Sendai Virus Research Articles

Rejuvenation of human mesenchymal stem cells using a nonintegrative and conditionally removable Sendai virus vector

Human mesenchymal stem cells (hMSCs) with extended lifespan and differentiation potential that can recapitulate in vivo characteristics could significantly contribute to basic research, drug development, and cell therapy. Specifically, they could ensure a stable supply of specific cellular resources, and possibly extracellular vesicles. Here, we established a technology for extending the lifespan while maintaining differentiation potential, termed “rejuvenation,” of hMSCs (rej-hMSCs) using nonintegrative and conditionally removable temperature-sensitive Sendai virus (SeV) vectors. Various immortalizing factors (i.e., Bmi-1, hTERT, SV40T, and/or HPV E6/E7) were first introduced by the SeV vector into the cells. A combination of three SeVs with Bmi-1, hTERT, or SV40T conferred markedly improved cell proliferation and cloning ability while maintaining differentiation potential and a normal karyotype. An extended lifespan was also demonstrated in other cell types. The rejuvenation of long-passaged or aged hMSCs was also confirmed. SeV vectors were rapidly removed as a function of cell doubling by increasing the temperature from 35 °C to 37 °C or higher, while proliferative ability was maintained. Following FACS sorting, the complete removal of SeV vectors was confirmed by qPCR analyses. Therefore, our cell rejuvenation technology could contribute to research and clinical applications by enabling the supply of modified cells without damaging host chromosomes.

Dose-escalation, tolerability, and efficacy of intratumoral and subcutaneous injection of hemagglutinating virus of Japan envelope (HVJ-E) against chemotherapy-resistant malignant pleural mesothelioma: a clinical trial

The hemagglutinating virus of Japan envelope (HVJ-E) is an inactivated Sendai virus particle with antitumor effect and inducing antitumor immunity. However, its dosage and efficacy have not been verified. We conducted a phase I clinical study on chemotherapy-resistant malignant pleural mesothelioma (MPM) aiming to determine the recommended dosage for a phase II study through dose-limiting toxicity and evaluate HVJ-E’s preliminary efficacy. HVJ-E was administered intratumorally and subcutaneously to the patients with chemotherapy-resistant MPM. While no serious adverse events occurred, known adverse events of HVJ-E were observed. In the preliminary antitumor efficacy using modified response evaluation criteria in solid tumors (RECIST) criteria, three low-dose patients exhibited progressive disease, while all high-dose patients achieved stable disease, yielding disease control rates (DCRs) of 0% and 100%, respectively. Furthermore, the dose-dependent effect of HVJ-E revealed on DCR modified by RECIST and the baseline changes in target lesion size (by CT and SUL-peak; p < 0.05). Comparing targeted lesions receiving intratumoral HVJ-E with non-injected ones, while no clear difference existed at the end of the study, follow-up cases suggested stronger antitumor effects with intratumoral administration. Our findings suggest that HVJ-E could be safely administered to patients with chemotherapy-resistant MPM at both study doses. HVJ-E exhibited some antitumor activity against chemotherapy-resistant MPM, and higher doses tended to have stronger antitumor effects than lower doses. Consequently, a phase II clinical trial with higher HVJ-E doses has been conducted for MPM treatment. Trial registration number: UMIN Clinical Trials Registry (#UMIN000019345).

Establishment of a human induced pluripotent stem cell (iPSC) line (JUCTCi018-A) from a patient with Charcot-Marie-Tooth disease type 2EE (CMT2EE) due to a homozygous c.122G > A p.(Arg41Gln) mutation in the MPV17 gene

(Charcot-Marie-Tooth disease (CMT) is a genetic disorder affecting peripheral nerves. The human induced pluripotent stem cell (iPSC) line JUCTCi018-A was created using dermal fibroblasts from a Charcot-Marie-Tooth disease type 2EE (CMT2EE) patient with a homozygous missense mutation in the MPV17 gene (c. 122G > A, p.Arg41Gln). These fibroblasts were reprogrammed using Sendai viruses that encoded OCT4, SOX2, KLF4, and c-MYC reprogramming factors. The iPSCs demonstrated normal morphology and karyotype, expressed pluripotency markers, and the ability to differentiate into the three germ layers. This iPSC line is valuable for investigating the mechanisms underlying CMT2EE.

Generation of a control induced pluripotent stem cell line (CBRCULi014-A) derived from the lymphoblastoid cells of a pediatric individual

Lymphoblastoid cell lines serve as a readily and continuous resource for generating induced pluripotent stem cells (iPSCs), enabling the modeling of various genetic disorders in vitro. When investigating congenital and infantile diseases, age-match controls derived from pediatric individuals are typically necessary, yet they may be scarce or difficult to obtain. Here, the Sendai virus system was employed to introduce reprogramming factors into lymphoblastoid cells derived from an apparently healthy 4-year-old female. The generated iPSCs strongly expressed pluripotency cell markers and displayed robust trilineage differentiation. CBRCULi014-A is therefore a reliable control iPSC line for pediatric disease investigation.

Zinc-finger CCHC-type containing protein 8 promotes RNA virus replication by suppressing the type-I interferon responses.

Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective.IMPORTANCEThe innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses.

Varicella-zoster virus recapitulates its immune evasive behaviour in matured hiPSC-derived neurospheroids.

Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With Type-I interferon (IFN) signalling being an important first line cellular defence mechanism against VZV infection by the peripheral tissues, we here investigated the triggering of innate immune responses in a human neural-like environment. For this, we established and characterised 5-month matured hiPSC-derived neurospheroids (NSPHs) containing neurons and astrocytes. Subsequently, NSPHs were infected with reporter strains of VZV (VZVeGFP-ORF23) or Sendai virus (SeVeGFP), with the latter serving as an immune-activating positive control. Live cell and immunocytochemical analyses demonstrated VZVeGFP-ORF23 infection throughout the NSPHs, while SeVeGFP infection was limited to the outer NSPH border. Next, NanoString digital transcriptomics revealed that SeVeGFP-infected NSPHs activated a clear Type-I IFN response, while this was not the case in VZVeGFP-ORF23-infected NSPHs. Moreover, the latter displayed a strong suppression of genes related to IFN signalling and antigen presentation, as further demonstrated by suppression of IL-6 and CXCL10 production, failure to upregulate Type-I IFN activated anti-viral proteins (Mx1, IFIT2 and ISG15), as well as reduced expression of CD74, a key-protein in the MHC class II antigen presentation pathway. Finally, even though VZVeGFP-ORF23-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules upon long-term infection, as well as disruption of cellular integrity within the infected NSPHs. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, and have the capacity to recapitulate the strong immune evasive behaviour towards VZV.

The E3 ligase ASB3 downregulates antiviral innate immunity by targeting MAVS for ubiquitin-proteasomal degradation.

E3 ubiquitin ligases are very important for regulating antiviral immunity during viral infection. Here, we discovered that Ankyrin repeat and SOCS box-containing protein 3 (ASB3), an E3 ligase, are upregulated in the presence of RNA viruses, particularly influenza A virus (IAV). Notably, overexpression of ASB3 inhibits type I IFN (IFN-I) responses induced by Sendai virus (SeV) and IAV, and ablation of ASB3 restores SeV and H9N2 infection-mediated transcription of IFN-β and its downstream interferon-stimulated genes (ISGs). Interestingly, animals lacking ASB3 presented decreased susceptibility to H9N2 and H1N1 infections. Mechanistically, ASB3 interacts with MAVS and directly mediates K48-linked polyubiquitination and degradation of MAVS at K297, thereby inhibiting the phosphorylation of TBK1 and IRF3 and downregulating downstream antiviral signaling. These findings establish ASB3 as a critical negative regulator that controls the activation of antiviral signaling and describe a novel function of ASB3 that has not been previously reported.

Generation and genetic repair of two human induced pluripotent stem cell lines from patients with Epidermolysis Bullosa simplex associated with a heterozygous mutation in the translation initiation codon of KLHL24

Fibroblasts from two patients carrying a distinct heterozygous mutation in the KLHL24 gene (c.1 A>G and c.2T>C) were reprogrammed to obtain hiPSC lines. Non-integrating Sendai virus and CRISPR-Cas9 editing were respectively used to deliver the reprogramming factors and repair the mutation in the patient-hiPSCs to obtain isogenic control pairs. No off-target nuclease activity was detected with the top-predicted sites. Patient and isogenic hiPSCs displayed typical morphology, expressed markers of the undifferentiated state, were able to differentiate into the three germ layers and had normal karyotypes. These isogenic pairs will expand the panel of hiPSC lines to model KLHL24-associated conditions.

Sendai virus-mediated RNA delivery restores fertility to congenital and chemotherapy-induced infertile female mice.

Current infertility treatment strategies focus on mature gametes, leaving a significant proportion of cases with gamete progenitors that stopped complete differentiation. On the other hand, recent advancements in next-generation sequencing have identified many candidate genes that may promote maturation of germ cells. Although gene therapy has shown success in mice, concerns about the integration of DNA vectors into oocytes hinder clinical applications. Here, we present the restoration of fertility in female mice through Sendai virus (SeV)-mediated RNA delivery. Ovaries lacking Kitl expression exhibit only primordial follicles due to impaired signaling to oocytes expressing the KIT tyrosine kinase. Despite SeVs being immunogenic and larger than the blood-follicle barrier, the administration of Kitl-expressing SeVs reinitiated oogenesis in genetically infertile mice that have only primordial follicles, resulting in the birth of normal offspring through natural mating. This virus also effectively addressed iatrogenic infertility induced by busulfan, a widely used cancer chemotherapy agent. Offspring born through SeV administration and natural mating displayed normal genomic imprinting patterns and fertility. Since SeVs pose no genotoxicity risk, the successful restoration of fertility by SeVs represents a promising approach for treating congenital infertility with somatic cell defects and protecting fertility of cancer patients who may become infertile due to loss of oocytes during cancer therapy.

Generation of induced pluripotent stem cells from ADHD patients who do or do not respond to Methylphenidate treatment

As a neurodevelopmental multifactorial disorder whose prevalence has been increasing worldwide, attention-deficit hyperactivity disorder (ADHD) is considered a public health concern. Methylphenidate (MPH) is the drug of choice for ADHD; however, not all patients respond fully to this treatment. Therefore, exploring the underlying molecular mechanisms involved in ADHD and potential novel therapeutic targets is crucial. Here, we generated induced pluripotent stem cells (iPSCs) from Peripheral Blood Mononuclear Cells (PBMCs) retrieved from four ADHD patients (two MPH responders and two non-responders) using Sendai virus. These lines might be helpful for the in vitro investigation of ADHD pathophysiology in a patient-specific manner.

Diminished GALNS activity in induced pluripotent stem cells of mucopolysaccharidosis IVA caused by compound p. S162Y and p. C165F mutation.

Mucopolysaccharidosis (MPS) IVA is a lysosomal storage disorder caused by mutations in the gene encoding the galactosamine (N-acetyl)-6-sulfatase (GALNS) enzyme. Children with MPS IVA usually develop pectus carinatum, genu valgum, and multiple skeletal abnormalities. To establish a patient-derived induced pluripotent stem cell (iPSC) disease model to investigate the effects of two GALNS missense mutations. The medical history and clinical manifestations of a patient with MPS IVA were first inspected. The effects of the identified GALNS mutations were predicted through bioinformatic analysis. iPSCs were then generated by using Sendai virus to introduce Yamanaka reprogramming factors to urinary cells isolated from the patient. The pluripotency, karyotypic integrity, genetic mutations, and differentiation ability of the iPSCs were tested. The effects of the GALNS mutations were further experimentally characterized using patient-derived cells. The patient exhibited a typical MPS IVA phenotype. Enzyme replacement therapy could not correct her skeletal abnormalities. GALNS c.485C>A (p.S162Y) and c.494G>T (p.C165F) mutations, inherited from her father and mother respectively, were identified in the patient. These two mutations were predicted to disturb the hydrophobic core of the GALNS catalytic domain. Patient-derived iPSCs were successfully generated, and further characterization indicated that the two missense mutations significantly diminished GALNS activity without affecting its amount at both the RNA and protein levels. We established a novel clinically relevant MPS IVA disease model that will be useful not only for investigating the pathogenic mechanisms of MPS IVA variants but also for drug screening and preclinical evaluation of novel therapies.

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases.

Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose‑d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

The generation of human induced pluripotent stem cell lines from individuals of Black African ancestry in South Africa

The lack of equitable representation of African diversity in scientific resources, such as genome-wide association studies and human induced pluripotent stem cell (hiPSC) repositories, has perpetuated inequalities in the advancement of health research. HiPSCs could be transformative in regenerative and precision medicine, therefore, the generation of diverse lines is critical in the establishment of African-relevant preclinical cellular models. HiPSC lines were derived from two healthy donors of Black African ancestry using Sendai virus reprogramming of dermal fibroblasts, and characterised to confirm stemness markers, trilineage differentiation, and genetic integrity. These hiPSCs represent a valuable resource for modelling African relevant disease biology.

Generation of an induced pluripotent stem cell line (NCHi016-A) from a 5-year-old female with pulmonary atresia with intact ventricular septum and one-and-half ventricle palliation

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare congenital heart defect characterized by underdeveloped pulmonary valve and right ventricular hypoplasia. Neonates undergoing surgery to open pulmonary valve have a range of post-surgical ventricular recovery: single-ventricle (1v) palliation, one-and-half ventricle (1.5v) palliation, and bi-ventricular (2v) repair. PA-IVS-1.5v typically requires surgical intervention to install cavopulmonary shunt and entails partial right ventricle recovery. NCHi016-A is an iPSC line derived from a 5-year-old female with PA-IVS-1.5v using Sendai Virus reprogramming. This iPSC line shows typical iPSC morphology, has normal karyotype, expresses pluripotency markers, and has potential to differentiate into three germ layers.

Identification of a novel caspase cleavage motif AEAD

Infections of many viruses induce caspase activation to regulate multiple cellular pathways, including programmed cell death, immune signaling and etc. Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation. Here, we identified and analyzed a novel caspase cleavage motif AEAD, and confirmed its caspase dependent cleavage activity in natural substrate, such as nitric oxide-associated protein 1 (NOA1). Fusing the enhanced green fluorescent protein (EGFP) with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria. Upon the activation of caspase triggered by Sendai virus (SeV) or herpes simplex virus type 1 (HSV-1) infection, EGFP diffusely localized to the cell due to the caspase-mediated cleavage, thus allowing visual detection of the virus-induced caspase activation. An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed, which significantly inhibited the activities of caspases-1, -3, -6, -7, -8 and -9, exhibiting a broad caspase inhibition effect. The inhibitor further prevented caspases-mediated cleavage of downstream substrates, including BID, PARP1, LMNA, pro-IL-1β, pro-IL-18, GSDMD and GSDME, protecting cells from virus-induced apoptotic and pyroptotic cell death. Together, our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.

Generation of an induced pluripotent stem cell line (BIORTCi001-A) from a healthy adult indigenous Nigerian participant

Genetic backgrounds influence cellular phenotypes, drug responses, and health outcomes, yet most human iPSC lines are derived from individuals of European descent, with lines from indigenous Africans particularly scarce. Addressing this gap, we generated iPSCs from dermal fibroblasts of a healthy 60-year-old indigenous Nigerian male of the Babur ethnic group using Sendai virus. The iPSC line displayed a normal karyotype, was characterized for pluripotency markers and differentiated into neural progenitor cells and astrocytes. To enhance African representation in research, this iPSC line will be available to the scientific community, with ongoing efforts focused on creating an open-access African iPSC biobank.Resource Table:

Immunogenicity and Protective Efficacy of a Single Intranasal Dose Vectored Vaccine Based on Sendai Virus (Moscow Strain) against SARS-CoV-2 Variant of Concern.

The mouse paramyxovirus Sendai, which is capable of limited replication in human bronchial epithelial cells without causing disease, is well suited for the development of vector-based intranasal vaccines against respiratory infections, including SARS-CoV-2. Using the Moscow strain of the Sendai virus, we developed a vaccine construct, Sen-Sdelta(M), which expresses the full-length spike (S) protein of the SARS-CoV-2 delta variant. A single intranasal delivery of Sen-Sdelta(M) to Syrian hamsters and BALB/c mice induced high titers of virus-neutralizing antibodies specific to the SARS-CoV-2 delta variant. A significant T-cell response, as determined by IFN-γ ELISpot and ICS methods, was also demonstrated in the mouse model. Mice and hamsters vaccinated with Sen-Sdelta(M) were well protected against SARS-CoV-2 challenge. The viral load in the lungs and nasal turbinates, measured by RT-qPCR and TCID50 assay, decreased dramatically in vaccinated groups. The most prominent effect was revealed in a highly sensitive hamster model, where no tissue samples contained detectable levels of infectious SARS-CoV-2. These results indicate that Sen-Sdelta(M) is a promising candidate as a single-dose intranasal vaccine against SARS-CoV-2, including variants of concern.

Hunnivirus structural protein VP2 inhibits beta interferon production by targeting the IRF3 essential modulator

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-β and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-β promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-β promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNβ induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.

Nonstructural Protein A238L of the African Swine Fever Virus (ASFV) Enhances Antiviral Immune Responses by Activating the TBK1-IRF3 Pathway.

African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation.

Modelling of a RYR2-associated left ventricular non-compaction and sudden cardiac death overlap syndrome in hiPSC-EHTs

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ESC training grant; DZHK Background Genetic cardiomyopathies contribute to a significant proportion of morbidity and mortality among young people. Over the past decades, a wide range of underlying molecular mechanisms has been discovered. The current project arose from the clinical observation of a left ventricular non-compaction and sudden cardiac death overlap syndrome in a large family in northern Italy. Whole exome sequencing performed on family members of different generations revealed a single nucleotide polymorphism in the RYR2 gene (RYR2 c.5654G&amp;gt;A homozygous – RyR2 p.G1885E - exon37:missense) with an autosomal-recessive inheritance pattern. The mutation is localized in the DR3 region of the RyR2 protein, adjacent to the FKBP12.6 binding site, a protein that is known to stabilize RyR2, preventing aberrant activation of the channel. Methods and Results Patient-derived, human induced pluripotent stem cells (hiPSCs), carrying the variant homo- and heterozygously, were generated by reprogramming patient-derived peripheral blood mononuclear cells using a non-integrative Sendai virus protocol. The lines were fully characterized and expressed high levels of pluripotency markers at the RNA and protein level. Patient lines were all able to efficiently differentiate into cardiomyocytes. Subsequently, 3D strip-format, force-generating Engineered Heart Tissues (EHTs) were cast and structurally and functionally characterized. Contraction analysis over 60 days showed differences in force development of the patient-derived lines. In particular, the EHTs carrying the homozygous pathogenic variant showed progressive reduction in both beating rate and force over time, increased spontaneous arrhythmogenicity and reduced inotropic response after exposure to isoprenaline, compared to the heterozygous line and unrelated healthy controls. Starting from day 30 after casting, patient-derived EHTs developed morphological changes with areas of marked cellular overgrowth and reduced troponin expression. Transcriptomic and proteomic analysis at 30 and 60 days after EHTs generation, as well as in 2D cardiomyocytes, complement the phenotyping and provide molecular insights into the underlying pathophysiology. To directly evaluate pathogenicity of the genetic variant identified, we generated isogenic homo- and heterozygously corrected hiPSCs, using CRISPR/ Cas9 technology, starting from the homozygous patient-line. The characterization of these corrected clones is currently ongoing. The genetically corrected 3D tissue models will be critical not only in defining the role of the novel RyR2 variant in the familial phenotype described, but also to unveil mechanistic roles of ryanodine receptor in cardiac disease. Conclusion We describe a novel recessive RYR2 variant associated with a left ventricular non- compaction and sudden cardiac death overlap syndrome. 3D cardiac tissue modelling based on patient-derived hiPSCs uncovered a proarrhythmic and structural phenotype of the mutated lines.