Method of determining a cellular response to a biological agent

• US 7,923,206 B2
• Filed: 11/18/2005
• Issued: 04/12/2011
• Est. Priority Date: 11/22/2004
• Status: Expired due to Fees

• Current Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Original Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Inventors: Khvorova, Anastasia, Leake, Devin, Marshall, William S., Robertson, Barbara, Robinson, Kathryn
• Primary Examiner(s): MCGARRY, SEAN
• Application Number:
  US11/283,481
• Publication Number:
  US 20060110766A1
• Time in Patent Office: 1,971 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/12 in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12N 2330/31 Libraries, arrays

Abstract

A method of determining a cellular response to a biological agent can be preformed in the presence of gene silencing. Such a method can include the use of siRNA in order to silence various genes in the cell in order to monitor the cellular response to the biological agent when a gene has been silenced. Accordingly, the response of a cell having a silenced gene to a biological agent can be indicative of which genes may be responsible for normal or abnormal cellular responses to various synthetic and natural biological agents such as active agents or pathogens.

Claims

1. A method of determining a cellular response to a biological agent, the method comprising:

providing a well plate in a sealed, sterile package, the well plate being storable at a temperature of up to at least about 37°

C. while maintaining functionality of a dried siRNA deposited therein, the well plate having a plurality of wells, including;

a first control well with a dried control composition, wherein the dried control composition includes at least one of no siRNA, a positive control siRNA that is capable of targeting a known gene, a non-targeting negative control siRNA, an siRNA having at least one toxic motif, a non-functional siRNA, an siRNA that inhibits being taken in and processed by RISC, or a control siRNA that includes a fluorescent marker, the dried control composition being capable of being solublized or resuspended in an aqueous medium; and

a first test well with a dried test gene silencing composition deposited therein that includes a test siRNA that is capable of silencing a first target gene, the dried test gene silencing composition being capable of being solublized or resuspended in the aqueous medium, the test siRNA comprising;

a sense strand having 2′

-O-methyl modifications on the first and second 5′

nucleotides; and

an antisense strand having a 2′

-O-methyl modification on the second 5′

nucleotide and a 5′

end phosphate;

wherein the sense region and the antisense region are capable of forming a duplex of 18-26 base pairs of nucleotides that has at least 80% complementarity over the range of the duplex, and wherein nucleotides of the sense strand and antisense strand other than the first 5′

sense nucleotide, the second 5′

sense nucleotide, and the second 5′

antisense nucleotide include a 2′

-OH;

unsealing the package to expose the plurality of wells;

adding the aqueous medium to the first control well and the first test well;

solublizing or resuspending the dried test gene silencing composition and the dried control composition in the aqueous medium;

adding cells to the first test well under conditions that permit transfection;

adding cells to a first control well under conditions that permit transfection;

adding a biological agent to the first test well and first control well, the biological agent being at least one of a known drug, a pro-drug, a candidate drug, a nucleic acid, a plasmid DNA, a polypeptide, a protein, an enzyme, a cytokine, a hormone, a polymer, a known cellular toxin, a suspected toxin, a microorganism, a virus, a bacterium, a yeast, a protozoan, or a sensitizing agent; and

determining whether a first response to the biological agent by the cells in the first test well is different from a first control response to the biological agent by the cells in the first control well, wherein a condition of the first response being different from the first control response is an indication that the first target gene is involved in the response by the cell to the biological agent.

2. A method as in claim 1, further comprising culturing the cells in the first test well and first control well before adding the biological agent.

3. A method as in claim 2, wherein the culturing is from about 24 hours to about 48 hours.

4. A method as in claim 1, wherein the first test well and first control well each have a substantially flat well floor.

5. A method as in claim 1, wherein the cells express the first target gene.

6. A method as in claim 1, wherein the dried test gene silencing composition includes a pool of four siRNAs.

7. A method as in claim 1, wherein the test siRNA in the dried test gene silencing composition includes at least one of a conjugate or a hairpin structure.

8. A method as in claim 1, further comprising:

sensitizing cells to the biological agent; and

determining the effect of the biological agent on the sensitized cells.

9. A method as in claim 1, further comprising:

screening a biological agent for a synthetic lethal combination; and

identifying a synthetic lethal combination associated with the biological agent.

10. A method as in claim 1, wherein the biological agent is a microorganism.

11. A method as in claim 10, wherein the microorganism is a pathogen.

12. A method as in claim 11, wherein the pathogen is a virus.

13. A method as in claim 11, wherein the pathogen is added to the first test well and first control well in a lethal amount.

14. A method as in claim 11, wherein the first test well containing more living cells compared to the first control well is an indication that the first target gene is involved in at least one of pathogenic infection, pathogen replication, or pathogen release from the cells.

15. A method as in claim 12, wherein the virus includes a reporter vector.

16. A method as in claim 1, wherein a total amount of siRNA in the test siRNA in the dried test gene silencing composition is present in an amount for transfecting cells in only the first test well.

18. A method as in claim 1, further comprising adding at least one cationic polynucleotide carrier to the control well and/or the test well, wherein the cationic polynucleotide carrier is added either before adding the aqueous medium, with the aqueous medium, or after adding the aqueous medium.

19. A method as in claim 18, wherein the cationic polynucleotide carrier includes at least one of a cationic lipid, a polymer, a lipo-polymer, or a lipid-peptide mixture.

17. A method of determining a cellular response to an siRNA, the method comprising:

adding an aqueous medium to a first test well in a well plate having a substantially dry gene silencing composition, the gene silencing composition having a test siRNA that is capable of silencing a first target gene, the dried test gene silencing composition being capable of being solublized or suspended in the aqueous medium in an amount sufficient for transfecting cells in the first test well, the test siRNA comprising;

a sense strand having 2′

-O-methyl modifications on the first and second 5′

nucleotides; and

an antisense strand having a 2′

-O-methyl modification on the second 5′

nucleotide and a 5′

end phosphate;

wherein the sense region and the antisense region are capable of forming a duplex of 18-26 base pairs of nucleotides that has at least 80% complementarity over the range of the duplex, and wherein nucleotides of the sense strand and antisense strand other than the first 5′

sense nucleotide, the second 5′

sense nucleotide, and the second 5′

antisense nucleotide include a 2′

-OH;

solublizing or resuspending the dried test gene silencing composition in the aqueous medium;

adding cells to the first test well under conditions that permit transfection;

adding cells to a first control well with a dried control composition under conditions that permit transfection, wherein, the dried control composition in the first control well includes at least one of no siRNA, a positive control siRNA that is capable of targeting a known gene, a non-targeting negative control siRNA, an siRNA having at least one toxic motif, a non-functional siRNA, an siRNA that inhibits being taken in and processed by RISC, or a control siRNA that includes a fluorescent marker, the dried control composition being capable of being solublized or resuspended in an aqueous medium;

comparing the effect of the control composition and the test siRNAs on the cells in the first test well and the first control well.

20. A method as in claim 17, wherein the dried test gene silencing composition includes a pool of four siRNAs.

21. A method as in claim 17, wherein the test siRNA in the dried test gene silencing composition includes at least one of a conjugate or a hairpin structure.

22. A method as in claim 17, further comprising adding at least one cationic polynucleotide carrier to the control well and/or the test well, wherein the cationic polynucleotide carrier is added either before adding the aqueous medium, with the aqueous medium, or after adding the aqueous medium.

23. A method as in claim 22, wherein the cationic polynucleotide carrier includes at least one of a cationic lipid, a polymer, a lipo-polymer, or a lipid-peptide mixture.