# US 10,308,946 B2 · Expression cassette for transformation comprising a modified viral sequence driven by a suitable promoter

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- Number: US 10,308,946 B2
- Document ID: US10308946B2
- Status: Granted
- Kind: B2
- Application number: 11/621,850
- Filed: 2007-01-10
- Published: 2009-04-23
- Granted: 2019-06-04
- Earliest claimed: 1998-05-22
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Lada Rasochova; Thomas German; Paul Ahlquist
- Inventors: Lada Rasochova; Thomas German; Paul Ahlquist
- Web page: https://patentgrep.com/patent/US10308946B2

## CPC

- C12N 15/8203: {Virus mediated transformation}
- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are novel methods and materials directed to transforming a host cell and expressing exogenous RNA therein. Specifically disclosed are DNA-launching platforms used to introduce a replicating viral segment attached to an exogenous polynucleotide into a cell, whereby the exogenous polynucleotide is expressed in said cell and confers a detectable trait.

## Claims

### Claim 1

A method of genotypically or phenotypically modifying one or more plant cells, said plant cell or cells having been rendered transgenic by stably comprising heterologous DNA encoding a trans-acting viral replication element that is incapable of self replication, said method comprising the following steps:
a) obtaining a DNA transfection vector comprising:
1) a polynucleotide molecule encoding a viral RNA molecule that has been modified to be incapable of self replication, but which replicates when expressed in a plant cell in the presence of a viral trans-acting element necessary for replication of the RNA, said viral RNA molecule also having been modified to include an exogenous RNA segment,
2) a DNA-dependent RNA polymerase promoter located at the 5′ end region of the polynucleotide molecule encoding the viral RNA molecule, and
3) a nucleotide sequence located at the 3′ end region of the polynucleotide molecule encoding the viral RNA molecule, wherein the nucleotide sequence is selected from one of the following:
i) a nucleotide sequence encoding a ribozyme, wherein the ribozyme cleaves at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule,
ii) a transcription termination sequence, and
iii) a restriction site that directs cleavage at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule; and
b) transfecting said one or more cells with said DNA transfection vector, wherein said polynucleotide molecule is transcribed thereby forming a replicatable RNA transcript that does not self replicate but is replicatable in the presence of said trans-acting viral replication element, wherein expression of said RNAtranscript confers a genotype or phenotype modification in said one or more plant cells, wherein said trans-acting viral replication element and said viral RNA molecule are derived from the same virus, and wherein said DNA transfection vector remains separate from the genome of the plant cell or cells.

### Claim 2 (depends on 1)

The method of claim 1 wherein said trans-acting viral replication element is constitutively expressed.

### Claim 3 (depends on 1)

The method of claim 1 wherein said DNA transfection vector comprises a ribozyme sequence, wherein the ribozyme cleaves at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 4 (depends on 1)

The method of claim 1 wherein said DNA transfection vector comprises a termination sequence located at the 3′ end region of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 5 (depends on 1)

The method of claim 1 wherein said DNA transfection vector comprises a restriction site that directs cleavage at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 6

A method of producing a plant or plant tissue comprising at least one genotypically or phenotypically modified plant cell, said cell having been rendered transgenic by stably comprising heterologous DNA encoding a trans-acting viral replication element that is incapable of self replication, said method comprising:
a) transfecting cells of said plant or plant tissue with a DNA transfection vector, wherein said DNA transfection vector remains separate from the genome of the cells, and wherein said DNA transfection vector comprises:
1) a polynucleotide molecule encoding a viral RNA molecule that has been modified to be incapable of self replication, but which replicates when expressed in a plant cell in the presence of a viral trans-acting element necessary for replication of the RNA, said viral RNA molecule also having been modified to include an exogenous RNA segment, such that said polynucleotide is transcribed to form a replicatable RNA transcript that does not self replicate but is replicatable in the presence of said trans-acting viral replication element,
2) a DNA-dependent RNA polymerase promoter located at the 5′ end region of the polynucleotide molecule encoding the viral RNA molecule, and
3) a nucleotide sequence located at the 3′ end region of the polynucleotide molecule encoding the viral RNA molecule, wherein the nucleotide sequence is selected from one of the following:
i) a nucleotide sequence encoding a ribozyme, wherein the ribozyme cleaves at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule,
ii) a transcription termination sequence, and
iii) a restriction site that directs cleavage at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule;
wherein the polynucleotide molecule encoding the viral RNA molecule is not flanked by two inverted repeat nucleotide sequences;
wherein said trans-acting viral replication element and said viral RNA molecule are derived from the same virus, and whereby expression of said RNA transcript confers a genotypic or phenotypic modification in at least one of said transfected cells.

### Claim 7 (depends on 6)

The method of claim 6 wherein said DNA transfection vector comprises a ribozyme sequence, wherein the ribozyme cleaves at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 8 (depends on 6)

The method of claim 6 wherein said DNA transfection vector comprises a termination sequence located at the 3′ end region of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 9 (depends on 6)

The method of claim 6 wherein said DNA transfection vector comprises a restriction site that directs cleavage at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 10 (depends on 1)

A method of producing a genotypically or phenotypically modified plant comprising obtaining at least one modified cell produced by the method of claim 1; and subjecting said modified cell to conditions whereby a plant is regenerated therefrom.

### Claim 11 (depends on 6)

The method of claim 6, wherein said plant or plant tissue comprises one or more cells transformed with a polynucleotide molecule encoding at least one trans-acting viral replication element and wherein said polynucleotide molecule is constitutively expressed.

### Claim 12 (depends on 1)

The method of claim 1, wherein said trans-acting viral replication element and said viral RNA molecule are derived from brome mosaic virus.

### Claim 13 (depends on 1)

The method of claim 1, wherein the DNA-dependent RNA polymerase promoter is fused to the 5′ end of the polynucleotide molecule encoding the viral RNA molecule, and
wherein the nucleotide sequence located at the 3′ end region of the polynucleotide encoding, the viral RNA molecule is fused to the 3′ end of the polynucleotide molecule encoding the viral RNA molecule, and the nucleotide sequence is selected from one of the following:
i) a nucleotide sequence encoding a ribozyme, wherein the ribozyme cleaves at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule,
ii) a transcription termination sequence, and
iii) a restriction site that directs cleavage at the 3′end of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 14 (depends on 1)

The method of claim 1, wherein said DNA transfection vector further comprises a nucleotide sequence encoding a viral coat protein.

### Claim 15 (depends on 1)

The method of claim 1, wherein the trans-acting viral replication element comprises a viral replicase and/or viral RNA polymerase.

### Claim 16 (depends on 6)

The method of claim 6, wherein said trans-acting viral replication element and said viral RNA molecule are derived from brome mosaic virus.

### Claim 17 (depends on 6)

The method of claim 6, wherein the DNA-dependent RNA polymerase promoter is fused to the 5′ end of the polynucleotide molecule encoding the viral RNA molecule, and
wherein the nucleotide sequence located at the 3′ end region of the polynucleotide encoding the viral RNA molecule is fused to the 3′ end of the polynucleotide molecule encoding the viral RNA molecule, and the nucleotide sequence is selected from one of the following:
i) a nucleotide sequence encoding a ribozyme, wherein the ribozyme cleaves at the 3′ end of the polynucleotide molecule encoding the viral RNA molecule,
ii) a transcription termination sequence, and
iii) a restriction site that directs cleavage at the 3′end of the polynucleotide molecule encoding the viral RNA molecule.

### Claim 18 (depends on 6)

The method of claim 6, wherein said DNA transfection vector further comprises a nucleotide sequence encoding a viral coat protein.

### Claim 19 (depends on 6)

The method of claim 6, wherein the trans-acting viral replication element comprises a viral replicase and/or viral RNA polymerase.

### Claim 20 (depends on 6)

The method of claim 6, wherein said replicatable RNA transcript moves cell-to-cell throughout the plant tissue or within the plant tissue.

### Claim 21 (depends on 20)

The method of claim 20, wherein said replicable RNA transcript is expressed to produce a protein of interest in cells without DNA encoding the protein of interest.