Macrocarriers and methods for high throughput production

A carrier and culture medium technology, applied in the field of carriers and high-yield production of large carriers, can solve the problem that BAC is not allowed to be obtained

Pending Publication Date: 2022-05-06
KATHOLIEKE UNIV LEUVEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these BACs do not allow for high, commercially viable BAC yields

Method used

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  • Macrocarriers and methods for high throughput production
  • Macrocarriers and methods for high throughput production
  • Macrocarriers and methods for high throughput production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0218] Example 1: Comparison of pMB1(ColE1) Ori and iOri

[0219] In pMB1(ColE1)Ori (see SEQ ID NO 10), RNAII DNA is transcribed from its cognate upstream promoter RNAIIp to generate the RNAII transcript. RNAII was used as a primer to initiate DNA synthesis at the Ori+1 start position of plasmid DNA. Expression of RNAII is regulated post-transcriptionally by the incorporation of complementary antisense RNAI. RNAI is expressed by the RNAI gene, which is located within the RNAII gene in the reverse orientation from its own RNAIp promoter. The plasmid-encoded ROP protein stabilizes the RNAI-RNAII duplex and, in this way, further limits the replication of the plasmid DNA ( Figure 2-3 ).

[0220] In iOri, expression of the 5'-end truncated RNAII* is initiated from the heterologous LacUVp promoter. The RNAII sequence is truncated by deleting the entire RNAI gene, and RNAI is not expressed. Expression of RNAII* is regulated at the transcriptional level by the binding of the LAC...

Embodiment 2

[0221] Example 2: Effect of point mutations in RNAH* of iOri on plasmid production.

[0222] have as figure 1 A plasmid of the indicated structure (pViroVet-4-YFV17D with mutated iOri) or with figure 1 Plasmids of the structures shown, in which the mutated iOri is replaced by wild-type iOri (i.e. iOri as in figure 2 As shown, no point mutation C->T at nucleic acid position 397 and point mutation A->G at nucleic acid position 176) (unmutated iOri) were used to transform BL21 bacteria.

[0223] Phosphate buffered LB medium was inoculated with two different bacterial clones transformed with pViroVet-4-YFV17D (pVV* and pVV4) with mutated iOri and one bacterial clone transformed with pViroVet with unmutated iOri - 4-YFV17D transformation (pVV-wt iOri) and incubation overnight in shake flasks.

[0224] Measure the OD of the bacterial culture the next morning 600 , the bacteria were diluted to OD in fresh medium supplemented with 1% glycerol in a fermenter vessel 600 for 2. Ba...

Embodiment 3

[0226] Example 3: Effect of biomass level on yield of vectors as taught herein upon induction of replication

[0227] have as figure 1 Plasmids with the indicated structure (pViroVet-4-YFV17D) and containing iOri were used to transform BL21 bacteria and glycerol stocks were prepared using existing techniques, e.g. as in the transformation protocol for BL21 competent cells (DE3) at New England BioLabs Inc. described in, see https: / / international.neb.com / Protocols / 0001 / 01 / 01 / transformation-protocol-for-b121-de3-competent-cells-c2527 , and as in the addgene homepage https: / / www.addgene.org / protocols / create-glycerol-stock / The protocol for creating bacterial glycerol stocks for long-term storage of plasmids is described above.

[0228] Table 1 shows the effect of biomass level on the yield of plasmids of the invention upon induction of replication.

[0229] OD at inoculation 600

OD at induction 600

OD at harvest 600

Yieldmg / l 0.1 1 2.57 0...

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Abstract

Provided herein is a method for producing a vector of at least 16 kb in size from a bacterial cell, comprising the following consecutive steps: a) obtaining a bacterial cell comprising a vector of at least 16 kb in size, said vector comprising an inducible origin of replication, b) inoculating a culture medium with the bacterial cell comprising the vector, c) culturing the bacterial cell in the culture medium, the present invention relates to a method for producing plasmids from bacterial cells, comprising the steps of: a) adding one or more inducers of an inducible starting point of replication to a culture medium, d) adding one or more inducers of the inducible starting point of replication to the culture medium when the optical density (OD600) of the bacterial culture at 600 nm reaches at least 20, e) further culturing the bacterial cells in the culture medium, f) optionally isolating the bacterial cells from the culture medium, and g) recovering plasmids from the bacterial cells. Also provided herein are vectors having a size of at least 16 kb comprising an inducible origin of replication for use in such methods.

Description

technical field [0001] The present invention relates to vectors and methods for high yield production of large vectors. Background technique [0002] Gene therapy and DNA vaccines are promising tools for the prevention, treatment and cure of diseases such as cancer and acquired immunodeficiency syndrome (AIDS). Gene therapy and DNA vaccines require large quantities of highly purified plasmid DNA (pDNA), which should be homogeneous in terms of structural form and DNA sequence. In addition, gene therapy and DNA vaccines, such as those against complex pathogens, require plasmids that can tolerate large genomic DNA inserts. For biopharmaceutical applications, yield and quality are the main drivers of plasmid manufacturing methods. In pursuit of increased yield, plasmids were developed that produced high copy numbers, meaning they would result in multiple copies of the plasmid per cell (>500 copies per cell, eg pUC18, pUC19 vectors). Such high plasmid copies per cell can be...

Claims

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Application Information

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IPC IPC(8): C12N15/70
CPCC12N15/70C12N2820/55C12N15/72C12N2800/101C12N2820/002
Inventor 梅洛迪·扬森约里·奥威尔克斯凯·达尔梅尔尼古拉斯·翁奇纳伊塞德里克·万萨伦内斯亚·戈里斯
Owner KATHOLIEKE UNIV LEUVEN
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