# US 10,501,755 B2 · FSH nucleic acid molecules to control insect pests

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- Number: US 10,501,755 B2
- Document ID: US10501755B2
- Status: Granted
- Kind: B2
- Application number: 15/478,957
- Filed: 2017-04-04
- Published: 2017-10-05
- Granted: 2019-12-10
- Earliest claimed: 2016-04-05
- Assignees: Dow AgroSciences LLC
- Applicants: Dow AgroSciences LLC; Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
- Inventors: Kenneth E. Narva; Sarah Worden; Meghan Frey; Murugesan Rangasamy; Premchand Gandra; Wendy Lo; Elane Fishilevich; Rainer Fischer; Andreas Vilcinskas; Eileen Knorr
- Web page: https://patentgrep.com/patent/US10501755B2

## CPC

- C12N 15/8286: {for insect resistance}
- A01N 57/16: containing heterocyclic radicals
- C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
- C07K 14/43563: {from insects}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 2310/14: interfering nucleic acids [NA]
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This disclosure concerns nucleic acid molecules and methods of use thereof for control of insect pests through RNA interference-mediated inhibition of target coding and transcribed non-coding sequences in insect pests, including coleopteran and/or hemipteran pests. The disclosure also concerns methods for making transgenic plants that express nucleic acid molecules useful for the control of insect pests, and the plant cells and plants obtained thereby.

## Claims

### Claim 1

An isolated nucleic acid molecule comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide encodes a hairpin ribonucleic acid (hpRNA) molecule with a stem-and-loop structure that inhibits an endogenous gene in a Diabrotica virgifera insect when the hpRNA molecule is ingested by the insect, the polynucleotide comprising:
a first nucleotide sequence comprising at least 410 contiguous nucleotides of SEQ ID NO:1, the complement or reverse complement of at least 410 contiguous nucleotides of SEQ ID NO:1, at least 320 contiguous nucleotides of SEQ ID NO:3, or the complement or reverse complement of at least 320 contiguous nucleotides of SEQ ID NO:3;
a second nucleotide sequence; and
a third nucleotide sequence that is reverse complement of the first nucleotide sequence,
wherein ribonucleotide sequences in the dsRNA molecule encoded by the first and the third nucleotide sequences are comprised in the stem structure of the hpRNA molecule when the polynucleotide is transcribed.

### Claim 2 (depends on 1)

The nucleic acid molecule of claim 1, wherein the first nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO:7, the complement and reverse complement of SEQ ID NO:7, SEQ ID NO:8, and the complements and reverse complement of SEQ ID NO:8.

### Claim 3 (depends on 1)

The nucleic acid molecule of claim 1, wherein the molecule is a vector.

### Claim 4 (depends on 3)

The nucleic acid molecule of claim 3, wherein the heterologous promoter is functional in a plant cell.

### Claim 5 (depends on 1)

The hpRNA molecule encoded by the polynucleotide of the nucleic acid molecule of claim 1, the hpRNA comprising:
a first ribonucleotide sequence encoded by the first nucleotide sequence;
a second ribonucleotide sequence encoded by the second nucleotide sequence; and
a third ribonucleotide sequence encoded by the third nucleotide sequence, wherein the third ribonucleotide sequence is the reverse complement of the first ribonucleotide sequence,
wherein the second ribonucleotide sequence is positioned between the first and third ribonucleotide sequences in the hpRNA molecule, and
wherein the first and third ribonucleotides are comprised in a stem structure in the hpRNA molecule.

### Claim 6 (depends on 1)

The nucleic acid molecule of claim 1, wherein the heterologous promoter is functional in a plant cell.

### Claim 7 (depends on 1)

A cell comprising the nucleic acid molecule of claim 1.

### Claim 8 (depends on 7)

The cell of claim 7, wherein the cell is a prokaryotic cell.

### Claim 9 (depends on 7)

The cell of claim 7, wherein the cell is a eukaryotic cell.

### Claim 10 (depends on 6)

A transgenic plant cell comprising the nucleic acid molecule of claim 6.

### Claim 11 (depends on 6)

A transgenic plant material comprising the nucleic acid molecule of claim 6.

### Claim 12 (depends on 11)

The transgenic plant material of claim 11, wherein the heterologous promoter is functional in a cell of the plant material.

### Claim 13 (depends on 12)

The transgenic plant material of claim 12, wherein the plant material is a plant or seed.

### Claim 14 (depends on 5)

A commodity product that comprises a detectable amount of the hpRNA molecule of claim 5.

### Claim 15 (depends on 10)

The transgenic plant cell of claim 10, wherein the cell is a Zea mays or Glycine max cell.

### Claim 16 (depends on 12)

The transgenic plant material of claim 12, wherein the plant material is a Zea mays or Glycine max plant material.

### Claim 17 (depends on 5)

A method for controlling a Diabrotica virgifera insect pest population, the method comprising feeding insects of the population with an agent comprising the hpRNA molecule of claim 5.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the agent is a transgenic plant material expressing the hpRNA molecule.

### Claim 19 (depends on 17)

The method according to claim 17, wherein the agent is a sprayable composition or RNA bait.

### Claim 20 (depends on 13)

A method for improving the yield of a crop, the method comprising:
cultivating in the crop the transgenic plant or seed of claim 13, such that the hpRNA molecule is expressed.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the transgenic plant or seed is Zea mays or Glycine max.

### Claim 22 (depends on 6)

A method for producing a transgenic plant cell, the method comprising:
transforming a plant cell with the nucleic acid molecule of claim 6;
culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture comprising a plurality of transgenic plant cells; and
selecting a transgenic plant cell that has integrated the polynucleotide in its genome and expresses the hpRNA molecule.

### Claim 23 (depends on 10)

A method for producing a transgenic plant resistant to Diabrotica virgifera infection, the method comprising:
regenerating a transgenic plant from the transgenic plant cell of claim 10.

### Claim 24 (depends on 1)

The nucleic acid of claim 1, further comprising a polynucleotide encoding an insecticidal polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp.

### Claim 25 (depends on 24)

The nucleic acid of claim 24, wherein the insecticidal polypeptide is selected from the group consisting of Cry1B, Cry 1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

### Claim 26 (depends on 10)

The transgenic plant cell of claim 10, wherein the cell comprises a polynucleotide encoding an insecticidal polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp.

### Claim 27 (depends on 26)

The transgenic plant cell of claim 26, wherein the insecticidal polypeptide is selected from the group consisting of Cry1B, Cry1I, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

### Claim 28 (depends on 11)

The transgenic plant material of claim 11, wherein the plant material comprises a polynucleotide encoding an insecticidal polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp.

### Claim 29 (depends on 28)

The transgenic plant material of claim 28, wherein the insecticidal polypeptide is selected from the group consisting of Cry1B, Cry1l, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

### Claim 30 (depends on 17)

The method according to claim 17, further comprising feeding the insects with an insecticidal polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp.

### Claim 31 (depends on 30)

The method according to claim 30, wherein the insecticidal polypeptide is selected from the group consisting of Cry1B, Cry 1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

### Claim 32 (depends on 13)

The transgenic plant or seed of claim 13, wherein the plant or seed is Zea mays or Glycine max.

### Claim 33

An isolated nucleic acid molecule comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide encodes a hairpin ribonucleic acid (hpRNA) molecule with a stem-and-loop structure that inhibits an endogenous gene in a Diabrotica virgifera insect when the hpRNA molecule is ingested by the insect, the polynucleotide comprising:
a first nucleotide sequence comprising SEQ ID NO:5, the complement or reverse complement of SEQ ID NO:5, SEQ ID NO:6, or the complement or reverse complement of SEQ ID NO:6;
a second nucleotide sequence; and
a third nucleotide sequence that is reverse complement of the first nucleotide sequence,
wherein ribonucleotide sequences in the hpRNA molecule encoded by the first and the third nucleotide sequences are comprised in the stem structure of the hpRNA molecule when the polynucleotide is transcribed.