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

Davidsson, Marcus LU ; Díaz-Fernández, Paula; Torroba, Marcos; Schwich, Oliver D.; Aldrin-Kirk, Patrick LU ; Quintino, Luis LU ; Heuer, Andreas LU ; Wang, Gang LU ; Lundberg, Cecilia LU and Björklund, Tomas LU (2018) In RNA 24(5). p.673-687
Abstract

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... (More)

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.

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Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Barcoding, Plasmid library, Trans-splicing, Viral vectors
in
RNA
volume
24
issue
5
pages
15 pages
publisher
Cold Spring Harbor Laboratory Press
external identifiers
  • scopus:85045516024
ISSN
1355-8382
DOI
10.1261/rna.063925.117
language
English
LU publication?
yes
id
6714d887-f248-45a7-bd05-abb9d7f5e92f
date added to LUP
2018-04-26 08:37:53
date last changed
2019-08-14 04:15:16
@article{6714d887-f248-45a7-bd05-abb9d7f5e92f,
  abstract     = {<p>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.</p>},
  author       = {Davidsson, Marcus and Díaz-Fernández, Paula and Torroba, Marcos and Schwich, Oliver D. and Aldrin-Kirk, Patrick and Quintino, Luis and Heuer, Andreas and Wang, Gang and Lundberg, Cecilia and Björklund, Tomas},
  issn         = {1355-8382},
  keyword      = {Barcoding,Plasmid library,Trans-splicing,Viral vectors},
  language     = {eng},
  month        = {05},
  number       = {5},
  pages        = {673--687},
  publisher    = {Cold Spring Harbor Laboratory Press},
  series       = {RNA},
  title        = {Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing},
  url          = {http://dx.doi.org/10.1261/rna.063925.117},
  volume       = {24},
  year         = {2018},
}