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Marcus Davidsson

Marcus Davidsson

Postdoctoral fellow

Marcus Davidsson

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

Summary, in 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.

Department/s

  • Molecular Neuromodulation
  • MultiPark: Multidisciplinary research focused on Parkinson´s disease
  • CNS Gene Therapy

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 (CSHL)

Topic

  • Medical Genetics

Keywords

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

Status

Published

Research group

  • Molecular Neuromodulation
  • CNS Gene Therapy

ISBN/ISSN/Other

  • ISSN: 1355-8382