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Minicircle DNA vector preparations and methods of making and using the same

A carrier and preparation technology, applied in the field of reducing the production cost of minicircles, can solve the problems of integration and mediated mutation of immunogenic virus complications

Inactive Publication Date: 2011-06-01
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages associated with viral vectors include immunogenicity, viral complications, and integration-mediated mutagenesis issues, among others

Method used

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  • Minicircle DNA vector preparations and methods of making and using the same
  • Minicircle DNA vector preparations and methods of making and using the same
  • Minicircle DNA vector preparations and methods of making and using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Reduction of impurity DNA in minicircle preparations from BW27783

[0137] In our initial protocol, minicircles were used to generate plasmids such as p2 C31.hFIX( figure 1 A) and Top 10 strains to produce minicircles (Chen ZY et al., Human Gene Therapy 16:126, 2005). In our minicircle preparation, however, we detected small but variable amounts of impurity DNA comprising the unrecombined parental plasmid and the plasmid backbone circle (plasmid BB). We observed that this impurity DNA was mainly due to the "all or nothing" phenomenon: that is, a bacterial subpopulation became unable to express the high load, low affinity L-arabinose transporter araE, and could not absorb L-arabinose and araC -expression under the control of the ABD regulatory system C31 and ISce I genes. Khlebnikov A and colleagues (Microbiology 147:3241, 2001) reported that expression of araE in strain BW27783 could be driven by the constitutive promoter cp8 to partially overcome this "all or not...

Embodiment 2

[0140] Removing the ENDA gene overcomes the DNA degradation problem

[0141] Since recA is known to affect the stability of plasmids (Khlebnikov A et al., J Bacteriol 182:7029, 2000), we inactivated the recA gene in BW27783, but found this did not help (data not shown), feeling that this was an endo caused by nuclease 1. We set out to delete the endA gene encoding this DNase. To this end, we generated the plasmid pkanR.endA containing the kanamycin resistance gene flanked by attB and attP sites and two PCR-generated endA gene targeting sequences ( figure 1 E); modified according to the scheme of Datsenko KA and Wanner BL (PNAS 97: 6640, 2000), the plasmid was cut with Pme 1 to obtain a linear targeting DNA sequence and integrated into the endA gene of BW27783 ( image 3 A). A first attempt with the proposed protocol using two 35-bp targeting sequences failed (data not shown); however we were successful later by extending the targeting sequences to 329- and 754-bp, respect...

Embodiment 3

[0144] Expression of the ISCE I gene in the bacterial genome

[0145] We assume that no The optimal method for C31- and ISce I-encoding DNA minicircles is to express recombinases from the bacterial genome C31 and restriction enzyme ISce1. We assume that in any culture there are dead bacteria and therefore contamination is unavoidable, these impurity DNAs can cause more harm as they are circular stable, physically indistinguishable from the minicircles and difficult to remove. Instead, put When C31 and ISce I are integrated into the bacterial genome, as linear DNA fragments of the bacterial genome, these harmful genes have less chance of contamination and are easier to remove, and can be degraded by host bacterial exonucleases, which are harmless or have little impact on the recipients . We therefore set out to relocate the BAD.ISce I cassette in the minicircle producing plasmid into the bacterial genome.

[0146] To this end, we generated the plasmid p3BAD.ISCe Ig.KanR...

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Abstract

The present invention provides minicircle nucleic acid vector formulations for use in administering to a subject, wherein the minicircle nucleic acid vectors include a polynucleotide of interest, a product hybrid sequence of a unidirectional site-specific recombinase, and are devoid of plasmid backbone bacterial DNA sequences. Also provided are methods of producing the subject formulations as well as methods for administering the minicircle nucleic acid vector formulations to a subject. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications.

Description

[0001] government rights [0002] This invention was made with government support under Federal Grant HL64274 awarded by the National Institutes of Health. The US Government has certain rights in this invention. Background of the invention [0003] The process of introducing exogenous nucleic acid sequences into cells known as "transformation" has exerted important benefits in various biotechnology and related applications, including research, synthesis and therapeutic applications. Research applications in which transformation plays a key role include the generation of transgenic cells and animals. Synthetic applications where transformation plays a key role include the production of peptides and proteins, as well as therapeutic RNAs such as interfering RNAs or ribozymes. Therapeutic applications in which transformation plays an important role include gene therapy applications. Because of the general utility of transformation for these and other applications, a variety of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00
CPCC12N2800/30C12N2800/80C12N2830/55C12N15/70C12N2830/002A61K48/0066A61K48/005C12N2800/108C07K14/195C12N15/85C12N15/66
Inventor Z·陈M·A·凯
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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