Methods and compositions for treating insects

• US 10,190,118 B2
• Filed: 11/07/2016
• Issued: 01/29/2019
• Est. Priority Date: 08/03/2009
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Ward, Donna T., Rhodes, Jason
• Primary Examiner(s): Bowman, Amy H
• Application Number:
  US15/330,843
• Publication Number:
  US 20170204411A1
• Time in Patent Office: 813 Days
• CPC Class Codes: A01K 67/0333 Genetically modified invertebrates, e.g. transgenic, polyploid
• CPC Class Codes: A01N 25/002 containing a foodstuff as carrier or diluent, i.e. baits
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/00 Applications; Uses
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2710/14043 viral genome or elements thereof as genetic vectore

Abstract

Provided herein are methods and compositions for modulating gene expression in insects by administering a composition comprising an RNA effector molecule and a delivery agent. Methods are provided for controlling pest populations by inhibiting insect growth, development, survival, reproduction and/or viability. Also provided herein are methods for treating or preventing disease in an insect caused by a pathogen or by external factors (e.g., pollution, environment, stress, weather, etc.).

Claims

1. A method for treating disease in an insect, the method comprising administering to the insect a composition comprising a double stranded RNA (dsRNA) molecule or a vector encoding a dsRNA molecule, and a delivery agent, wherein the dsRNA molecule is 19-24 nucleotides long, comprises a sense strand and an antisense strand, and modulates gene expression of an insect, wherein the double-stranded region of the dsRNA is about 19 nucleotides in length, and

wherein the dsRNA further comprises (i) at least one mismatch, (ii) a 5′

-terminal dinucleotide overhang, or a 3′

-terminal dinucleotide overhang, or both a 5′

- and 3′

-dinucleotide overhang, and (iii) at least one asymmetrical modification, and

wherein the dsRNA molecule comprises at least one nucleotide modification to increase stabilization of the dsRNA, the modification selected from the group consisting of;

(i) a 5′

-phosphorothioate or 5′

-phosphorodithioate modifications within the double stranded region,

(ii) a phosphorothioate modification within the nucleotides at the terminal overhang region,

(iii) a cationic modification of nucleotides 1 and 2 on the 5′

terminus, wherein the cationic modification is at the C5 position of pyrimidines and C2, C6 and C8, exocyclic N2 or exocyclic N6 of purines,

(iv) at least one 2′

-fluoro modified nucleotide comprising a nucleobase modification

(v) at least one 2′

-O-methyl/2′

-fluoro modified nucleotide comprising a nucleobase modification,

(vi) a 5′

-PuPu-3′

dinucleotide at the 3′

terminal of at least one of the strands in the dsRNA molecule wherein both nucleotides comprise a modified methoxyethyl (MOE) at 2′

-position, and

(vii) a 5′

-PuPu-3′

dinucleotide at the 5′

terminal of at least one of the strands in the dsRNA molecule wherein both nucleotides comprise a modified MOE at 2′

-position; and

wherein modulation of the gene treats the disease.

2. The method of claim 1, wherein the disease is caused by an insect pathogen selected from the group consisting of a virus, mite, nematode, bacteria, fungus, or parasite.

3. The method of claim 1, wherein the disease is caused by pollution, exposure to electromagnetic radiation, exposure to pesticides, environment, or stress.

4. The method of claim 1, wherein the dsRNA molecule inhibits or activates gene expression.

5. The method of claim 1, wherein said administering comprises providing a food source for the insect, wherein the food source comprises the composition.

6. The method of claim 5, wherein the food source comprises a virus, a bacterium, a fungus, a plant, or a yeast cell expressing the RNA effector molecule.

7. The method of claim 1, wherein said administering comprises contacting the insect with a solution comprising the composition.

8. The method of claim 7, wherein the composition is administered topically.

9. The method of claim 1, wherein the vector is a viral vector, an expression vector, or a plasmid.

10. The method of claim 9, wherein the viral vector comprises a baculoviral vector.

11. The method of claim 1, wherein the insect is a bee, wasp, butterfly, ant or ladybug.

12. The method of claim 1, wherein the composition is administered to adult insects.

13. The method of claim 1, wherein the composition is administered to a breeding or feeding locus.

14. The method of claim 1, wherein the insect is a hive bee or a forager bee, and the pathogen is selected from the group consisting of IAPV, Acute Bee Paralysis Virus and Kashmir Bee Paralysis Virus.

15. A method for modulating gene expression in an insect, the method comprising:

administering to the insect a composition comprising a dsRNA molecule or a vector encoding a dsRNA molecule and a delivery agent, wherein the dsRNA molecule is 19-24 nucleotides long, comprises a sense strand and an antisense strand, and modulates gene expression in the insect,

wherein the double-stranded region of the dsRNA is about 19 nucleotides in length, and

wherein the dsRNA further comprises (i) at least one mismatch, (ii) a 5′

-terminal dinucleotide overhang, or a 3′

-terminal dinucleotide overhang, or both a 5′

- and 3′

-dinucleotide overhang, and (iii) at least one asymmetrical modification, and

wherein the dsRNA molecule comprises at least one nucleotide modification to increase stabilization of the dsRNA, the modification selected from the group consisting of;

(i) a 5′

-phosphorothioate or 5′

-phosphorodithioate modifications within the double stranded region,

(ii) a phosphorothioate modification within the nucleotides at the terminal overhang region,

(iii) a cationic modification of nucleotides 1 and 2 on the 5′

terminus, wherein the cationic modification is at the C5 position of pyrimidines and C2, C6 and C8, exocyclic N2 or exocyclic N6 of purines,

(iv) at least one 2′

-fluoro modified nucleotide comprising a nucleobase modification

(v) at least one 2′

-O-methyl/2′

-fluoro modified nucleotide comprising a nucleobase modification,

(vi) a 5′

-PuPu-3′

dinucleotide at the 3′

terminal of at least one of the strands in the dsRNA molecule wherein both nucleotides comprise a modified methoxyethyl (MOE) at 2′

-position, and

(vii) a 5′

-PuPu-3′

dinucleotide at the 5′

terminal of at least one of the strands in the dsRNA molecule wherein both nucleotides comprise a modified MOE at 2′

-position; and

wherein modulation of the gene treats a disease.

16. The method of claim 15, wherein the insect is a hive-dwelling insect and modulation of gene expression in the insect is delayed until the insect returns to the hive.