3 RESULTS
3.4 Evaluation of 5’-‐trans-‐splicing ex vivo
3.4.2 Characterization of 5’-‐trans-‐splicing at mRNA level
3.4.2.1 Detection of repaired Mybpc3 mRNA
Based on the pilot experiments in 3.4.1 to determine the optimal MOI of AAV6 in isolated cardiomyocytes, PTM-‐mediated 5’-‐trans-‐splicing was investigated in cell culture. In order to determine the effect of MOI on PTM-‐mediated 5’-‐trans-‐splicing activity, KI NMCMs were transduced with AAV6-‐CMV-‐PTM1BD1 and the corresponding control AAV6-‐CMV-‐
Ctr.PTM1BD1 with MOI ranging from 1,000 to 30,000 for 7 days (Figure 32; upper panel).
PCR using primer pair FLAG/E9R to specifically amplify repaired mRNA revealed 5’-‐trans-‐
splicing in the sample transduced with AAV6-‐CMV-‐PTM1BD1 using MOI of 30,000, but not at lower MOI, and neither in untreated cells nor in cells transduced with corresponding control-‐
PTMs. Using the primer pair E1F/E9R the repaired Mybpc3 sequence was amplified, in addition to the three endogenous mutant Mybpc3 forms (total Mybpc3 mRNA). Non-‐
transduced cardiomyocytes showed only the mutant Mybpc3 mRNAs as indicated by arrowheads (Figure 32; middle panel). The band of 896 bp in AAV6-‐CMV-‐PTM1BD1 (MOI 30,000) corresponded to both, repaired and mutant-‐1 mRNAs. Moreover, mutant-‐2 (778 bp) and mutant-‐3 (824 bp) mRNAs were detected in treated cells as well. The level of total Mybpc3 mRNA did slightly differ between the samples and was very low in the AAV6-‐CMV-‐
Ctr.PTM1BD1 sample (MOI 1,000). However, the level of Myh6 mRNA, encoding alpha-‐
myosin heavy chain did not markedly differ between the samples, suggesting good vitality of the cells (Figure 32; lower panel). There was no genomic contamination detectable in the
samples, as shown in the -‐RT samples. Altogether, this experiment demonstrated that PTM1BD1 was able to generate detectable levels of repaired mRNA.
Figure 32: Detection of repaired Mybpc3 mRNA induced by 5’-‐trans-‐splicing in MOI-‐dependent manner. Total RNA was extracted from KI NMCMs transduced with AAV6-‐CMV-‐PTM1BD1 and AAV6-‐CMV-‐Ctr.PTM1BD1 using increasing MOI from 1,000 up to 30,000 for 7 days prior to harvesting and reverse transcribed into cDNA.
Samples without reverse transcriptase are indicated as (-‐). PCR analysis was performed using primer pairs FLAG/E9R and E1F/E9R to amplify repaired (921 bp) and total Mybpc3 (Mut-‐1/repaired, 896 bp; Mut-‐2, 778 bp;
Mut-‐3, 824 bp) mRNA, respectively. As a negative control H2O was added instead of cDNA. Amplification of Myh6 mRNA was used as control for quality and quantity of cDNA. The expected amplicons are indicated by arrowheads. Abbreviations: NT, non-‐transduced; RT, reverse transcriptase.
Given the observation that MOI of 30,000 gave the most promising 5’-‐trans-‐splicing rate, each of the generated PTMs and corresponding control-‐PTMs were tested for their 5’-‐trans-‐
splicing activity ex vivo with this MOI. KI NMCMs were transduced with AAV6-‐CMV-‐PTMxBDx and the corresponding controls AAV6-‐CMV-‐Ctr.PTMxBDx with MOI of 30,000 for 7 days. PCR amplifications revealed PTM-‐mediated 5’-‐trans-‐splicing in samples transduced with AAV6-‐
CMV-‐PTMxBDx (Figure 33; upper panel) under these conditions. As expected, untreated cells and the corresponding control-‐PTMs did not result in any 5’-‐trans-‐splicing. Overall, PTMs either targeting Mybpc3 intron 6 or 7 were capable to repair mutant mRNA. However, PTM1BD1 and PTM1BD2, both targeting Mybpc3 intron 6 showed markedly higher 5’-‐trans-‐
splicing efficiency than PTM2BD1 complementary to Mybpc3 intron 7. To evaluate whether cis-‐splicing is reduced, the primer pair E1F/E9R was used for RT-‐PCR to amplify total Mybpc3 mRNA (Figure 33; lower panel). After 7 days of infection there was no major difference between the samples. Moreover, there was no genomic contamination detectable in the samples, as shown in the -‐RT samples.
Figure 33: Detection of repaired Mybpc3 mRNA induced by 5’-‐trans-‐splicing ex vivo. Total RNA was extracted from KI NMCMs transduced with AAV6-‐CMV-‐PTMxBDx and AAV6-‐CMV-‐Ctr.PTMxBDx (MOI 30,000) for 7 days prior to harvesting and reverse transcribed into cDNA. Samples without reverse transcriptase are indicated as (-‐). PCR analysis was performed using primer pairs FLAG/E9R to amplify repaired (921 bp) and E1F/E9R to amplify total Mybpc3 (Mut-‐1/repaired, 896 bp; Mut-‐2, 778 bp; Mut-‐3, 824 bp) mRNA. As a negative control H2O was added instead of cDNA. The expected amplicons are indicated by arrowheads. Abbreviations: NT, non-‐
transduced; RT, reverse transcriptase.
In order to investigate whether 5’-‐trans-‐splicing did not display an undesired artefact as a consequence of aberrant splicing mechanisms due to the point mutation in Mybpc3 exon 6, WT NMCMs were transduced with AAV6 encoding PTM1BD2 or PTM2BD1 and the respective control-‐PTMs for 7 days. PTM1BD1 was not tested in WT NMCMs, since it covered the loxP site sequence, which remained in the DNA after Cre-‐mediated recombination in Mybpc3 KI intron 6, (Vignier et al., 2009). Furthermore ‘toxic’ molecules, containing the wild-‐type Mybpc3 sequences (Tox1, exons 1 to 6; Tox2, exons 1 to 7) with an artificial stop codon were tested in WT NMCMs as an additional negative control for 5’-‐trans-‐splicing, since they did not contain any binding domain. PCR using primer pair FLAG/E9R revealed the repaired mRNA product of 921 bp in PTM-‐transduced WT NMCMs (Figure 34; upper panel). As expected, non-‐transduced NMCMs and NMCMs treated with control-‐PTMs did not show any 5’-‐trans-‐splicing specific amplicons. Interestingly, the level of repaired mRNA appeared to be lower in WT than in KI NMCMs.
Figure 34: Detection of repaired Mybpc3 mRNA induced by 5’-‐trans-‐splicing in WT NMCMs. Total RNA was extracted from WT NMCMs transduced with indicated constructs for 7 days prior to harvesting and reverse transcribed into cDNA. Samples without reverse transcriptase are indicated as (-‐). PCR analysis was performed using primer pairs FLAG/E9R and E1F/E9R to amplify repaired (921 bp) and total Mybpc3 (wild-‐type and/or repaired, 896 bp) mRNA, respectively. As a negative control H2O was added instead of cDNA. Amplification of Casq2 mRNA was used as control for quality and quantity of cDNA. The expected amplicons are indicated by arrowheads. Abbreviations: NT, non-‐transduced; RT, reverse transcriptase.
To examine the efficiency of 5’-‐trans-‐splicing versus endogenous cis-‐splicing in WT NMCMs, the primer set E1F/E9R was used to amplify total Mybpc3 mRNA. In non-‐transduced and control-‐PTM-‐transduced samples the cis-‐spliced WT mRNA with 896 bp was detected, whereas WT NMCMs transduced with PTMs showed the same band corresponding to repaired and wild-‐type Mybpc3 mRNA (Figure 34; middle panel). The level of Casq2 mRNA encoding calsequestrin 2 did not markedly differ between the samples and confirmed the normal transcription of this gene in the cells under treatment (Figure 34; lower panel). There was no genomic contamination detectable in the samples, as shown in the -‐RT samples.
Thus, PTMs are capable to 5’-‐trans-‐splice both KI and WT Mybpc3 mRNA.
3.4.2.2 Evaluation of 5’-‐trans-‐splicing in absence of the polyadenylation signal in