US 5,670,370 A · Biological containment

Abstract

A replicon, in which a nucleotide sequence encoding a cell killing function is regulatably expressed when the replicon is harboured in one type of host cell (primary host cell), so that cells harbouring the replicon are killed under conditions under which the cell killing function is expressed, and the nucleotide sequence encoding the cell killing function is regulatably or constitutively expressed when the replicon is harboured in another type of host cell (secondary host cell), so that cells harbouring the replicon are invariably killed or killed under conditions under which the cell killing function is expressed, may be used in a method of active biological containment of cells under defined environmental conditions. The biological containment principle may be utilized in the industrial production of a biosynthetic product by recombinant DNA techniques, when deliberately releasing a genetically engineered microorganism to the natural environment or in the preparation of a live vaccine. The expression of the cell killing function may be regulated by means of a promoter.

Claims49

Dependent claims

A recombinant replicon which comprises a first gene, whose expression results in the formation of a toxic product which has a toxic effect on Enterobacteriaceae cells in which said replicon can replicate, and an invertible promoter which regulates the expression of said first gene, whereby, when said replicon is introduced into host Enterobacteriaceae cells in which said promoter is functional, under suitable conditions of expression said invertible promoter is repeatedly inverted, leading to stochastic expression of the first gene and to formation of said toxic product, thereby stochastically limiting the life of said replicon-bearing host cell, where said first gene comprises a coding sequence which encodes a polypeptide selected from the group consisting of R1 Hok, F Hok and the polypeptide encoded by relB-orf3.

A method of biologically containing bacterial cells growing in a first, controllable environment, which cells could escape to a second and physically distinct environment, which comprises:

(a) providing in said cells a recombinant replicon, said replicon comprising a first gene, expressed under the control of a regulatable promoter functional in said cells, whose expression results in formation of a toxic product which has a toxic effect on said cells, said cells natively containing or being modified to contain a second gene whose product inhibits expression of said first gene, said inhibition being regulatable by an environmental factor, the level of said environmental factor in the second environment being such that said first gene is expressed and said toxic product is formed, whereby cells in the second environment which harbor said replicon are killed, and
(b) manipulating the first environment such that the level of the environmental factor therein is such that expression of said first gene is inhibited, whereby cells bearing said replicon are able to grow in said first environment but not in said second environment, the toxic effect being such that at least 99.9% of the cells are killed in said second environment, which but for said effect is an environment in which said cells can grow, where said bacterial cells are selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide selected from the group consisting of R1 Hok, F Hok and the polypeptide encoded by relB-orf3.

A method of biologically containing bacterial cells growing in an initial environment subject to physical or chemical change resulting in a changed environment, so that said cells are able to grow in the initial environment but not in the changed environment, which comprises

providing in said cells a recombinant replicon, said replicon comprising a first gene, expressed under the control of a regulatable promoter functional in said cells, whose expression results in formation of a toxic product which has a toxic effect on said cells, said cells natively containing or being modified to contain an inhibitory gene whose product inhibits expression of said first gene, said inhibition being regulatable by an environmental factor, the level of said environmental factor in said changed environment being such that said first gene is expressed and said toxic product is formed, whereby said cells in said changed environment which harbor said replicon are killed, the level of the environmental factor in said initial environment being such that expression of said first gene is inhibited, whereby cells bearing said replicon are able to grow in the initial environment but not in the changed environment, the toxic effect being such that at least 99.9% of the cells are killed in said changed environment, which but for said effect is an environment in which said cells can grow, said cells being selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells,
where said first gene comprises a coding sequence which encodes a polypeptide selected from the group consisting of R1 Hok, F Hok and the polypeptide encoded by relB-orf3.

Dependencies are inferred from the claim text.

Biological containment

Life of this patent

Biological containment

Life of this patent

Abstract

Claims49