5' trans-splicing repair of the PLEC1 gene

J Invest Dermatol. 2008 Mar;128(3):568-74. doi: 10.1038/sj.jid.5701152. Epub 2007 Nov 8.

Abstract

The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient transfection of EBS-MD fibroblasts with a 5' pre-trans-splicing molecule encoding wild-type exons 2-9 led to specific replacement of the mutated 5' portion of the endogenous PLEC1 transcript through trans-splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Transformed
  • Epidermolysis Bullosa Simplex / genetics
  • Epidermolysis Bullosa Simplex / therapy*
  • Fibroblasts / cytology
  • Fibroblasts / physiology*
  • Gene Expression
  • Genetic Therapy / methods*
  • Humans
  • Plectin / genetics*
  • Polymerase Chain Reaction
  • RNA Precursors / genetics
  • RNA Splice Sites
  • RNA Splicing*
  • Retroviridae / genetics

Substances

  • PLEC protein, human
  • Plectin
  • RNA Precursors
  • RNA Splice Sites