Harnessing a High Cargo-Capacity Transposon for Genetic Applications in Vertebrates

Balciunas et al. (2006). PLoS Genetics DOI: 10.1371/journal.pgen.0020169 Citations: 343

Transposons and viruses lost activity when cargo exceeded 8 kb, limiting gene therapy. This paper defined minimal Tol2 sequences (miniTol2) transposing DNA over 10 kb into human cells. MiniTol2 corrected FAH deficiency in a tyrosinemia type 1 mouse model.

Parts used

IRES

Key findings

  • The Tol2 transposon was characterized with minimal sequences (miniTol2: 261 bp 5' + 202 bp 3' sequences) that showed activity statistically indistinguishable from full-length Tol2 in zebrafish somatic assays and human cells—10-kb Tol2 transposons mediated gene transfer nearly as efficiently as 5-kb elements in HT1080 cells, demonstrating high cargo-capacity independent of size.
  • Tol2 lacks overexpression inhibition observed with Sleeping Beauty transposon—peak gene transfer rates were similar between systems despite Tol2's larger native size (5 kb vs 2 kb), with Tol2 maintaining activity across transposon:transposase ratios of 1:1 to 1:10 while SB activity decreased at high transposase concentrations.
  • Hydrodynamic injection of Tol2/FAHIL (8.5 kb) or miniTol2/FAHIL (5.8 kb) transposons corrected hereditary tyrosinemia type 1 in FAH-knockout mice—animals co-injected with transposase maintained body weight without NTBC treatment and showed 100-fold increase in liver luciferase expression over 2 months, with 36 integration sites confirmed via inverse PCR displaying 8-bp target duplications.

Abstract

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.