Embryonal cardiac muscle cells, their preparation and their use

• US 5,928,943 A
• Filed: 08/22/1997
• Issued: 07/27/1999
• Est. Priority Date: 11/22/1994
• Status: Expired due to Term

• Current Assignee: Cardion AG
• Original Assignee: Institut Fur Pflanzengenetik Und Kulturpflanzenforschung
• Inventors: Franz, Wolfgang-Michael, Wobus, Anna M.
• Primary Examiner(s): Degen, Nancy
• Application Number:
  US08/849,706
• Time in Patent Office: 704 Days
• Field of Search: 435/325, 435/29, 435/363, 435/34, 435/366, 435/375, 514/44
• US Class Current: 435/363
• 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 2506/02 from embryonic cells
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5061 Muscle cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The invention concerns embryonal (cardiomyocytary and cardiomyoblastary) cardiac muscle cells, their preparation and their use, in particular for cell-mediated gene transplant. The areas of application of the invention are medicine and genetic engineering. The embryonal cardiac muscle cells according to the invention contain two gene constructs comprising: a) a regulatory, 1.2-kb long DNA sequence of the ventricle-specific myosin light-chain-2 (MLC-2v) promoter, the selectable marker gene β-galactosidase in fusion with the reporter gene neomycin; and b) a regulatory DNA sequence of the herpes simplex virus thymidine kinase promoter and the selectable marker gene hygromycin, and optionally immortalizing genes and/or optionally genes inactivated by homologous recombination and/or optionally one or a plurality of therapeutic genes. The cells can advantageously be used for cell-mediated gene transplant, in particular for constructing healthy tissue and assisting contractile functions, for investigating substances, in particular for pharmacological investigations and for the transfer of therapeutic genes into the myocardium.

Claims

1. An embryonal cardiac muscle cell, essentially consisting of two gene constructs of two different regulatory DNA sequences and two different selectable marker genes.

2. The embryonal cardiac muscle cell according to claim 1, essentially consisting of two gene constructs comprising

a) a regulatory, 2.1 kb long DNA sequence of the ventricle-specific myosin light-chain-2(MLC-2v) promoter, the selectable marker gene β

-galactosidase, and the reporter gene neomycin, and

b) a regulatory DNA sequence of the herpes simplex virus thymidine kinase promoter and the selectable marker gene hygromycin.

3. The embryonal cardiac muscle cell according to claim 1, further comprising sequences of the adenovirus (Ad) or of the adeno-associated virus (AAV).

4. A vector, comprising MLC-2 promoter, selectable marker gene β

-galactosidase and the reporter gene neomycin as fusion gene "β

geo", and SV40-Poly A-Tail.

5. The vector according to claim 4, further comprising

a position for receiving immortalizing genes.

6. A vector, essentially consisting of a regulatory DNA sequence of the herpes simplex virus thymidine kinase promoter and the selectable marker gene hygromycin.

7. A method for determining the effects of pharmacological substances comprising

contacting pharmacological substances with embryonal cardiac muscle cells;

and

determining the effects of said substances on said cells.