A Novel Minimal-Size Vector (MIDGE) Improves Transgene Expression in Colon Carcinoma Cells and Avoids Transfection of Undesired DNA
Authors: Frank Schakowski, Marcus Gorschlüter, Claas Junghans, Matthias Schroff, Peter Buttgereit, Carsten Ziske, Björn Schöttker, Sven A. König-Merediz, Tilman Sauerbruch, Burghardt Wittig, Ingo G.H. Schmidt-Wolf
Journal: Molecular Therapy (2001)

Viral and plasmid vectors may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes contained in their sequence. Furthermore, replication-defective viral vectors carry the potential risk of recombination with wild-type viruses or activation of oncogenes. A new vector type for minimalistic, immunologically defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal-size gene transfer unit containing the expression cassette, including promoter, gene, and RNA-stabilizing sequence, flanked by two short hairpin oligonucleotide sequences. The resulting vector is a small, linear, covalently closed, dumbbell-shaped molecule. DNA not encoding the desired gene is reduced to a minimum. Here, we transfected colon carcinoma cell lines using cationic lipid, cationic polymer, and electroporation with several MIDGE vectors and corresponding plasmids containing transgenes encoding enhanced green fluorescent protein (eGFP) and human interleukin-2 (hIL-2). Transfection efficiency as measured qualitatively and quantitatively with eGFP was found to be comparable for both vector types. However, hIL-2 secretion and eGFP expression were approximately two- to fourfold higher in most cells transfected with these transgenes using MIDGE vectors compared to the plasmid control. This report demonstrates the advantages of this new vector type and its prospects for ex vivo gene therapy studies. Viral and plasmid vectors may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes contained in their sequence. Furthermore, replication-defective viral vectors carry the potential risk of recombination with wild-type viruses or activation of oncogenes. A new vector type for minimalistic, immunologically defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal-size gene transfer unit containing the expression cassette, including promoter, gene, and RNA-stabilizing sequence, flanked by two short hairpin oligonucleotide sequences. The resulting vector is a small, linear, covalently closed, dumbbell-shaped molecule. DNA not encoding the desired gene is reduced to a minimum. Here, we transfected colon carcinoma cell lines using cationic lipid, cationic polymer, and electroporation with several MIDGE vectors and corresponding plasmids containing transgenes encoding enhanced green fluorescent protein (eGFP) and human interleukin-2 (hIL-2). Transfection efficiency as measured qualitatively and quantitatively with eGFP was found to be comparable for both vector types. However, hIL-2 secretion and eGFP expression were approximately two- to fourfold higher in most cells transfected with these transgenes using MIDGE vectors compared to the plasmid control. This report demonstrates the advantages of this new vector type and its prospects for ex vivo gene therapy studies.