# US 5,702,916 A · Biological Containment

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- Number: US 5,702,916 A
- Document ID: US5702916A
- Status: Granted
- Kind: A
- Application number: 08/449,958
- Filed: 1995-05-25
- Granted: 1997-12-30
- Earliest claimed: 1984-05-15
- Assignees: GX BioSystems A/S
- Inventors: S.o slashed.ren Molin; Poul Kirketerp Andersson; Kenn Axo Gerdes; Per Klemm
- Web page: https://patentgrep.com/patent/US5702916A

## CPC

- C12N 15/69: Increasing the copy number of the vector
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/68: Stabilisation of the vector
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

A replicon, in which a nucleotide sequence encoding a cell killing function is regulatably expressed when the replicon is harbored in one type of host cell (primary host cell), so that cells harboring 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 harbored in another type of host cell (secondary host cell), so that cells harboring 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.

## Claims

### Claim 1

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 having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

### Claim 2 (depends on 1)

The replicon of claim 1 wherein the invertible promoter is the promoter of the fimA gene or a functional homologue thereof.

### Claim 3 (depends on 1)

The replicon of claim 1, wherein the invertible promoter is operably linked to said first gene but not natively associated therewith.

### Claim 4 (depends on 1)

The replicon of claim 1, said promoter having a phase switch responsive to "on" and "off" gene products, being caused to transcribe said first gene by an "on" gene product and caused not to transcribe said first gene by an "off" gene product, whereby the frequency of expression of said first gene may be controlled by modulating the relative levels of expression of said "on" and "off" gene products.

### Claim 5 (depends on 4)

The replicon of claim 4, further comprising a gene encoding an "on" gene product which directs the phase switch of said invertible promoter into the "on" position.

### Claim 6 (depends on 4)

The replicon of claim 4, further comprising a gene encoding an "off" gene product which directs the phase switch of said invertible promoter into the "off" position.

### Claim 7 (depends on 5)

The replicon of claim 5 wherein the "on" product encoding gene is the fimB gene or a functional homologue thereof.

### Claim 8 (depends on 5)

The replicon of claim 5 wherein the "off" product encoding gene is the fimE gene or a functional homologue thereof.

### Claim 9 (depends on 1)

The replicon of claim 1 wherein said coding sequence encodes a polypeptide in which the Cys residues at amino acid positions 16 and 31 of the R1 Hok or F Hok sequence are conserved.

### Claim 10 (depends on 1)

The replicon of claim 1 in which said coding sequence encodes a polypeptide in which at least about 60% of the charged amino acid residues of R1 Hok are conserved.

### Claim 11 (depends on 1)

The replicon of claim 1 wherein said coding sequence encodes a polypeptide selected from the group consisting of the gene products of R1 hok, F hok, and relB-orf3.

### Claim 12 (depends on 1)

A transformed bacterial cell population comprising Enterobacteriaceae cells harboring the replicon of claim 1, said replicon being capable of replicating in said cells, said invertible promoter being functional in said cells, the expression of said first gene having a toxic effect on said cells.

### Claim 13 (depends on 12)

The cell population of claim 12 where the first gene is the R1 hok or F hok gene.

### Claim 14 (depends on 12)

The cell population of claim 12 where the cells are E. coli cells.

### Claim 15 (depends on 12)

The cell population of claim 12 wherein the invertible transcriptional promoter is the promoter of the fimA gene.

### Claim 16 (depends on 12)

The transformed cell population of claim 12 wherein said replicon further comprises a gene expressing a fusion protein, said fusion protein comprising an outer surface protein of said cell and an epitope of interest not native to said cell, whereby said fusion protein serves as a means for transporting the epitope, when expressed, to the outer surface of the cell.

### Claim 17 (depends on 1)

A method of stochastically limiting an Enterobacteriaceae cell population which comprises transforming the cells of said cell population with a replicon according to claim 1, said replicon being capable of replicating in said cells, the expression of said first gene having a toxic effect on said cells, the frequency of the inversion of said invertible promoter in said cells being such as to stochastically limit the survival of said cell population.

### Claim 18 (depends on 17)

The method of claim 17 wherein the invertible promoter is a fimA promoter.

### Claim 19 (depends on 17)

The method of claim 17, wherein said toxic effect is such that if said promoter remained "on", at least about 99.9% of the cells would be killed.

### Claim 20 (depends on 17)

The method of claim 17 wherein said toxic effect is such that at least 99.9% of the cells are killed in one hour if said promoter remains "on".

### Claim 21 (depends on 17)

The method of claim 17 wherein the invertible promoter has substantially the same inversion frequency as the fimA promoter.

### Claim 22 (depends on 17)

The method of claim 17 wherein one of the manifestations of said toxic effect is the transformation of at least some of the bacterial cells into ghost cells.

### Claim 23 (depends on 17)

The method of claim 17 wherein the first gene encodes a polypeptide which is expressed in nature in Enterobacteriaceae, Pseudomonadaceae, or Bacillaceae cells.

### Claim 24

A transformed bacterial cell of a first kind of cell, said transformed cell comprising:
(a) a recombinant extrachromosomal replicon comprising a first gene, expressed under the control of a regulatable promoter, whose expression results in formation of a toxic product which has a lethal effect on both cells of said first kind and on bacterial cells of a second kind with which said first kind of cells is capable of naturally exchanging genetic information; and
(b) a second replicon comprising a second gene which encodes a gene product which inhibits expression of such first gene, said second gene being lacking in cells of said second kind, said first and second kinds of cells both being selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

### Claim 25 (depends on 24)

The transformed cell of claim 24, wherein the second gene encodes a repressor polypeptide which inhibits transcription of said first gene.

### Claim 26 (depends on 25)

The transformed cell of claim 25 wherein the second gene encodes trp repressor and the first gene is operably linked to a trp promoter including the operator site for trp repressor.

### Claim 27 (depends on 24)

The transformed cell of claim 24 in which the second replicon is a recombinant chromosomal replicon.

### Claim 28

A method of containing an extrachromosomal recombinant replicon to a first kind of bacterial cells, where said replicon could be naturally transferred to a second kind of bacterial cells, which comprises providing on the recombinant extrachromosomal replicon a first gene, expressed under the control of a regulatable promoter which is functional in both kinds of cells, whose expression results in formation of a toxic product which has a lethal effect on the first and second kind of cells, said first kind of cells having or being modified to have a second replicon comprising a second gene which encodes a gene product which inhibits the expression of said first gene and thereby protects said first kind of cells, said second gene being lacking in said second kind of cells, whereby if a cell of the second kind receives said extrachromosomal recombinant replicon said first gene is expressed and said toxic product is formed, which has a toxic effect thereon said first and second kinds of cells both being selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

### Claim 29 (depends on 28)

The method of claim 28 in which said second replicon is a chromosomal replicon.

### Claim 30 (depends on 28)

The method of claim 28, wherein said second gene encodes a repressor polypeptide which inhibits transcription of said first gene.

### Claim 31 (depends on 30)

The method of claim 30, wherein said repressor polypeptide is the trp repressor and said first gene is operably linked to a trp promoter including the operator site for said trp repressor.

### Claim 32 (depends on 29)

The method of claim 29 wherein the chromosomal replicon is recombinant and the second gene is not natively associated with said chromosomal replicon.

### Claim 33 (depends on 28)

The method of claim 28 wherein said lethal effect is such that at least 99.9% of the cells are killed in one hour if not protected by said inhibitory gene product.

### Claim 34 (depends on 28)

The method of claim 28 wherein the cells are gramnegative bacterial cells.

### Claim 35 (depends on 28)

The method of claim 28 wherein one of the manifestations of said toxic effect is the transformation of at least some of the bacterial cells into ghost cells.

### Claim 36 (depends on 28)

The method of claim 28 wherein the cells are Enterobacteriaceae cells.

### Claim 37 (depends on 28)

The method of claim 28 wherein the first gene encodes a polypeptide which is expressed in nature in Enterobacteriaceae, Pseudomonadaceae, or Bacillaceae cells.

### Claim 38 (depends on 28)

The method of claim 28, wherein the first gene is the R1 hok or F hok gene.

### Claim 39 (depends on 28)

The method of claim 28 wherein said coding sequence encodes a polypeptide in which the Cys residues at amino acid positions 16 and 31 of the R1 Hok or F Hok sequence are conserved.

### Claim 40 (depends on 28)

The method of claim 28 in which said coding sequence encodes a polypeptide in which at least about 60% of the charged amino acid residues of R1 Hok are conserved.

### Claim 41 (depends on 28)

The method of claim 28 wherein said coding sequence encodes a polypeptide selected from the group consisting of the gene products of R1 hok, F hok, and relB-orf3.

### Claim 42 (depends on 24)

The transformed bacterial cell of claim 24 wherein said coding sequence encodes a polypeptide in which the Cys residues at amino acid positions 16 and 31 of the R1 Hok or F Hok sequence are conserved.

### Claim 43 (depends on 24)

The transformed bacterial cell of claim 24 in which said coding sequences encodes a polypeptide in which at least about 60% of the charged amino acid residues of R1 Hok are conserved.

### Claim 44 (depends on 24)

The transferred bacterial cell of claim 24 wherein said coding sequence encodes a polypeptide selected from the group consisting of the gene products of R1 hok, F hok, and relB-orf3.

### Claim 45

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 cell a recombinant replicon, said replicon comprising a first gene, expressed under the control of a regulatable promoter which is 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 having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

### Claim 46 (depends on 45)

The method of claim 45, wherein the environmental factor is temperature.

### Claim 47 (depends on 45)

The method of claim 45, wherein the environmental factor is the concentration of a regulator chemical.

### Claim 48 (depends on 45)

The method of claim 45, wherein said second gene expresses a repressor polypeptide which inhibits transcription of said first gene.

### Claim 49 (depends on 47)

The method of claim 47, wherein the environmental factor is the concentration of tryptophan, the promoter is the trp promoter, the second gene is the trp repressor gene, and sufficient tryptophan is added to said first environment to provide an inhibitory concentration thereof.

### Claim 50 (depends on 45)

The method of claim 45, wherein the second gene is located on the same replicon as said first gene.

### Claim 51 (depends on 45)

The method of claim 45, wherein the second gene is located on a chromosomal replicon and the first gene on an extrachromosomal replicon.

### Claim 52 (depends on 45)

The method of claim 45, wherein the toxic effect is such that at least 99.9% of the cells are killed within one hour of excape into the second environment.

### Claim 53 (depends on 45)

The method of claim 45 wherein the cells are gramnegative bacterial cells.

### Claim 54 (depends on 45)

The method of claim 45 wherein one of the manifestations of said toxic effect is the transformation of at least some of the bacterial cells into ghost cells.

### Claim 55 (depends on 45)

The method of claim 45 wherein the cells are Enterobacteriaceae cells.

### Claim 56 (depends on 45)

The method of claim 45 wherein the first gene encodes a polypeptide which is expressed in nature in Enterobacteriaceae, Pseudomonadaceae, or Bacillaceae cells.

### Claim 57 (depends on 45)

The method of claim 45 wherein said coding sequence encodes a polypeptide in which the Cys residues at amino acid positions 16 and 31 of the R1 Hok or F Hok sequence are conserved.

### Claim 58 (depends on 45)

The method of claim 45 in which said coding sequences encodes a polypeptide in which at least about 60% of the charged amino acid residues of R1 Hok are conserved.

### Claim 59 (depends on 45)

The method of claim 45 wherein said coding sequence encodes a polypeptide selected from the group consisting of the gene products of R1 hok, F hok, and relB-orf3.

### Claim 60

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 which is 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 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 Entero-bacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where said first gene comprises a coding sequence which encodes a polypeptide having an amino acid sequence wherein at least about 60% of the residues are identical to or conservative modifications of the corresponding residues of the amino acid sequence of the R1 Hok and/or F Hok polypeptides and/or the polypeptide encoded by relB-orf3.

### Claim 61 (depends on 60)

The method of claim 60 wherein the environmental factor is the concentration of a chemical in the environment.

### Claim 62 (depends on 60)

The method of claim 60, wherein the toxic effect is such that at least 99.9% of the cells are killed within one hour of said physical or chemical change in the environment.

### Claim 63 (depends on 60)

The method of claim 60 wherein the cells are gramnegative bacterial cells.

### Claim 64 (depends on 60)

The method of claim 60 wherein one of the manifestations of said toxic effect is the transformation of at least some of the bacterial cells into ghost cells.

### Claim 65 (depends on 60)

The method of claim 60 wherein the cells are Enterobacteriaceae cells.

### Claim 66 (depends on 60)

The method of claim 60 wherein the first gene encodes a polypeptide which is expressed in nature in Enterobacteriaceae, Pseudomonadaceae, or Bacillaceae cells.

### Claim 67 (depends on 60)

The method of claim 60 wherein said coding sequence encodes a polypeptide in which the Cys residues at amino acid positions 16 and 31 of the R1 Hok or F Hok sequence are conserved.

### Claim 68 (depends on 60)

The method of claim 60 in which said coding sequences encodes a polypeptide in which at least about 60% of the charged amino acid residues of R1 Hok are conserved.

### Claim 69 (depends on 60)

The method of claim 60 wherein said coding sequence encodes a polypeptide selected from the group consisting of the gene products of R1 hok, F hok, and relB-orf3.