Cell-specific molecule and method for importing DNA into a nucleus

• US 6,130,207 A
• Filed: 11/05/1997
• Issued: 10/10/2000
• Est. Priority Date: 11/05/1997
• Status: Expired due to Fees

• Current Assignee: South Alabama Medical Science Foundation
• Original Assignee: South Alabama Medical Science Foundation
• Inventors: Dean, David Andrew, Zimmer, Warren Eugene
• Primary Examiner(s): Priebe, Scott D.
• Assistant Examiner(s): Nguyen, Dave Trong
• Application Number:
  US08/964,700
• Time in Patent Office: 1,070 Days
• Field of Search: 514/44, 424/93.21, 435/320.1, 435/455, 435/325, 435/69.1, 435/458, 536/23.1, 536/21.1, 530/350
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/90 avian

Abstract

The invention provides a cell-specific nuclear targeting molecule having a nucleic acid sequence which includes a binding site for a nuclear DNA binding protein expressed only in a specific cell type. The invention further provides a plasmid for targeting a DNA molecule into the nuclei of a specific cell type. The plasmid comprises the cell-specific nuclear targeting molecule and a DNA molecule to be targeted to the nuclei of the specific cell type. This plasmid of the subject invention can be introduced into various host cells, and the cell-specific nuclear targeting molecule will target the DNA molecule to the nuclei of the specific cell type. Thus, the invention further provides a method of targeting a DNA molecule into the nuclei of a specific cell type. The method comprises providing a plasmid (the plasmid comprising the cell-specific nuclear targeting molecule and the DNA molecule to be targeted) and introducing the plasmid into the cytoplasm of the specific cell type. In this method, the cell-specific nuclear targeting molecule targets the DNA molecule into the nuclei of the specific cell type.

Claims

1. A nuclear targeting molecule having a nucleic acid sequence as shown in SEQ ID NO:

1 or SEQ ID NO;

2.

2. A plasmid for targeting an exogenous DNA molecule into nuclei of smooth muscle cells, the plasmid comprising:

a nuclear targeting molecule having a first nucleic acid sequence as shown in SEQ ID NO;

1 or SEQ ID NO;

2, the first nucleic acid sequence including a binding site for a transcription factor, the transcription factor being unique to smooth muscle cells; and

an exogenous DNA to be targeted to the nuclei of smooth muscle cells.

3. The plasmid of claim 2 wherein the plasmid further comprises nucleic acid encoding a selection marker.

4. The plasmid of claim 2 wherein the plasmid further comprises a bacterial origin of replication.

5. The plasmid of claim 2 wherein the plasmid further comprises a promoter, expression of the exogenous DNA molecule to be targeted to the nuclei being under control of the promoter.

6. The plasmid of claim 2 wherein the plasmid further comprises a second nucleic acid sequence to direct integration of the exogenous DNA molecule into the genome of the smooth muscle cells.

7. The plasmid of claim 6 wherein the second nucleic acid sequence to direct integration is a viral integration sequence.

8. The plasmid of claim 2 wherein the plasmid further comprises unique restriction sites for insertion of additional nucleic acid sequences.

9. The plasmid of claim 8 wherein the unique restriction sites are provided by a polylinker.

10. An isolated host cell comprising the plasmid of claim 2.

11. The isolated host cell of claim 10 wherein the isolated host cell is a non-dividing cell.

12. The isolated host cell of claim 11 wherein the non-dividing cell is a quiescent cell.

13. The isolated host cell of claim 11 wherein the non-dividing cell is a terminally differentiated cell.

14. A method of targeting an exogenous DNA into nuclei of smooth muscle cells, the method comprising:

providing a plasmid for targeting an exogenous DNA into nuclei of smooth muscle cells, the plasmid comprising a nuclear targeting molecule having a nucleic acid sequence as shown in SEQ ID NO;

1 or SEQ ID NO;

2, the nucleic acid sequence including a binding site for a transcription factor, the transcription factor being unique to smooth muscle cells, and the plasmid further comprising an exogenous DNA to be targeted to the nuclei of smooth muscle cells; and

introducing the plasmid into the cytoplasm of smooth muscle cells, wherein the nuclear targeting molecule targets the exogenous DNA into the nuclei of the smooth muscle cells.

15. The method of claim 14 wherein the smooth muscle cells are non-dividing cells.

16. The method of claim 15 wherein the non-dividing cells are quiescent cells.

17. The method of claim 15 wherein the non-dividing cells are terminally differentiated cells.