3 RESULTS
3.5 Evaluation of 5’-‐trans-‐splicing in vivo
3.5.2 Evaluation of 5’-‐trans-‐splicing in adult Mybpc3-‐targeted KI mouse
The feasibility of PTM-‐driven 5´-‐trans-‐splicing was eventually investigated in KI mice in vivo.
To this aim, PTMΔpA under the control of the cardiomyocyte-‐specific TNNT2 promoter was packaged in AAV9. Besides, AAV9 encoding TNNT2-‐driven Renilla luciferase (RLuc) transgene was used for visualizing the distribution of protein expression in the living animal. AAV9-‐
TNNT2-‐PTM∆pA (1.04 x 1011 vg) and AAV9-‐TNNT2-‐RLuc (1.37 x 1011 vg) were administered systemically in the tail vein of 7-‐week-‐old animals. The control mouse received NaCl.
3.5.2.1 Characterization of cardiac function
To investigate the cardiac phenotype of KI mice that had received AAV9-‐TNNT2-‐PTM∆pA, AAV9-‐TNNT2-‐RLuc or NaCl compared to WT controls, longitudinal echocardiographic analyses were performed during one month after injection. Compared to WT mice, KI mice treated with AAV9 and NaCl exhibited higher left ventricular mass (LVM), increased left-‐
ventricular-‐mass-‐to-‐body-‐weight ratio (LVM/BW) and lower fractional shortening (FAS) (Figure 50). No major difference in body weight (BW) was found between the groups at the age of 7 to 10 weeks. The cardiac function was not improved after AAV9-‐TNNT2-‐PTM∆pA administration at the different time points and even displayed a deteriorated phenotype at the age of 10 weeks with higher LVM and LVM/BW and lower FAS than the NaCl KI control.
In summary, longitudinal echocardiographic analysis did not display major differences in cardiac function between mice that received either AAV9-‐TNNT2-‐PTM∆pA or NaCl.
BW
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KI + NaCl KI + PTMΔpA WT KI + Rluc
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Figure 50: Determination of the cardiac phenotype in KI mice after treatment with AAV9-‐TNNT2-‐PTM∆pA.
7-‐week-‐old KI mice received AAV9-‐TNNT2-‐PTM∆pA, AAV9-‐TNNT2-‐RLuc or NaCl by systemic administration into the tail vein and were analyzed by transthoracic echocardiography at 7 to 10 weeks of age. Untreated WT mice were used as controls. Body weight (BW), left ventricular mass (LVM), LVM to BW ratio (LVM/BW) and fractional area shortening (FAS) are shown. Number of animals is indicated in the bars.
3.5.2.2 In vivo bioluminescence imaging
Renilla luciferase (RLuc) expression was evaluated by in vivo bioluminescence imaging. Non-‐
invasive optical reporter gene imaging can be applied to study vector-‐mediated gene delivery and expression in living animals (Bhaumik and Gambhir, 2002). Thereby Renilla luciferase is commonly used as bioluminescent reporter. It catalyzes the oxidation of coelenterazine to yield coelenteramide and visible flashlight of 480 nm. The signal increases with increasing coelenterazine dose upon time up to a limit and extinguishes within 10 minutes after injection of the substrate. In vivo bioluminescence imaging of TNNT2 driven RLuc expression was performed 4 weeks after virus administration. Coelenterazine was injected simultaneously in the peritoneal cavity of the RLuc-‐ and NaCl-‐treated animals.
Luminescence was clearly recorded in the heart of the mouse transduced with the reporter gene luciferase and was moderately higher than in the NaCl animal (Figure 51). The unspecific background signal at the site of injection was due to autoluminescence of the substrate itself and low levels of light emitted from the animal even though there is no bioluminescent light. In conclusion, this optical imaging study confirmed that the combination of AAV9 and cardiac-‐specific promoter preferentially restricted the luciferase gene expression, and therefore of other genes of interest, to the heart.
Figure 51: Optical imaging of Renilla luciferase reporter gene expression. Mice were anesthetized, placed in the chamber of the Xenogen In Vivo Imaging System and ventilated with a respirator. In vivo bioluminescence imaging was performed 4 weeks after AAV9-‐TNNT2-‐RLuc administration by i.p. injection of 2.5 mg/kg body weight coelenterazine in mice. Light emission was recorded specifically in the heart of the mouse that received AAV9-‐TNNT2-‐RLuc. The region of interest was manually selected and the signal intensity was recorded within constant 3 minutes scans. The positive signal at the site of i.p. injection in both NaCl-‐ and AAV9-‐TNNT2-‐RLuc-‐
injected mice corresponded to autofluorescence of the substrate. Abbreviations: i.p., intraperitoneal; ROI, region of interest.
3.5.2.3 Characterization of 5’-‐trans-‐splicing at mRNA level
The characterization of 5’-‐trans-‐splicing at mRNA level was conducted in animals 4 weeks after AAV9-‐TNNT2-‐PTM∆pA administration. Total RNA was extracted from heart and liver of all mice and reverse transcribed to cDNA. PTM-‐driven-‐5’-‐trans-‐splicing was detected by PCR using Mybpc3 primers FLAG/E9R only in the PTM∆pA-‐transduced heart and not in other mice or organs (Figure 52; upper panel). The amplified repaired Mybpc3 PCR product was confirmed by sequencing (data not shown). Using the primer set E1F/E9R to analyze total Mybpc3 transcripts, repaired Mybpc3 sequence was amplified in addition to the three endogenous cis-‐spliced Mybpc3 mutants (Figure 52; upper middle panel). In contrast to the ex vivo data, 5’-‐trans-‐splicing with the PTM∆pA did not visibly reduce cis-‐splicing.
Figure 52: Detection of repaired Mybpc3 mRNA induced by 5’-‐trans-‐splicing in vivo. Heart and liver were extracted 4 weeks after administration of AAV9-‐TNNT2-‐PTM∆pA, AAV9-‐TNNT2-‐RLuc or NaCl in 7-‐week-‐old mice and total RNA was 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. Luciferase-‐specific primers were used to amplify luciferase mRNA. As a negative control H2O was added instead of cDNA.
Amplification of Gapdh mRNA was used for quality and quantity of cDNA. The expected amplicons are indicated by arrowheads.
The Renilla luciferase mRNA was overexpressed in the heart of the mouse that received AAV9-‐TNNT2-‐RLuc and to a very low extent in its liver, validating efficient cardiotropic transduction with AAV9 (Figure 52; lower middle panel). Moreover, there was only low genomic contamination detectable in the samples, as shown in the -‐RT samples. In order to estimate the repaired Mybpc3 mRNA level as percentage of total Mybpc3 mRNA in PTM∆pA-‐
transduced heart semi-‐quantitative RT-‐PCR analysis was performed as described earlier (see 3.4.2.4). The final comparison was 5’-‐trans-‐ (repaired) versus cis-‐plus 5’-‐trans-‐splicing (total) and based on the intensity of the amplified bands (Figure 53; i.e. lane 4 of repaired Mybpc3 mRNA and lane 11 of total Mybpc3 mRNA). The level of repaired Mybpc3 mRNA reached up to 0.05% of total Mybpc3 mRNA. Thus, the amount of AAV9 encoding PTM∆pA was suitable to produce detectable levels of repaired Mybpc3 product in the heart.
Figure 53: Determination of the percentage of repaired Mybpc3 mRNA by semi-‐quantitative RT-‐PCR. Total RNA was extracted from heart of AAV9-‐TNNT2-‐PTM∆pA-‐transduced mouse and reverse transcribed into cDNA.
cDNA was used to amplify total (E1F/E9R primers) or only repaired Mybpc3 mRNA (FLAG/E9R) by PCR (25 cycles). PCR products were column-‐purified and serially diluted (1:3). In a second round of PCR, a common primer pair (E1F/E2R) was used to amplify a 242-‐bp fragment in all samples. The percentage of Mybpc3 mRNA that was repaired was estimated by comparing bands of similar intensities (black rectangles).
3.5.2.4 Characterization of 5’-‐trans-‐splicing at protein level
Despite hardly discernible levels of repaired Mybpc3 mRNA, Western blot analyses were performed using an antibody directed against the FLAG-‐tagged cMyBP-‐C protein. To detect repaired and endogenous cMyBP-‐C protein in parallel, protein analysis was performed using the anti-‐cMyBP-‐C antibody targeting the MyBP-‐C motif. As already observed in the ex vivo experiment the 150-‐kDa FLAG-‐tagged repaired cMyBP-‐C protein was undetectable by standard Western blot (Figure 54; upper panel). Even FLAG-‐immunoprecipitation (IP) followed by protein analysis did not enrich the repaired cMyBP-‐C protein in the PTM∆pA-‐
transduced cardiac sample (data not shown).
Figure 54: Western blot analysis after AAV9-‐mediated transduction of KI mice. Heart and liver were extracted 4 weeks after administration of AAV9-‐TNNT2-‐PTM∆pA, AAV9-‐TNNT2-‐RLuc or NaCl in 7-‐week-‐old mice and protein lysates were analyzed. The Western blot membrane was stained with antibody directed against the FLAG epitope. The expected protein molecular weights are indicated by arrowheads. As a positive control for the FLAG signal HEK293 cells transiently transfected (48 h) with pGG2-‐CMV-‐FLAG-‐WT-‐Mybpc3 plasmid (HEK293 Mybpc3) were used. Lamin A/C was used as a loading control. Abbreviations: MW, molecular weight; NT, non-‐
transduced.
Total cMyBP-‐C protein (150 kDa) was stained with a specific antibody only in ventricular tissue of the three mice and not in the livers (Figure 55; upper panel). The RLuc (36 kDa) was hugely overexpressed only in the heart of the corresponding mouse (Figure 55; middle panel). The expression of the transgene driven by TNNT2 promoter was restricted to the heart. The faint total cMyBP-‐C protein signal for the AAV9-‐TNNT2-‐Rluc-‐transduced mouse was due to the overexpression of RLuc in the corresponding myocardial tissue sample (see also decreased signal for GAPDH; Figure 55; lower panel), since the same amount of total protein is loaded on the gel.
Figure 55: Determination of the total full-‐length cMyBP-‐C and RLuc protein levels in vivo. Heart and liver were extracted 4 weeks after administration of AAV9-‐TNNT2-‐PTM∆pA, AAV9-‐TNNT2-‐RLuc or NaCl in 7-‐week-‐old mice and protein lysates were analyzed. Western blot membrane was stained with an antibody directed against MyBP-‐C motif and another directed against RLuc. The expected protein molecular weights are indicated by arrowheads. As a positive control for the RLuc signal HEK293 cells transiently transfected (48 h) with Rluc plasmid (HEK293 Rluc) were used. GAPDH was used as a loading control.