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Cell free biosynthesis of high-quality nucleic acid and uses thereof

A high-quality, cell-free technology that can be used in fermentation and other directions to solve problems such as being unsuitable for cell-free nucleic acid production, and achieve the effects of effective absorption, simplified purification steps, and increased fidelity.

Inactive Publication Date: 2008-01-09
CYTOGENIX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0024] Currently, the RCA system is not suitable for cell-free production of nucleic acids for gene therapy, DNA vaccines, or other therapeutic applications

Method used

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  • Cell free biosynthesis of high-quality nucleic acid and uses thereof
  • Cell free biosynthesis of high-quality nucleic acid and uses thereof
  • Cell free biosynthesis of high-quality nucleic acid and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 - Synthesis and cell-free amplification of β-galactosidase (LacZ-DU)

[0094] a) Plasmid-based template pSV-β-galactosidase vector (Promega, Madison, WI, USA) was partially digested with EcoR I and Pst I. An approximately 4.2 kb fragment (LacZ-DU) containing the CMV promoter, Lac Z ORF and SV40 small T antigen termination sequence was isolated, blunt-ended with T4 DNA polymerase and cloned into pGEM TM -Sma I site of 7Zf(+) (Promega, Madison, WI, USA), resulting in pGEM-LacZ-DU vector. LacZ-DU was then excised from pGEM-LacZ-DU with Xba I, gel purified and circularized with T4 DNA ligase (New England Biolabs, Beverly, MA, USA) following the manufacturer's recommendations.

[0095] b) PCR-based template using a forward primer (5'-CG GGATCC GACTCTTCGCGATGTAC-3'), reverse primer (5'-CG GGATCC CAGCATGCCTGC-3'), by pVAX TM The 200-GW / lacZ vector (Invitrogen Carlsbad, CA, USA) amplifies LacZ-DU. Containing 200ng of each primer, 10ngpVAX TM 200-GW / lacZ vector...

Embodiment 2

[0103] Example 2 - Synthesis and cell-free expansion of luciferase (Luc-DU)

[0104] The pGL3 vector (Promega Corp. Madison, WI, USA) was digested with Sal I and Xho I. A 2.17 kb fragment containing the SV40 promoter, luciferase ORF, and SV40 small T antigen termination sequence (Luc-DU) was isolated, purified, and treated with T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) following the enzyme manufacturer's recommendations. ) re-circularized (re-circularized).

[0105] a) Cell-free amplification will contain six base primers (hexamers) 5'-ApApTpTp with a concentration of 400pmol s Gp s C-3' and 5'-ApGpCpAp s AP s T-3' and 10ng / 25μl cyclic Luc-DU reaction in 40mM Tris-HCl pH8, 10mM MgCl 2 Heat to 95°C for 3 minutes and cool to room temperature. Add Phi29 DNA polymerase (10U, NewEngland Biolabs, Beverly, MA, USA), 1mM dNTPs (25 / 25 / 25 / 25), 5% glycerol, 0.7U yeast inorganic pyrophosphatase (Sigma, St.Louis, MO, USA) and 100 μg / ml BSA. At 30°C, in 50mM Tris-HCl pH7.5, 10mM...

Embodiment 3

[0106] Example 3 - Expression of amplified DNA in human cells

[0107] at 37°C and 5% CO 2 Human lung cancer cell A549 (ATCC) was cultivated in Dulbecco's modified Eagle medium (DMEM; Invitrogen Carlsbad, CA, USA) under environmental conditions, which supplemented 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA). ), 100 U / ml penicillin, 100 μg / ml streptomycin (Invitrogen Carlsbad, CA, USA).

[0108] a) DNA transfection The day before transfection, A549 cells were treated with 1×10 5 Densities of cells / ml were seeded in 6-well plates. GenePORTER2 transfection reagent (Gene Therapy System, San Diego, CA, USA) was used for cell transfection according to the manufacturer's instructions. Shortly thereafter, cell-free amplified DU or parental plasmid DNA (Promega Corp. Madison, WI, USA) was mixed with 2 μg vector pssXE DNA (Chen and McMicken, Gene Ther 10:1776-1780, 2003) in 50 μl of DNA dilution B. ) were mixed and incubated at room temperature for 5 min. The ...

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Abstract

The invention provides an improved cell free amplification method capable of producing large quantities of therapeutic-quality nucleic acids and methods of using the synthesized nucleic acid in research, therapeutic and other applications- The methods combine several different state-of-the-art procedures and coordinate their applications to affordably synthesize nucleic acids for therapeutic purposes. It combines in vitro rolling circle amplification, high fidelity polymerases, high affinity primers, and streamlined template specifically designed for particular applications. For expression purposes, the templates contain an expression cassette including a eukaryotic promoter, the coding sequence for the gene of interest, and a eukaryotic termination sequence. Following amplification, concatamers are subsequently processed according to their intended use and may include: restriction enzyme digestion for the production of short expression cassettes (SECs); ligation steps to circularize the SEC (CNAs); and / or supercoiling steps to produce sCNAs. The final product contains nearly non-detectable levels of bacterial endotoxin.

Description

Technical field [0001] The present invention relates to a method for producing high-quality nucleic acid and its use. Background technique [0002] The emergence of DNA-based therapeutics in gene transfer, gene therapy, and DNA vaccines has increased the demand for DNA that meets stringent quality standards in terms of purity, potency, efficacy, and safety. Because gene expression is relatively less efficient and of relatively short duration in the target tissue, large amounts of DNA are typically required for this type of application. [0003] The current state of the art relies on the growth of plasmids in bacterial cultures and on expensive purification techniques to produce therapeutic-quality nucleic acids. Typical methods for purifying plasmids from bacteria and other cellular sources include the use of organic, mutagenic, and toxic compounds, including phenols, ethidium bromide, and cesium chloride, and enzymes such as lysozyme, proteinase K, and ribose. Nuclease A....

Claims

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Application Information

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IPC IPC(8): C12P19/34
Inventor 陈寅弗雷德里克·肯德希弗兰克·瓦兹奎斯马尔科姆·斯考尼克
Owner CYTOGENIX INC
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