Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-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 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: barcoding; plasmid library; trans-splicing; viral vectors.

FIGURE 1.

Generation and validation of the splice acceptor plasmid library. (A) Schematics showing the split-GFP trans-splicing screening approach. LV derived splice donor expressing N-terminal GFP and full Synapsin I intron 9–10 and splice acceptor expressing intron fragment, C-terminal GFP and DNA barcode (BC). The trans-spliced mRNA is a hybrid between donor and acceptor and the result is the full open reading frame of GFP. By deep sequencing of molecular barcodes and intron fragments from the plasmid library prior to the cell culture or in vivo screening assay, a look-up table can be created to create a link between the two. (B) A schematic overview of the process used for the preparation of intron fragments for library cloning by using dUTP-based fragmentation via Uracil DNA Glycosylase and NaOH followed by end-repair and A-tailing. (C) Plot of the overall coverage of the Synapsin I intron with the negative (active) strand in red and positive (inactive) strand orientation in blue. (D) Length distribution of all inserted intron fragments. (See Supplemental Fig. S1 for additional information.)

FIGURE 2.

trans-splicing screening assay in cell culture based of split-GFP fluorescence. (A,B) FACS plot showing trans-splicing positive cells expressing GFP and mRFP of cells from C (A) and from D (B). (C–D″) Confocal image of stable cell lines expressing splice acceptor either from the LV intron fragment library (C–C″) or the negative control containing the scrambled sequence (D–D″) (both expressing C-GFP), transfected with splice donor (expressing N-GFP). Successful trans-splicing is indicated by GFP expression and both constructs constitutively express mRFP for assessment of transduction efficacy and FACS enrichment of transduced cells. (E–I) Flowchart of the single cell assessment assay of trans-splicing efficacy based on fluorescence intensity. (E) FACS plot showing the distribution of GFP+ cells used for single cell sorting. Cells were sorted and analyzed in FACS based on GFP/mRFP double fluorescence. (F) Expansion of single sorted cells from 96-well to 24-well. (G) Single sorted cells were after expansion subjected to a second round of transfection with splice donor, and GFP fluorescence was analyzed in a flow cytometer. (H) Correlation between first (FACS) and second (flow cytometer) round of transfection for each single sorted and expanded cell. Fluorescence was quantified using the MESF standard beads. Dots with full circle are cells with splice acceptors containing intron fragments, and dots with white center are cells expressing a splice acceptor containing only the scrambled sequence. (I) DNA from cells in F was extracted and PCR amplified and sent for Sanger sequencing. The sequenced intron fragment was then mapped to trans-splicing efficacy based on the fluorescence data in H. Top part shows trans-splicing efficacy and bottom part shows each fragment's position in the Synapsin I intron, color-coded based on the achieved fluorescence intensity.

FIGURE 3.

Results from three trans-splicing screening assays based both in cell culture and in vivo. (A–D) Trans-splicing efficacy over Synapsin I intron 9–10 plotted as trans-splicing efficacy for each base in the intron. (A) The collected results from Figure 2, i.e., the screening based on fluorescence in HEK293T cells transduced with LV-intron fragment library and transfected with splice donor. Data now normalized based on the relative distribution of each fragment in the complete library to allow for comparison to the mRNA base screening assays below. (B–D) Results from screening based on mRNA sequencing of barcodes. Efficacy calculated from fragments in reverse orientation are shown in blue, and fragments in forward orientation are shown in gray. Fragments selected for further validations P1, P2, N1, and N2 are shown by vertical gray lines. (B) Screening in HEK293T cells transfected with intron fragment library and splice donor. (C) Screening in HEK293T cells were transduced with intron fragment library and transfected with splice donor. (D) Screening in C57BL/6 mice injected in striatum with LV-intron fragment library. (E–G) Trans-splicing efficacy over Synapsin I intron 9–10 plotted as individual fragments. Plots E–G correspond to A–C. Recovered fragments in reverse orientation are again shown in blue and recovered fragments in forward orientation are shown in gray. (H) Validation of trans-splicing efficacy for selected fragments P1, P2, N1, and N2 as well as intron fragment library and scrambled sequence. HEK293T cells were transfected with splice acceptor and splice donor, and trans-splicing efficacy (GFP expression) was assessed by flow cytometry. GFP expression was normalized to both iRFP (splice donor) and mRFP (splice acceptor).

FIGURE 4.

Validation of trans-splicing in cell culture and removal of aberrant cis-splicing in lentiviral vectors. (A–C) Validation of trans-splicing efficacy for the fragment P1, selected in the screening assay, compared to library and scrambled sequence. HEK293T cells stably expressing splice donor were transfected with P1 (A–A″), library (B–B″), and scrambled sequence (C-C″), respectively, and analyzed 48 h post-transfection by confocal microscopy. A–C shows trans-spliced GFP, A′–C″ shows the transfection control mRFP, and A″–C″ shows the pseudo colored overlay image. (D,E) Validation of LV-P1 (D) compared to LV-Scr (E) in HEK293T cells. Cells were transduced with LV, enriched by FACS and expanded, and then transfected with splice donor. Analysis was done using flow cytometry plotting trans-spliced GFP fluorescence against the transduction control mRFP. (F,G) Validation of LV-2.0 vectors after PCR confirmation. LV-P1 2.0 and LV-Scr 2.0 were used to transduce HEK293T cells. After enrichment and expansion, cells were transfected with splice donor and analyzed by flow cytometry. No improvement on trans-splicing efficacy was observed.

FIGURE 5.

Validation of a splice acceptors expressing full-length GFP trans-splicing in vivo in the mouse brain. (A) Schematic of the improved approach to the trans-splicing event between TagBFP[+intron] as splice donor and LV/AAV with full-length GFP as splice acceptor (1). After trans-splicing, this again forms a mature mRNA (2), but in this version, the full GFP is inserted in the reading frame of the N-TagBFP. Through the insertion of the P2A ribosome skipping sequence, the N-TagBFP is split from the GFP at the ribosomal translation into protein (3). (B,C) Transfection of stable cell lines expressing TagBFP[−intron] (B) and TagBFP[+intron] (C). Cell lines were transfected with LV-P1-fuP2A and analyzed using confocal microscopy. Some scattered cells in TagBFP[+intron] were positive for GFP meaning successful trans-splicing (C). dsRed was used as a transfection control (B′–C″). (D) Quantification of LV-P1 and AAV-P1 in HEK293 cells. Cells were transduced with either LV or AAV and trans-splicing efficacy was assessed by RT-qPCR with forward primer targeting TagBFP and reverse primer targeting GFP. Control sample was AAV transduction of cells expressing TagBFP[−intron]. (E–G). Trans-splicing in vivo. WT mice were injected with scrambled (Scr) acceptor vector with a furin-P2A (fu-P2A) cleavage site AAV-Scr|fu-P2A (E), an active construct AAV-P1|fu-P2A (F), or AAV-GFP (G) in striatum. Sections were stained for GFP using immunohistochemistry developed into a brown precipitation staining using the DAB-peroxidase reaction. The figure shows representative images from striatum (Str) (E–G) and globus pallidus (GP) (E′–G′). Scale bar in G′ represents 50 µm in E–G′.