Restoration of Normal NF1 Function with Antisense Morpholino Treatment of Recurrent Pathogenic Patient-Specific Variant c.1466A>G; p.Y489C

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with almost 3000 different disease-causing variants within the NF1 gene identified. Up to 44% of these variants cause splicing errors to occur within pre-mRNA. A recurrent variant in exon 13, c.1466A>G; p.Y489C (Y489C) results in the creation of an intragenic cryptic splice site, aberrant splicing, a 62 base pair deletion from the mRNA, and subsequent frameshift. We investigated the ability of phosphorodiamidate morpholino oligomers (PMOs) to mask this variant on the RNA level, thus restoring normal splicing. To model this variant, we have developed a human iPS cell line homozygous for the variant using CRISPR/Cas9. PMOs were designed to be 25 base pairs long, and to cover the mutation site so it could not be read by splicing machinery. Results from our in vitro testing showed restoration of normal splicing in the RNA and restoration of full length neurofibromin protein. In addition, we observe the restoration of neurofibromin functionality through GTP-Ras and pERK/ERK testing. The results from this study demonstrate the ability of a PMO to correct splicing errors in NF1 variants at the RNA level, which could open the door for splicing corrections for other variants in this and a variety of diseases.

Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

The potential of COVID-19 patients’ sera to cause antibody-dependent enhancement of infection and IL-6 production

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35–40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

eIF2B-capturing viral protein NSs suppresses the integrated stress response

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction Test

Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0–75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.

BSG/CD147 and ACE2 receptors facilitate SARS-CoV-2 infection of human iPS cell-derived kidney podocytes

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood. Method: In this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing. Result: Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. Conclusion: These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro. These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

The First-in-Human Clinical Trial of iPSC-Derived Platelets (iPLAT1): Autologous Transfusion to an Aplastic Anemia Patient with Alloimmune Platelet Transfusion Refractoriness

Introduction: Platelet transfusion have saved lives of patients with thrombocytopenia through preventing or treating bleeding complications. Currently, platelet products are provided from blood banks which collect blood from healthy donors. However, our ageing society bears the risk of supply in the future. Furthermore, although the rate is decreasing, alloimmune platelet transfusion refractoriness (allo-PTR) is still found in 5% of platelet transfusion patients. Gestation and previous platelet transfusion cause sensitization to produce alloantibodies mostly against class I human leukocyte antigens (HLA-I) and less frequently against human platelet antigens (HPA), resulting in allo-PTR. In these cases, platelets from compatible donors are transfused, but for patients with rare HLA or HPA, donors are difficult to find. As a possible solution, we proposed the application of platelets from induced pluripotent stem cells (iPSC-platelets), which we have succeeded in the ex vivo production at clinical scale. iPSC-platelets are produced from expandable megakaryocyte cell lines (imMKCLs) as master cells and using a "turbulent flow" bioreactor and various new drugs. imMKCLs are established from iPSCs during megakaryocytic differentiation by overexpression of c-MYC, BMI1 and BCL-XL under the doxycycline control promoter. Switching off these transgenes leads to the maturation of imMKCLs. Turbulent flow was found to be a crucial factor in efficient ex vivo production of healthy iPSC-platelets from imMKCLs. We also developed TA-316, a thrombopoietin mimetic compound, KP-547, an ADAM17 inhibitor that inhibits CD42b shedding and stores platelet function, and also found the combination of AhR antagonist and ROCK inhibitor enables feeder-free liquid culture in imMKCL maturation. Aim: To evaluate the safety of autologous iPS-platelets administered to an aplastic anemia patient with allo-PTR due to anti-HPA alloantibody, who experienced systemic post-transfusion purpura-like complication and have no compatible donor in Japan. Methods and Results: Preclinical studies showed that iPSC-platelets were competent in in vitro assays and mouse and rabbit models for circulation and hemostasis, and pathogen and tumorigenicity free. The clinical study was approved by the Certified Special Committee for Regenerative Medicine of the Kyoto University and by the Health Sciences Council of the Japan Ministry of Health, Labour and Welfare as meeting the Act on the Safety of Regenerative Medicine. The clinical study started in March 2019. Using imMKCL master cells derived from the patient, iPS-platelets were manufactured at the Facility for iPS Cell Therapy (FiT) in Center for iPS Cell Research and Application (CiRA), Kyoto University according to the GMP standard. Three doses of 1x10^10, 3x10^10 and 1x10^11 were administered in a dose escalation single-center open-label uncontrolled study at Kyoto University Hospital. The primary endpoint was safety as measured by frequency and extent of adverse events, which were evaluated by the Efficacy Safety Assessment Committee for each dose cohort. Three doses of the administration of autologous iPS-platelets have been completed. No significant adverse event was observed during the full observation period of one year after the last dose. Conclusion: The iPLAT1 study completed the administration of iPSC-platelets for the first time and confirmed the safety in an allo-PTR patient who would otherwise have no HPA-compatible platelet product. The insights gained from the current study should further contribute to development of allogeneic iPSC-platelet products that can be readily administered to wide range of patients. (Japan Registry of Clinical Trials number jRCTa050190117) Figure 1 Figure 1. Disclosures Sugimoto: Astellas Pharma Inc.: Honoraria; Ebara Corporation: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Megakaryon Co: Consultancy, Honoraria; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria. Kanda: Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; SymBio Pharmaceuticals, Ltd.: Membership on an entity's Board of Directors or advisory committees; Sanofi K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Ono Pharma Inc.: Honoraria; Novartis Pharma K.K.: Honoraria; NextGeM Inc: Patents & Royalties; Megakaryon Co: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; DAIICHI SANKYO Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; CHUGAI PHARMACEUTICAL Co., Ltd.: Honoraria; Bristol-Myers Squibb Co: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Amgen Astellas BioPharma: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA LIMITED.: Honoraria. Kondo: Asahi Kasei Pharmaceutical: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Chugai Pharma: Honoraria; MSD Pharmaceutical: Honoraria; Sumitomo Dainippon Pharma: Honoraria. Shimizu: Megakaryon co: Consultancy. Hirai: Kyowa Kirin: Patents & Royalties, Research Funding; Bristol-Myers K.K.: Research Funding; CSL Behring: Research Funding; Mitsubishi Tanabe Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Novartis Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding. Takaori-Kondo: Bristol-Myers K.K.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Celgene: Research Funding. Eto: Megakaryon co: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Kirin Co., Ltd.: Honoraria; Takeda Pharmaceutical Co Ltd: Honoraria; Kowa Co Ltd: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; TEIJIN PHARMA LIMITED.: Honoraria; Kyoto Manufacturing Co., Ltd.: Research Funding.

Human iPS cell derived RPE strips for secure delivery of graft cells at a target place with minimal surgical invasion

Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.