Producing improved viral and non-viral nanoplasmid vectors

A plasmid, origin of replication technology, applied in the direction of viruses/phages, viruses, vectors, etc.

Pending Publication Date: 2020-12-29
阿尔德夫隆有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0038] No method has been reported for high-yield production of pFAR vectors; this system utilizes a plasmid-borne suppressor tRNA gene to complement the TAG amber nonsense mutation of the thyA gene to complement thymidine auxotrophy and allow cells to grow on minimal media ( Marie et al., Supra, 2010)

Method used

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  • Producing improved viral and non-viral nanoplasmid vectors
  • Producing improved viral and non-viral nanoplasmid vectors
  • Producing improved viral and non-viral nanoplasmid vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0287] Example 1: pUC and R6K origin of replication plasmid replication and production

[0288] pUC origin vector replication and production background: The vast majority of therapeutic plasmids use the pUC origin, which is a high-copy derivative of the pMB1 origin (closely related to the ColE1 origin). For pMB1 replication, plasmid DNA synthesis is unidirectional and does not require a plasmid-borne initiator protein. The pUC origin is a high-copy derivative of the pMB1 origin that deletes the helper ROP(rom) protein and has an additional temperature-sensitive mutation that destabilizes the RNAI / RNAII interaction. Moving cultures containing these origins from 30°C to 42°C resulted in an increase in plasmid copy number. The pUC plasmid can be produced in many E. coli cell lines.

[0289] RNA-OUT No Antibiotic Selectable Marker Background: Antibiotic-free selection was performed in an E. coli strain containing pCAH63-CAT RNA-IN-SacB (P5 / 66 / 6) chromosomally integrated at t...

Embodiment 2

[0305] Example 2: Production of pUC and R6K origin vectors

[0306] Shake flask production: Shake flask production using proprietary plasmid + shake flask medium. Seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 μg / mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). Plates were grown at 30-32°C; cells were resuspended in medium and used to provide approximately 2.5 OD for 500 mL plasmid + shake flasks 600 The shake flask contained 50 μg / mL of antibiotics for ampR or kanR selection plasmids or 0.5% sucrose for selection of RNA-OUT plasmids. The flasks were grown to saturation at the growth temperatures shown in Tables 5, 6, 7 and 9 with shaking.

[0307] Fermentation production: Fermentations were performed in New Brunswick BioFlo 110 bioreactors using a proprietary fed-batch medium (NTC3019, HyperGRO medium) as described (Carnes and Williams, Supra, 2011). Se...

Embodiment 3

[0324] Embodiment 3: Construction and preparation of pUC and R6K starting point structure carrier

[0325] The R6Kγ origin (SEQ ID NO: 1; Figure 1E )-RNA-OUT (SEQ ID NO: 5; Figure 1B ) bacterial replication selection region (SEQ ID NO: 8; Figure 1C ) were cloned into the polylinker region of various pUC57-based vectors to generate pNTC-NP1, pNTC-NP2, pNTC-NP3, pNTC-NP4, pNTC-NP5, pNTC-NP6, pNTC-NP7 vectors. Each vector has different flanking restriction sites that can be used to engineer the destination vector for R6K replication-RNA-OUT selection. Table 4 shows the 5' and 3' polylinker sequences flanking the R6K-RNA-OUT insert in the pNTC-NP1-7 vector. A 1 CpG R6Kγ origin-2 CpG RNA-OUT bacterial replication selection region based on pUC57 was also created (SEQ ID NO: 9; Figure 1D ) version (pNTC-3xCpGNP1), as shown in Table 4.

[0326] The R6Kγ origin (SEQ ID NO: 1) is the modified R6K origin of the 6-repeat ( Figure 1E ). A 7-repeat based R6K gamma origin (SEQ ID...

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Abstract

A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I-dependent origin of replication, and an insert including a structured DNA sequence selected from the group consisting of inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication or eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction ofreplication. The method also includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, whereby the resultant Pol III-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinantDNA molecule is also provided.

Description

[0001] Statement Regarding Federally Sponsored Research or Development [0002] Not applicable. [0003] Cross References to Related Applications [0004] This application claims priority to U.S. Provisional Patent Application No. 62 / 645,892, filed March 21, 2018, entitled "Generation of Improved Viral and Nonviral Nanoplasmid Vectors," which is incorporated by reference in its entirety into this article. technical field [0005] The present invention relates to recombinant DNA molecules (i.e., vectors) that can be used in viral and non-viral gene therapy, viral and non-viral cell therapy, and more specifically, for improving the yield and quality of viral and non-viral vectors, reducing transfection-related Toxicity, improved transposition of non-viral transposon vectors, improved packaging titers of viral vectors, improved expression of genes encoded by viral and non-viral vectors, and for elimination of viral and non-viral vector-mediated gene transfer of antibiotic selec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/86C12N2750/14143C12N2800/101C12N2800/24C12N2800/90C12N2820/55C12N15/85C12N2740/15043C12N2740/15052C12N2750/14152
Inventor 詹姆斯·A·威廉姆斯
Owner 阿尔德夫隆有限责任公司
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