J Bacteriol Virol.  2012 Mar;42(1):57-62. 10.4167/jbv.2012.42.1.57.

Recombinant AAV Vector with MITF-M Promoter for Melanoma Gene Therapy

• Park SW
• Paik SY

• KMID: 1434773
• DOI: http://doi.org/10.4167/jbv.2012.42.1.57

Abstract

We have developed the recombinant adeno-associated virus (AAV) carrying the EGFP gene under the control of the microphtalmia-associated transcription factor-M (MITF-M) promoter region for melanoma-specific expression. MITF-M distal enhancer (MDE) region enhances the specific expression of the reporter gene specifically in cultured melanoma cells. Expression of EGFP protein was very high in AAV-CMV-EGFP infected cells but relatively low in cells infected with AAV-Mitf(Enh/Pro)-EGFP. After an in vitro infection by a recombinant AAV carrying the EGFP gene under the control of human MITF-M promoter, the reporter gene was expressed in MITF-M producing melanoma cell lines (SK-28 and G361), but not in MITF-M non-producing cell lines (HaCat). These results suggest that the utilization of the MITF-M promoter in a recombinant AAV vector could provide benefits in gene therapy applications.

Keyword

Adeno-associated virus expression vector; Melanoma gene therapy; Microphtalmia-associated transcription factor-M; Tissue-specific promoter

MeSH Terms

Cell Line
Dependovirus
Genes, Reporter
Genetic Therapy
Green Fluorescent Proteins
Humans
Lifting
Melanoma
Promoter Regions, Genetic
Green Fluorescent Proteins

Figure

• 

  Figure 1 Cloning of the recombinant AAV transfer vectors. (A) pAAV-MCS plasmid DNA vector contained multi cloning sites under the control of the CMV promoter. The gene cassette was terminated by a bovine growth hormone (bGH) poly (A) addition site. (B) The pAAV-CMV-EGFP plasmid DNA was generated by inserting expression cassette encoding the EGFP reporter gene into the standard AAV transfer vector pAAV-MCS. (C) Next, the recombinant pAAV-Mitf(Enh/Pro)-EGFP DNA was generated by inserting melanocyte-specific enhancer/promoter region into the pAAV-CMV-EGFP plasmid instead of CMV promoter.

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  Figure 2 Cell type specific gene expression by MITF-M enhancer/promoter. (A) EGFP protein was very highly expressed in AAV-CMV-EGFP infected cells but relatively low detected in cells infected with AAV-Mitf(Enh/Pro)-EGFP. (B) Two melanoma cell lines were infected with AAV-Mitf(Enh/Pro)-EGFP and AAV-CMV-EGFP at an m.o.i. of 10. The EGFP activity was detected in two melanoma cells (SK-28 and G361). However, EGFP gene expression was not detected in MITF-M no producing cell line (HaCat). Cells were photographed by epifluorescence microscopy at maximum expression point.

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