Apparatus and system having dry gene silencing compositions

• US 7,935,811 B2
• Filed: 11/18/2005
• Issued: 05/03/2011
• 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): VIVLEMORE, TRACY ANN
• Application Number:
  US11/283,484
• Publication Number:
  US 20060115461A1
• Time in Patent Office: 1,992 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• 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 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

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 a first siRNA which silences a first target gene. The gene silencing composition can be configured such that the at least first siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the well. Additionally, the at least first siRNA can include a modification or a conjugate. 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 reverse transfection apparatus for introducing siRNA into a cell to effect gene silencing, the apparatus comprising:

a well plate having a well for transfecting cells, the well plate being provided in a sealed, sterile package such that the well plate is storable for an extended period of time at a temperature of up to at least 37°

C. while maintaining functionality of an siRNA deposited therein, wherein the well is substantially devoid of a cationic polynucleotide carrier selected from the group consisting of lipids, polymers, lipo-polymers, lipid-peptide mixtures, and combinations thereof; and

a substantially dry gene silencing composition in the well, the gene silencing composition having at least a first siRNA which silences at least a first target gene, the gene silencing composition being substantially devoid of a cationic polynucleotide carrier selected from the group consisting of lipids, polymers, lipo-polymers, lipid-peptide mixtures, and combinations thereof such that the at least first siRNA in the substantially dry gene silencing composition is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the well, the at least first 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 are a 2′

OH.

2. An apparatus as in claim 1, further comprising a conjugate coupled to the siRNA at the 5′

terminal nucleotide or 3′

terminal nucleotide on one of the sense strand or antisense strand.

3. An apparatus as in claim 1, wherein the total concentration of siRNA is less than about 100 nM when solubilized or suspended in the aqueous medium.

4. An apparatus as in claim 1, wherein the total concentration of siRNA is less than about 1 nM when solubilized or suspended in the aqueous medium.

5. A kit for introducing siRNA into a cell to effect gene silencing using reverse transfection, the kit comprising:

a well plate having a well for transfecting cells, the well plate being provided in a sealed, sterile package such that the well plate is storable for an extended period of time at a temperature of up to at least 37°

C. while maintaining functionality of an siRNA deposited therein, wherein the well is devoid of a cationic polynucleotide carrier selected from the group consisting of lipids, polymers, lipo-polymers, lipid-peptide mixtures, and combinations thereof, the well plate including;

a substantially dry gene silencing composition in the well, the gene silencing composition consisting essentially of at least one ribonucleic acid siRNA configured to silence at least one target gene, the gene silencing composition being disposed in the well such that the siRNA in the substantially dry gene silencing composition is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the well, wherein the ribonucleic acid siRNA comprises;

(a) a sense strand and an antisense strand, wherein said sense strand and said antisense strand together form an RNA duplex region consisting of 19 base pairs, and wherein the sense strand has 2′

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

nucleotides, and the antisense strand has a 2′

-O-alkyl modification on the second 5′

nucleotide and a 5′

end phosphate and wherein all other nucleotides on the sense and antisense strands are 2′

OH; and

(b) either zero overhang regions, or one or more overhang regions,

wherein each overhang region is two or fewer nucleotides in length;

an aqueous medium configured for solubilizing or resuspending the substantially dry gene silencing composition; and

a polynucleotide carrier compound configured to be added to the substantially dry gene silencing composition in the well either before the aqueous medium, with the aqueous medium, or after the aqueous medium in order to facilitate reverse transfection,

wherein the polynucleotide carrier includes at least one of a lipid, a polymer, a lipo-polymer, or a lipid-peptide mixture.

6. The kit recited in claim 5, the gene silencing composition optionally including at least two ribonucleic acid siRNAs configured to silence the first target gene.

7. The kit recited in claim 5, the gene silencing composition optionally including at least four ribonucleic acid siRNAs configured to silence the first target gene.

8. The kit recited in claim 5, wherein the 2′

-O-alkyl modifications are independently selected from the group consisting of 2′

-O-methyl, 2′

-O-ethyl, 2′

-O-propyl, 2′

-O-isopropyl, 2′

-O-butyl, 2′

-O-isobutyl, 2′

-O—

CH₂CH₂OCH₃, 2′

-O—

CH₂CH₂OH, 2′

-orthoester, 2′

-ACE group orthoester, and combinations thereof.