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Muscle-targeted minicircle DNA gene therapy

A gene and recombinant gene technology, applied in the field of microcircular DNA gene therapy, can solve the problems of unsustainable expression and low transfection efficiency, and achieve the effect of overcoming the limited half-life

Inactive Publication Date: 2020-01-14
SYNO MINICIRCLE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest defect of plasmid vectors is the low transfection efficiency in vivo, and the expression is difficult to sustain

Method used

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  • Muscle-targeted minicircle DNA gene therapy
  • Muscle-targeted minicircle DNA gene therapy
  • Muscle-targeted minicircle DNA gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, minicircle DNA parent plasmid construction

[0038] 1) Synthesize full-length FIX and BDD-FVIII gene DNA fragments (as shown in SEQ ID NO: 9-13).

[0039] 2) The above DNA fragments were subcloned into the minicircle DNA empty vector pMC.DTS-CMVmax (map as figure 1 shown), and constructed into a minicircle DNA master plasmid.

Embodiment 2

[0040] Embodiment 2, microcircle DNA preparation

[0041] 1) The minicircular DNA master plasmid was transformed into genetically engineered E coli bacteria ZYCY10P3S2T (Nature Biotechnology 2010, 28:1287-9).

[0042] 2) Inoculate the ZYCY10P3S2T containing the minicircle master plasmid into the TB medium containing kana, culture it on a shaker at 37°C for 12-16h, and the bacteria will produce a large amount of minicircle DNA master plasmid.

[0043] 3) Add the induction medium containing arabinose, and under the induction of arabinose (32° C. shaker culture for 8 h), ZYCY10P3S2T expresses the ΦC31 recombinase and the endonuclease that recognizes the I-SceI site. Under the action of ΦC31 recombinase, the minicircle DNA parent plasmid undergoes DNA recombination at the attB / attP recombination site to form two small circular DNA molecules: i) minicircle DNA (only contains the target gene expression cassette and 36-bp attR site); ii) a small circle composed of plasmid backbone D...

Embodiment 3

[0045] Example 3, In vivo expression verification of microcircle DNA in animals

[0046] 1. Experimental steps

[0047] 1) FIX microcircle DNA and BDD-FVIII microcircle DNA were intramuscularly injected into C57BL / 6 mice.

[0048] 2) Collect blood regularly according to the predetermined time point, and separate the plasma.

[0049] 3) ELISA kit to detect the expression of plasma FIX factor and BDD-FVIII factor (see image 3 ).

[0050] 2. Experimental results

[0051] After a single intramuscular injection of microcircles (MC.FIX and MC.BDD-FVIII), FIX and BDD-FVIII were continuously expressed in mice for more than 40 days, the plasma FIX concentration was maintained at about 30ng / mL, and the plasma BDD-FVIII was maintained at normal 2-3% of the level.

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Abstract

The invention relates to muscle-targeted minicircle DNA gene therapy, in particular to a minicircle DNA vector for long-term efficient expression of a therapeutic gene product in muscle. The minicircle DNA vector is combined with a muscle targeted delivery technology, and can efficiently express the therapeutic gene product in muscle for a long time, thereby overcoming the defects of limited half-life period of protein drugs and unsustainable efficacy. The minicircle DNA vector can be used for treating common hereditary genetic defects or chronic diseases, and has the advantages of safety, effectiveness and low cost.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a minicircle (Minicircle, MC) DNA carrier, more specifically a muscle-targeted minicircle DNA gene therapy. Background technique [0002] Protein drugs (including hormones and peptides) have been widely used in the clinical treatment of various monogenic disorders (Monogenic Disorders) and chronic diseases. However, protein / hormone / polypeptide drugs have poor thermal stability and are inconvenient to prepare and store; and their half-life in vivo is limited, requiring repeated administration. Especially for hereditary gene defect diseases and chronic diseases, long-term or even life-long medication is often required, and the use of protein drugs is even more inconvenient and unaffordable. Gene therapy can continuously express the target gene product in the body, and conveniently achieve the purpose of long-term treatment. The essence of gene therapy is to deliver the gene carrier car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N15/57C12N15/12A61K48/00A61K38/37A61K38/48A61P43/00A61P7/04
CPCA61K38/37A61K38/4846A61K48/005A61K48/0075A61P7/04A61P43/00C07K14/755C07K2319/02C12N9/644C12N15/85C12Y304/21022
Inventor 谌平谢亦武陈志英
Owner SYNO MINICIRCLE BIOTECH CO LTD
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