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Method of Regulating Transcription in a Cell and Methods of Modulating Gene Expression (S00004.pdf)

Inventors
Beverly Emerson and Shilpa Kadam

Applications
Drug Discovery, Regulation of Gene Expression, Chromatin Remodeling
Chromatin based technology to define novel and highly specific protein targets for selective gene modulation

Appropriate differentiation and development of higher organisms require precisely regulated expression of multiple genes. The packaging of DNA into chromatin within the eukaryotic nucleus is highly organized and plays a critical role in regulating gene expression. The various types of chromatin structures that form on individual genes determine whether a gene is turned on or off. Regulation of chromatin structure is a critical component of gene regulation which affects tissue differentiation and cell function related to aging, cancer and a variety of diseases caused by changes in gene expression. Drug discovery related to modulation of gene expression, either to down regulate an overexpressed gene or to up regulate a silenced gene cannot be done at the DNA level alone. Chromatin based assays allow for the natural state of DNA regulation and provide optimal targets for therapeutic intervention. One important family of mammalian chromatin remodeling complexes, SWI/SNF (switch/sniff) is targeted to individual genes to specifically regulate its expression. SWI/SNF interacts with only certain classes of transcription factors and this property enables SWI/SNF to be selectively recruited to particular promoters. For example, proteins containing zinc finger DNA-binding domains (ZF DBDs), which is the largest class of eukaryotic transcription factors, interact with the BRG1 subunit of human SWI/SNF complex and recruit this complex to target promoters rather than the other catalytic subunit of SWI/SNF, BRM complexes. One invention provides for screening assays that identify small molecules that enhance or block the association between chromatin remodeling complexes and the specific transcription factors with which they interact. Specifically, the assays utilize several families of chromatin remodeling complexes which play key roles in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man. These in vitro assays can be developed for high-throughput screening to identify small molecule drugs that alleviate specific diseases caused by gene over or under expression. The second invention relates to three new findings of commercial potential. First, ZF DBDs or peptides are sufficient to direct SWI/SNF to a repressed promoter and create an accessible chromatin structure which enables other transcription factors to interact and activate the gene. In the absence of the ZF DBD, SWI/SNF and the other factors do not interact and the gene remains silent. Second, SWI/SNF BRG1 complexes, but not BRM, bind to the CREB transcription factor only when phosphorylated. CREB is the critical regulator of cAMP-response genes and thus a direct link between this signaling pathway and targeted chromosomal binding and gene activation by a specific form of SWI/SNF is established. Third, the opposite specificity exists for another type of signaling pathway. In this case, BRM SWI/SNF, but not BRG1, interacts with critical regulators of the Notch signaling pathway and is recruited to Notch target genes. Thus it is possible to identify drugs that selectively inhibit one type of pathway without affecting others..

References
S00004: Cell 95(1):93-104(October 1998)
S02019: Molecular Cell 11:377-389 (February 2003)

Patent Status:
U.S. Patent Application published as 2002-0022021
U.S. Patent Application published as 2005-0079512

License Terms:
Non-Exclusive Licenses Negotiable

Reference_Number: S00004
Contact: Dave Odelson, Ph.D. o Senior Licensing Executive o 858.453.4100 x1223 o dodelson@salk.edu

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Products Useful for Modulating Expression of Exogenous Genes in Mammalian Systems (S98001.pdf)

Inventors
Ronald Evans and David No

Applications
Gene Expression
Modulation of exogenous gene expression in mammalian and other systems using modified ecdysone receptors

This invention relates to various methods for modulating the expression of an exogenous gene in a mammalian subject employing modified ecdysone receptors. The invention method is useful in a wide variety of applications where inducible in vivo expression of an exogenous gene is desired, such as in vivo therapeutic methods for delivering recombinant proteins into a variety of cells within a patient. Advantages of ecdysteroid use include the lipophilic nature of the compounds, short half-lives, and favorable pharmacokinetics that prevent storage and expedite clearance. The invention also relates to modified ecdysone receptors, nucleic acids encoding modified ecdysone receptors, modified ecdysone response elements, gene transfer vectors, recombinant cells, and transgenic animals containing nucleic acids encoding modified ecdysone receptor..

References
Proc. Natl. Acad. Sci. 93: 3346-3351 (1996)

Patent Status:

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A Molecular Switch Regulating Neurogenesis (S04010.pdf)

Inventors
Fred Gage and Tomoko Kuwabara

Applications
CNS, Modulation of Gene Expression
A "master switch" that determines whether the genetic program that mediates neural differentiation is repressed or activated

During the initiation of neurogenesis, neural stem cells exit the undifferentiated state and commit to becoming neuroblasts, the first stage in the differentiation toward neurons. Previous efforts have been unsuccessful in revealing the mechanism which controls this neural phenotype development. This invention describes the transcriptional regulatory mechanism that functions as a "master switch" in neural development, determining whether the genetic program that mediates neural differentiation is repressed or activated. The mechanism relates to recently identified LEF/Sox overlapping response elements within the upstream regulatory sequences of neural specific genes such as NeuroD1. Activation via this molecular switch induces neurogenesis in neural stem cells and controls the irreversible commitment step from stem cells to neuroblasts. Compositions and methods are described for directing neural specific expression of polynucleotide sequences. These can be used to modulate differentiation of stem cells into neural lineage cells, or, for the prevention/inhibition of differentiation-for example, to maintain a stem cell in an undifferentiated state. New screening methods have also been identified that are particularly suited for high-throughput evaluation of large composition libraries to identify agents that modulate differentiation of stem cells..

References
No publications to date

Patent Status:
Application pending

License Terms:
Exclusive, Partially Exclusive, Nonexclusive license negotiable

Reference_Number: S04010
Contact: Mike White, Ph.D., CLP o Director, OTM o 858.453.4100 x1703 o mwhite@salk.edu

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Hormone Receptor Functional Dimers and Methods of Use (S98032.pdf)

Inventors
Fred Gage, Steven Suhr, Elad Gil and Marie-Claude Senut

Applications
Gene Expression.


This invention relates to chimeric proteins comprising at least two functional protein units. The chimeric proteins can fold under crystallization conditions to form functional entities. The DNA binding characteristics of the invention functional entities differ from those of wild-type complexes formed between "monomeric" receptors and their binding partners. Some functional entities, e.g. dimers expressed as fusion proteins, transactivate responsive promoters in a manner similar to wild-type complexes, while others do not promote transactivation and function instead essentially as constitutive repressors..

References
Published PCT Application WO01/36447

Patent Status:
U.S. Application pending, PCT filed Oct. 2000

License Terms:
Exclusive, Partial Exclusive or Nonexclusive license negotiable

Reference_Number: S98032
Contact: Mike White, Ph.D., CLP o Director, OTM o 858.453.4100 x1703 o mwhite@salk.edu

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Modular Assembly Retroviral Vectors and Uses Therefor (S96013.pdf)

Inventors
Fred H. Gage and Steven T. Suhr

Applications
Gene Expression/Gene Therapy
Gene transfer vectors with the capacity for prolonged or modulated transgene expression

This invention relates to novel retroviral vectors containing modified long terminal repeats (LTR) which enable high level and ligand-modulatable expression of a desired gene product, even after prolonged periods of cellular quiescence. These novel vectors overcome proviral transcriptional inactivation which occurs in cultured primary cells that are growth arrested due to environmental constraints such as contact inhibition and/or nutrient starvation. These vectors represent a class of retroviral vectors in which LTR-promoted proviral expression in infected cells may be maintained or increased, even in situations generally considered to be non-permissive for retroviral vectors. This invention can be applied: as gene transfer vectors with the capacity for prolonged or modulated transgene expression for either in vivo or ex vivo gene therapy; as gene transfer vectors for efficient production of transgenic animals; as vectors for efficient gene transfer to developing embryos; and as vectors with inducible high titers..

References
No publications to date

Patent Status:

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Regulation of Tyrosine Hydroxylase Expression (S98035.pdf)

Inventors
Kazuhiro Sakurada, Theo Palmer and Fred H. Gage

Applications
Gene Expression, CNS
Nurr1 polypeptide upregulates tyrosine hydroxylase activity and promotes expression of DOPA, norepinenephrine and epinephrine.

The invention describes methods to regulate tyrosine hydroxylase expression and the treatment of catecholamine-related diseases including Parkinson's disease, manic depression and schizophrenia. The basis for the invention is the discovery that expression of Nurr1 polypeptide induces tyrosine hydroxylase in both undifferentiated and differentiated mammalian cells including adult hippocampal progenitor cells. Specifically, the invention provides cells that contain exogenous nucleic acid as well as methods and materials for inducing tyrosine hydroxylase expression, treating catecholamine-related deficiencies and identifying tyrosine hydroxylase-related deficiencies. Neural progenitor cells, neural cells and neural stem cells which contain the exogenous nucleic acid and express the Nurr1 polypeptide are upregulated for tyrosine hydroxylase activity and thus promote the expression of DOPA, norepinephrine and epinephrine. These cells can be used to treat catecholamine deficiency diseases directly. In addition, these diseases can be treated by transfection using the genetic construct for Nurr1 expression..

References
Development. 126(18): 4017-26 (Sept. 1999)

Patent Status:

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Stimulation of Transgene Expression by RNA Export Elements (S97031.pdf)

Inventors
Thomas Hope, Romain Zufferey, Didier Trono, John Edward Donello

Applications
Gene Expression
Enhance transgene expression by promoting correct processing and transport of RNA from the cell nucleus to the cytoplasm.

High levels of transgene expression are desired for most gene therapy protocols. This invention is directed to cis-acting RNA export elements and the use of such elements to enhance transgene expression. Specifically, the RNA export elements of the invention enhance transgene expression by promoting correct processing and transport of RNA from the cell nucleus to the cytoplasm. Nuclear export of RNAs containing invention cis acting RNA export elements is mediated by cellular factors which interact with structural elements contained within the cis acting elements. Accordingly, such export requires no exogenous factors. Introduction of a cis-acting RNA export element of the invention into a retroviral vector substantially increases the expression of transgene contained in the vector. For example, introduction of an export element derived from the woodchuck hepatitis virus (WHV) downstream of GFP and luciferase cDNAs, within the context of an HIV-based vector, increased expression of these reporters from 5- to 10-fold compared to homologous vectors which did not contain the export element. Stimulation of transgene expression by the export element is vector and promoter independent. Similar stimulatory effects are seen when the transgene is delivered in the widely utilized MLV based vector system. Moreover, the invention export element derived from the WHV stimulates transgene expression to similar levels when stable cell populations are generated by transfection. Transgene expression utilizing the export element derived from WHV is also stimulated when either the strong CMV promoter or the weaker thymidine kinase promoter are utilized to drive transgene expression. The posttranscriptional regulatory element isolated from WHV, namely WPRE, is notable because it is an extremely efficient RNA export element with the ability to activate a reporter for RNA export up to levels achieved with HIV-1 Rev and the Rev-responsive element (RRE)..

References
Journal of Virology, Vol. 73 No. 4: p. 2886-2892 (April 1999)
Human Gene Therapy 10:2295-2305 (September 1999)
Journal of Virology Vol. 72 No. 6: p. 5085-5092 (June 1998)
Molecular and Cellular Biology, Vol. 22 No.7, p. 2057-2067 (April 2002)
Methods Mol Biol. 183: 331-40 (2002)

Patent Status:
U.S. Patent No. 6,287,814 issued Sept. 11, 2001
U.S. Patent No. 6,312,912 issued Nov. 6, 2001
PCT/US98/19441

License Terms:
Non-exclusive Field of Use licenses available

Reference_Number: S97031
Contact: Mike White, Ph.D., CLP o Director, OTM o 858.453.4100 x1703 o mwhite@salk.edu

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pCL Vector System: Rapid Production of Helper-Free, High-Titer, Recombinant Retroviruses (pCL.pdf)

Inventors
Inder Verma

Applications
Gene Expression
Maximize recombinant-retrovirus titers in a simple, robust, and flexible experimental system.

Production, cloning, and characterization of helper-free retrovirus vectors takes about two months when conventional murine retrovirus packaging cell lines are used. The pCL vector system includes retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 106 to 5 X 106 within 48 hours. The pCL vectors have been designed to maximize recombinant-retrovirus titers in a simple, robust, and flexible experimental system. By selecting vectors designed to express genes from one of four promoters, the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal and include an added safety modification that renders them self-inactivating..

References
J. of Virology Vol. 70, No. 8, p. 5701-5705 (1996)

Patent Status:
No Application filed

License Terms:
Nonexclusive license negotiable

Reference_Number: pCL
Contact: Mike White, Ph.D., CLP o Director, OTM o 858.453.4100 x1703 o mwhite@salk.edu

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FLP-Mediated Gene Modification in Mammalian Cells, and Compositions and Cells Useful Therefor (S95048.pdf)

Inventors
Geoff Wahl and Stephen O'Gorman

Applications
Gene Expression
Rapid and precise site-specific recombination in mammalian cells

The invention relates to methods for the site-specific recombination of DNA in mammalian cells or host mammalian organisms and is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. The invention also relates to novel DNA constructs, as well as compositions, cells and host organisms containing such sequences. FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. FLP can be used to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development..

References
Science Vol. 251, pp. 1351-1355 (1991)

Patent Status:
U.S. Patent No. 5,677,177 issued October 14, 1997

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Site-Specific Recombination in Eukaryotes and Constructs Useful Therefor (S97025.pdf)

Inventors
Geoff Wahl and Stephen O'Gorman

Applications
Gene Expression
A fusion of the protamine 1 gene promoter with the Cre recombinase gene for efficient manipulation of chromosomal sequences in mammalian systems

The invention relates to the expression of site-specific recombinases in combination with germ-line promoters and embryonic stem cells to markedly facilitate the production of altered chromosomal alleles in intact organisms. For example, by this method it is possible to create alleles in mice that have been homologously recombined and then recombined by a site-specific recombinase in no more time, and with the expenditure of no more resources, than it would typically take to create a simple, homologously recombined allele. In one application of this method the mouse protamine-1 gene promoter was fused to the Cre recombinase gene and inbred strain 129 transgenic mice were prepared. Males that contained the transgenes and a Cre recombination target efficiently recombined the target in their germ cells, but not in other tissues. More than 80% of the progeny of such males inherited the Cre-recombined target. Mouse embryonic stem cells (129 strain) have been derived from one protamine-Cre transgenic line. A variety of target genes have been homologously recombined in these cells using targeting vectors that contain loxP-flanked selectable markers. When targeted embryonic stem cells have been used to prepare chimeras, good germline transmission has been routinely obtained and the selectable marker has been efficiently excised in the germline of the chimeras; most progeny inherit the Cre-recombined allele..

References
Proc Natl Acad Sci USA Vol. 94, pp. 14602-14607 (December 1997)
Published PCT Application WO9910488

Patent Status:
U.S. Application pending; No Foreigns

License Terms:
Nonexclusive field of use licenses negotiable; Also requires Cre-Lox license from Bristol-Myers Squibb

Reference_Number: S97025
Contact: Dave Odelson, Ph.D. o Senior Licensing Executive o 858.453.4100 x1223 o dodelson@salk.edu