Apparatus and system having dry gene silencing pools

• US 7,923,207 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: Marshall, William S., Robertson, Barbara, Robinson, Kathryn, Khvorova, Anastasia, Leake, Devin
• Primary Examiner(s): MCGARRY, SEAN
• Application Number:
  US11/283,482
• Publication Number:
  US 20060110829A1
• Time in Patent Office: 1,971 Days
• Field of Search: None
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

A reverse transfection apparatus can be used for introducing siRNA into a cell to effect gene silencing. Such an apparatus can include a well plate having a well configured for transfecting cells. The well can include a substantially dry gene silencing composition that has at least two siRNAs which silences at least a first target gene. The gene silencing composition can be configured such that the at least two siRNAs are each capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the well. Additionally, the siRNAs can include a hairpin structure, modification, or a conjugate. Also, the at least siRNAs can be rationally designed. The reverse transfection apparatus can be provided as a kit or system that additionally includes cells, polynucleotide carriers, reverse transfection reagents, and the like.

Claims

1. A method of reverse transfection for introducing siRNA into a cell to effect gene silencing, the method comprising:

providing a well plate, the well plate having a plurality of wells with a dried gene silencing composition deposited in at least one well having at least one siRNA which is capable of silencing a target gene, the at least one 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, 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;

adding an aqueous medium to the at least one well so as to suspend or solubilize the at least one siRNA into the aqueous medium; and

adding cells to the at least one well having the suspended or solubilized at least one siRNA under conditions that permit the at least one siRNA to be introduced into the cell and to effect gene silencing.

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

selecting a suitable polynucleotide carrier, wherein selecting is a function of at least one of transfection technique, siRNA composition, or cell type being transfected;

and adding a cationic polynucleotide carrier to the at least one well so as to form a siRNA-carrier complex, wherein the polynucleotide carrier is added either before, during, or after adding the aqueous medium to the at least one well, and wherein the siRNA-carrier complex is suspended or solubilized in the aqueous medium.

3. A method as in claim 2, wherein the polynucleotide carrier includes a lipid.

4. A method as in claim 1, further comprising silencing production of a target polypeptide by at least 50%.

5. A method as in claim 4, further comprising silencing production of the target polypeptide by at least 80%.

6. A method as in claim 1, wherein the cells are added in an amount of about 2×

10³ to about 3×

10⁴ cells per 0.35 cm² of cell growth surface area.

7. A method as in claim 1, wherein the well plate has been stored in a sealed package.

8. A method as in claim 7, wherein the sealed package contains an inert gas therein.

9. A method as in claim 7, wherein the sealed package is vacuum sealed.

10. A method as in claim 7, wherein the well plate has been stored in the sealed package at room temperature.

11. A method as in claim 10, wherein the well plate is shipped in the sealed package at ambient temperature.

12. A method as in claim 1, wherein the dried gene silencing composition is characterized as being spotted on a floor of a well of the well plate such that the dried gene silencing composition does not flow or displace from the well floor when the well plate is tilted or inverted.

13. A method of reverse transfection for introducing siRNA into a cell to effect gene silencing, the method comprising:

providing a well plate contained in a sealed, sterile package, said well plate having a plurality of wells containing a dried gene silencing composition, the dried gene silencing composition comprising;

at least one siRNA that is capable of silencing a target gene, said siRNA being capable of being solubilized or suspended in an aqueous medium in a known amount for transfecting cells when deposited in an individual well of the well plate, the at least one 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, 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;

adding an aqueous medium to at least one of the wells having the dried gene silencing composition so as to suspend or solubilize the at least one siRNA into the aqueous medium; and

adding cells to the at least one well having the suspended or solubilized at least one siRNA under conditions that permit the at least one siRNA to be introduced into the cell and to effect gene silencing.

14. The method of claim 13 further comprising, adding a cationic polynucleotide carrier either before, during, or after adding the aqueous medium to the at least one well, wherein the polynucleotide carrier includes at least one compound selected from the group consisting of cationic polymers, lipids, lipopolymers, lipid-peptide mixtures, and cholesterol.

15. The method as in claim 13, wherein the providing includes obtaining a well plate that has been stored in the sealed, sterile package at a temperature of up to at least about 37°

C. while maintaining functionality of the at least one siRNA in the at least one well.

16. A method of reverse transfection for introducing siRNA into a cell to effect gene silencing, the method comprising:

providing a well plate contained in a sealed package, said well plate having a plurality of wells, each well containing a substantially dried gene silencing composition having at least one siRNA which is capable of silencing a target gene, said siRNA being capable of being solubilized or suspended in an aqueous medium in a known amount for transfecting cells when deposited in an individual well of the well plate, the at least one 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, 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 wells;

adding an aqueous medium containing a cationic polynucleotide carrier compound to at least one of the wells so as to suspend or solubilize the at least one siRNA into the aqueous medium; and

adding cells to the at least one well having the suspended or solubilized at least one siRNA under conditions that permit the at least one siRNA to be introduced into the cell and to effect gene silencing.

17. The method of claim 16, wherein the cationic polynucleotide carrier compound includes at least one compound selected from the group consisting of cationic polymers, lipids, lipopolymers, lipid-peptide mixtures, and cholesterol.

18. The method as in claim 17, wherein the cationic polynucleotide carrier compound includes a lipid.

19. The method as in claim 16, wherein the providing includes obtaining a well plate that has been stored in the sealed, sterile package at a temperature of up to at least about 37°

C. while maintaining functionality of the at least one siRNA in the at least one well.