Compositions and Methods to Monitor RNA Delivery to Cells

• US 20090317802A1
• Filed: 12/08/2006
• Published: 12/24/2009
• Est. Priority Date: 12/09/2005
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Chen, Alice A., Bhatia, Sangeeta N., Derfus, Austin M.
• Application Number:
  US12/096,344
• Publication Number:
  US 20090317802A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 49/0002 General or multifunctional contrast agents, e.g. chelated agents
• CPC Class Codes: A61K 49/0043 Fluorescein, used in vivo
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61K 49/0067 quantum dots, fluorescent nanocrystals
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

Methods and compositions for tracking or monitoring uptake of siRNA by mammalian cells are provided. The methods and compositions may be used to monitoring the silencing activity of the internalized siRNA. The compositions contain an siRNA, an optically or magnetically detectable nanoparticle such as a quantum dot and, optionally, a transfection reagent. Cells are contacted with an siRNA and an optically or magnetically detectable nanoparticle, optionally in the presence of a transfection reagent. Detection of internalized nanoparticles is indicative of siRNA uptake. The invention allows analysis, identification, processing, etc., of cells that have efficiently taken up siRNA. In one embodiment, cells are sorted into at least two populations based on the amount of siRNA taken up.

Claims

1. An isolated composition comprising an optically or magnetically detectable nanoparticle and an RNAi agent.

2. The composition of claim 1, wherein the nanoparticle is physically associated with the RNAi agent.

3. The composition of claim 1, wherein the nanoparticle is not conjugated to the RNAi agent.

4. The composition of claim 1, wherein the nanoparticle is conjugated to the RNAi agent.

5. The composition of claim 1, wherein the RNAi agent is an siRNA.

6. The composition of claim 1, wherein the nanoparticle comprises a quantum dot.

7. The composition of claim 1, wherein the nanoparticle comprises a plasmon resonant particle.

8. The composition of claim 1, wherein the nanoparticle comprises a fluorescent or luminescent moiety.

9. The composition of claim 1, further comprising a transfection reagent comprising one or more materials selected from the group consisting of:

cationic lipids, non-cationic lipids, cationic polymers, non-cationic polymers, dendrimers, polysaccharides, dextran, and translocation peptides.

10. The composition of claim 1, further comprising a transfection reagent, wherein the nanoparticle, the RNAi agent, and the transfection reagent form a complex.

12. The composition of claim 1, wherein the composition comprises (i) at least first and second optically or magnetically detectable nanoparticles having distinguishable optical or magnetic properties, and (ii) at least first and second RNAi agents, wherein the first optically or magnetically detectable nanoparticle is physically associated with the first RNA agent and the second optically or magnetically detectable nanoparticle is physically associated with the second RNAi agent.

13. The composition of claim 1, further comprising (i) at least one additional optically or magnetically detectable nanoparticle having optical or magnetic properties distinguishable from those of the first and second nanoparticles, and (ii) at least one additional RNAi agent, wherein each additional RNAi agent is associated with a nanoparticle having optical or magnetic properties distinguishable from those of the nanoparticles that are physically associated with the other RNAi agents.

15. The composition of claim 1, wherein the optically or magnetically detectable nanoparticle, the RNAi agent, or both, has an endosomal escape agent attached thereto.

16. The composition of claim 1, wherein the optically or magnetically detectable nanoparticle, the RNAi agent, or both, has a cell targeting agent attached thereto.

17. The composition of claim 1, wherein the optically or magnetically detectable nanoparticle is attached to the RNAi agent by a cleavable linkage.

11. (canceled)

14. (canceled)

18-30. -30. (canceled)

31. A kit comprising:

an optically or magnetically detectable nanoparticle and an RNAi agent.

32-40. -40. (canceled)

41. A method of monitoring delivery of a functional RNA to a cell comprising steps of:

(a) contacting the cell with an optically or magnetically detectable nanoparticle and a functional RNA; and

(b) analyzing the cell to detect the presence, absence, or amount of the nanoparticle in the cell, wherein presence of the nanoparticle in the cell is indicative of presence of the functional RNA in the cell.

43. The method of claim 41, wherein the amount of the nanoparticle in the cell is indicative of the amount of the functional RNA in the cell.

44. The method of claim 41, wherein the amount of the nanoparticle in the cell correlates with activity of the functional RNA in the cell.

45. The method of claim 41, wherein the functional RNA is an RNAi agent and the amount of the nanoparticle in the cell correlates with gene silencing activity of the RNAi agent in the cell.

49. The method of claim 41, wherein the step of analysing comprises performing FACS, imaging, or fluorescence microscopy.

42. (canceled)

46-48. -48. (canceled)

50-69. -69. (canceled)

70. A method of sorting cells comprising steps of:

(a) contacting cells with an optically or magnetically detectable nanoparticle and a functional RNA;

(b) analyzing the cells to detect the presence, absence, or amount of the nanoparticle in the cells; and

(c) identifying the cells as belonging to one of at least two populations based on the presence, absence, or amount of the nanoparticle in the cells.

74. The method of claim 70, further comprising:

physically separating the cells into at least two populations based on the presence, absence, or amount of the nanoparticle in the cells.

71-73. -73. (canceled)

75-82. -82. (canceled)

83. A method of monitoring gene silencing in a cell comprising steps of:

(a) contacting the cell with an optically or magnetically detectable nanoparticle and an RNAi agent targeted to a gene; and

(b) analyzing the cell to detect the presence, absence, or amount of the nanoparticle in the cell, wherein presence of the nanoparticle in the cell is indicative of silencing of the gene by the RNAi agent.

84. The method of claim 83, wherein the amount of the nanoparticle in the cell is indicative of the degree of silencing of the gene.

86. The method of claim 83, wherein the amount of the nanoparticle in the cell is indicative of the amount of the RNAi agent in the cell.

87. The method of claim 83, wherein the RNAi agent is an siRNA and the amount of the nanoparticle in the cell correlates with gene silencing activity of the siRNA in the cell.

85. (canceled)

88-115. -115. (canceled)