Molecular barcoding of viral vectors enables mapping and optimization of mRNAtrans-splicing

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 -splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determiningsplicing are reproducible regardless of the screening system. With this screening, we have located the most permissive 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, thesplicing 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.

Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation

“…Similar to other studies, we also addressed the lack of reliable established methods for quality control of barcoded plasmid libraries. 27 , 28 , 29 , 32 , 33 In particular, plating out a small aliquot of the transformed bacteria and counting the colonies might lead to variable results, as it depends on (1) the quality of the agar plate, (2) the person performing the experiment, (3) the plating out technique, and (4) the homogeneity of the bacterial concentration in the transformation mixture. Our approach is based on an accumulation curve using NGS data.…”

A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism

“…It is important to realize that the principle behind the BRAVE approach is not limited to the screening of capsid variants carrying modifications restricted to a single, small domain, such as the HS-binding motif used in this study (36, 50). It is possible that the BRAVE screening approach can be used where rational changes are introduced at any site of the 3 capsid proteins.…”

Next-Generation Gene Therapy for Parkinson’s Disease Using Engineered Viral Vectors

“…With this approach, we can be sure that the protein sequence selected will be efficiently synthesized in human cells, and the potential for being infective cross-species is increased. The same library can then be characterized across numerous model systems such as hESC-derived dopamine (DA) cells, organoids, transplanted human neurons in the rat brain, and many more [ 30, 31 ]. The readout from each model system can then be integrated to identify novel capsid variants to be highly efficient and translational to the human brain.…”