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Docket #: S02-190

Minicircle DNA for gene delivery and protein expression

Minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The pC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB).

The researchers have demonstrated that minicircular DNAs devoid of bacterial sequences expressed 45-, and 560-fold more serum human factor IX, and alpha 1 - antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver. Gene expression was persistent for at least 6 months. The minicircles do not contain any bacterial DNA, previously shown to result in transcriptional silencing of the transgene in vivo.

These minicircles are capable of expressing high and persistent levels of therapeutic products in vivo, and have a great potential to serve as episomal vectors for treatment of a wide variety of diseases. They can also be used as a research tool to achieve non-insertional gene expression in quiescent tissues.

More recently, the Stanford researchers have developed a bacterial strain that 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. More details about this bacterial strain can be found under Stanford Docket S08-108.

Applications

  • Expression of high and persistent levels of therapeutic products in vivo.
  • Great potential to serve as episomal vectors for treatment of a wide variety of diseases.
  • Efficient method for in vivo target validation.
  • Tissues other than liver are being investigated.

Advantages

  • Large scale production of 1mg/l bacterial culture. Prior to purification, the yield of minicircles is 96% (the other 4% are circles containing bacterial DNA).
  • Persistent gene expression for at least 6 months (length of the study).
  • No risk of insertion-specific mutations due to episomal location of the miniplasmid.
  • 560-fold more alpha 1 - antitrypsin in serum, compared to standard plasmid DNAs transfected into mouse liver.
  • 45-fold more human factor IX in serum, compared to standard plasmid DNAs transfected into mouse liver.

Publications

Patents

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