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BS-513-01 SUPPRESSION-REPLACEMENT GENE THERAPY FOR SCN5A-MEDIATED TYPE 3 LONG QT SYNDROME

      Background

      Congenital long QT syndrome (LQTS) is an autosomal dominant disorder characterized by delayed repolarization of the myocardium with a prolonged QT interval on electrocardiogram that may manifest as syncope, seizure, or sudden cardiac arrest/death. Long QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the SCN5A-encoded Nav1.5 sodium channel. No current therapies target the molecular cause of LQT3.

      Objective

      To develop an SCN5A suppression-replacement (SupRep) gene therapy to rescue the prolonged cardiac action potential duration (APD) in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a patient with LQT3.

      Methods

      Custom designed shRNAs targeting SCN5A were tested for knockdown efficiency using TSA201 cells and RT-qPCR. A dual-component “suppression-and-replacement” (SupRep) SCN5A gene therapy was created by cloning into a single construct a custom-designed SCN5A shRNA that produces ∼90% knockdown (suppression) and a “shRNA-immune” (shIMM) SCN5A cDNA (replacement). Patient-specific SCN5A-F1760C iPSC-CMs were generated form a patient with severe LQT3 (QTc > 680ms). FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90).

      Results

      Six unique shRNAs targeting SCN5A were tested, and one candidate shRNA was identified that suppressed the endogenous SCN5A alleles in TSA201 cells with about 92% knockdown efficiency. Compared to control iPSC-CMs, the baseline APD90 was significantly prolonged in SCN5A-F1760C iPSC-CM cells [680 ± 20 ms (n=30) vs 342 ± 16 ms (n=20), p<0.0001]. Following treatment with SCN5A-SupRep gene therapy, the APD90 was significantly decreased in F1760C iPSC-CMs compared to untreated cells [F1760C: 680 ± 20 ms (n=30) vs F1760C + SupRep: 470 ± 18 ms (n=39), p<0.0001]. This strategy demonstrated that the SCN5A-SupRep gene-therapy can rescue the pathologically prolonged APD in LQT3 patient-derived iPSC-CMs.

      Conclusion

      We provide the first proof-of-principle gene therapy for correction of LQT3. Akin to our sentinel discovery of SupRep gene therapy for LQT1, SCN5A-SupRep gene therapy successfully corrected/normalized the pathologic APD90, thereby eliminating the pathognomonic feature of LQT3.