Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression

A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo ( Nicol et al., 2002 ; Chen et al., 2004 ). Emerging evidence indicates that plasmid bacterial backbone sequences are responsible for the transcriptional repression and this process is independent of CpG methylation ( Chen et al., 2008 ). Dumbbell-shaped DNA vectors consisting solely of essential elements for transgene expression have been developed to circumvent these drawbacks. This novel non-viral vector has been shown to improve transgene expression in vitro and in vivo ( Schakowski et al., 2001 and 2007). Here we describe a novel method for fast and efficient production of minimised small RNA-expressing dumbbell vectors. In brief, the PCR-amplified promoter sequence is ligated to a chemically synthesized hairpin RNA coding DNA template to form the covalently closed dumbbell vector. This new technique may facilitate applications of dumbbell-shaped vectors for preclinical investigation and human gene therapy.