polymerase III.

### Claim 15 (depends on 2)

The chimeric DNA of claim 2, wherein the target specific DNA region which is transcribed to form the unpolyadenylated hairpin RNA lacks a DNA region involved in 3′ end formation and polyadenylation.

### Claim 16 (depends on 15)

The chimeric DNA of claim 15, wherein the target specific DNA region comprises a 3′ end formation signal and lacks a polyadenylation signal.

### Claim 17

An animal cell in culture comprising a chimeric DNA which comprises a promoter operably linked to a target specific DNA region which encodes an unpolyadenylated hairpin RNA, wherein the target specific DNA region comprises a target specific sense nucleotide sequence and a target specific antisense nucleotide sequence, wherein the target specific antisense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of a complement of a part of an RNA molecule transcribed or produced from a nucleic acid of interest in the animal cell, wherein the target specific sense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of the part of the RNA molecule transcribed or produced from the nucleic acid of interest, wherein the target specific sense nucleotide sequence and the target specific antisense nucleotide sequence are separated and linked by a spacer sequence, such that the unpolyadenylated hairpin RNA resulting from transcription of the target specific DNA region comprises the spacer sequence located between sense and antisense nucleotide sequences in the unpolyadenylated hairpin RNA, and wherein the sense and antisense nucleotide sequences are complementary to each other so as to form the unpolyadenylated hairpin RNA.

### Claim 18 (depends on 17)

The animal cell in culture of claim 17, wherein the unpolyadenylated RNA is expressed in the cell.