METHODS USING GENETIC PACKAGE DISPLAY FOR DETECTING AND IDENTIFYING PROTEIN-PROTEIN INTERACTIONS THAT FACILITATE INTERNALIZATION AND TRANSGENE EXPRESSION AND CELLS OR TISSUES COMPETENT FOR THE SAME AND METHODS FOR EVOLVING GENE DELIVERY VECTORS

• US 6,723,512 B2
• Filed: 05/24/2001
• Issued: 04/20/2004
• Est. Priority Date: 08/29/1997
• Status: Expired due to Fees

• Current Assignee: Cardium Biologics, Inc.
• Original Assignee: Selective Genetics, Inc.
• Inventors: Baird, Andrew, Larocca, David, Kassner, Paul
• Primary Examiner(s): Ponnaluri, Padmashri
• Application Number:
  US09/866,073
• Publication Number:
  US 20020068272A1
• Time in Patent Office: 1,062 Days
• Field of Search: 435/5, 435/6, 435/69.1, 435/69.8, 435/320.1, 435/DIG.2, 435/DIG.4, 435/DIG.14, 435/DIG.15, 435/DIG.35, 536/23.1, 536/23.4, 536/25.32
• US Class Current: 506/10
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

A genetic package display system and methodology for probing protein-protein interactions that lead to cell transduction, selecting and/or identifying internalizing ligands, target cells and tissues which internalize known or putative ligands, and cell transduction facilitating peptides is provided. A ligand displaying genetic package that carries a selectable marker (e.g., reporter, selection, etc.) and presents a ligand on its surface is utilized to identify internalizing ligands, tranduction facilitating peptides, and/or a variety of cells and tissue types for the ability to be successfully transduced by the ligand displaying genetic package. Also provided are methods for evolving a ligand displaying package to facilitate gene delivery or delivery of any desired agent (e.g., pharmaceutical, polypeptide, peptide, etc.) into a cell and/or targeting cellular compartments such as the nucleus, endosome, chloroplast, mitochondria, etc.

Claims

1. A method of selecting ligands displayed on a phage that internalize and facilitate transgene expression, comprising:

(a) contacting one or more ligand displaying phage with a cell(s), wherein said phage carry a transgene encoding a detectable product which is expressed upon internalization of the phage, and

(b) detecting product expressed by the cell(s) and encoded by the transgene;

(c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) expressing the detectable product by polymerase chain reaction (PCR) or Hirt extraction, and

thereby selecting a ligand displayed on a phage that internalize and facilitate transgene expression.

2. The method of claim 1, further comprising isolating the cell(s) that express the detectable product.

3. The method of claim 2, wherein the cell(s) are isolated by flow cytometry.

4. The method of claim 1, wherein the ligand displaying phage comprise a library of ligand displaying phage.

5. The method of claim 4, wherein the library is a cDNA library.

6. The method of claim 4, wherein the library is an antibody gene library.

7. The method of claim 4, wherein the library is a random peptide gene library.

8. The method of claim 4, wherein the library is a mutein library.

9. The method claim 1, wherein the detectable product is selected from the group consisting of green fluorescent protein, β

-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, luciferase, human growth hormone and neomycin phosphotransferase.

10. The method of claim 1, wherein the phage are filamentous phage.

11. The method of claim 10, wherein the phase carries a genome vector.

12. The method of claim 10, wherein the phage carries a hybrid vector.

13. The method of claim 12, wherein the hybrid vector comprises AAV or SV40 genome nucleic acid sequences.

27. The method of any one of claims 1 or 14, further comprising contacting the cells with a genotoxic agent.

28. The method of any one of claims 1 or 14, further comprising subjecting the cells to heat shock.

29. The method of any one of claims 1 or 14, further comprising contacting the cells with a genotoxic agent and subjecting the cells to heat shock.

30. The method of any one of claims 1 or 14, wherein said phage displays at least two differing heterologous sequences on its surface.

31. The method of any one of claims 1 or 14, wherein the phage comprise phagemid particles.

32. The method of claim 31, wherein said phagemid are multivalent for the ligand.

33. The method of claim 31, wherein the phagemid are monovalent for the ligand.

14. A high throughput method of identifying a ligand displayed on a phage that internalizes and facilitates transgene expression, comprising;

(a) contacting one or more ligand displaying phage with a cell(s) in an array, wherein said phage carry a transgene encoding at least one detectable product which is expressed upon internalization of the phage; and

(b) detecting product(s) expressed by the cell(s) in the array, and

thereby identifying a ligand displayed on a phage that internalizes and facilitates transgene expression.

15. The method of claim 14, further comprising recovering a nucleic acid molecule encoding a ligand from the cells expressing the product by PCR or Hirt extraction.

16. The method of claim 14, wherein the ligand displaying phage comprises a library of ligand displaying phage.

17. The method of claim 16, wherein the library is a cDNA library.

18. The method of claim 16, wherein the library is an antibody gene library.

19. The method of claim 16, wherein the library is a random peptide gene library.

20. The method of claim 16, wherein the library is a mutein library.

21. The method of claim 14, wherein the detectable product is selected from the group consisting of green fluorescent protein, β

-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, luciferase, human growth hormone and neomycin phosphotransferase.

22. The method of claim 14, wherein the phage are filamentous phage.

23. The method of claim 14, wherein the phage are lambdoid phage.

24. The method of claim 14, wherein the phage carries a hybrid vector.

25. The method of claim 24, wherein the hybrid vector comprises SV40 O or AAV genome nucleic acid sequences.

26. The method of claim 14, wherein the phase carries a genome vector.