Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system

Abstract

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.

Gene Therapy Progress and Prospects: Nonviral vectors

“…65 Sleeping Beauty has been used to accomplish stable chromosomal integration of functioning genes in somatic cells of adult mice. 66 In addition, several phage integrases and their corresponding recognition elements, which can mediate integration into mammalian chromosomes, were reported by Calos's group. [67][68][69][70] Although the integration efficiency of integration system is still low, this technology may one day enable site-specific and high-efficiency integration into the host chromosome without the potential for mutagenesis.…”

Hydroporation as the mechanism of hydrodynamic delivery

“…1,2 This method, called the hydrodynamicsbased procedure, has been widely utilized by the gene therapy community for evaluating therapeutic activities of various genes (see, for reviews, Liu and Knapp 3 and Herweijer and Wolff 4 ). Other reported applications of this technique include studies to define the regulatory functions of DNA sequences, [5][6][7][8][9] investigations to evaluate gene suppression activity of siRNA, [10][11][12] and experiments to establish animal models for viral infection. 13,14 Despite many desirable features of the hydrodynamics-based procedure such as simplicity, convenience, and high efficiency, further improvement and new applications of this procedure require a full understanding of the mechanisms underlying this technique.…”

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer

“…Among such approaches is plasmidbased gene transfer mediated by the Sleeping Beauty (SB) synthetic transposable element. [6][7][8] SB is an ancestral fish member of the Tc1/mariner family. 9 SB transposition integrates incoming DNA into the genome using a 'cutand-paste' mechanism requiring short inverted repeat (IR) sequences flanking the insert and the SB transposase protein.…”

“…10 SB transposase has been shown to facilitate gene integration into mammalian cell lines and murine liver sufficient to support long-term gene delivery. 6,7 In spite of these attractive features, transposon-mediated gene transfer has not yet been reported capable of achieving stable genetic correction of human tissue from patients with characterized genetic disorders.…”