Methods for phototransfecting nucleic acids into live cells

Patent metadata

Field	Value
Publication number	US-10646590-B2
Application number	US-8647106-A
Country	US
Kind code	B2
Filing date	Dec 12, 2006
Priority date	Dec 13, 2005
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

The present invention includes methods for transferring a multigenic phenotype to a cell by transfecting, preferably by phototransfection, and locally transfecting a cell or a cellular process with a laser while the cell is bathed in a fluid medium comprising two or more nucleic acids, thereby introducing the nucleic acid into the interior of the cell. Expression of the nucleic acids results in a multigenic phenotype in the transfected cell.

First claim

What is claimed is:

1. A method of transferring a phenotype of a first cell to a second cell, said method comprising
transfecting the second cell with an mRNA transcriptome of the first cell a first time, wherein transfecting the second cell comprises irradiating the second cell with a laser, wherein said second cell is bathed in a fluid medium comprising the mRNA transcriptome of the first cell, wherein said second cell is irradiated ex vivo or in vitro, wherein said laser porates a cellular membrane of said second cell and said mRNA transcriptome enters said second cell, wherein said mRNA transcriptome is functional in the second cell, and
transfecting the second cell with an mRNA transcriptome of the first cell at least a second time, wherein transfecting the second cell comprises irradiating the second cell with a laser, wherein said second cell is bathed in a fluid medium comprising the mRNA transcriptome of the first cell, wherein said second cell is irradiated ex vivo or in vitro, wherein said laser porates a cellular membrane of said second cell and said mRNA transcriptome enters said second cell wherein said mRNA transcriptome is functional in the second cell,
thereby initiating a change in physiology and morphology of the second cell, wherein the change in physiology and morphology yields a phenotype of the second cell that is indicative of the first cell
wherein the first cell and second cell are selected from the group consisting of:
a) wherein the second cell is a fibroblast, and wherein the first cell is selected from the group consisting of an astrocyte, a cardiomyocyte, and a stem cell;
b) wherein the second cell is a neuron, and wherein the first cell is selected from the group consisting of an astrocyte and a cardiomyocyte; and
c) wherein the second cell is an astrocyte and wherein the first cell is a cardiomyocyte.

2. The method of claim 1 , wherein said mRNA transcriptome is isolated from a cell.

3. The method of claim 1 , wherein said mRNA transcriptome is prepared by a method selected from the group consisting of mRNA isolation from a cell, in vitro transcription or chemical synthesis.

4. The method of claim 1 , wherein the fluid medium further comprises at least one isolated DNA molecule.

5. The method of claim 1 , wherein the method further comprises transfecting the second cell with one or more RNAs of the first cell, wherein the one or more RNAs are selected from the group consisting of siRNA, miRNA, hnRNA, tRNA and combinations thereof.

6. The method of claim 1 , wherein said mRNA transcriptome comprises a mixture of different RNAs encoding two or more different polypeptides, wherein each different RNA is present at a relative abundance in said mixture that is essentially the same as a relative abundance of each different RNA present in the first cell that is in a different physiological state than the second cell.

7. The method of claim 6 , wherein the fluid medium further comprises an isolated inhibitory nucleic acid.

8. The method of claim 1 , wherein said laser is a titanium sapphire laser.

9. The method of claim 1 , wherein said fluid medium is a liquid.

10. The method of claim 1 , wherein said second cell is bathed with said fluid medium comprising the mRNA transcriptome at a discrete location on the cell's surface.

11. The method of claim 1 , wherein said second cell is selected from the group consisting of a human cell, a non-human primate cell, a mouse cell, a rabbit cell, a rat cell, a goat cell, a guinea pig cell, and a horse cell.

12. The method of claim 1 , wherein the second cell is a fibroblast, and wherein the first cell is selected from the group consisting of an astrocyte, a cardiomyocyte, and a stem cell.

13. The method of claim 1 , wherein the second cell is a neuron, and wherein the first cell is selected from the group consisting of an astrocyte and a cardiomyocyte.

14. The method of claim 1 , wherein the second cell is an astrocyte and wherein the first cell is a cardiomyocyte.

15. The method of claim 1 , wherein the second cell is cultured for at least 5 days after transfection of the mRNA transcriptome.

Assignees

• Eberwine James
• Haydon Philip G
• Sul Jai Yoon
• Takano Hajime
• Wu Chia Wen Kitty
• Zeng Fanyi
• Univ Pennsylvania

Inventors

• Eberwine James
• Haydon Philip G
• Sul Jai-Yoon
• Takano Hajime
• Wu Chia-Wen Kitty
• Zeng Fanyi

Classifications

• C12N15/1079

  Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086) · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• A61K48/0083 Primary

  characterised by an aspect of the administration regime · CPC title