Heterologous transcription factors

Details Heterologous transcription factors Heterologous transcription factors

1996-06-27

2001-10-23

Martin, Jill D. (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C435S455000, C435S069100, 57, C536S023100, C536S023400, C536S023500

Reexamination Certificate

active

06306649

ABSTRACT:

INTRODUCTION
A large number of biological and clinical protocols, among others, gene therapy, production of biological materials, and biological research, depend on the ability to elicit specific and high-level expression of genes encoding RNAs or proteins of therapeutic, commercial, or experimental value. Achieving a sufficiently high level of expression for clinical or other utility in genetically engineered cells within whole organisms has often been a limiting problem. Various approaches for addressing this problem, including the search for stronger transcriptional promoters or higher transfection efficiencies, have in many cases not met with success. Meanwhile, in various lines of research with transcription factors, promising results in transient transfection models have not been borne out with chromosomally integrated reporter gene constructs. Furthermore, overexpression of transcription factors is commonly associated with toxicity to the host cell. Despite those precedents, this invention takes a novel approach to the challenge of optimizing heterologous gene expression through new uses of, and new designs for, transcription factor proteins which are expressed within the engineered cells containing the target gene. The invention provides improved methods and materials for achieving high-level expression of a target gene in genetically engineered cells, including genetically engineered cells within whole organisms.
SUMMARY OF THE INVENTION
This invention involves protein transcription factors, DNA sequences encoding such proteins, transcription control sequences responsive to the transcription factors, target gene constructs containing a target gene operably linked to such a transcription control sequence, cells engineered to contain a target gene construct and to express such the transcription factor, organisms containing such cells and the use of these materials in gene therapy, production of biological materials, and biological research. In order to achieve constitutive expression of a target gene in a cell, preferably a cell within a host organism, one introduces into the organism cells which contain (a) a transcription factor construct containing a first heterologous DNA sequence encoding and capable of expressing a transcription factor capable of activating transcription of a gene linked to a trancription control sequence responsive to the transcription factor, and (b) a target gene construct containing a second heterologous DNA sequence comprising a target gene operably linked to a transcription control sequence comprising a DNA promoter sequence and one or more copies of a DNA recognition sequence permitting gene transcription responsive to the presence of the transcription factor.
Generally the cells are animal cells, preferably syngeneic to the host organism into which the cells are introduced. Host organisms of particular interest are mammals, i.e., post-implantation embryos and especially post-natal mammals. The invention is considered to be of particular significance to the practice of gene therapy with human subjects. In human gene therapy applications the engineered cells will typically be of mammalian origin, preferably human and in some cases autologous to the host.
The transcription factor may be a naturally occurring protein, especially if it is heterologous to the cell type to be engineered. Currently preferred embodiments, however, involve the use of a chimeric transcription factor containing at least two mutually heterologous peptide sequences. The transcription factor will contain one or more DNA-binding domains and one or more transcription activation domains, each of which containing peptide sequence often derived from naturally occurring transcription factors. For example, a fusion protein containing the well-known Herpes simplex virus transcription activation domain, VP16, linked to the bacterial DNA binding domain, GAL4, constitutes such a chimeric transcription factor. Preferably, however, the peptide sequence of each of the domains will be derived from a naturally occurring human peptide sequence. In some embodiments the DNA-binding domain and/or the transcription activation domain comprises a composite domain containing mutually-heterologous and/or reiterated subdomains.
The peptide sequence spanning positions 450 through 550 of human NF-kB p65, for instance, constitutes a transcription activation domain of human origin which may be used in transcription factors of this invention. In some embodiments, a novel, extended p65 sequence, spanning residues 361 through 550, is used. That peptide sequence is referred to herein as “p65(361-550)”. In various embodiments the transcription factor contains multiple copies of the transcription activation domain and/or a plurality of different transcription activation domains, subdomains or potentiating motifs. Transcription activation domains comprising a plurality of different and/or reiterated peptide sequences constitute composite transcription activation domains. One illustrative class of composite transcription activation domains comprise one or more copies of (a) the full sequence of p65(361-550), (b) one or more portions of that sequence, or (c) a combination of (a) and (c), together with one or more copies of one or more transcription activation potentiating motifs. Such motifs may be selected or derived from the so-called “proline-rich”, “glutamine-rich” and “acidic” activation motifs such as the VP16 V8 motif (DFDLDMLG)[SEQ ID NO:1], the related “V9” motif (DFDLDMLGG) [SEQ ID NO:2] or a human activation motif such as the 14 amino acid acidic motif of human heat shock factor.
Various DNA binding domains may be incorporated into the design of the transcription factor so long as a corresponding DNA “recognition” sequence is known or can be identified to which the domain is capable of binding. One or more copies of the recognition sequence are incorporated into the transcription control sequence of the target gene construct. Again, peptide sequence of human origin is preferred for the DNA binding domain(s). Composite DNA binding domains provide a means for achieving novel sequence specificity for the protein-DNA binding interaction. An illustrative composite DNA binding domain containing component peptide sequences of human origin is ZFHD-1 which is described in detail below. Individual DNA-binding domains may be further modified by mutagenesis to decrease, increase, or change the recognition specificity of DNA binding. These modifications could be achieved by rational design of substitutions in positions known to contribute to DNA recognition (often based on homology to related proteins for which explicit structural data are available). For example, in the case of a homeodomain, substitutions can be made in amino acids in the N-terminal arm, first loop, second helix, and third helix known to contact DNA. In zinc fingers, substitutions can be made at selected positions in the DNA recognition helix. Alternatively, random methods, such as selection from a phage display library could be used to identify altered domains with increased affinity or altered specificity. Individual DNA-binding domains may be further modified by mutagenesis to decrease, increase, or change the recognition specificity of DNA binding. These modifications could be achieved by rational design of substitutions in positions known to contribute to DNA recognition (often based on homology to related proteins for which explicit structural data are available). For example, in the case of a homeodomain, substitutions can be made in amino acids in the N-terminal arm, first loop, second helix, and third helix known to contact DNA. In zinc fingers, substitutions can be made at selected positions in the DNA recognition helix. Alternatively, random methods, such as selection from a phage display library could be used to identify altered domains with increased affinity or altered specificity.
In one embodiment, the DNA sequence encoding the transcription factor and the DNA sequence encoding the target gene are both ope

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