Methods for transfecting nucleic acid into live cells

Patent metadata

Field	Value
Publication number	US-10583204-B2
Application number	US-201213426422-A
Country	US
Kind code	B2
Filing date	Mar 21, 2012
Priority date	Dec 13, 2005
Publication date	Mar 10, 2020
Grant date	Mar 10, 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 tranfected cell.

First claim

What is claimed is:

1. A method of transferring a phenotype of a donor cell to a recipient cell, the method comprising:
transfecting a recipient cell with a transcriptome of a donor cell a first time, wherein the cell type of the donor cell is different than the cell type of the recipient cell, wherein the recipient cell is transfected in vitro or ex vivo, wherein the RNAs of the transcriptome enter the recipient cell, wherein the RNAs are functional in the recipient cell, and
transfecting the recipient cell with a transcriptome of the donor cell at least a second time, wherein the recipient cell is transfected in vitro or ex vivo, wherein the RNAs of the transcriptome enter the recipient cell, wherein the RNAs are functional in the recipient cell,
thereby initiating a change in physiology and morphology of the recipient cell, wherein the change in physiology and morphology yields a phenotype of the recipient cell that is indicative of the donor cell
wherein the donor cell and recipient cell are selected from the group consisting of:
a) wherein the recipient cell is a fibroblast, and wherein the donor cell is selected from the group consisting of an astrocyte, a cardiomyocyte, and a stem cell;
b) wherein the recipient cell is a neuron, and wherein the donor cell is selected from the group consisting of an astrocyte and a cardiomyocyte; and
c) wherein the recipient cell is an astrocyte and wherein the donor cell is a cardiomyocyte.

2. The method of claim 1 , wherein the method further comprises transfecting the recipient cell with at least one DNA of the donor cell.

3. The method of claim 1 , wherein the RNA is at least one selected from the group consisting of: mRNA, siRNA, miRNA, hnRNA, and tRNA.

4. The method of claim 1 , wherein the RNAs are expressed by the recipient cell.

5. The method of claim 1 , wherein the RNAs are isolated from a donor cell.

6. The method of claim 1 , wherein RNAs are in vitro transcribed.

7. The method of claim 1 , wherein the RNAs are prepared by a method selected from the group consisting of: mRNA isolation from a donor cell, in vitro mRNA transcription or mRNA chemical synthesis.

8. The method of claim 1 , wherein the phenotype of the recipient cell comprises two or more characteristics selected from the group consisting of: expression of the donor cell nucleic acid, protein expression of donor cell protein, expression of donor cell immunological markers, donor cell morphology, donor cell physiology, donor cell bioproduct synthesis, and donor cell membrane lipid composition.

9. The method of claim 1 , wherein the phenotype-of the recipient cell comprises three or more phenotype characteristics selected from the group consisting of: expression of the donor cell nucleic acid, protein expression of donor cell protein, expression of donor cell immunological markers, donor cell morphology, donor cell physiology, donor cell bioproduct synthesis, and donor cell membrane lipid composition.

10. The method of claim 1 , wherein the phenotype-of the recipient cell comprises four or more phenotype characteristics selected from the group consisting of: expression of the donor cell nucleic acid, protein expression of donor cell protein, expression of donor cell immunological markers, donor cell morphology, donor cell physiology, donor cell bioproduct synthesis, and donor cell membrane lipid composition.

11. The method of claim 1 , wherein the phenotype-of the recipient cell comprises the phenotype characteristics of donor cell morphology and donor cell physiology.

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

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

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

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

• C12N13/00

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

• A61K48/0083 Primary

  characterised by an aspect of the administration regime · CPC title

• C12N15/87

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

• C12N15/1079

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