Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal to manufacture...
Reexamination Certificate
2001-02-27
2002-09-03
Yucel, Remy (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal to manufacture...
C800S018000
Reexamination Certificate
active
06444871
ABSTRACT:
FIELD OF THE INVENTION
The present invention is concerned with compositions and methods that rely upon the tetracycline resistance (tet) operator and repressor to control transcription in mammalian cells. It encompasses methods for recombinantly producing proteins and the vectors and host cells utilized in such methods. In addition, the present invention is directed to viruses which are recombinantly engineered so that their replication is controlled by the tet operator/repressor system. These viruses may serve as vehicles for gene transfer both in vitro and in vivo; as agents for immunization; and as a means for delivering nucleic acid therapeutic agents to cells.
BACKGROUND OF THE INVENTION
The ability to specifically regulate transgene expression has been a central concern in molecular biology for many years. In the case of mammalian cells, the in vitro regulation of recombinant genes has most often been accomplished through the use of inducible promoters that respond to agents such as heavy metal ions (Brinster, et al.,
Nature
296:3942 (1982); heat shock (Nover, in
Heat Shock Response
, pp. 167-220, CRC, Fla. (1991)); and hormones (Klock, et al.,
Nature
329:734-736 (1987)). Unfortunately, these promoters generally provide only a relatively a low level of expression even in the presence of inducer and most of the inducers that have been used in vitro.have unacceptable side effects in vivo.
As an alternative to inducible promoters, attempts have been made to control mammalian gene expression using well-characterized prokaryotic regulatory elements. In most cases, regulatory systems have relied upon strong interactions between prokaryotic operators and repressor proteins as a means for either targeting eukaryotic transcription modulators to specific sites within a host cell genome (see e.g., Labow, et al.,
Mol. Cell. Biol.
10:3343-3356 (1990)) or in attempts to directly inhibit gene expression using the prokaryotic repressor (see e.g., Brown, et al.,
Cell
49:603-612 (1987)).
In the case of prokaryotic elements associated with the tetracycline resistance (tet) operon, systems have been developed in which the tet repressor protein is fused with polypeptides known to modulate transcription in mammalian cells. The fusion protein has then been directed to specific sites by the positioning of the tet operator sequence. For example, the tet repressor has been fused to a transactivator (VP16) and targeted to a tet operator sequence positioned upstream from the promoter of a selected gene (Gussen, et al.,
Proc. Nat'l Acad. Sci. USA
89:5547-5551 (1992); Kim, et al.,
J. Virol.
69:2565-2573 (1995); Hennighausen, et al.,
J. Cell. Biochem.
59:463472 (1995)). The tet repressor portion of the fusion protein binds to the operator thereby targeting the VP16 activator to the specific site where the induction of transcription is desired. An alternative approach has been to fuse the tet repressor to the KRAB repressor domain and target this protein to an operator placed several hundred base pairs upstream of a gene. Using this system, it has been found that the chimeric protein, but not the tet repressor alone, is capable of producing a 10 to 15-fold suppression of CMV-regulated gene expression (Deuschle, et al.,
Mol. Cell. Biol.
15:1907-1914 (1995)). The main problem with these types of systems is that the portion of fusion proteins corresponding to the mammalian transactivator or repressor tends to interact with cellular transcriptional factors and cause pleiotropic effects.
Ideally, a system for regulating mammalian gene expression should be highly specific for a selected gene and subject to induction by factors suitable for use both in vitro and in vivo. The present invention discloses such a system and describes how it can be used to regulate transgene expression. In addition, the invention describes how this system can be adapted to engineer viruses to serve as vectors, therapeutic agents and vaccines.
SUMMARY OF THE INVENTION
The present invention is directed to a number of different compositions and methods which share the common feature of having gene expression regulated by the tet operator/repressor system.
A. Compositions and Methods for the Production of Recombinant Protein
In its first aspect, the invention is directed to a recombinant DNA molecule which contains a mammalian promoter sequence with a TATA element; at least one tet operator sequence; and a gene sequence operably linked to the promoter and lying downstream from the operator. The exact positioning of the operator sequence (or sequences) relative to the TATA element is critical to the invention. In order to be effective at controlling transcription, the operator must begin at least 6 nucleotides downstream from the last nucleotide in the TATA element and, when a gene encoding a protein is expressed, the operator should be positioned before the translation initiation codon. In general, the operator should not begin more than about 100 nucleotides downstream and, preferably, it should begin within 6 to 24 nucleotides downstream of the TATA element. When positioned in this manner, it has been found that the binding of the repressor protein causes an essentially complete shutdown in transcriptional activity. This is true even for very strong and highly promiscuous promoters such as the human CMV immediate early promoter.
It is expected that the recombinant DNA molecule described above will, most typically, be incorporated into mammalian cells that constitutively express the tet repressor protein. Suitable cells may be developed by transforming a mammalian cell line, e.g., U2OS cells or Vero cells, with a vector containing the tet repressor protein gene operably linked to a promoter active in the cells (e.g., a CMV promoter, HSV-1 promoter or SV40 promoter). Alternatively, the DNA molecule may contain, in addition to the elements already discussed, a second promoter, preferably constitutive, operably linked to the tet repressor gene sequence. The invention encompasses, not only the DNA molecules, but also the host cells transformed with the DNAs and the recombinant proteins made by the cells.
The present invention is also directed to a method for recombinantly producing protein in which mammalian host cells are transformed with a vector containing a mammalian promoter sequence having a TATA element; at least one tet operator sequence positioned at least 6 nucleotides 3′ to the TATA element; and a gene lying 3′ to the operator and operably linked to the promoter. The gene 3′ to the operator may encode an antisense nucleic acid that inhibits the expression of a selected gene, a therapeutically active agent (e.g. a tumor suppressor or a transdominant negative mutant polypeptide of a cellular protein), a protein of interest for experimental purposes or simply a protein whose isolation is desired. In all cases where the gene encodes a protein, the operator sequence will be positioned before the translation initiation codon of the gene. The transformed cells should constitutively express the repressor protein and recombinant gene expression may be induced in the cells by introducing tetracycline. Typically, the tet operator sequence will be located between 6 and 100 nucleotides (preferably between 6 and 24 nucleotides) 3′ to the last nucleotide in the TATA element. The preferred promoter is the human CMV immediate-early promoter. It has been found that this system allows for the very tight regulation of gene expression, i.e., expression is essentially completely shut off until the inducer, tetracycline, becomes available.
The method can be used to produce recombinant protein in cultured mammalian cells or in the cells of a transgenic or non-transgenic animal. When a transgenic animal is used for production, it will most typically be a mouse and it is necessary that the cells transformed with the vector described above be embryonic stem cells. The stem cells may be engineered to express the tetracycline repressor by transforming them with the repressor gene operably linked to a promoter
Brigham and Women's Hospital
Fitch Even Tabin & Flannery
Qian Celine
Sanzo Michael A.
Yucel Remy
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