Correction of DNA protein kinase deficiency by spliceosome-mediated RNA trans-splicing and sleeping beauty transposon delivery

Mol Ther. 2007 Jul;15(7):1273-9. doi: 10.1038/sj.mt.6300178. Epub 2007 Apr 24.

Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Catalytic Domain
  • Cell Line
  • Humans
  • Mice
  • Mutation / genetics
  • Polynucleotide 5'-Hydroxyl-Kinase / deficiency*
  • Polynucleotide 5'-Hydroxyl-Kinase / metabolism*
  • RNA, Messenger / genetics
  • Spliceosomes / genetics*
  • Trans-Splicing / genetics*
  • Transcription, Genetic / genetics
  • Transposases / genetics*

Substances

  • RNA, Messenger
  • Polynucleotide 5'-Hydroxyl-Kinase
  • Transposases
  • sleeping beauty transposase, human