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

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Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing. / Davidsson, Marcus; Díaz-Fernández, Paula; Torroba, Marcos; Schwich, Oliver D.; Aldrin-Kirk, Patrick; Quintino, Luis; Heuer, Andreas; Wang, Gang; Lundberg, Cecilia; Björklund, Tomas.

In: RNA, Vol. 24, No. 5, 01.05.2018, p. 673-687.

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Davidsson, Marcus ; Díaz-Fernández, Paula ; Torroba, Marcos ; Schwich, Oliver D. ; Aldrin-Kirk, Patrick ; Quintino, Luis ; Heuer, Andreas ; Wang, Gang ; Lundberg, Cecilia ; Björklund, Tomas. / Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing. In: RNA. 2018 ; Vol. 24, No. 5. pp. 673-687.

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TY - JOUR

T1 - Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing

AU - Davidsson, Marcus

AU - Díaz-Fernández, Paula

AU - Torroba, Marcos

AU - Schwich, Oliver D.

AU - Aldrin-Kirk, Patrick

AU - Quintino, Luis

AU - Heuer, Andreas

AU - Wang, Gang

AU - Lundberg, Cecilia

AU - Björklund, Tomas

PY - 2018/5/1

Y1 - 2018/5/1

N2 - 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.

AB - 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.

KW - Barcoding

KW - Plasmid library

KW - Trans-splicing

KW - Viral vectors

U2 - 10.1261/rna.063925.117

DO - 10.1261/rna.063925.117

M3 - Article

VL - 24

SP - 673

EP - 687

JO - RNA

T2 - RNA

JF - RNA

SN - 1355-8382

IS - 5

ER -