Figure 1.
Trans-splicing strategy for 3′ replacement.
(A) The murine dystrophin minigene consists of exons 22 to 24 (boxes E22 to E24) with natural introns (lines). The splice sequences are illustrated by black balls: 5′ splice sites (5′SS), branching points (BP), polypyrimidine tracts (PPT) and 3′ acceptor sites (3′SS). The cross represents the nonsense mdx mutation in E23. The trans-splicing (TS) molecule AS-E24 comprises a 150 nt antisense sequence (AS) complementary to intron 22 as well as a spacer, a strong conserved yeast branch point sequence, a polypyrimidine tract, a 3′ acceptor site and E24. The sequence of the spacer, BP, PPT and 3′SS is given with the BP, PPT and 3′SS in bold and illustrated by black balls. The E24 sequence from minigene (white box) and TS molecule (black box) are identical, only the downstream sequence differs in length. TS constructs were made with three different antisense sequences, AS1 to AS3. Arrows indicate the positions of the forward A and reverse B PCR primers in the minigene and the TS molecule. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and trans-splicing, and the predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT-PCR analysis using PCR primers A and B of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7), pSMD2-E24 (AS-), pSMD2-AS1-E24 (AS1), pSMD2-AS2-E24 (AS2) and pSMD2-AS3-E24 (AS3). RT- AS2: samples containing dystrophin minigene and pSMD2-AS2-E24 without reverse transcription; H2O: PCR negative control. Representative results from three independent transfection experiments. (D) An exact E22-E24 junction was confirmed by sequencing of the 304bp product.
Figure 2.
Exon replacement approach on dystrophin minigene transcripts.
(A) The exon exchange molecule (ExCh) AS-E24-AS' comprises the same elements as the TS molecule (see Fig. 1A) followed by a 5′ donor splice site (5′SS) and a second antisense sequence (AS') of 150 nt complementary to intron 23. The sequence of 5′SS is given in bold and is illustrated by a black ball. ExCh constructs were made with five different AS' antisense sequences, AS4 to AS8. Arrows indicate the positions of the forward A and reverse C PCR primers in the minigene. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and exon exchange, and predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7), the TS constructions pSMD2-AS1-E24 (AS1), pSMD2-AS2-E24 (AS2) and ExCh molecules pSMD2-AS-E24-AS' containing AS1 or AS2 and AS4 to AS8. AS2-2XAS4, ExCh plasmid pSMD2-AS2-E24-2XAS4 containing two AS4 copies; H20: PCR negative control. Representative results from two independent transfection experiments. (D) Accurate E22-E24 and E24-E24 junctions were confirmed by sequencing of the 408bp product.
Figure 3.
Effect of intronic splice enhancer sequences on exon replacement efficiency.
(A) Exon exchange molecules AS-E24-AS' with intronic splice enhancers ISE or DISE sequences. (B) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructs pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7) and the following ExCh plasmids with AS4 or AS8: pSMD2-AS2-E24-AS' (-), pSMD2-AS2-ISE-E24-AS' (ISE), pSMD2-AS2-E24-DISE-AS' (DISE) and pSMD2-AS2-E24-2XAS' (2XAS'). H20: PCR negative control.
Figure 4.
Efficiency of dystrophin exon exchange induced by AS4 containing ExCh molecules.
(A) Expected amplicons from PCR1 with E23-F & E23-R primers and (B) from PCR2 with E24-F & E24-R primers. (C) Histogram showing numbers of parental and exchanged transcripts evaluated by absolute quantitative real-time RT-PCR. PCR1 corresponds to the parental transcripts whereas PCR2 to the repaired transcripts. PCR1+PCR2 corresponds to the totality of the dystrophin minigene transcripts. (D) ExChange efficiency estimated by calculating the ratio between levels obtained for repaired transcripts (PCR2) and whole dystrophin minigene transcripts (PCR1+PCR2). Representative results from two independent transfection experiments.
Figure 5.
Dosing study of AS2-E24-DISE-AS4 molecule.
(A) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with 250 ng of dystrophin minigene and 100, 200, 400 and 750 ng of pSMD2-AS2-E24-DISE-AS4 plasmids. (B) Histogram showing numbers of parental and exchanged transcripts evaluated by absolute quantitative real-time RT-PCR illustrated in Fig. 4A. PCR1 corresponds to the parental transcripts whereas PCR2 to the repaired transcripts. PCR1+PCR2 corresponds to the totality of the dystrophin minigene transcripts. (C) ExChange efficiency of the dosing study estimated by calculating the ratio between levels obtained for repaired transcripts (PCR2) and whole dystrophin minigene transcripts (PCR1+PCR2). Representative results from two independent transfection experiments.
Figure 6.
Exon 23 correction by exon replacement.
(A) The ExCh AS2-E23-DISE-AS4 molecule comprises the same elements as AS2-E24-DISE-AS4, except E23 instead of E24. E23 is the wild-type murine dystrophin E23 carrying a HindIII restriction site absent in the mdx minigene E23. (B) Expected transcripts generated by cis-splicing and E23 exchange, and predicted size of the corresponding RT-PCR products using primers A and C. (C) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl) and ExCh plasmid pSMD2-AS2-E23-DISE-AS4; H20: PCR negative control. Representative results from two independent transfection experiments. (D) Cloning results of the RT-PCR amplicons obtained in (C) with AS2-E23-DISE-AS4 molecule. The clone numbers corresponding to parental and repaired inserts are described.