University of California

City of Hope

Other

Chiron Viagene, Inc.

San Diego Regional Cancer Center

Theodore Friedmann

Jiing-Kuan Yee

Fred Harrison Gage

Atsushi Miyanohara

Michael B Rosenberg

Shin-Tai Chen

Jon Asher Wolff

Jasodhara Ray

Jane C Burns

Malcolm Schinstine

Michael D Kawaja

Douglas J Jolly

Wen-Hwa Lee

Jack R Barber

Charles E Prussak

Robert E Sobol

Ivor Royston

Eva Y H P Lee

Nobuhiko Emi

Huei-Jen Sue Huang

Akihiro Abe

A method for gene therapy for cancers wherein chromosomal location of an inactive or defective cancer suppressing gene is established, a replacement gene which is preferably cloned is then used to replace the inactive or defective cancer suppressing gene in the chromosome. In addition to its uses in therapy, the present invention provides a means for prophylactically treating individuals having a genetic predisposition to cancer and provides an animal model for testing for carcinogenicity of environmental substances.

Products and methods for controlling the suppression of the neoplastic phenotype

The invention provides a system for production of retroviruses which are replication incompetent. In the system, gag and pol retroviral structural proteins are expressed separately by different plasmids in a packaging cell line. Separate gag and pol expressing plasmids are provided, as are packaging cell lines containing such plasmids. Retrovirus products of the retroviral vector production system, including chimeric retroviruses, are also provided.

Method for retrovirus vector production by separated gag and pol expression

The present invention features compositions and methods for the inducible expression of a polypeptide, especially a polypeptide normally cytotoxic to the eukaryotic host cell in which it is to be expressed. A nucleotide sequence encoding a polypeptide of interest is operably linked to an inducible promoter. Expression from the inducible promoter is regulated by a multi-chimeric transactivating factor, composed of a first ligand-binding domain that negatively regulates transcription, a transcriptional activation domain, and a second ligand-binding domain that positively regulates the transcriptional activation function of the transactivator. Transcription of the nucleotide sequence under control of the inducible promoter is activated by the multi-chimeric transactivator when both the ligand that binds the first ligand-binding domain is absent and the ligand that binds the second ligand-binding domain is present. This inducible expression system is particularly useful in the expression of the cytotoxic protein VSV G for the production of pseudotyped retroviral vectors.

Inducible expression system

Transfection of host cells cultured in the presence of serum is increased by adding VSV-G or polybrene to a nucleic acid-lipid complex or culture medium prior to transfection.

Transfection in serum-containing media

The present invention features compositions and methods for the inducible expression of a polypeptide, especially a polypeptide normally cytotoxic to the eukaryotic host cell in which it is to be expressed. A nucleotide sequence encoding a polypeptide of interest is operably linked to an inducible promoter (e.g, a promoter composed of a minimal promoter linked to multiple copies of tetO, the binding site for the tetracycline repressor (tetR) of the Escherichia coli tetracycline resistance operon Tn10). Expression from the inducible promoter is regulated by a multi-chimeric transactivating factor, composed of a first ligand-binding domain that negatively regulates transcription (e.g., a prokaryotic tetracycline repressor polypeptide), a transcriptional activation domain, and a second ligand-binding domain that positively regulates the transcriptional activation function of the transactivator (e.g., a ligand-binding domain of a steroid receptor, preferably an estrogen receptor (ER)). Transcription of the nucleotide sequence under control of the inducible promoter is activated by the multi-chimeric transactivator when both the ligand that binds the first ligand-binding domain (e.g., tetracycline) is absent and the ligand that binds the second ligand-binding domain (e.g., a steroid) is present. This inducible expression system is particularly useful in the expression of the cytotoxic protein VSV G for the production of pseudotyped retroviral vectors.

An enveloped vector particle contains gag and pol proteins from a retrovirus, a nucleic acid sequence and an envelope that includes VSV G envelope glycoprotein. The vector particle can be used to introduce nucleic acids into cells.

VSV G pseusdotyped retroviral vectors

Methods of genetically modifying donor cells by gene transfer for grafting into the central nervous system to treat defective, diseased or damaged cells are disclosed. The modified donor cells produce functional molecules that effect the recovery or improved function of cells in the CNS. Methods and vectors for carrying out gene transfer and grafting are described.

Method of grafting genetically modified cells to treat defects, disease

The present invention features packaging cell lines and recombinant retroviral particles produced thereby, particularly pseudotyped retroviral particles. Preferably, the packaging cell lines are derived from HeLa, Cf2Th, D17, MDCK, or BHK cells, most preferably from Cf2Th cells. Retroviral particles are produced by inducibly expressing an envelope protein of interest (e.g., a retroviral envelope or the envelope protein of vesicular stomatitis virus (VSV G)). Inducible expression of the envelope protein is accomplished by operably linking an envelope protein-encoding nucleotide sequence to an inducible promoter (e.g., a promoter composed of a minimal promoter linked to multiple copies of tetO, the binding site for the tetracycline repressor (tetR) of the Escherichia coli, tetracycline resistance operon Tn10). Expression from the inducible promoter is regulated by a multi-chimeric transactivating factor, composed of a first ligand-binding domain that negatively regulates transcription from the inducible promoter (e.g., a prokaryotic tetracycline repressor polypeptide (tetR)), a transcriptional activation domain, and a second ligand-binding domain (e.g., a ligand-binding domain of a steroid receptor, preferably an estrogen receptor (ER)).

Packaging cell lines for pseudotyped retroviral vectors

A novel method of tumor immunotherapy is described comprising the genetic modification of cells resulting in the secretion of cytokine gene products to stimulate a patient's immune response to tumor antigens. In one embodiment, autologous fibroblasts genetically modified to secrete at least one cytokine gene product are utilized to immunize the patient in a formulation with tumor antigens at a site other than an active tumor site. In another embodiment, cells genetically modified to express at least one tumor antigen gene product and to secrete at least one cytokine gene product are utilized in a formulation to immunize the patient at a site other than an active tumor site.

Cancer immunotherapy with carrier cells

The present application discloses retrovirus-derived vectors in which the retroviral envelope glycoprotein has been replaced by the G glycoprotein of vesicular stomatitis virus, and the use of these vectors in the transfer of exogenous genes into the cells of a wide variety of non-mammalian organisms. Also disclosed is a method for the generation of retroviral vectors in high titers, wherein a recombinant, stable host cell line is provided which harbors the retroviral vector of interest without envelope protein. High-titer retroviral vector production is initiated by introducing nucleic acid encoding a functional membrane-associated protein into the cell line. The vectors disclosed in the present application can be concentrated by ultracentrifugation to titers greater than 10.sup.9 cfu/ml which are especially useful in human gene therapy trials, and can also infect cells, such as hamster and fish cells, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein.

Growing community of inventors

La Jolla, CA, United States of America

Theodore Friedmann