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EP-4553161-B1 - ANTIBIOTIC-FREE PLASMID PRODUCTION SYSTEM AND USE THEREOF

EP4553161B1EP 4553161 B1EP4553161 B1EP 4553161B1EP-4553161-B1

Inventors

  • SHI, Jinxiu
  • LIN, YINGJUN
  • LAHN, Bruce

Dates

Publication Date
20260513
Application Date
20240910

Claims (15)

  1. Plasmid production system, characterized in that it comprises host cell expression cassettes and a plasmid; wherein the host cell expression cassette comprises a toxic gene expression cassette and an anti-toxic gene expression cassette; wherein the toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing a toxic gene product; wherein the anti-toxic gene expression cassette comprises a promoter, an operator and a nucleic acid fragment 2 for transcribing an anti-toxic gene product; wherein the anti-toxic gene product has an inhibition effect on the toxicity produced by the toxic gene product; and wherein the plasmid comprises a nucleic acid fragment 3 for transcribing a small RNA and an origin of replication, wherein the nucleic acid fragment 3 is reversely complementary to the nucleic acid fragment 1 to produce a steric effect, thereby inhibiting the function of the toxic gene product.
  2. Plasmid production system according to claim 1, characterized in that the nucleic acid fragment 3 is reversely complementary to the nucleic acid fragment 1 completely, or has at least 20% reverse complementarity or at least 10 continuous bp reverse complementarity.
  3. Plasmid production system according to claim 1 or 2, characterized in that the nucleic acid fragment 1 comprises a UTR sequence and a toxic gene.
  4. Plasmid production system according to any one of claims 1 to 3, characterized in that the toxic gene is selected from any one of ccdB, ParE, MazF, Kid, HicA, RelE, VapC, Doc, RatA, HipA, Zeta, ToxN, YeeV, CptA, GhoT, Hok, TisB, SymE and PasA; the nucleic acid fragment 2 is an anti-toxic gene, and the anti-toxic gene is selected from any one of ccdA, ParD, MazE, Kis, HicB, RelB, VapB, Phd, RatB, HipB, Epsilon, ToxI, YeeU, CptB, GhoS, Sok, IstR-1, SymR and PasB/C.
  5. Plasmid production system according to any one of claims 1 to 4, characterized in that the operon is any one of arabinose operon, Lac operon, rhamnose catabolism operon, tryptophan operon, gab operon and Gal operon.
  6. Plasmid production system according to any one of claims 3 to 5, characterized in that the small RNA transcribed from the nucleic acid fragment 3 is reversely complementary to the UTR sequence in the nucleic acid fragment 1 and produces a steric effect.
  7. Plasmid production system according to any one of claims 3 to 6, characterized in that the UTR sequence and the toxic gene are expressed in fusion.
  8. Plasmid production system according to any one of claims 1 to 7, characterized in that the number of the anti-toxic gene expression cassettes and the number of the toxic gene expression cassettes are both selected from integers ≥1.
  9. Plasmid production system according to any one of claims 1 to 8, characterized in that the host cell expression cassette further comprises a Rep protein.
  10. Plasmid production system according to any one of claims 1 to 9, characterized in that the origin of replication is selected from ColE1, pBR322, pMB1, R6K, pUC, F1, p15A, 2 µ ori or oriV.
  11. Plasmid production system according to any one of claims 1 to 10, characterized in that the plasmid further comprises a target gene.
  12. Plasmid production system according to any one of claims 1 to 11, characterized in that the plasmid comprises a promoter, a target gene, an origin of replication and a nucleic acid fragment 3 for transcribing a small RNA ligated in sequence.
  13. Plasmid production system according to any one of claims 1 to 12, further comprising a host bacterium or a host cell, wherein the host bacterium and the host cell are derived from Escherichia coli, Agrobacterium, Bacillus, Caulobacter or a yeast.
  14. Use of the plasmid production system according to any one of claims 1 to 13 in producing a plasmid or in preparing a product for cell gene therapy.
  15. Method for producing a plasmid using the plasmid production system according to any one of claims 1 to 13, characterized in that a host integrated with the host cell expression cassette is transferred with the plasmid and is cultured.

Description

Technical Field The invention relates to the field of biotechnology, and in particular to a resistance-free plasmid production system and the use thereof. Background In recent years, gene therapy methods have attracted more and more attention, especially DNA or RNA vaccines, which have overcome the defects of traditional vaccines or viral vector vaccines in many aspects. Whether it is a DNA or RNA vaccine or a viral vector vaccine, the demand for plasmids is usually very large. Traditional plasmid systems have many defects in the application of gene therapy. On the one hand, traditional plasmids carry antibiotic marker genes, but the overexpression of resistance genes will have certain side effects on the metabolism and growth of Escherichia coli, and the spread of antibiotics in the environment will cause widespread antibiotic resistance in the population. At the same time, the residual antibiotics in the plasmids may also cause allergic reactions in sensitive individuals. On the other hand, traditional plasmids carry prokaryotic bacterial backbones, most of which contain unmethylated CpG sequences, which are easy to induce the innate immune response (CpG oligodeoxynucleotide and products of genes on the prokaryotic backbone of the plasmid can be immunogens). Whether antibiotic marker genes or prokaryotic bacterial backbones are carried, the sequences outside the exogenous gene expression cassette will account for too large a proportion of the plasmid, increasing the size of the plasmid, which not only affects the yield of the plasmid, but also leads to a decrease in transfection efficiency, which is a negative impact on gene therapy. Smaller supercoiled plasmids can reach cell nucleus more effectively and enable the exogenous gene to be expressed continuously and persistently. Therefore, plasmids that lack resistance genes and have shorter prokaryotic backbones have greater advantages. In order to improve the safety of gene therapy, a new generation of plasmid backbone systems without antibiotic resistance markers have been developed, mainly including the following: 1. Complementation of malnutrition strains: The strain is modified by introducing a deletion or mutation into a gene essential for maintaining bacterial growth, resulting in malnutrition of the strain and inability to grow; then a plasmid carrying the deleted gene is introduced into the strain to restore the growth of the strain. 2. Toxicity-antitoxicity system: A toxic gene is inserted into the bacterial genome to make the bacteria unable to grow, and an anti-toxic gene is cloned into the plasmid and introduced into the strain to restore the growth of the strain. 3. Operator/repressor system: A certain repressor protein is introduced upstream of a growth-essential gene in the bacterial genome to inhibit the expression of the essential gene, and a plasmid comprising one or more operator gene sequences is introduced into the bacteria to competitively titrate the repressor, thereby allowing the expression of the gene. 4. Overexpression of growth-essential genes: Overexpression of certain growth-essential genes (fabl or murA) in Escherichia coli can reduce the sensitivity to antibacterial compounds thereof, but inhibitors are present in the culture medium and must be removed from the purified plasmid DNA. Although the above-mentioned novel resistance-free plasmid systems have solved the problem of using antibiotic marker genes, there are still two major problems: 1. The sequence outside the target gene expression cassette accounts for too large a proportion in the plasmid, resulting in an increase of plasmid length. Therefore, under the same unit weight, part of the plasmid yield is contributed by the ineffective prokaryotic sequence backbone, and the copy number of the target gene expression cassettes accounts for a relatively reduced proportion, affecting the yield of the target expression cassettes. 2. Although some systems do not require the use of antibiotics, other auxiliary inhibitors still need to be added, and these inhibitors cannot be guaranteed to be 100% removed in the final plasmid product, which will not only increase the cost of plasmid preparation, but also the impact of the remain thereof in gene therapy is uncontrollable. Therefore, these resistance-free vector systems cannot be effectively popularized at present. Therefore, there remains a need in the art to develop a plasmid production system that does not require the use of antibiotics and other auxiliary inhibitors. Chen Zhe et. al., Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system", Metabolic Engineering, vol. 79, 1 September 2023 (2023-09-01), pages 86-96, XP093194256,AMSTERDAM, NL) discloses an antibiotic-free plasmid vector with a minimized backbone utilizing a toxin-antitoxin (TA) system in which the Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Rose-ivirga sp. The toxin gene is integrated into t