J Korean Diabetes Assoc.  2007 Nov;31(6):465-471. 10.4093/jkda.2007.31.6.465.

Comparison of Minicircle with Conventional Plasmid for the Non-viral Vascular Endothelial Growth Factor (VEGF) Gene Therapy

• Kwon M
• Lee S
• Chung H
• Yoon C
• Kim M
• Park J

• KMID: 2177559
• DOI: http://doi.org/10.4093/jkda.2007.31.6.465

Abstract

BACKGROUND: Delayed wound healings in diabetic patients are related with the impairment of the expressions of various growth factors. Treatments using growth factors have been attempted on diabetic foot ulcer. VEGF (vascular endothelial growth factor) accelerates neo-angiogenesis on the early phase of the wound healing and exerts chemo-attractive effect for the other growth factors and cytokines. Non-viral gene transfer strategies are attractive tool for the gene therapy due to the safety and the versatility, but the low efficiency has been the serious problem.
METHODS
We performed the VEGF gene therapy using reconstructed minicircle MINI-pbetaVEGF DNA with a polymeric carrier, polyethylenimine (PEI, 25 kDa) in HEK293, CHO, and NIH3T3 cell lines, and compared its efficiency with the conventional VEGF plasmid pbetaVEGF.
RESULTS
The levels of expressed VEGF were higher in the groups using BPEI (branched PEI) as a gene carrier than naked plasmid transfer in all cell lines (P < 0.05). The minicircle MINI-pbetaVEGF DNA showed much higher VEGF expression than conventional plasmid pbetaVEGF (P < 0.05).
CONCLUSION
Minicircle DNA MINI-pbetaVEGF showed much higher transfection efficiency than conventional plasmid pbetaVEGF. It might be used in actual human clinical trial due to its higher efficiency and possible safety for the treatment of diabetic foot ulcer.

Keyword

Gene therapy; Minicircle DNA; Vascular endothelial growth factor (VEGF)

MeSH Terms

Cell Line
Cytokines
Diabetic Foot
DNA
Genes, vif
Genetic Therapy*
Humans
Intercellular Signaling Peptides and Proteins
Plasmids*
Polyethyleneimine
Polymers
Transfection
Ulcer
Vascular Endothelial Growth Factor A*
Wound Healing
Cytokines
DNA
Intercellular Signaling Peptides and Proteins
Polyethyleneimine
Polymers
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Production of minicircle pβVEGF. A, L-(+)-arabinose (Sigma) was added on E.coli DH5a transformed with pBAD-pieC31-βVEGF plamid and the additional incubation for the integration of DNAs at 30℃ by times; B, The minicircle pβVEGF was separated by linearized the remnant bacterial backbone using EcoRV restriction enzyme.

• 

  Fig. 2 Reconstruction of the minicircle pβVEGF. The expression cassette of pβVEGF comprised with chicken beta actin promoter, the VEGF165 cDNA and d SV40 poly adenylation signal sequence was excised with Bgl II and Cla I and ligated bluntly between attB and attP in plasmid pBAD-pieC31. The minicircle pβVEGF was produced by C31 integrase recombinating the att structure.

• 

  Fig. 3 Luciferase activity assay in HEK293 cell line. Luciferase activities were increased on cells transfected with DNA/BPEI polyplexes than naked plasmids (P < 0.05). And it was increased more one hundred times on minicircle pβVEGF than conventional pβVEGF on groups using BEPI (branched polyethylenimine), cataionic gene carrier.

• 

  Fig. 4 VEGF protein level by ELISA in HEK293, NIH3T3, and CHO cell line. VEGF proteins were more expressed on groups of BPEI complexes than naked plasmids in all cell lines (P < 0.05). VEGF expressions were more increased about 2.5, 2 and 1.25 times on minicircle pβVEGF group than conventional pβVEGF using BEPI in HEK293(A), NIH3T3(B), and CHO(C) cell line.

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