Non-integrating Sendai Virus Research Articles

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.

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>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.

Generation of human induced pluripotent stem cell line (XWHNi004-A) from a male with APOE gene mutation

Genetic polymorphism of apolipoprotein E (APOE) confers differential susceptibility to Alzheimer's disease (AD), and APOE ɛ4 variants is the most powerful risk factor for this disease. Here, we report the generation of a human induced pluripotent stem cell (iPSC) line carrying the APOE ɛ4/ɛ4 genotype from peripheral blood mononuclear cells (PBMCs) isolated from a male with a family history of AD utilizing non-integrative Sendai virus vector. The iPSC maintains their original genotype, highly express endogenous pluripotency markers, displays a normal karyotype, and retains the ability to differentiate into cells representative of the three germ layers.

Generation of an induced pluripotent stem cell line (KNIHi001-A) by reprogramming peripheral blood mononuclear cells isolated from a patient with Parkinson's disease

Parkinson's disease is a degenerative brain disorder characterized by dopamine neuronal degeneration and dopamine transporter loss. In this study, we generated an induced pluripotent stem cell (iPSC) line, KNIHi001-A, from the peripheral blood mononuclear cells (PBMCs) of a 76-year-old man with Parkinson's disease. The non-integrating Sendai virus was used to reprogram iPSCs. iPSCs exhibit pluripotent markers, a normal karyotype, viral clearance, and the ability to differentiate into the three germ layers.

Safety of GMP-compliant iPSC lines generated by Sendai virus transduction is dependent upon clone identity and sex of the donor.

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.

Characterization of two human induced pluripotent stem cell lines derived from Batten disease patient fibroblasts harbouring CLN5 mutations

The neuronal ceroid lipofuscinoses (NCLs) are a group of common inherited neurodegenerative disorders of childhood. All forms of NCLs are life-limiting with no curative treatments. Most of the 13 NCL genes encode proteins residing in endolysosomal pathways, such as CLN5, a potential lysosomal enzyme. Two induced pluripotent stem cell lines (hiPSCs) were generated from skin fibroblasts of CLN5 disease patients via non-integrating Sendai virus reprogramming. They demonstrate typical stem cell morphology, express pluripotency markers, exhibit trilineage differentiation potential and also successfully differentiate into neurons. These hiPSCs represent a potential resource to model CLN5 disease in a human context and investigate potential therapies.

Generation of a human induced pluripotent stem cell line (OGIi001) from peripheral blood mononuclear cells of a healthy male donor

We have successfully derived a novel human induced pluripotent stem cell (hiPSC) line using non-integrative Sendai virus. This hiPSC line was generated from a healthy male adult donor, aged 55, and subjected to thorough characterization and extensive quality control. The analysis confirmed the expression of undifferentiated stem cell markers, demonstrated the ability to differentiate into the three germ layers, and revealed the absence of any chromosomal abnormalities.

Generation of a rhesus macaque induced pluripotent stem cell line (riPSC05) under feeder-free conditions

We generated and characterized a rhesus macaque induced pluripotent stem cell (iPSC) line using induced reprogramming of fibroblasts isolated from a rhesus macaque fetus. The fibroblasts were expanded and then reprogrammed using non-integrating Sendai virus technology. This line is available as riPSC05. The authenticity of riPSC05 was confirmed through the expression of pluripotent and self-renewal markers, in vitro-directed differentiation towards three germ layers (ectoderm, mesoderm, and endoderm), karyotyping, and STR analysis.

Generation of the human iPSC lines AKOSi011-A carrying the mutation p.Pro65Ser/p.Asp35T and AKOSi012-A, carrying the mutation p.Tyr231His, derived from FAHN patient fibroblasts

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a hereditary neurodegenerative disease caused by mutations in the FA2H gene. Patients show a wide range of neurological symptoms and an abnormal myelination. Here we describe the generation of the human induced pluripotent stem cell (hiPSC) lines AKOSi011-A and AKOSi012-A, derived from FAHN-patient fibroblasts, carrying the compound heterozygous mutation p.Pro65Ser/p.Asp35Tyr and the homozygous mutation p.Tyr231His, respectively. The hiPSC lines were generated using a non-integrating Sendai virus. The obtained hiPSCs show an unobtrusive karyotype, carry the mutations of the original fibroblasts, express pluripotency markers and can differentiate into cells of the three germ layers.

Abstract P1033: Derivation Of Large Biorepository Of Human Ipsc Lines For Open Access Sharing Among Academic Investigators

Background: Human induced pluripotent stem cells (iPSCs) and their ability to differentiate into numerous cell types have revolutionized biomedical and translational research in many fields such as cardiovascular disease, development, aging, regeneration, cancer, personalized medicine, and many rare diseases. The need for obtaining iPSC lines with the associated donors’ clinical data and from patients of diverse genetic backgrounds has been greater than ever. Greenstone Biosciences Inc. (GSB) has built a curated biorepository to address this demand as an asset for the scientific community utilizing iPSCs. Methods: The GSB biobank curates clinical data, isolates peripheral blood mononuclear cells (PBMCs), reprograms and banks patient-specific iPSC lines, and performs quality assurance. Reprogramming of PBMCs to iPSCs is carried out using non-integrating Sendai virus encoding for Oct4, Sox2, Klf4, and c-Myc. We then rigorously validate all iPSCs for pluripotency using immunofluorescence of stemness markers, RT-PCR, and SNP-based virtual karyotyping along with quality control methods established as a standard protocol. Results: We have a growing repository of >1,000 iPSC lines with various disease backgrounds across demographics such as age, sex, race, and ethnicity. The diseases with genetic mutations include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), long QT syndrome (LQTS), arrhythmogenic right ventricular dysplasia / cardiomyopathy (ARVD/C), Brugada syndrome (BrS), Duchenne’s muscular dystrophy (DMD), Spinal muscular atrophy (SMA), and many other rare diseases and syndromes. We have also established a simple material transfer agreement (MTA) for open access by academic investigators. Conclusions: We introduce the Greenstone Biosciences iPSC Biobank, a biorepository containing >1,000 human iPSC lines with de-identified patient clinical information and genetic backgrounds. This invaluable resource will help support iPSC research among academic investigators.

Generation of induced pluripotent stem cell line (XWHNi002-A) from a female with APP gene mutation

The β-amyloid precursor protein (APP) is a crucial pathogenic gene linked to Alzheimer's disease (AD). A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) isolated from a female with APP gene mutation utilizing non-integrative Sendai virus. The iPSC line exhibits high expression of pluripotency markers, retains the APP mutation, displays a normal karyotype, and has the ability to differentiate into normal teratoma tissue. This iPSC line represents a valuable cell model for investigating the pathological mechanisms and therapeutic strategies of AD.

Generation and characterisation of a human induced pluripotent stem cell line, NIMHi007-A, from peripheral blood mononuclear cells derived from an adult healthy female

We report the generation and characterisation of a human induced pluripotent stem cell (iPSC) line, NIMHi007-A, derived from peripheral blood mononuclear cells (PBMCs) of a healthy female adult individual. PBMCs were reprogrammed using the non-integrating Sendai virus consisting of Yamanaka reprogramming factors- SOX2, cMYC, KLF4, and OCT4. The iPSCs displayed a normal karyotype, express pluripotency markers, and could generate into three germ layers, endoderm, mesoderm, and ectoderm, in-vitro. This iPSC line, NIMHi007-A, can be used as a healthy control for various in-vitro disease models and study their underlying pathophysiological mechanisms.

Derivation of Breast Cancer Patient Derived Human Induced Pluripotent Stem Cell Line (YBLi006-A) with FANC-BRCA Gene Mutations: A Resource for Precision & Personalized Medicine

Fanconi anemia complementation group I (FANCI) is located on the chromosome 15q26.1 locus and becomes ubiquitinated following DNA damage. 3.06% of patients with breast cancer have altered FANCI gene. We generated an iPSC line (YBLi006-A) from peripheral blood mononuclear cells (PBMCs) of a patient carrying a mutation in FANCIgene (NM_001376911.1, NM_001376910.1, NM_001113378.2; c.80G > T, c.257C > T, c.2225G > C; p.Gly27Val, p.Ala86Val, p.Cys742Ser) using non-integrating Sendai virus technology. This unique breast cancer patient-derived-iPSC line will be resourceful to analyze the entire coding sequence and splicing sites ofFANCIin high-risk familial breast cancer.

Generation of iPSC lines from peripheral blood mononuclear cells from five healthy donors

We describe the generation and characterisation of five human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, and pluripotency was validated by flow cytometry and immunofluorescence of pluripotency markers, and their differentiation into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.

Generation of an induced pluripotent stem cell line from a Huntington’s disease patient with a long HTT-PolyQ sequence

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by an abnormal length of CAG repeats in the gene HTT, leading to an elongated poly-glutamine (poly-Q) sequence in huntingtin (HTT). We used non-integrative Sendai virus to reprogram fibroblasts from a patient with juvenile onset HD to induced pluripotent stem cells (iPSCs). Reprogrammed iPSCs expressed pluripotency-associated markers, exhibited a normal karyotype, and following directed differentiation generated cell types belonging to the three germ layers. PCR analysis and sequencing confirmed the HD patient-derived iPSC line had one normal HTT allele and one with elongated CAG repeats, equivalent to ≥180Q.

Characterization of human induced pluripotent stem cells line (PNUSCRi004-A) from a Parkinson’s disease patient carrying L483P, A495P and V499V mutations

The hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) collected from a female patient with young onset Parkinson’s disease (PD), carrying on heterozygous c.1448 T > C (L483P), c1483 G > C (A495P) and c.1497 G > C (V499V) mutations in the GBA gene. The PBMCs was reprogrammed into an induced pluripotent stem cell (iPSC) line (GBA PD8 or PNUSCRi004-A hiPSCs) using non-integrative Sendai virus. The cell line, PNUSCRi004-A displayed a normal karyotype and expression of pluripotency markers capable of producing derivatives of three germ layers (Ectoderm, Endoderm and Mesoderm).

Generation of mesenchymal stromal cells from urine-derived iPSCs of pediatric brain tumor patients.

Human induced pluripotent stem cells (iPSCs) provide a virtually inexhaustible source of starting material for next generation cell therapies, offering new opportunities for regenerative medicine. Among different cell sources for the generation of iPSCs, urine cells are clinically relevant since these cells can be repeatedly obtained by non-invasive methods from patients of any age and health condition. These attributes encourage patients to participate in preclinical and clinical research. In particular, the use of urine-derived iPSC products is a convenient strategy for children with brain tumors, which are medically fragile patients. Here, we investigate the feasibility of using urine samples as a source of somatic cells to generate iPSC lines from pediatric patients with brain tumors (BT-iPSC). Urinary epithelial cells were isolated and reprogrammed using non-integrative Sendai virus vectors harboring the Yamanaka factors KLF4, OCT3/4, SOX2 and C-MYC. After reprogramming, BT-iPSC lines were subject to quality assessment and were compared to iPSCs obtained from urine samples of non-tumor pediatric patients (nonT-iPSC). We demonstrated that iPSCs can be successfully derived from a small volume of urine obtained from pediatric patients. Importantly, we showed that BT-iPSCs are equivalent to nonT-iPSCs in terms of morphology, pluripotency, and differentiation capacity into the three germ layers. In addition, both BT-iPSCs and nonT-iPSCs efficiently differentiated into functional mesenchymal stem/stromal cells (iMSC) with immunomodulatory properties. Therefore, this study provides an attractive approach to non-invasively generate personalized iMSC products intended for the treatment of children with brain tumors.

Successful Ex Vivo Telomere Elongation with Exg-001 in a Patient with a Dyskeratosis Congenita

We report a first-in-human gene therapy strategy to extend telomere length in patients with telomere biology disorders. Patients with telomere biology disorders, including dyskeratosis congenita, have extremely short telomeres leading to premature cellular senescence and increased risk of bone marrow failure. Zinc Finger and SCAN Domain Containing 4 (ZSCAN4) is a protein that regulates telomere elongation, enhances genome stability, and is expressed transiently in preimplantation embryos, germ cells during meiosis, embryonic stem cells, and rare adult tissue stem cells. EXG-001 is a non-integrating temperature-sensitive non-transmissible Sendai virus vector encoding for human ZSCAN4. We demonstrated that the transient exposure to ZSCAN4 via EXG-001 extended telomeres of primary fibroblast cells derived from dyskeratosis congenita patients as well as healthy human CD34+ cells. However, we did not know whether EXG-001 can extend short telomeres of CD34+ cells of dyskeratosis congenita patients. We translated this work in a first-in-human clinical trial (NCT04211714), in which autologous CD34+ hematopoietic stem cells (HSC) were collected by pheresis from an adult subject with dyskeratosis congenita over two days after mobilization with plerixafor and filgrastim with IRB approval and consent. 2.8x106 CD34/kg were collected and treated with EXG-001 ex vivo in a functionally closed tubing system using the CliniMACS Prodigy (Miltenyi Biotec). EXG-001 treatment increased telomere length of the CD34+ cells of study subject by 1.24-fold, bringing them into the healthy normal control range of CD34+ cell telomere length (Figure 1A). To address the concern of effects on terminal colony forming unit studies were performed and demonstrated maintenance of normal differentiation potential after treatment (Figure 1B; EXG34217). 0.85x106 CD34/kg autologous treated HSC (EXG34217) were reinfused into the subject in February 2022 without conditioning regimen and further in vivo studies are in progress. This novel approach is genotype and mutation independent and uses modified autologous stem cells in the absence of conditioning regimen or immunosuppression which is highly desirable in this radiation and chemotherapy sensitive population. These data support further investigation and translation of telomere elongation via ZSCAN4 exposure by EXG-001 in patients with telomere biology disorders as a potential therapeutic intervention for the prevention or treatment of bone marrow failure. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Generation of induced pluripotent stem cell(iPSC)line CJUHi001-A derived peripheral blood mononuclear cells of spinocerebellar ataxia type 1(SCA1) the CAG repeat mutation in ATXN1 gene

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by CAG repeat mutations in the ATXN1 gene. In this study, we generated an induced pluripotent stem cell line (iPSC) by using non-integrating Sendai virus (SeV) from peripheral blood mononuclear cells(PBMCs)of SCA1 patient harboring a CAG repeat mutation in the ATXN1 gene. The induced patient-specific iPSC line with a normal karyotype and expresses pluripotent markers, it also shows differentiation totipotency and tridermogenesis in vitro. It may be an excellent model for studying spinocerebellar ataxia type 1 (SCA1) in vitro and will be beneficial for studying SCA1 pathogenesis and therapeutic intervention strategies.

Establishment of the iPSC line CUIMCi005-A from a patient with Stargardt disease for retinal organoid culture

Pathogenic variation in the ABCA4 gene is the underlying cause of Stargardt disease, the most common inherited retinal degeneration. We established an induced pluripotent stem cell line for retinal organoid research from a patient with mild disease features who is compound heterozygous for the frequent c.5882G>A (p.Gly1961Glu) missense variant and a c.4947delC (p.Glu1650Argfs*12) frameshift variant. Peripheral blood mononuclear cells were reprogrammed using a non-integrating Sendai virus approach. G-banded karyotyping was normal (46, XY) and mycoplasma testing was negative. Immunohistochemistry and RT-qPCR were performed to verify the expression of pluripotency and stemness markers (LIN28, NANOG, OCT4 and SOX2) and trilineage differentiation.