Citations

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Pfizer Background Type 1 long QT syndrome (LQT1) is a genetic channelopathy characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs. Patients with LQT1 may manifest QT prolongation and ventricular arrhythmias that can culminate in sudden cardiac death. Purpose With this project, we aim to investigate whether our mutation independent, KCNQ1-specific suppression-replacement (SupRep) gene therapy can restore the deficient K+-channel and thereby restore the healthy phenotype. Methods Our proprietary dual component SupRep gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo AAV9-mediated KCNQ1SupRep gene therapy was performed by targeted intra-aortic root construct injection (1E12 vg/kg body weight) during balloon occlusion using a Swan Ganz catheter. 2 weeks after in vivo gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 (KCNQ1-Y315S, loss-of IKs) and wild-type (WT) rabbits to determine the effect of SupRep therapy on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes to evaluate the effect of SupRep on the cardiomyocyte’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) both, in vitro – after plasmid transfection, and in vivo – after AAV9-mediated gene therapy. AAV9-KCNQ1-construct expression was verified with immunohistochemistry targeting the GFP-tag. Results After injection of 3 LQT1 rabbits so far, no significant changes were observed in the LQT1 rabbits’ QTc before and after in vivo AAV9-mediated gene therapy. However, at the cellular level, LQT1 cardiomyocytes transfected in vitro with the SupRep-plasmid demonstrated a significant reduction of APD90 (ms, 1Hz stimulation at 37°) compared to LQT1 control cells (SupRep-in-vitro, 358±21 vs. control 447±22, p<0.05). No differences were observed between sham-plasmid transfected cardiomyocytes and LQT1 control cells after one day of culturing. Similarly, cardiomyocytes isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening of both APD90 and Ca2+90 compared to LQT1 controls, leading to levels similar to WT controls (APD90, 1Hz, 37°: LQT1: 530±18, WT: 445±18, LQT1-SupRep: 375±25, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT) (Ca2+90: LQT1: 487±23, WT: 346±16, LQT1-SupRep: 393±19, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT). Conclusion In vivo KCNQ1-suppression-replacement gene therapy normalizes cellular action potential and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the cardiomyocyte level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.

Background: Long-QT syndrome (LQTS) is characterized by QT prolongation and increased risk for syncope, seizures, and sudden cardiac death. The majority of LQTS stems from pathogenic mutations in KCNQ1 , KCNH2 , or SCN5A . However, ≈10% of patients with LQTS remain genetically elusive. We utilized genome sequencing to identify a novel LQTS genetic substrate in a multigenerational genotype-negative LQTS pedigree. Methods: Genome sequencing was performed on 5 affected family members. Only rare nonsynonymous variants present in all affected family members were considered. The candidate variant was characterized functionally in patient-derived induced pluripotent stem cell and gene-edited, variant corrected, isogenic control induced pluripotent stem cell–derived cardiomyocytes. Results: A missense variant (p.G6S) was identified in ALG10B -encoded α-1,2-glucosyltransferase B protein. ALG10B (alpha-1,2-glucosyltransferase B protein) is a known interacting protein of KCNH2 -encoded K v 11.1 (HERG [human Ether-à-go-go–related gene]). Compared with isogenic control, ALG10B-p.G6S induced pluripotent stem cell–derived cardiomyocytes showed (1) decreased protein expression of ALG10B (p.G6S, 0.7±0.18, n=8 versus control, 1.25±0.16, n=9; P <0.05), (2) significant retention of HERG in the endoplasmic reticulum ( P <0.0005), and (3) a significantly prolonged action potential duration confirmed by both patch clamp (p.G6S, 531.1±38.3 ms, n=15 versus control, 324.1±21.8 ms, n=13; P <0.001) and multielectrode assay ( P <0.0001). Lumacaftor—a compound known to rescue HERG trafficking—shortened the pathologically prolonged action potential duration of ALG10B-p.G6S induced pluripotent stem cell–derived cardiomyocytes by 10.6% (n=31 electrodes; P <0.001). Conclusions: Here, we demonstrate that ALG10B-p.G6S downregulates ALG10B, resulting in defective HERG trafficking and action potential duration prolongation. Therefore, ALG10B is a novel LQTS-susceptibility gene underlying the LQTS phenotype observed in a multigenerational pedigree. ALG10B mutation analysis may be warranted, especially in genotype-negative patients with an LQT2-like phenotype.

Background: KCNH2 -mediated arrhythmia syndromes are caused by loss-of-function (type 2 long QT syndrome [LQT2]) or gain-of-function (type 1 short QT syndrome [SQT1]) pathogenic variants in the KCNH2 -encoded K v 11.1 potassium channel, which is essential for the cardiac action potential. Methods: A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 short hairpin RNA with ~80% knockdown (suppression) and a “short hairpin RNA-immune” KCNH2 cDNA (replacement). Induced pluripotent stem cell-derived cardiomyocytes and their CRISPR-Cas9 variant-corrected isogenic control (IC) induced pluripotent stem cell-derived cardiomyocytes were made for 2 LQT2- (G604S, N633S) and 1 SQT1- (N588K) causative variants. All variant lines were treated with KCNH2-SupRep or non-targeting control short hairpin RNA (shCT). The action potential duration (APD) at 90% repolarization (APD 90 ) was measured using FluoVolt voltage dye. Results: KCNH2-SupRep achieved variant-independent rescue of both pathologic phenotypes. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD 90 to near curative (IC-like) APD 90 levels (G604S IC, 471±25 ms; N633S IC, 405±55 ms) compared with treatment with shCT (G604S: SupRep-treated, 452±76 ms versus shCT-treated, 550±41 ms; P <0.0001; N633S: SupRep-treated, 399±105 ms versus shCT-treated, 577±39 ms, P <0.0001). Conversely, for the SQT1-causative variant, N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD 90 (IC: 429±16 ms; SupRep-treated: 396±61 ms; shCT-treated: 274±12 ms). Conclusions: We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. KCNH2-SupRep gene therapy successfully normalized the pathologic APD 90 , thereby eliminating the pathognomonic feature of both LQT2 and SQT1.

Abstract Abstract 3110 High-dose therapy followed by autologous stem cell transplantation (ASCT) is standard therapy for patients with relapsed or refractory Hodgkin Lymphoma (HL), although the optimal conditioning regimen remains uncertain. The three drug alkylating agent regimen, BuMelTt, has been established as our institutional regimen. We performed a retrospective review of outcomes obtained with BuMelTt compared to those achieved with other standard conditioning regimens. 133 patients with relapsed/refractory HL who underwent ASCT between January 1990 and December 2009 were analyzed. 62 patients received BuMelTt and 71 patients received other standard preparative regimens (Standard) consisting of CBV (44), CyVp16TBI (20), BEAM (4), CyTBI (2), and Mel (1). The median follow-up (range) was 4.3 years (0.01–14.24) in BuMelTt and 3.9 years (0.07–20.21) in Standard. The two groups (BuMelTt vs Standard) were balanced for gender (65% male vs 65% male), median age (29 vs 33), median number of prior regimens (2 vs 2), and pre-transplant disease status. Disease status was categorized as PR2/CR2 (48% vs 42%), primary induction failure sensitive (16% vs 17%), and Other which included PIF-refractory, 1st relapse refractory, and greater than 1st relapse (35% vs 41%). The 5 year overall survival (OS) was superior for patient who received BuMelTt conditioning (74% vs 54%, p = 0.03). There was also a trend for improved 5 year event free survival (EFS) for BuMelTt at 56% vs 39% for Standard (p=0.07). Other risk factors for outcome after ASCT were also analyzed and included disease status, more than 2 chemotherapy regimens before ASCT, Karnofsky score < 90% at ASCT, time from diagnosis to ASCT < 12 months, prior extranodal involvement, and age > 40 years. By univariate analysis, >2 chemotherapy regimens before ASCT (HR 1.7, p=0.05) and disease status of Other (HR 2.8, p<0.01) were predictive for lower OS. There was a trend for influence on OS for BuMelTt (HR 0.6, p=0.07) and PIF-sensitive (HR 1.9, p=0.09). By multivariate analysis, disease status of PIF-sensitive or Other predicted worse outcomes for OS (HR 2.4 & 2.9, p 0.03 & <0.01, respectively), and there was still a trend for benefit with BuMelTt (HR 0.6, p=0.09), although >2 chemotherapy regimens lost significance (p=0.22). For EFS, disease status of PIF-sensitive or Other were prognostic in univariate analysis (HR 2.1 or 2.3, p=0.02 or <0.01, respectively), and there was a trend for improved EFS with BuMelTt (HR 0.66, p=0.08). In multivariate analysis, disease status of PIF-sensitive or Other remained prognostic (p=0.03 & 0.004, respectively), while BuMelTt was not significant (p=0.27). There was no significant difference in 100 day non-relapse related mortality for BuMelTt vs Standard conditioning (5% vs 3%, p=0.88). More patients developed NCI CTCAE grade 3/4 mucositis with BuMelTt than Standard (89% vs 61%, p<0.01), but fewer patients had bacteremia with BuMelTt than Standard (7% vs 23%, p=0.02). There was no difference in duration of hospitalization, episodes of febrile neutropenia, incidence of veno-occlusive disease, time to neutrophil engraftment, or time to platelet engraftment. Our results suggest that BuMelTt may improve OS and EFS compared to standard conditioning regimens with increased but manageable toxicity. A case matched control study is planned. Disclosures: No relevant conflicts of interest to declare.