# US 9,040,676 B2 · Vigilant cells

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- Number: US 9,040,676 B2
- Document ID: US9040676B2
- Status: Granted
- Kind: B2
- Application number: 10/567,298
- Filed: 2004-08-11
- Published: 2007-05-24
- Granted: 2015-05-26
- Earliest claimed: 2003-08-11
- Assignees: University of South Florida
- Applicants: M. Ian Phillips; Yao Liang Tang
- Inventors: M. Ian Phillips; Yao Liang Tang
- Web page: https://patentgrep.com/patent/US9040676B2

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C12N 2510/02: Cells for production
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2800/00: Nucleic acids vectors
- C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12N 2830/85: mammalian
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention concerns a system for modulating tissue physiology, for example, to prevent or reverse tissue damage caused by disease. The system utilizes vigilant cells that include stable vectors containing a gene switch/biosensor and a gene amplification system. The vectors allow expression of a transgene (such as a cardioprotective gene) in the vigilant cells to be regulated in response to a physiological signal, to be switched on or off, and to provide sufficient levels of the transgene product to achieve a desired result, e.g., prevention or reversal of myocardial cell damage. In addition to myocardial infarction, the vectors can be used to treat cells in a number of other disease states, including diabetes, cancer, stroke, and atherosclerosis. These approaches to stem cell-based gene therapy provide a novel strategy not only for treatment but for prevention of cell destruction.

## Claims

### Claim 1

A genetically modified stem or progenitor cell comprising:
(a) a first exogenous polynucleotide comprising a gene switch/biosensor operatively linked to a promoter, wherein said gene switch/biosensor encodes an oxygen-sensitive chimeric transactivator; and
(b) a second exogenous polynucleotide comprising a gene amplification system, wherein said gene amplification system comprises a nucleic acid sequence encoding a therapeutic product, wherein said therapeutic product is heme oxygenase-1 (HO-1);
wherein said cell comprises a non-viral vector comprising said first and second exogenous polynucleotides.

### Claim 2 (depends on 1)

The cell of claim 1, wherein said gene amplification system further comprises a GAL4 upstream activating sequence (UAS) linked to said nucleic acid sequence encoding said therapeutic product.

### Claim 3 (depends on 2)

The cell of claim 2, wherein said physiological stimulus is a signal associated with a pathological condition, and wherein said chimeric transactivator of said first exogenous polynucleotide binds to said GAL4 UAS of the second exogenous polynucleotide in response to the signal associated with the pathological condition, resulting in expression of the therapeutic nucleic acid sequence encoding the therapeutic product.

### Claim 4 (depends on 3)

The cell of claim 3, wherein said signal is hypoxia associated with ischemia.

### Claim 5 (depends on 1)

The cell of claim 1, wherein said gene switch/biosensor comprises an operatively linked promoter, wherein said oxygen-sensitive chimeric transactivator comprises a GAL4 DNA-binding domain (DBD), an oxygen-dependent degradation domain (ODD), and a p65 activation domain (p65AD); and wherein said nucleic acid sequence encoding a therapeutic product comprises a cardioprotective gene linked to a GAL4 upstream activating sequence (UAS).

### Claim 6 (depends on 1)

The cell of claim 1, wherein said cell is a pluripotent or totipotent stem cell.

### Claim 7 (depends on 1)

The cell of claim 1, wherein said cell is selected from the group consisting of a hematopoietic stem cell, a mesenchymal stem cell (MSC), a muscle derived stem cell, and a bone marrow mesenchymal progenitor cell (MPC).

### Claim 8 (depends on 1)

The cell of claim 1, wherein said therapeutic product is a polypeptide that is heterologous to said cell.

### Claim 9 (depends on 1)

The cell of claim 1, wherein said therapeutic product is a polypeptide that is endogenous to said cell.

### Claim 10

A modified mammalian tissue, wherein said tissue comprises a genetically modified mammalian stem or progenitor cell, wherein said cell comprises:
(a) a first exogenous polynucleotide comprising a gene switch/biosensor operatively linked to a promoter, wherein said gene switch/biosensor encodes an oxygen-sensitive chimeric transactivator; and
(b) a second exogenous polynucleotide comprising a gene amplification system, wherein said gene amplification system comprises a nucleic acid sequence encoding a therapeutic product, wherein said therapeutic product is heme oxygenase-1 (HO-1);
wherein said cell comprises a non-viral vector comprising said first and second exogenous polynucleotides.

### Claim 11 (depends on 10)

The tissue of claim 10, wherein said tissue is human tissue and said cell is a human cell.

### Claim 12 (depends on 10)

The tissue of claim 10, wherein said cell is autologous to said tissue.

### Claim 13 (depends on 10)

The tissue of claim 10, wherein said tissue is myocardium, mesenchymal tissue, pancreatic tissue, liver tissue, or brain tissue.

### Claim 14 (depends on 10)

The tissue of claim 10, wherein said cell is a hematopoietic stem cell, a mesenchymal stem cell (MSC), a muscle derived stem cell, or a bone marrow mesenchymal progenitor cell (MPC).

### Claim 15 (depends on 1)

The cell of claim 1, wherein said promoter is a cytomegalovirus (CMV) promoter.

### Claim 16 (depends on 10)

The tissue of claim 10, wherein said promoter is a cytomegalovirus (CMV) promoter.