Plasmid stabilization

Details Plasmid stabilization Plasmid stabilization

1999-11-12

2004-06-01

Wang, Andrew (Department: 1635)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S006120, C435S325000, C435S375000, C435S041000, C536S024100

Reexamination Certificate

active

06743780

ABSTRACT:

FIELD OF THE INVENTION
The invention relates in general to stable maintenance of a plasmid, and in particular to that of a plasmid containing a gene useful in gene therapy.
BACKGROUND OF THE INVENTION
The stable maintenance of a plasmid, particularly at high copy number, is important for the preparation of plasmid DNA. However, extrachromosomal DNA carried in host cells is inherently unstable in cell culture because cultured cells which contain plasmids usually have an increased metabolic burden compared to plasmid-free segregant cells. In efforts to maintain plasmid stability and decrease metabolic burden, plasmids engineered to contain dominant selectable markers have been routinely used. During scale-up fermentation of bacterial or yeast host strains, the presence of the selecting agent prevents plasmid loss and overgrowth by cells not burdened by the effort of replication and maintenance of plasmid DNA.
Antibiotic resistance genes, for example encoding resistance to antibiotics such as ampicillin, kanamycin or tetracycline, are the most common dominant selectable markers used in molecular biology cloning and fermentation procedures for the production of recombinant proteins or plasmid DNA. For continuous fermentation in the presence of an antibiotic, selective pressure is lost because the antibiotic loses activity over time due to culture dilution or degradation by the host cell. Therefore, some of the more successful methods for maintaining plasmids do not utilize antibiotic selection but rather rely on a mutant host which is unable to synthesize an amino acid, inserting the gene which provides for this synthesis in the plasmid. Other solutions which prevent the takeover of a culture by plasmid-free segregant involve placing a gene coding for a toxic product in the chromosome and then including a corresponding repressor system in the plasmid. Plasmid-free cells are effectively killed upon segregation.
Even with selective pressure, however, plasmid-free cells may continue to grow due to leakage of the complementing product of the selective gene from plasmid-bearing cells, lowering the total plasmid productivity of the culture. In addition, the use of genes for antibiotic resistance or other dominant selectable markers on vectors intended for gene therapy has raised potential problems related to expression of those genes in the target mammalian cell or host mammalian organism. Promiscuous expression of plasmid-borne genes, such as drug-resistance or nutritional markers, of the host cell (e.g. a yeast or bacteria) in the target mammalian cell may lead to its destruction and/or to an antigenic response to the gene product in the mammal. There are also concerns regarding contamination of the final product with the antibiotic used for plasmid selection in culture, with the potential induction of a severe immune response to the antibiotic, e.g., anaphylactic shock. The widespread use of bacterial genes for antibiotic resistance also will ultimately result in their transfer to the bacterial population as a whole.
The stable maintenance of plasmids at high copy number in transfected cells is also of importance in ex vivo gene therapy. Following the administration of a transfected cell, such as a microorganism or other cell, to a recipient organism (e.g., a mammal) for therapy, it becomes difficult or impossible to maintain plasmid DNA within the transplanted cells because it is difficult to select for a plasmid in vivo. For example, use of the in vitro selection compound may be contraindicated in the recipient, as would be the deletion of a given biochemical constituent (such as an amino acid) from the enviroment surrounding the transplanted cells, were such a feat of biological engineering as the latter technically feasible. As is true for in vivo nucleic acid delivery methods, it is advantageous to remove from the plasmids bearing the therapeutic- or other gene of interest genes that are not relevant to the therapeutic application, in order to minimize potential risks stemming from transmission of their products to the recipient, which may provoke side effects such as a toxic or anaphylactic response.
There is, therefore, a need for a method of plasmid maintenance that does not require the presence of extraneous plasmid-borne host genes or antibiotic selection.
SUMMARY OF THE INVENTION
The invention encompasses a transformed host cell containing a plasmid comprising an operator susceptible to binding by a repressor expressed in trans, a first chromosomal gene encoding the repressor, and a second chromosomal gene that is functionally associated with an operator and essential for cell growth, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth.
As used herein, “functionally associated” or “operatively associated”, with respect to an operator sequence and an associated gene, means that the operator is linked in cis to the gene such that expression of the gene is susceptible to repression upon binding of a repressor to the operator. It will be understood by one of skill in the art that the operator sequence present on the plasmid need not be a sequence that is identical to the operator sequence on the chromosomal gene, in that the plasmid operator need only consist of the minimal sequences necessary for binding the repressor that represses transcription of the chromosomal gene. It will also be understood that mutated operator sequences are also useful according to the invention, for example, sequences having one or more nucleotides inserted, deleted, or substituted which result in increased or decreased affinity for the corresponding repressor. As used herein, “cell growth” refers to increasing numbers of cells in a culture medium over time, and also refers to cell survival where the number of cells does not increase over time, but rather the number of live cells does not decrease over time.
Preferably, the repressor gene encodes one of the lac repressor, the &lgr; repressor, the
E. coli
trp repressor, the
E. coli
galR repressor, and the
E. coli
araC repressor. As described above, each repressor is operative in trans with a trans-associated operator sequence that is present both in the chromosome and on the plasmid. The invention contemplates the presence of one or more repressor genes on the host chromosome, e.g., one, two or three repressor genes, in order to ensure plasmid stability where one chromosomal repressor gene becomes mutated or deleted.
Preferred operator sequences therefore include the lac operator, the &lgr; operator, the trp operator, the gal operator, and the ara operator. If desired, the corresponding promoter may be functionally associated with its operator. Note that bacterial repressor/operator systems are of use in yeast including, but not limited to, the Lac repressor/operator pair, which may be used to block access of positive regulators of yeast transcription to their respective binding sites (e.g. the binding of Gal4p to the sequence CGGN
5
(A/T)N
5
CCG [SEQ ID NO: 1].
In other preferred embodiments, the cell is a bacterial cell that may be either gram negative or positive, for example,
E. coli
, Listeria, Shigella, Clostridium, Salmonella, Bacillus or Lactococcus. Alternatively, the host cell may be a yeast, mycobacterial, slime mold, algal or fungal cell, or other cell of the Domain Arachaea (including archaebacteria), phylum Protista, or animal or plant kingdom.
More than one different essential chromosomal gene may be present in the cell chromosome, wherein two or more essential genes are linked to an operator and are therefore susceptible to repression by the repressor; in this way, accidental de-repression of a single essential gene (e.g. through mutation of its associated operator) cannot result in the growth of plasmid-free cells, since at least one other essential gene remains repressed. In one preferred embodiment of the invention, the gene encoding the repressor protein is present in two or three copies at different lo

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