Menu

Javascript is not activated in your browser. This website needs javascript activated to work properly.
You are here

Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing

Author:
  • Marcus Davidsson
  • Paula Díaz-Fernández
  • Marcos Torroba
  • Oliver D. Schwich
  • Patrick Aldrin-Kirk
  • Luis Quintino
  • Andreas Heuer
  • Gang Wang
  • Cecilia Lundberg
  • Tomas Björklund
Publishing year: 2018-05-01
Language: English
Pages: 673-687
Publication/Series: RNA
Volume: 24
Issue: 5
Document type: Journal article
Publisher: Cold Spring Harbor Laboratory Press

Abstract english

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA trans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determining trans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissive trans-splicing sequences targeting an intron in the Synapsin I gene. Using viral vectors, we were able to splice full-length fluorophores into the mRNA while retaining very low off-target expression. Furthermore, this approach also showed evidence of functionality in the mouse striatum. However, in its current form, the trans-splicing events are stochastic and the overall activity lower than would be required for therapies targeting loss-of-function mutations. Nevertheless, the herein described barcode-based screening assay provides a unique possibility to screen and map large libraries in single animals or cell assays with very high precision.

Keywords

  • Medical Genetics
  • Barcoding
  • Plasmid library
  • Trans-splicing
  • Viral vectors

Other

Published
  • Molecular Neuromodulation
  • CNS Gene Therapy
  • ISSN: 1355-8382
Marcus Davidsson
E-mail: marcus [dot] davidsson [at] med [dot] lu [dot] se

Molecular Neuromodulation unit
Wallenberg Neuroscience Center
Department of Experimental Medical Science
BMC A10, 221 84 Lund, Sweden
 
Phone: +46 46 222 68 36
e-Mail: tomas [dot] bjorklund [at] med [dot] lu [dot] se