Minicircle vector for non-insertional transgene expression in quiescent cells and tissues

The minicircle is a non-viral DNA vector for non-insertional transgene expression. A typical minicircle contains a transgene expression cassette, and is free of all other plasmid DNA elements, including an antibiotic resistance gene and a plasmid DNA replication origin. This reduces the potential for silencing and inflammatory effects associated with these sequences.

The minicircle produced with this method provides a stable and persistent expression of the transgene expression cassette in quiescent cells and tissues, and reduces CpG inflammatory responses observed in some plasmid transfection schemes.

This invention improves the production of minicircle DNA by making a versatile bacterial strain. This bacterium is genetically engineered to express (1) multiple copies of an inducible phiC31 gene, the recombinase which mediates the formation of a minicircle expression cassette from the parental plasmid; (2) multiple copies of an inducible ISce1 gene, the restriction enzyme, which when expressed destroys the unwanted plasmid bacterial backbone DNA, and (3) two different genes encoding transporters of L-arabinose, which is the inducer of the araC.BAD system controlling the expression of both the recombinase phiC31 and the restriction enzyme ISce1.

Consequently, the new system has the following advantages:

(1) Reduces contamination of parental plasmid and its derivatives from 5 -15% to 1%.
(2) Reduces the time of minicircle preparation to that of routine plasmid preparations
(3) Reduces the amount of arabinose required by over 100-fold.
(4) Reduces the risk of phiC31/ISceI genes by removing them from the parental plasmid to the bacterial genome making the final process GMP compatible.