Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-13
2002-11-12
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S325000
Reexamination Certificate
active
06479653
ABSTRACT:
BACKGROUND OF THE INVENTION
Activation of transcription of a eukaryotic gene involves the interaction of a variety of proteins to form a complex that is recruited to the gene through protein:DNA interactions. Key protein domains on one or more of the components include transcription activation domains and DNA binding domains. Elucidating the mechanism of transcription, identifying and characterizing components of the transcriptional machinery and in some cases harnessing some of those components have been the subject of extensive research. (See, e.g., Brent and Ptashne, 1985; Hope and Struhl, 1986; Keegan et al. 1986., Fields and Song, 1989; Spencer et al, 1993, Belshaw et al, 1996 and Rivera et al, 1996)(A Bibliography is provided just prior to the Examples, below.)
Transcription activation domains are thought to function by recruiting a number of proteins with specific functions to the promoter (Lin and Green, 1991; Goodrich et al, 1993; Orphanides et al. 1996 and references cited therein; Ptashne and Gann, 1997 and references cited therein). Among the large number of activation domains that have been characterized to date, the acidic-activation domain of the Herpes Simplex virus encoded protein, VP16, is considered to be a very strong inducer of transcription and is widely used in biological research (Sadowski et al, 1988, Ptashne and Gann, 1997). The transcription activation domain of the p65 subunit of the human transcription factor NF-kB is also a very potent stimulator of gene expression, and in certain contexts can induce transcription more strongly than VP16 (Schmitz and Baeuete, 1991; Ballard et al, 1992; Moore at al, 1993, Blair et al, 1994; Natesan et al, 1997). Both the VP16 and p65 activation domains are thought to function by interacting with and recruiting a number of proteins to the promoter (Cress and Triezenberg, 1990; Scmitz at al, 1994; Uesugi et at, 1997).
One of the remarkable features of such activation domains is that “fusing” them to heterologous protein domains seldom affects their ability to activate transcription when recruited to a wide variety of promoters. The high degree of functional independence exhibited by these activation domains makes them valuable tools in various biological assays for analyzing gene expression and protein-protein or protein-RNA or protein-small molecule drug interactions (Fields and Song, 1989; Senguptha et al, 1996; Rivera et al, 1996; Triezenberg, 1995 and references cited therein). The ability to activate gene expression strongly and when recruited to a wide range of promoters makes both p65 and VP16 attractive candidates for activation of gene transcription in gene therapy and other applications. However, even more potent activation domains, if available, would be useful for achieving higher levels of transcription on a per cell basis, and for improving the efficiency of the many biological assays that rely upon activation of transcription of a reporter gene.
Several strategies to improve the potency of activation domains and thereby the expression of genes under their control have been reported (Emami and Carey, 1992; Gerber at al, 1994; Ohashi et al, 1994; Blair at al, 1996; Tanaka et al, 1996). These approaches generally involve increasing the number of copies of activation domains fused to the DNA binding domain or generating activators containing synergizing combinations of activation domains. Although some activators generated by these methods have been shown to be more potent, a number of limitations preclude their widespread application. First, potent activators comprising reiterated activation domains do not increase the absolute levels of reporter gene expression when tested on promoters with multiple binding sites for the activator (Emami and Carey, 1992). Second, a number of synergistic combinations of activation domains reported in the literature involve weak activation domains and the absolute levels of gene expression induced by these synergizing activation domains are much lower compared to potent acidic activation domains from VP16 or p65 (Gerber at al, 1994; Tanaka et al, 1996). Third, it is not known whether any of these potent activation domains are capable of inducing gene transcription strongly when they are non-covalently linked to the DNA binding domain. Fourth, many potent activators containing multiple copies of VP16 or other acidic activators are highly toxic and/or accumulate to only low levels in the cell.
As mentioned at the outset, a variety of important applications involving gene transcription require or would benefit from higher levels of gene expression. As noted above, however, efforts to improve the potency of activation domains have been disappointing. Moreover, expression of various transcription activators revealed that observed levels of more potent activators, such as the p65 unit of NF-kB, are lower than expected. Without wishing to be bound by any one theory, we suggest that the more potent the activation domain, the more toxic it is to the cell, the more disfavored is its expression and/or the less of it is observed to accumulate in cells. How, then, is it possible to increase levels of heterologous gene expression? Remarkably, we have found that it is still possible to outmaneuver these facts of nature to improve heterologous gene expression and have in fact done so using the principles of “bundling”, the engineering of the transcription activation domain, and combinations thereof, as described below.
SUMMARY OF THE INVENTION
This document discloses new improvements in the design and delivery of transcription activation domains and provides improved materials and methods for regulating the transcription of a target gene. Aspects of the invention are applicable to systems involving either covalent or non-covalent linking of the transcription activation domain to a DNA binding domain.
Key features of the invention include “bundling” domains, fusion proteins containing them, recombinant nucleic acids encoding such fusion proteins, systems involving bundles of such fusion proteins, and other materials and methods involving such bundling domains. Key fusion proteins of the invention contain at least two mutually heterologous domains, one of which being a bundling domain. An important design concept is that the fusion proteins do not need to act alone. Instead, they find and bind to each other (or with other proteins containing the bundling domain) to form a posse to accomplish their mission. In practice, cells are engineered by the introduction of recombinant nucleic acids encoding the fusion proteins, and in some cases with additional nucleic acid constructs, to render them capable of ligand-dependent regulation of transcription of a target gene. Administration of the ligand to the cells then regulates (positively, or in some cases, negatively) target gene transcription.
Detailed information concerning bundling domains, guidance on their use and illustrative examples are provided below. Generally speaking, bundling domains include any domain that induces proteins that contain it to form multimers (“bundles”) through protein-protein interactions with each other or with other proteins containing the bundling domain. Examples of bundling domains that can be used in the practice of this invention include domains such as the lac repressor tetramerization domain, the p53 tetramerization domain, a leucine zipper domain, and domains derived therefrom which retain observable bundling activity. Proteins containing a bundling domain are capable of complexing with one another to form a bundle of the individual protein molecules. Such bundling is “constitutive” in the sense that it does not require the presence of a cross-linking agent (i.e., a cross-linking agent which doesn't itself contain a proteinaceous bundling domain) to link the protein molecules.
Illustrative (non-limiting) examples of heterologous domains which can be included along with a bundling domain in various fusion proteins of this invention include transcription regulatory domains (i.e., transcription activ
Gilman Michael Z.
Natesan Sridaran
ARIAD Gene Therapeutics, Inc.
Berstein David L.
Loeb Bronwer M.
Yucel Remy
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