Sangamo Biosciences, Inc.

Amgen Inc.

The United States of America, As Represented by the Secretary, Department of Health and Human Services

University of Colorado

The University of North Carolina at Chapel Hill

The United States of America As Represented by the Department of Health

Synergen, Inc.

Amgen Boulder Inc.

Nuron Biotech, Inc.

Cytokine Sciences, Inc.

Stephen P Eisenberg

Casey Christopher Case

Eric E Jarvis

George N Cox

Edward J Rebar

Andrew Jamieson

Sharon Kaye Spratt

Robert C Thompson

Qiang Liu

Pei-Qi Liu

Alan P Wolffe

Gary L Stetler

Pradip K Bandyopadhyay

Sharon M Wahl

Jeffrey L Cleland

Mary S Rosendahl

Andreas Sommer

Charles H Hannum

Matthew B Seefeldt

Michael T Brewer

Charles H Kirkpatrick

William P Arend

Fenneke G Joslin

Elizabeth J Wolffe

George Cox, Iii

David J Dripps

Robert Hageman

David Dripps

Ronald Evans

Henryk Cudny

Walker H Busby, Jr

David R Clemmons

Martin J McDermott

The present invention provides methods for modulating expression of endogenous cellular genes using recombinant zinc finger proteins.

Regulation of endogenous gene expression in cells using zinc finger proteins

High pressure to treat aggregated interferons, particularly recombinant human interferon-&#x3b2;, to reduce the aggregate content of interferon material. Highly pure, soluble monomeric recombinant interferon-&#x3b2; is prepared in representative embodiments. Multiple strategies may be used in combination that make nonglycosylated IFN-&#x3b2; more amenable to high pressure treatment. It has been found that refolding yields of high pressure treatment can be significantly improved by use of a combination of strategies, including, or example a pre-treatment of the IFN-&#x3b2; that involves solubilizing and then precipitating the protein. This pre-treatment is particularly effective with respect to recombinant IFN-&#x3b2; inclusion bodies recovered from host cells such ascells. According to another strategy, refolding under high pressure is much more effective when the refolding reagent incorporating the IFN-&#x3b2; incorporates a zwitterionic surfactant and/or a cholate salt. When a solubilization and precipitation pre-treatment is used, the effectiveness of the high pressure treatment is further enhanced when the refolding reagent incorporating the protein incorporates a disulfide shuffling chemistry such as cysteine/cystine. According to still yet another strategy, high pressure treatment is more effective when using atypically high treatment pressures. When coupled with purification techniques, these strategies singly or in combination provide a low aggregate or substantially aggregate free, biologically active solution. Biologically active solutions comprising nonglycosylated interferon, said interferon comprising less than about 5 weight percent of protein aggregation has been found to exhibit improved PK/PD characteristics.

High pressure treatment of aggregated interferons

Repressing endogenous CCR5 gene expression in cells using engineered zinc finger proteins

Provided herein are a variety of methods and compositions for regulating angiogenesis, such methods and compositions being useful in a variety of applications where modulation of vascular formation is useful, including, but not limited to, treatments for ischemia and wound healing. Certain of the methods and compositions accomplish this by using various zinc finger proteins that bind to particular target sites in one or more VEGF genes. Nucleic acids encoding the zinc finger proteins are also disclosed. Methods for modulating the expression of one or more VEGF genes with the zinc finger proteins and nucleic acids are also disclosed. Such methods can also be utilized in a variety of therapeutic applications that involve the regulation of endothelial cell growth. Pharmaceutical compositions including the zinc finger proteins or nucleic acids encoding them are also provided.

Regulation of angiogenesis with zinc finger proteins

The invention provides criteria and methods for selecting optimum subsequence(s) from a target gene for targeting by a zinc finger protein. Some of the methods of target site selection seek to identify one or more target segments having a DNA motif containing one or more so-called D-able subsites having the sequence 5'NNGK3&#x2032;. Other methods of the invention are directed to selection of target segments within target genes using a correspondence regime between different triplets of three bases and the three possible positions of a triplet within a nine-base site. In another aspect, the invention provides methods of designing zinc finger proteins that bind to a preselected target site. These methods can be used following the preselection of target sites according to the procedures and criteria described above. The methods of design use a database containing information about previously characterized zinc finger proteins.

Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites

Disclosed herein are compositions comprising an engineered zinc finger protein and a nuclear hormone receptor transcriptional control domain, polynucleotides encoding one or more components of the compositions, methods of making the compositions and methods of using these compositions, for example to modulate gene expression and/or to generate transgenic plants or animals.

Ligand-controlled regulation of endogenous gene expression

The present invention provides methods for modulating expression of endogenous cellular genes using engineered zinc finger proteins.

Growing community of inventors

Boulder, CO, United States of America

Stephen P Eisenberg