# US 9,434,925 B2 · Genetically modified mesenchymal stem cell that express an exogenous cytotoxic protein

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- Number: US 9,434,925 B2
- Document ID: US9434925B2
- Status: Granted
- Kind: B2
- Application number: 13/264,084
- Filed: 2010-04-13
- Published: 2012-04-12
- Granted: 2016-09-06
- Earliest claimed: 2009-04-13
- Assignees: Apceth GmbH & Co. KG
- Applicants: Peter Nelson
- Inventors: Peter Nelson
- Web page: https://patentgrep.com/patent/US9434925B2

## CPC

- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- C12N 15/86: Viral vectors
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C12N 2510/00: Genetically modified cells
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

This invention provides a method for treating a subject afflicted with a tumor using genetically modified mesenchymal stem cells, wherein each genetically modified mesenchymal stem cell contains an exogenous nucleic acid comprising (i) a cytotoxic protein-encoding region operably linked to (ii) a promoter or promoter/enhancer combination, whereby the cytotoxic protein is selectively expressed when the genetically modified mesenchymal stem cells come into proximity with the tumor's stromal tissue. This invention further provides genetically modified mesenchymal stem cells for use in this method.

## Claims

### Claim 1

A method for treating a subject afflicted with a tumor comprising introducing into the subject's bloodstream a therapeutically effective number of genetically modified mesenchymal stem cells, wherein each genetically modified mesenchymal stem cell contains an exogenous nucleic acid comprising (i) a cytotoxic protein-encoding region operably linked to (ii) a RANTES promoter.

### Claim 2 (depends on 1)

The method of claim 1, wherein the subject is human.

### Claim 3 (depends on 1)

The method of claim 1, wherein the tumor is metastatic.

### Claim 4 (depends on 1)

The method of claim 1, wherein the tumor is vascularized.

### Claim 5 (depends on 1)

The method of claim 1, wherein the tumor is not vascularized.

### Claim 6 (depends on 1)

The method of claim 1, wherein the genetically modified mesenchymal stem cells are CD34− stem cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the genetically modified mesenchymal stem cells are allogenic with respect to the subject.

### Claim 8 (depends on 1)

The method of claim 1, wherein the genetically modified mesenchymal stem cells are autologous with respect to the subject.

### Claim 9 (depends on 1)

The method of claim 1, wherein the tumor's stromal tissue comprises fibroblast-like cells.

### Claim 10 (depends on 1)

The method of claim 1, wherein the introduction of the genetically modified mesenchymal stem cells is not preceded, accompanied or followed by myeloablation.

### Claim 11 (depends on 1)

The method of claim 1, wherein the tumor is selected from the group consisting of a prostate tumor, a breast tumor, a pancreatic tumor, a squamous cell carcinoma, a breast tumor, a melanoma, a basal cell carcinoma, a hepatocellular carcinoma, testicular cancer, a neuroblastoma, a glioma or a malignant astrocytic tumor, a colorectal tumor, an endometrial carcinoma, a lung carcinoma, an ovarian tumor, a cervical tumor, an osteosarcoma, a rhabdo/leiomyosarcoma, a synovial sarcoma, an angiosarcoma, an Ewing sarcoma/PNET and a malignant lymphoma.

### Claim 12 (depends on 1)

The method of claim 1, wherein the tumor is a pancreatic tumor.

### Claim 13 (depends on 1)

The method of claim 1, wherein the cytotoxic protein is Herpes simplex viral thymidine kinase, and the subject is treated with ganciclovir in a manner permitting the Herpes simplex viral thymidine kinase to render the ganciclovir cytotoxic.

### Claim 14 (depends on 1)

The method of claim 1, wherein the therapeutically effective number of genetically modified mesenchymal stem cells is from about 1×105 to about 1×109 cells/kg body weight.

### Claim 15 (depends on 1)

The method of claim 1, wherein the therapeutically effective number of genetically modified mesenchymal stem cells is from about 1×106 to about 1×108 cells/kg body weight.

### Claim 16 (depends on 15)

The method of claim 15, wherein the therapeutically effective number of genetically modified mesenchymal stem cells is from about 5×106 to about 2×107 cells/kg body weight.

### Claim 17

A genetically modified mesenchymal stem cell comprising an exogenous nucleic acid comprising (i) a cytotoxic protein-encoding region operably linked to (ii) a RANTES promoter.

### Claim 18 (depends on 17)

The stem cell of claim 17, wherein the stem cell is a human stem cell.

### Claim 19 (depends on 17)

The stem cell of claim 17, wherein the stem cell is a CD34− stem cell.

### Claim 20 (depends on 17)

The stem cell of claim 17, further comprising a (iii) selection marker gene operably linked to (iv) a constitutive promoter or promoter/enhancer combination.

### Claim 21 (depends on 20)

The stem cell of claim 20, wherein the selection marker gene comprises an antibiotic resistance gene or a gene encoding a surface marker protein.

### Claim 22 (depends on 20)

The stem cell of claim 20, further comprising an (v) insulator sequence located between the cytotoxic protein-encoding region and a surface marker gene.

### Claim 23 (depends on 17)

The stem cell of claim 17, wherein the cytotoxic protein-encoding region operably linked to the RANTES promoter is integrated into the stem cell genome.

### Claim 24 (depends on 20)

The stem cell of claim 20, wherein the selection marker gene operably linked to the constitutive promoter or promoter/enhancer combination is integrated into the stem cell genome.

### Claim 25 (depends on 20)

The stem cell of claim 20, wherein the cytotoxic protein-encoding region operably linked to the RANTES promoter and the selection marker gene operably linked to the constitutive promoter or promoter/enhancer combination are a part of a proviral sequence integrated into the stem cell genome.

### Claim 26 (depends on 25)

The stem cell of claim 25, wherein the proviral sequence is a lentiviral, alpha-retroviral or gamma-retroviral sequence.

### Claim 27 (depends on 17)

The stem cell of claim 17, wherein the cytotoxic protein is Herpes simplex viral thymidine kinase or cytosine deaminase.

### Claim 28

An isolated genetically modified human CD34− stem cell comprising an exogenous nucleic acid comprising (i) a Herpes simplex viral thymidine kinase-encoding region operably linked to (ii) a RANTES promoter.

### Claim 29 (depends on 11)

The method of claim 11, wherein the glioma or malignant astrocyte is a glioblastoma multiforme.

### Claim 30 (depends on 1)

The method according to claim 1, wherein the therapeutically effective number of genetically modified mesenchymal stem cells is introduced into the subject's bloodstream via intravenous injection.