Evaluation of MYBPC3 trans -Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes

Figure 1. Phenotypic Characterization of HCM hiPSC-Derived Cardiomyocytes Patient-specific (HCM) and control (Ctrl) hiPSC-derived cardiomyocytes (hiPSC-CMs) were seeded in a confluent monolayer, cultured for 7 days, and prepared for subsequent analysis. (A) Representative immunofluorescence images of HCM and control hiPSC-CMs. Both cell lines were stained with antibodies directed against cMyBP-C (red), α-actinin (green), and with Hoechst33342 for nuclear staining (blue; scale bars, 50 μm). Higher magnification images of a few sarcomeres are shown on the right panels. (B) Quantification of CM cell size. Control and HCM hiPSC-CMs were seeded in 96-well plates at a density of 10,000 cells/well, cultured for 7 days, and stained with an antibody against α-actinin. Cell size was measured in 27–51 fields/well in both groups. Control hiPSC-CMs were evaluated from 33 wells of three independent experiments and HCM hiPSC-CMs from 21 wells of two independent experiments, for a total of 28,336 and 12,874 CMs, respectively. Images were taken with the Opera High-Content Screening System (PerkinElmer), and the analyses were performed with the Columbus Image Data Management and Analysis System. (C)…

Figure 2. 5′ trans-Splicing in Control hiPSC-Derived Cardiomyocytes (A) Schematic representation of 5′ trans-splicing. 5′ pre-trans-splicing molecules (5′ PTMs) carry a 5′ FLAG-tagged wild-type (WT) MYBPC3 cDNA sequence from exon 1 to exon 21, conserved 5′ splice donor site (5′SS) sequences, and a binding domain (BD) targeting intron 21. Upon successful binding of 5′ PTM to the endogenous MYBPC3 pre-mRNA, 5′ trans-splicing can occur and results in a trans-spliced MYBPC3 mRNA. (B) Schematic illustration of primer pairs used to amplify either only trans-spliced or trans-spliced and/or endogenous (total) MYBPC3 mRNAs. The primer pair FLAG-F/E23-R was used to generate a 2,180-bp fragment corresponding to the trans-spliced MYBPC3 mRNA. trans-spliced and/or endogenous MYBPC3 mRNA was amplified either using the primer pair E1-F/E23-R (2,138-bp fragment) or the primer pair E15-F/E16-R (193-bp fragment). (C) hiPSC-derived cardiomyocytes (hiPSC-CMs) were transduced with AAV-5′ PTM or AAV-Mock (TNNT2 promoter without insert) at an MOI of 10,000 and cultured in 2D for 7 days prior to harvesting. Representative agarose gel of RT-PCR, using specific primer pairs for trans-spliced and total MYBPC3 mRNA,…

Figure 3. 3′ trans-Splicing in Control hiPSC-Derived Cardiomyocytes (A) Schematic illustration of 3′ trans-splicing. 3′ pre-trans-splicing molecules (3′ PTMs) contain a BD complementary to MYBPC3 intron 21, conserved 3′ splice donor site sequences (3′SS), and a 3′-FLAG-tagged wild-type (WT) MYBPC3 cDNA sequence from exon 22 to exon 34. Upon successful binding of the 3′ PTM to the endogenous MYBPC3 pre-mRNA, 3′ trans-splicing can occur and results in trans-spliced MYBPC3 mRNA. (B) Schematic representation of primer pairs used to amplify either only trans-spliced MYBPC3 mRNA or trans-spliced and/or endogenous (total) MYBPC3 mRNA. The primer pair E21-F/FLAG-R amplifies a 1,874-bp fragment specific of trans-spliced MYBPC3 mRNA. The primer pairs E21-F/E33-R and E33-F/E33-R amplify in both trans-spliced and endogenous MYBPC3 mRNA a 1,812-bp and a 157-bp fragment, respectively. (C) Control hiPSC-CMs were transduced with AAV-3′ PTM or AAV-Mock (MOI 10,000) for 1 week prior to harvesting. Representative agarose gel of RT-PCR, using specific primer pairs for trans-spliced and total MYBPC3 mRNA, is shown (n = 9 from four independent transductions). (D) Determination of the percentage of trans-spliced…

Figure 4. Proof-of-Concept of MYBPC3 Gene Replacement in Human iPSC-Derived Cardiomyocytes (A) Schematic illustration of full-length wild-type (WT) exogenous and total MYBPC3 cDNA/mRNA sequence (exons 1–34). Location of primer pairs and size of amplicons are shown. The primer pair FLAG-F/E2-R amplifies exclusively the exogenous MYBPC3 sequence (amplicon size: 193 bp). The primer pair E1-F/E2-R is suitable for amplification of both exogenous and endogenous (total) MYBPC3 sequence (amplicon size: 151 bp). (B) Control and HCM hiPSC-CMs were transduced with AAV-FLAG-MYBPC3 (GR) or AAV-Mock (Mock) at an MOI of 10,000 and cultured in 2D for 7 days before harvesting. Representative RT-PCR using primers shown in (A) for specific amplification is shown (n = 3, one well each from three independent transductions). (C) Evaluation of MYBPC3 transcript levels in GR-transduced control and HCM hiPSC-CMs by qRT-PCR with a common primer pair (n = 3–5, with n = number of wells of one transduction experiment). (D) Determination of the percentage of exogenous MYBPC3 mRNA by qRT-PCR. In a first round of RT-PCR, either exogenous (FLAG-F/E2-R primers) or total (E1-F/E2-R primers) MYBPC3 transcripts were amplified…

Figure 5. Phenotypic Characterization of Control and HCM hiPSC-Derived Cardiomyocytes after MYBPC3 Gene Transfer (A) Representative images of non-transduced (NT) and transduced (GR) control (Ctrl) and HCM hiPSC-CMs after immunofluorescence staining with anti-FLAG and anti-α-actinin antibodies. Images were taken with a Zeiss LSM 800 microscope (scale bars, 20 μm). Higher magnification images of a few sarcomeres are shown on the right panel. (B) Quantification of CM cell size. NT and GR control and HCM hiPSC-CMs were seeded in 96-well plates at a density of 10,000 cells/well, cultured for 1 week, and stained with antibodies against FLAG-tag and α-actinin. Low-resolution images of >100 cells in each group from six wells of one experiment each (Ctrl NT n = 108; Ctrl GR n = 110; HCM NT n = 114; HCM GR n = 133) were taken with the Zeiss LSM 800 microscope and analyzed with Fiji software (ImageJ). Data are expressed as mean ± SEM (***p < 0.001, two-way ANOVA plus Bonferroni post-test; ###p < 0.001, unpaired Student’s t test).

Figure 6. Gene Expression Analysis of HCM hiPSC-Derived Cardiomyocytes in the Absence of or after MYBPC3 Gene Transfer The mRNA levels of transduced (GR) and non-transduced (NT) HCM hiPSC-CMs were determined with the nanoString nCounter Elements technology, normalized to housekeeping genes, and related to NT CMs. Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001, unpaired Student’s t test; n = 3, with n = number of wells from one transduction experiment).