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KR-20260066119-A - Non-resistant plasmid production system and its applications

KR20260066119AKR 20260066119 AKR20260066119 AKR 20260066119AKR-20260066119-A

Abstract

The present invention relates to a system for producing tolerant plasmids. The present invention relates to constructing a plasmid comprising a nucleic acid fragment 3 encoding a small RNA by combining a toxic transcript/antitoxic transcript, an operon-related element, and a small RNA, and a host cell expression cassette comprising a toxic gene expression cassette and an antitoxic gene expression cassette, wherein the toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing the toxic transcript, and the antitoxic gene expression cassette comprises a promoter, an operon, and a nucleic acid fragment 2 for transcribing the antitoxic transcript. Since the inverse complementarity of nucleic acid fragment 3 and nucleic acid fragment 1 causes a steric effect that inhibits the function of the toxic transcript, the selection and production of plasmids can be realized without the need to add antibiotics or other inhibitors throughout the entire plasmid production process, thereby resolving the risk of antibiotics and inhibitors to plasmids in cell gene therapy.

Inventors

  • 시, 진시우
  • 린, 잉준
  • 란, 브루스

Assignees

  • 윤저우 바이오사이언시스 (광저우) 컴퍼니 리미티드

Dates

Publication Date
20260512
Application Date
20240910
Priority Date
20230911

Claims (15)

  1. A plasmid production system characterized by comprising a host cell expression cassette and a plasmid, Here, the host cell expression cassette includes a toxic gene expression cassette and an antitoxic gene expression cassette; The above toxic gene expression cassette comprises a promoter and nucleic acid fragment 1 for transcribing a toxic transcript; The above antitoxic gene expression cassette comprises a promoter, an operator, and nucleic acid fragment 2 for transcribing an antitoxic transcript; wherein the antitoxic transcript has an inhibitory effect on the toxicity generated by the toxic transcript; The above plasmid production system comprises nucleic acid fragment 3 for transcribing small RNA and an Ori replication origin, wherein nucleic acid fragment 3 exerts a steric effect in reverse complementary to nucleic acid fragment 1 to inhibit the function of toxic transcripts.
  2. A plasmid production system characterized in that, in the claim, nucleic acid fragment 3 is completely inverse complementary to nucleic acid fragment 1, has at least 20% inverse complementary, or has at least 10 consecutive bp inverse complementary.
  3. A plasmid production system according to claim 1 or 2, characterized in that nucleic acid fragment 1 comprises a UTR sequence and a toxic gene.
  4. A plasmid production system according to any one of claims 1 to 3, wherein the toxicity 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; and nucleic acid fragment 2 is an antitoxicity gene, and the antitoxicity 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. A plasmid production system according to any one of claims 1 to 4, wherein the operon is one of an arabinose operon, a Lac operon, a rhamnose catabolic operon, a tryptophan operon, a gab operon, and a Gal operon.
  6. A plasmid production system characterized in that, in any one of claims 3 to 5, the small RNA transcribed from nucleic acid fragment 3 is inversely complementary to the UTR sequence of nucleic acid fragment 1 and causes a stereochemical effect.
  7. A plasmid production system characterized in that, in any one of claims 3 to 6, the UTR sequence and the toxic gene are fused and expressed.
  8. A plasmid production system characterized in that, in any one of claims 1 to 7, the number of antitoxic gene expression cassettes and the number of toxic gene expression cassettes are both selected from integers of 1 or more.
  9. A plasmid production system characterized in that, in any one of claims 1 to 8, the host cell expression cassette further comprises a Rep protein.
  10. A plasmid production system according to any one of claims 1 to 9, characterized in that the replication origin is selected from ColE1, pBR322, pMB1, R6K, pUC, F1, p15A, 2μori, or oriV.
  11. A plasmid production system characterized in that, in any one of claims 1 to 10, the plasmid further comprises a target gene.
  12. A plasmid production system according to any one of claims 1 to 11, characterized in that the plasmid comprises a promoter, a target gene, a replication origin, and a nucleic acid fragment 3 for transcribing small RNA in sequence.
  13. In any one of claims 1 to 12, the host bacteria or host cells are further included, said host bacteria and host cells are Escherichia coli , Agrobacterium , Bacillus , or Caulobacter A plasmid production system characterized by being derived from yeast.
  14. Use of a plasmid production system according to any one of claims 1 to 13 in the production of plasmids or the manufacture of products for cell gene therapy.
  15. A method for producing a plasmid using a plasmid production system according to any one of claims 1 to 13, characterized by delivering the plasmid to a host integrated with a host cell expression cassette and culturing it.

Description

Non-resistant plasmid production system and its applications The present invention relates to the field of biotechnology, in particular to a resistance-free plasmid production system and its uses. Recently, gene therapies, particularly DNA or RNA vaccines, which overcome the shortcomings of conventional vaccines or viral vector vaccines in many aspects, are receiving increasing attention. Regardless of whether it is a DNA or RNA vaccine or a viral vector vaccine, the demand for plasmids is very high. Existing plasmid systems have many flaws in the application of gene therapy. On the one hand, while existing plasmids contain antibiotic marker genes, the overexpression of resistance genes will cause specific adverse effects on the metabolism and growth of E. coli, and the diffusion of antibiotics into the environment will lead to widespread antibiotic resistance in the population. At the same time, residual antibiotics in the plasmids may trigger allergic reactions in sensitive individuals. On the other hand, most existing plasmids contain unmethylated CpG sequences and possess a prokaryotic skeleton that can easily induce innate immune responses. Whether antibiotic marker genes or a prokaryotic skeleton are included, sequences outside the foreign gene expression cassette occupy an excessively high proportion of the plasmid, increasing its size. This leads not only to reduced plasmid yield but also to decreased transformation efficiency, negatively impacting gene therapy. Smaller, supercoordinated plasmids reach the cell nucleus more effectively, enabling the sustained and permanent expression of foreign genes. Therefore, plasmids without resistance genes and with shorter prokaryotic skeletons offer significant advantages. To improve the safety of gene therapy, next-generation plasmid scaffold systems free of antibiotic resistance markers have been developed and primarily include the following: 1. Nutritional deficiency strain compensation: After making the strain nutritionally deficient and unable to grow by modifying it through the introduction of deletions or mutations into genes essential for maintaining bacterial growth; then, the growth is restored by introducing a plasmid containing the deleted gene into the strain. 2. Toxic-antitoxicity system: The growth of the strain is restored by inserting a toxic gene into the bacterial genome to prevent bacterial growth, and then cloning an antitoxic gene into a plasmid and introducing it into the strain. 3. Operator/repressor system: The expression of the essential gene is suppressed by introducing a specific repressor protein upstream of a gene essential for growth in the bacterial genome, and then, the gene is expressed by introducing a plasmid containing one or more operator gene sequences into the bacteria to competitively titrate to the repressor. 4. Overexpression of Growth-Essential Genes: Overexpression of specific growth-essential genes (fabl or murA) in E. coli can reduce its susceptibility to antimicrobial compounds; however, since inhibitors are present in the culture medium, these genes must be removed from the purified plasmid DNA. While the new resistance-free plasmid systems mentioned above have addressed the issue of using antibiotic marker genes, two major problems still remain: 1. Sequences outside the target gene expression cassette occupy a large proportion of the plasmid, leading to an increase in plasmid length. Consequently, a portion of the plasmid yield per unit weight is contributed by the ineffective prokaryotic sequence backbone, and the copy number of the target gene expression cassette accounts for a relatively reduced proportion, affecting the yield of the target expression cassette. 2. Although some systems do not require the use of antibiotics, other co-inhibitors must still be added. Since there is no guarantee that these inhibitors will be 100% removed from the final plasmid product, this not only increases the cost of plasmid manufacturing but also makes it impossible to control the impact of the residues on gene therapy. Therefore, these resistance-free vector systems are not currently being effectively distributed. Figure 1 shows an example map of the functional elements of the miniVec stable strain; Figure 2 shows a map of the miniVec plasmid; Figure 3 shows a diagram of the synergistic effect of VecSeqA/VecSeqB and araBAD-ccdB/cddA; Figure 4 shows the map of the intermediate vector 1; Figure 5 shows the map of the intermediate vector 2; Figure 6 shows a map of pHelper vector 1; Figure 7 shows the map of pHelper vector 2; Figure 8 shows the map of the intermediate vector 3; Figure 9 shows the map of the intermediate vector 4; Figure 10 shows a map of the intermediate vector 5; Figure 11 shows a map of the intermediate vector 6; Figure 12 shows a map of the intermediate vector 7; Figure 13 shows a map of the intermediate vector 8. details The present invention provides a refractory plasmid production system and its uses. T