# US 6,723,512 B2 · 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

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- Number: US 6,723,512 B2
- Document ID: US6723512B2
- Status: Granted
- Kind: B2
- Application number: 09/866,073
- Filed: 2001-05-24
- Published: 2002-06-06
- Granted: 2004-04-20
- Earliest claimed: 1997-08-29
- Assignees: Selective Genetics, Inc.
- Inventors: David Larocca; Paul Kassner; Andrew Baird
- Web page: https://patentgrep.com/patent/US6723512B2

## CPC

- 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
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12Q 2600/158: Expression markers
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 4)

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

### Claim 7 (depends on 4)

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

### Claim 8 (depends on 4)

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

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 10)

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

### Claim 12 (depends on 10)

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

### Claim 13 (depends on 12)

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

### Claim 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.

### Claim 15 (depends on 14)

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.

### Claim 16 (depends on 14)

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

### Claim 17 (depends on 16)

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

### Claim 18 (depends on 16)

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

### Claim 19 (depends on 16)

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

### Claim 20 (depends on 16)

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

### Claim 21 (depends on 14)

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.

### Claim 22 (depends on 14)

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

### Claim 23 (depends on 14)

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

### Claim 24 (depends on 14)

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

### Claim 25 (depends on 24)

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

### Claim 26 (depends on 14)

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

### Claim 27 (depends on 1, 14)

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

### Claim 28 (depends on 1, 14)

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

### Claim 29 (depends on 1, 14)

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.

### Claim 30 (depends on 1, 14)

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

### Claim 31 (depends on 1, 14)

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

### Claim 32 (depends on 31)

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

### Claim 33 (depends on 31)

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