Complete remission of intracranial lymphocytic vasculitis following resection: illustrative case

Abstract: Lymphocytic vasculitis (LV), a histopathological subtype of primary angiitis of the CNS, is a rare inflammatory disorder that often presents as mass-like lesions mimicking malignant brain tumors. Owing to its rarity, the characteristics of LV remain poorly understood. The authors report the case of a young woman who presented with progressive headache. MRI revealed a solitary, ring-enhancing lesion with marked perifocal edema. Fluorodeoxyglucose positron emission tomography (FDG-PET) demonstrated relatively low uptake at the lesion margin, whereas fluciclovine-PET showed mild uptake. Given the high intracranial pressure and uncertain diagnosis, gross-total resection was performed. Histopathological analysis confirmed LV, characterized by perivascular lymphocytic infiltration, fibrin exudation, and the absence of neoplastic or demyelinating features. The patient did not receive additional immunosuppressive therapy and remained disease free for 12 months. To the best of the authors' knowledge, this is the first reported case of a solitary intracranial LV that was successfully managed with resection alone. This case suggests that resection may be a possible therapeutic option for select patients, particularly those with solitary lesions and negative autoimmune profiles. FDG-PET and fluciclovine-PET with MRI may provide supportive information for distinguishing LV from malignant neoplastic tumors. https://thejns.org/doi/10.3171/CASE25705.

Yazawa O
Tateishi K
Ikegaya N
Kawai T
Harada J
Kato A
Morihara K
Yamanaka S
Utsunomiya D
Fujii S
Tanaka F
Yamamoto T


Real-Time Sub-Molecular Visualization of DNA Folding Using a Hybrid High-Speed AFM and Optical Tweezers System

Abstract: Optical tweezers have contributed to elucidating the folding mechanisms associated with biomolecules. By combining them with fluorescence imaging techniques, imaging can also be performed while measuring or applying forces that couple to biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this limitation, here, we developed a hybrid high-speed atomic force microscopy (HS-AFM) and optical tweezers system. To resolve the challenge of incompatible instrumental configurations, we designed a customized optical tweezers system optimized for HS-AFM. Using this platform, we applied external forces to synthetic DNA secondary structures and directly visualized duplex dissociation and spontaneous reannealing upon force release, demonstrating reversible control of folding. We also captured reversible DNA overstretching and transient secondary structure formation in ssDNA. These findings were further analyzed using molecular dynamics simulations and viscoelastic modeling. This integrated approach provides a powerful platform for investigating folding dynamics and force-coupled mechanisms in biomolecules.

The journal of physical chemistry letters

Umeda K
Yamanaka S
Imamura M
Nagae F
Fukuda S
Watanabe H
Uchihashi T
Takada S
Ando T

Optical Tweezers
Microscopy, Atomic Force
Nucleic Acid Conformation
DNA
Molecular Dynamics Simulation
DNA, Single-Stranded


Prognostic impact of systolic blood pressure and antithrombotic strategy in patients with atrial fibrillation and stable coronary artery disease: a post-hoc analysis of the AFIRE trial

Abstract: The AFIRE (Atrial Fibrillation and Ischemic Events with Rivaroxaban in Patients with Stable Coronary Artery Disease) trial demonstrated that rivaroxaban monotherapy was non-inferior in efficacy and superior in safety compared to rivaroxaban plus single antiplatelet therapy in patients with atrial fibrillation (AF) and stable coronary artery disease (CAD). This study examined whether systolic blood pressure (SBP) affects clinical outcomes and modifies the impact of antithrombotic therapy. In this post hoc analysis, participants were stratified based on median SBP at baseline: >126 mmHg (High SBP group, n = 1042) and ≤126 mmHg (Low SBP group, n = 1093). The primary efficacy endpoint was a composite of cardiovascular events and all-cause death. The primary safety endpoint was major bleeding. The mean SBP was 139 mmHg and 114 mmHg in the High and Low SBP groups, respectively. In the propensity score-matched cohort (n = 1684), the Low SBP group had a significantly higher incidence of the primary efficacy endpoint (hazard ratio [HR], 1.38; 95% confidence interval [CI], 1.01-1.88; p = 0.039), while the primary safety endpoint was comparable between groups. In the Low SBP group, rivaroxaban monotherapy was associated with lower risks of both the primary efficacy (HR, 0.60; 95% CI, 0.41-0.86; p = 0.006) and safety endpoints (HR, 0.40; 95% CI, 0.22-0.74; p = 0.003) compared with combination therapy, whereas no significant differences were observed in the High SBP group. Lower SBP was associated with increased risk of cardiovascular events and all-cause death. Rivaroxaban monotherapy demonstrated more favorable efficacy and safety outcomes particularly patients with lower SBP.

Hypertension research : official journal of the Japanese Society of Hypertension

Yamanaka S
Noda T
Nochioka K
Higuma T
Kaikita K
Akao M
Ako J
Matoba T
Nakamura M
Miyauchi K
Hagiwara N
Kimura K
Matsui K
Ogawa H
Yasuda S
AFIRE Investigators

Humans
Atrial Fibrillation
Male
Female
Coronary Artery Disease
Aged
Blood Pressure
Rivaroxaban
Middle Aged
Prognosis
Fibrinolytic Agents
Factor Xa Inhibitors
Platelet Aggregation Inhibitors
Treatment Outcome

Journal Article
Randomized Controlled Trial
Multicenter Study
Research Support, Non-U.S. Gov't


C-C chemokine receptor 4 is a candidate for regulatory T-cell-depletion immunotherapy in differentiated thyroid cancer

Abstract: Immunotherapy using immune checkpoint inhibitors is a standard treatment option for many types of malignancies but is not effective for differentiated thyroid cancer (DTC). Regulatory T-cells (Tregs), which are one of the main suppressive factors in the tumor microenvironment, are another important target for immunotherapy. This study investigated the characteristics of Tregs in DTC with the aim of further developing immunotherapy. Peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs) were obtained from 12 patients with DTC and 12 patients with goiter (benign control group) who underwent primary surgery between February 2017 and September 2022. Flow cytometry analyses were performed for CD4, CD45RA, FOXP3, CC chemokine receptor 4 (CCR4; a candidate Treg-targeting molecule), and immune checkpoint molecules in order to characterize the features of Tregs. In the DTC patients, age ranged from 39 to 84 years, and the male:female ratio was 7:5. Pathological staging was pT1 or pT2 in 4 patients, pT3 in 8, pN0 in 5, and pN1 in 7, and extrathyroidal extension was observed in 5 patients. Effector Treg (eTreg) frequency in CD4+T-cells in TILs and PBLs was significantly higher in DTC than in goiter. Extrathyroidal extension was associated with a higher eTreg frequency in TILs. A positive correlation was found between eTreg frequency and the expression of immune checkpoint molecules (PD-1, TIM3, GITR, and OX40) on eTregs in TILs from DTC patients. These findings suggest that Tregs are activated in DTC and are important in the creation of an immunosuppressive microenvironment. In addition, the mean positive rate (±standard deviation) of CCR4 on eTregs was high in PBLs (95.8 % ± 3.8 %) and TILs (92.8 % ± 9.8 %) from DTC patients, suggesting that CCR4 is a potential target for eTreg-depletion immunotherapy for DTC. Tregs were increased and activated in DTC tumor tissue, indicating that they play an important role in creating an immunosuppressive microenvironment in DTC. The results suggest that eTreg-depletion immunotherapy using an anti-CCR4 antibody (mogamulizumab) might be effective for treating DTC.

Sano R
Nakamura H
Suzuki S
Inukai D
Okamoto H
Yamanaka S
Takahara T
Satou A
Fujimoto Y
Tsuzuki T
Ueda R
Ogawa T

Humans
Male
Female
T-Lymphocytes, Regulatory
Thyroid Neoplasms
Middle Aged
Adult
Aged
Receptors, CCR4
Aged, 80 and over
Immunotherapy
Lymphocytes, Tumor-Infiltrating
Forkhead Transcription Factors
Tumor Microenvironment
Leukocyte Common Antigens


Comparison of Myeloablative Busulfan/Cyclophosphamide and Busulfan/Fludarabine for Allogeneic Hematopoietic Stem Cell Transplantation: A Systematic Review and Meta-Analysis

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for many hematological malignancies. The busulfan plus cyclophosphamide (Bu/Cy) combination is a standard myeloablative conditioning (MAC) regimen, but it is associated with significant regimen-related toxicity and non-relapse mortality (NRM). To mitigate these issues, a regimen combining high-dose busulfan with fludarabine (Bu/Flu) was developed, aiming to reduce toxicity while preserving efficacy. While a previous meta-analysis suggested Bu/Flu may reduce early mortality, it included non-randomized studies, and several large-scale trials have been published since. Therefore, an updated, robust comparison of these two widely used MAC regimens is clinically essential. The primary objective of this study was to conduct a systematic review and meta-analysis of randomized controlled trials (RCTs) to comprehensively evaluate and compare the efficacy and safety profiles of Bu/Flu versus Bu/Cy as MAC regimens for adults undergoing allo-HSCT for hematological malignancies. This systematic review and meta-analysis was conducted following PRISMA guidelines, with a protocol registered on PROSPERO. We systematically searched PubMed, EMBASE, and CENTRAL databases for RCTs comparing Bu/Flu with Bu/Cy in adult patients with hematological malignancies receiving myeloablative busulfan doses. The primary outcome was NRM at 100 d and 1-yr. Secondary outcomes included all-cause mortality, relapse, and grade 3 to 5 adverse events. For statistical analysis, we calculated pooled risk ratios (RR) with 95% confidence intervals (CIs) using fixed- or random-effects models. The certainty of evidence was assessed using the Grades of Recommendations, Assessment, Development, and Evaluations methodology. Five RCTs met the inclusion criteria, encompassing a total of 975 patients. The meta-analysis demonstrated that the Bu/Flu regimen was associated with a significantly lower NRM at 1-yr compared to Bu/Cy (RR 0.49; 95% CI 0.31 to 0.79; I² = 0%; moderate certainty; 629 patients, 2 trials). Furthermore, Bu/Flu resulted in a significantly reduced incidence of grade 3 to 5 adverse events (RR 0.83, 95% CI 0.74 to 0.94; I² = 40%; high certainty; 385 patients, 2 trials). There were no statistically significant differences between the two regimens in terms of all-cause mortality at the end of the study (RR 0.99, 95% CI 0.83 to 1.19; I² = 48%; moderate certainty; 965 patients, 5 trials) or relapse rates (RR 1.11, 95% CI 0.86 to 1.44; I² = 20%; moderate certainty; 972 patients, 5 trials). Bu/Flu and Bu/Cy demonstrate comparable efficacy concerning long-term overall survival and relapse control in the setting of myeloablative allo-HSCT. However, the Bu/Flu regimen presents a superior safety profile, evidenced by a significant reduction in both NRM at 1-yr and severe adverse events. Bu/Flu should be considered a safer MAC alternative to Bu/Cy, particularly for patients at an increased risk of treatment-related toxicity.

Shimada T
Masaki T
Koyamada R
Ishikawa K
Yamanaka S
Sato K
Ota E
Yamashita T
Mori S

Humans
Busulfan
Hematopoietic Stem Cell Transplantation
Vidarabine
Cyclophosphamide
Transplantation Conditioning
Transplantation, Homologous
Myeloablative Agonists
Hematologic Neoplasms

Journal Article
Systematic Review
Meta-Analysis
Comparative Study


Inhibition of glycolysis and stimulation of mitochondrial biogenesis lead to increased ROS levels and cell death in HNF-1ß positive clear cell carcinoma

Abstract: Ovarian clear cell carcinoma is characterized by HNF-1ß overexpression and is known to be resistant to chemotherapy. An inhibitor screening that specifically targets HNF-1ß led us to identify Actinonin as a candidate for cancer treatment. Actinonin, which is known to inhibit aminopeptidase M, has also been recognized for its antibacterial properties. We confirmed that GSK-3ß interference/inhibition, as a component of the HNF-1ß pathway, combined with Actinonin, has a highly potent antitumor effect compared to monotherapy. The same effect was observed in renal clear cell carcinoma lines expressing HNF-1ß. Actinonin promoted mitochondrial production by suppressing aerobic respiration, which decreased AMPK levels and increased ROS production. However, it also elevated GADD45α expression and induced mitophagy. GSK-3ß inhibition suppressed glycolysis and shifted energy production to OXPHOS, leading to increased ROS production. Furthermore, this combination produced excess ROS beyond metabolic capacity, which accumulated in lipid bilayers, leading to a further increase in CHOP gene expression and suppression of mitochondrial turnover. The GSK-3ß inhibitor and Actinonin combination demonstrated a powerful tumor-suppressive effect in vivo without severe side effects. Combining GSK-3ß inhibition with Actinonin can effectively eliminate cancer cells with HNF-1ß overexpression by inhibiting glycolysis and promoting mitochondrial turnover, highlighting new options for cancer therapy.

Kawahara N
Kuniyasu H
Mori S
Kishi S
Sugimoto S
Maehana T
Yamanaka S
Kawaguchi R
Kimura F

Reactive Oxygen Species
Humans
Glycolysis
Cell Line, Tumor
Mitochondria
Female
Animals
Adenocarcinoma, Clear Cell
Glycogen Synthase Kinase 3 beta
Organelle Biogenesis
Cell Death
Mice
Carcinoma, Renal Cell
Mice, Nude
Ovarian Neoplasms
Kidney Neoplasms


Malignant peritoneal mesothelioma as a secondary cancer following radiotherapy for cervical cancer: a case report and literature review

Abstract: Malignant peritoneal mesothelioma (MPM) as a secondary malignancy following radiotherapy is extremely rare. We report a case of MPM that developed as a secondary malignancy after initial concurrent chemoradiotherapy (CCRT) for cervical cancer.Case Presentation.A 52-year-old woman with no history of asbestos exposure underwent definitive CCRT for stage IIB cervical cancer, as classified by the International Federation of Gynecology and Obstetrics (FIGO) staging system. Over 10 years after initial treatment, she developed ascites. Diagnostic laparoscopy led to a pathological diagnosis of peritoneal mesothelioma. Given the absence of asbestos exposure and her medical history, the tumor was considered a radiation-induced secondary cancer. The patient received two lines of chemotherapy, but her performance status progressively declined, and she was transitioned to best supportive care. She died 1 year and 8 months after the MPM diagnosis. This case highlights the importance of long-term surveillance following radiotherapy for cervical cancer. Including this case, only four reports exist of MPM developing as a secondary malignancy following pelvic radiotherapy for cervical cancer. In all cases, patients presented with abdominal symptoms and ascites. The case findings underscore the need for clinicians to consider secondary malignancies such as MPM when ascites is detected during follow-up and to pursue a thorough diagnostic workup.

Otsubo M
Aichi M
Kawashima Y
Yamanaka S
Imai Y
Miyagi E
Mizushima T


Correction: Generation of Human Melanocytes from Induced Pluripotent Stem Cells

Abstract: [This corrects the article DOI: 10.1371/journal.pone.0016182.].

Ohta S
Imaizumi Y
Okada Y
Akamatsu W
Kuwahara R
Ohyama M
Amagai M
Matsuzaki Y
Yamanaka S
Okano H
Kawakami Y


A true brachial artery aneurysm in a 1-year-old child: A case report

Pediatrics international : official journal of the Japan Pediatric Society


Treatment of initially unresectable local advanced pancreatic adenocarcinoma with FOLFIRINOX: A prospective study YCOG1403 (C-FLAP study)

Abstract: This study aimed to provide evidence regarding the treatment of patients with unresectable pancreatic cancer. We conducted a prospective single-arm phase II trial using the FOLFIRINOX regimen. After completing 4-8 cycles, patients underwent surgical resection when feasible. The primary endpoint was R0 resection rate. Fifteen patients were enrolled in this study. A median of six courses of FOLFIRINOX chemotherapy was administered, and a partial response or R0 resection was achieved in 26.7% and 33% of the patients, respectively. Severe adverse events due to chemotherapy and major surgical complications were observed in 33.3% and 6.7% of patients, respectively. The median overall survival of patients who underwent R0 resection or with R1 or unresectable disease was 47.8 months (95% confidence interval (CI), 22.5-73.1) or 14.5 months (95% CI, 11.8-17.2), respectively (P = 0.031). Well-selected patients with unresectable locally advanced pancreatic cancer treated with FOLFIRINOX achieved relatively high R0 resection rates and prolonged survival. Therefore, induction with FOLFIRINOX is feasible and well tolerated for locally advanced, initially unresectable pancreatic cancer and may be effective in facilitating R0 resection and prolonging survival.

Kobayashi N
Yabushita Y
Mori R
Takahashi T
Miyake K
Sawada Y
Homma Y
Matsuyama R
Okubo N
Katsuta E
Kubota K
Yamanaka S
Ichikawa Y
Endo I

Antineoplastic Combined Chemotherapy Protocols

Journal Article
Clinical Trial, Phase II


Embryonal rhabdomyosarcoma of the uterine corpus in a postmenopausal Woman: A case report and literature Review

Abstract: Rhabdomyosarcoma (RMS) is a rare malignant mesenchymal tumor, primarily affecting children. Occurrence in the uterine corpus of adults, particularly postmenopausal women, is extremely uncommon. A 60-year-old postmenopausal nulligravid woman presented with abnormal genital bleeding for three months. Pelvic examination revealed a friable mass extending from the uterine corpus. Biopsy confirmed embryonal RMS. She underwent modified radical hysterectomy with bilateral salpingo-oophorectomy for complete surgical resection. One cycle of adjuvant VAC chemotherapy (vincristine, actinomycin D, and cyclophosphamide) was administered but discontinued at her request. No residual disease was observed postoperatively, and she remains disease-free 12 months after surgery. Adult-onset uterine corpus embryonal RMS is rare, and standardized treatment protocols are lacking. Early diagnosis, complete surgical resection, and individualized therapy are essential. A literature review highlights clinical characteristics, treatment strategies, and prognostic considerations.

Omuro M
Imai Y
Ogawara Y
Mizushima T
Muraoka E
Yamanaka S
Fujii S
Miyagi E


Observation of a guest-free Si(46) clathrate-I framework from Ba(8-x)Si(46) upon in situ vacuum heating

Abstract: Silicon and its composites are key materials owing to their extensive use in the semiconductor industry. While the diamond-structured form dominates, other allotropes with superior properties at ambient conditions remain of interest. Toward this, Si46 clathrate type-I crystals containing alkali/alkaline-earth metals have been extensively studied, but the experimental observation of a guest-free Si46 structure has been challenging. Using advanced electron microscopy, we show experimental evidence of guest-free clathrate-I Si46 framework from Ba8-xSi46 under in situ heating. We reveal the stepwise Ba evacuation process, starting with loss of Ba1 from the smaller cages to form Ba6Si46, followed by removal of Ba2 in larger cages to reach Si46 that appears in the thin region of the nanocrystal with a thickness around 4-6 nm at 500 °C. Calculations give a quasi-direct bandgap of 1.89 eV and support the preferential evacuation of Ba1. The observation of this guest-free Si46 framework opens up possibilities for applications in high-speed transistors, optoelectronic devices or solar cell technologies.

Zhou Y
Zhang Q
Mayoral A
Spackman PR
Matsumoto T
Li J
Gale JD
Anderson MW
Fukuoka H
Yamanaka S
Terasaki O


New-onset anemia and its association with ventricular arrhythmias and sudden cardiac death in patients with heart failure with preserved ejection fraction

Abstract: Anemia is a common comorbidity associated with adverse outcomes in patients with heart failure with preserved ejection fraction (HFpEF). However, the clinical relevance of new-onset anemia to sudden cardiac death (SCD) in patients with HFpEF remains unclear. This study investigated the association between new-onset anemia with ventricular arrhythmias (VAs) and SCD. Anemia was defined as a hemoglobin (Hb) level of <13 g/dL in men and <12 g/dL in women. Patients with Hb levels above these thresholds were categorized as without anemia. We analyzed data of 686 patients with symptomatic HFpEF (ejection fraction ≥ 50 %, New York Heart Association class II-IV) without anemia at baseline from a multicenter prospective observational CHART-2 study. The primary endpoint was a composite of ventricular tachycardia, ventricular fibrillation, and SCD. At the 1-year follow-up, 109 patients developed new-onset anemia (median Hb, 11.9 g/dL), whereas 577 remained without anemia (median Hb, 14.0 g/dL). Over a median follow-up of 9.2 years, patients with new-onset anemia had a significantly higher incidence of composite outcomes (12.8 % vs. 5.2 %, P = 0.008). After adjusting for potential confounders, new-onset anemia was associated with an elevated risk of the composite outcome (adjusted hazard ratio 2.20, 95 % confidence interval 1.10-4.42, P = 0.027). The association between new-onset anemia and lethal arrhythmias was independent of heart failure hospitalization or myocardial infarction occurring before the primary endpoint. New-onset anemia was significantly associated with an increased risk of VAs and SCD in patients with HFpEF, underscoring the importance of monitoring Hb levels for risk stratification.Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00418041.

International journal of cardiology. Heart & vasculature

Ito T
Nochioka K
Noda T
Shiroto T
Yamanaka S
Yamamoto N
Sato H
Chiba T
Nakano M
Inoue T
Susukita K
Takahama H
Takahashi J
Miyata S
Shimokawa H
Yasuda S


Incidence and clinical implications of microbubble formation during pulsed field ablation

Yano S
Takatsuki S
Kotajima T
Sasajima K
Yamanaka S
Himeno Y
Yamashita S
Yamaoka K
Katsumata Y
Nishiyama T
Kimura T
Ieda M


Successful Treatment of Refractory Immune-Mediated Colitis and Duodenitis with Tofacitinib

Abstract: Immune-mediated colitis (IMC) is a common and potentially severe immune-related adverse event of immune checkpoint inhibitors (ICIs). Although corticosteroids and biologics are standard treatments, some cases remain refractory. Tofacitinib, a Janus kinase inhibitor, has shown promise in refractory IMC; however, evidence regarding its treatment effects remains limited. Here, we present a case of pembrolizumab-induced IMC and duodenitis refractory to corticosteroids, infliximab, and vedolizumab. The patient was successfully treated after receiving a 30-day course of tofacitinib, which resulted in rapid symptom resolution and mucosal healing. No recurrence of colitis was observed 3 months after treatment cessation. As ICIs are increasingly used, the incidence of refractory IMC and other immune-related toxicities is expected to rise. This case highlights the need for further studies to establish the optimal use of tofacitinib in refractory IMC and duodenitis.

Misawa N
Higurashi T
Inoue K
Suzuki H
Tamura S
Yoneda M
Hori C
Yamanaka S
Nakajima A


A Case of Renal Metastasis of Urothelial Carcinoma With Trophoblastic Differentiation Successfully Treated With Robot-Assisted Partial Nephrectomy

Abstract: There are no previous reports of solitary renal metastases from urothelial carcinoma with trophoblastic differentiation, a rare bladder cancer subtype that is pathologically hCGβ positive. A 77-year-old male with urothelial carcinoma with trophoblastic differentiation underwent robot-assisted radical cystectomy following neoadjuvant chemotherapy. Pathological examination revealed urothelial carcinoma, classified as ypT2b and ypN0 with detection of focal hCGβ positivity. Postoperatively, serum hCGβ levels decreased from 1.6 to < 0.2 mIU/mL. At the 9-month follow-up, serum hCGβ was elevated to 20.1 mIU/mL with no recurrence on PET-CT. Gemcitabine-cisplatin chemotherapy was initiated; however, a solitary renal tumor was detected. Partial nephrectomy confirmed that the tumor was a renal metastasis of bladder cancer. Serum hCGβ levels decreased and remained < 0.2 mIU/mL, even 20 months after partial nephrectomy. We report a case of urothelial carcinoma with trophoblastic differentiation and elevated serum hCGβ levels, in which a solitary renal metastasis was successfully resected by robot-assisted partial nephrectomy.

Honda S
Uemura K
Ito H
Muraoka E
Tatenuma T
Ito Y
Muraoka K
Hasumi H
Kawano N
Yamanaka S
Makiyama K


In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID

Abstract: Heterobifunctional molecules, such as proteolysis-targeting and autophagy-targeting chimera, represent new drug concepts. They are composed of two protein binders that can induce proximity interactions between two proteins and protein catalysis. Currently, cereblon (CRBN)- and von Hippel-Lindau (VHL)-binders with thalidomide- and VH032-backbones are widely used as E3 ligase binders. Here, we developed a method to validate proteins that interact with heterobifunctional molecules in cells using AirID, a proximity biotinylation enzyme. Interactome of target proteins was validated for six heterobifunctional molecules. ThBD-AirID, a fusion of the thalidomide-binding domain (ThBD) of CRBN and AirID, effectively biotinylated the target proteins. AirID fused to full-length VHL also exhibited highly effective biotinylation. Heterobifunctional molecules with the same target binder but different E3 binders showed different proximity interactome profiles in cells. Analysis using ThBD-AirID revealed a nuclear interaction between androgen receptor and ARV-110. AirID-fused ThBD and VHL could be useful for validating the heterobifunctional molecular interactome in cells.

Yamada K
Yamanaka S
Yamakoshi H
Kohyama A
Iwabuchi Y
Kosako H
Sawasaki T

Von Hippel-Lindau Tumor Suppressor Protein


[A Case of Liver and Axillary Lymph Node Metastasis 32 Years after Breast Cancer Surgery]

Abstract: unknown, but she remained symptom-free for 32 years. However, a CT scan performed for an unrelated condition revealed 2 liver tumors with calcifications located in S8(10 cm)and S6(3 cm). Given her medical history, liver metastasis from breast cancer was suspected. Core needle biopsies of the liver tumor and a left axillary lymph node detected on PET-CT, confirmed metastatic breast cancer that was estrogen receptor-positive, progesterone receptor-positive, and human epidermal growth factor receptor 2-negative. Upon diagnosis, chemotherapy with paclitaxel and bevacizumab was initiated. Follow-up CT scans at 6 and 15 months showed continued tumor regression in the liver and axilla. After 15 months of treatment, both tumors continued to shrink. Although late recurrence is common with breast cancer, recurrence more than 30 years post-surgery is rare. This case of late-stage metastatic breast cancer is presented alongside a literature review.

Gan to kagaku ryoho. Cancer & chemotherapy

Aikawa A
Oshi M
Kawashima K
Sasamoto M
Fujiwara Y
Adachi S
Narui K
Takase H
Yamanaka S
Fujii S
Yamada A
Endo I

Journal Article
Case Reports
English Abstract


Root-associated ectomycorrhizal fungal communities in and around aggregated retention patches left in logged areas of Abies sachalinensis planted forests

Abstract: An aggregated retention system retains several groups of trees within cutblocks to maintain public functions such as biodiversity conservation. We examined ectomycorrhizal (EcM) fungal communities associated with regenerating Abies sachalinensis seedlings and their surrounding trees at different locations; inside and at the edge of the retained patches, and in clear-cut areas 10 and 50 m from the edge. The EcM fungi on the roots were grouped into operational taxonomic units (OTU) based on the similarity of their ribosomal DNA internal transcribed spacer sequences. Higher OTU richness was found inside (63 OTUs) and at the edge of the patches (59 OTUs) compared to clear-cut areas (33 or 25 OTUs). The ordination analysis inferred that location may influence the EcM fungal communities. However, further studies with more site replications are needed to clarify the effects of the patches on shaping EcM fungal communities.


Surgical Integration of Metanephros-bladder Composite Tissue Transplants in Adult Pigs

Kinoshita Y
Yamanaka S
Iwai S
Kume H
Yokoo T
Kobayashi E


Transcriptome landscapes induced by estetrol, estradiol, and ethinylestradiol in primary human endometriotic stromal cells

Abstract: To compare the transcriptomes induced by estrogens used in combined oral contraceptives (COCs): estetrol (E4), ethinylestradiol (EE), and estradiol (E2) in human primary endometriotic stromal cells. Endometriotic stromal cells were cultured in the presence of E4 (10-6 mol/L), E2 (10-8 mol/L) or EE (10-9 mol/L) for 24 hours. Transcriptomes induced by E4 were compared with those induced by E2 and EE. Endometriotic stromal cells were obtained from patients with ovarian endometriomas. Transcriptomes were examined using RNA sequencing analysis, and messenger RNA levels were measured using reverse transcription-quantitative polymerase chain reaction. Estetrol treatment resulted in 114 differentially expressed genes compared with the control (|log2fold change| >0.2). The number was higher than those differentially expressed between E4 and E2 (82 genes) and between E4 and EE (75 genes). Of the differentially expressed genes between E4 and the control, 79% (90 genes) changed in the same manner as in EE and E2. In contrast, 21% (24 genes) of the genes changed in an E4-preferential manner. The present study elucidated that although E4 induces the expression of specific unique genes in endometriotic stromal cells distinct from those regulated by other estrogens, the overall gene expression profile is largely analogous to that induced by other estrogens. When differences in progestin are not considered, it is suggested that the COC containing E4 exhibits effects on endometriosis comparable with those of other COCs.

Nakajima M
Izumi G
Koga K
Silvana V
Elsherbini M
Okumura A
Wada M
Yamanaka S
Hirota Y
Osuga Y

Humans
Ethinyl Estradiol
Female
Stromal Cells
Estradiol
Transcriptome
Estetrol
Endometriosis
Endometrium
Cells, Cultured
Adult


A novel mouse model for investigating the long-term impact of reduced nephron numbers on renal function and salt sensitivity

Abstract: Nephrogenesis occurs during the fetal period; because nephrogenesis does not occur after birth, preterm infants have low nephron numbers. However, whether a reduction in the number of pure nephrons alone affects renal function in conventional animal models remains unclear. Therefore, we aimed to examine whether differences in nephron number alone could lead to differences in future renal function by specifically ablating the nephrons. Mice transgenic for Cre recombinase in the nephron progenitor cells were crossed with the locus of X-over P1-diphtheria toxin receptor transgenic mice. Diphtheria toxin was administered on embryonic day 13.5 to suppress fetal nephrogenesis. Various renal function assessments were performed until the maximum age of 1 year. Some mice received a high-salt diet (HSD). The mouse model showed a glomerular loss of approximately half per cross-sectional area and no or mild renal damage at 1 year of age. Furthermore, HSD induced the early onset and exacerbation of renal impairment. The new mouse model used in this study showed HSD-induced early onset and exacerbation of renal damage, suggesting that appropriate salt management can prevent the onset and exacerbation of renal impairment in preterm infants known to have low nephron numbers. We established a new method for generating mice with low nephron numbers without affecting growth. The mouse model did not develop kidney disease but could develop mild kidney disease; however, kidney damage is exacerbated by a high-salt diet. Adequate salt management may prevent the future development of renal damage in infants with low nephron counts when born prematurely.

Inage Y
Matsumoto K
Haruhara K
Hirano D
Matsui K
Kinoshita Y
Morimoto K
Koda N
Yamamoto S
Fujimoto T
Fukunaga S
Yamanaka S
Oishi K
Kobayashi E
Yokoo T

Animals
Nephrons
Mice, Transgenic
Mice
Sodium Chloride, Dietary
Kidney
Female
Disease Models, Animal
Diphtheria Toxin
Male


Impact of body mass index on D-dimer diagnostic utility for deep vein thrombosis in patients with cancer: a single-center retrospective analysis

Abstract: Deep vein thrombosis (DVT) is a common complication in cancer patients associated with significant morbidity and mortality. D-dimer is a widely used screening tool for suspected DVT; however, its accuracy may be influenced by body mass index (BMI). We evaluated whether BMI modified the diagnostic utility of D-dimer levels in patients with cancer and DVT. We retrospectively analyzed 439 patients with newly diagnosed solid cancers suspected of having DVT between January 2013 and December 2020. DVT was confirmed or excluded by using computed tomography or echography. D-dimer was measured, and patients were classified by BMI as low- (< 18.5 kg/m2), normal- (18.5-24.9 kg/m2), or high-BMI (≥ 25.0 kg/m2). D-dimer levels, positive and negative predictive values (PPV and NPV), and overall survival (OS) were compared. Of 439 patients, 175 (39.9%) had DVT. BMI did not differ significantly between the DVT-positive and DVT-negative patients. In the normal and high BMI groups, D-dimer levels were significantly higher in patients with DVT than those without (p < 0.01), whereas patients with a low BMI did not show this difference (p = 0.12). Using a 1 µg/mL cut-off, PPV was 32% in low-, 52% in normal-, and 49% in high-BMI patients; NPV was 83%, 91%, and 97%, respectively. OS did not differ among the BMI groups, and no deaths were directly related to DVT. Diagnostic performance of D-dimer level may diminish in underweight patients with cancer. Clinicians should carefully interpret the D-dimer levels in low-BMI populations and consider additional diagnostic strategies.

International journal of clinical oncology

Masuda Y
Konishi T
Yakushijin Y
Yamanaka S
Hasebe S
Yamanouchi J
Takenaka K

Humans
Fibrin Fibrinogen Degradation Products
Venous Thrombosis
Male
Female
Retrospective Studies
Neoplasms
Body Mass Index
Middle Aged
Aged
Adult
Aged, 80 and over


Intrathecal Baclofen Therapy Improves Refractory Status Dystonicus in Neuro-hepatic Wilson's Disease: A Case Report

Abstract: Wilson's disease is an autosomal recessive disorder of copper metabolism. A current unresolved issue is the worsening of neurological symptoms during the initial treatment phase, particularly with chelation therapy. This phenomenon, termed "early neurological worsening," is attributed to the rapid mobilization and redistribution of copper during treatment initiation. We report the case of a 10-year-old boy, with neuro-hepatic Wilson's disease who developed treatment-refractory generalized dystonia, which improved with intrathecal baclofen therapy. The patient experienced walking discomfort 5 months before referral to our hospital, with rapid progression to dysphagia and a 3 kg weight loss. Initially, he presented with dystonia, including foot inversion. Wilson's disease was diagnosed based on physiological, clinical, and imaging findings, with confirmation of a homozygous mutation in the ATP7B gene. The patient was treated with trientine hydrochloride, followed by zinc monotherapy. Despite appropriate chelation therapy, dystonia progressed to severe axial torsion involving the trunk. His condition deteriorated to status dystonicus, with high-grade fever, elevated creatine phosphokinase levels, and dehydration, requiring midazolam sedation. These symptoms were attributed to "early neurological worsening." A trial of intrathecal baclofen injection provided symptom relief, leading to the implantation of a baclofen pump, which significantly reduced the status dystonicus. At discharge, the patient had a modified Rankin Scale score of 5. Three years later, although wheelchair-dependent, his oral intake and speech are progressively improving with training. This is the first reported case of status dystonicus in Wilson's disease successfully treated with intrathecal baclofen, highlighting its potential as a viable treatment option for Wilson's disease-associated debilitating dystonia.

Yamanaka S
Hanada T
Higashi T
Matsunaga M
Yonee C
Maruyama S
Hanaya R


Clinical and plasma proteomic characterization of heart failure with supranormal left ventricular ejection fraction: An emerging entity of heart failure

Abstract: The clinical guidelines categorize heart failure (HF) based on left ventricular ejection fraction (LVEF). However, the current LVEF cutoffs, 40% and 50%, may not fully address the underlying characteristics and cardiovascular risk of HF, particularly for HF with higher LVEF. This study aimed to characterize HF with supranormal ejection fraction (HFsnEF) using different LVEF cutoffs (35%, 55%, and 70% for men, and 40%, 60%, and 75% for women). This study divided 442 patients from the CHART-Omics study into four groups: HF with reduced ejection fraction (HFrEF) (n = 55, 65.5 years), HF with mildly reduced ejection fraction (HFmrEF) (n = 125, 69.3 years), HF with normal ejection fraction (HFnEF) (n = 215, 69.0 years) and HFsnEF (n = 47, 67.1 years). When clinical backgrounds were adjusted and HFnEF served as the reference, HFsnEF carried an increased hazard ratio (HR) for the composite of cardiovascular death and HF hospitalization of 2.71 (95% confidence interval [CI] 1.10-6.66, p = 0.030), while HFrEF had a HR of 3.14 (95% CI 1.36-7.23, p = 0.007). HFsnEF was characterized by an increase in relative left ventricular wall thickness and a decrease in left ventricular dimensions, whereas increased left ventricular mass and dimensions characterized HFrEF. Quantitative analysis of 4670 plasma proteins showed essential differences between HFsnEF and HFrEF, for example, 'protein synthesis' versus 'cell morphology', 'cellular assembly and organization' and 'nucleic acid metabolism' for underlying pathophysiology, and 'energy production' versus 'connective tissue disorders' and 'cell-to-cell signalling and interaction' for prognostication. Heart failure with supranormal ejection fraction, an unnoticed but emerging entity in HF, carries a similarly increased cardiovascular risk as HFrEF but has unique structural and plasma proteomic characteristics.

Sakata Y
Nochioka K
Yasuda S
Ishida K
Shiroto T
Takahashi J
Kasahara S
Abe R
Yamanaka S
Fujihashi T
Hayashi H
Kato S
Horii K
Teramoto K
Tomita T
Miyata S
Sugimura K
Waga I
Nagasaki M
Shimokawa H

Humans
Heart Failure
Stroke Volume
Female
Male
Aged
Proteomics
Ventricular Function, Left
Middle Aged
Prognosis


Author Correction: BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans

Şahin U
Muik A
Vogler I
Derhovanessian E
Kranz LM
Vormehr M
Quandt J
Bidmon N
Ulges A
Baum A
Pascal KE
Maurus D
Brachtendorf S
Lörks V
Sikorski J
Koch P
Hilker R
Becker D
Eller AK
Grützner J
Tonigold M
Boesler C
Rosenbaum C
Heesen L
Kühnle MC
Poran A
Dong JZ
Luxemburger U
Kemmer-Brück A
Langer D
Bexon M
Bolte S
Palanche T
Schultz A
Baumann S
Mahiny AJ
Boros G
Reinholz J
Szabó GT
Karikó K
Shi PY
Fontes-Garfias C
Perez JL
Cutler M
Cooper D
Kyratsous CA
Dormitzer PR
Jansen KU
Türeci Ö


Reducing cell intrinsic immunity to mRNA vaccine alters adaptive immune responses in mice

Abstract: The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment, but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1Ψ) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1Ψ-modified mRNA (D1-modRNA), and D1-modRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an up-regulation of Stat1 and RnaseL, and increased systemic inflammation. This result correlated with significantly reduced antigen-specific antibody responses and reduced protection against influenza virus infection compared with D1-modRNA and cC1-modRNA. Incorporation of m1Ψ alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1Ψ and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material.

Wang Z
Jacobus EJ
Stirling DC
Krumm S
Flight KE
Cunliffe RF
Mottl J
Singh C
Mosscrop LG
Santiago LA
Vogel AB
Kariko K
Sahin U
Erbar S
Tregoning JS


ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model

Abstract: Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

Daly O
Mahiny AJ
Majeski S
McClintock K
Reichert J
Boros G
Szabó GT
Reinholz J
Schreiner P
Reid S
Lam K
Lepper M
Adler M
Meffen T
Heyes J
Karikó K
Lutwyche P
Vlatkovic I


Progress in vaccine development for infectious diseases-a Keystone Symposia report

Abstract: The COVID-19 pandemic has taught us many things, among the most important of which is that vaccines are one of the cornerstones of public health that help make modern longevity possible. While several different vaccines have been successful at stemming the morbidity and mortality associated with various infectious diseases, many pathogens/diseases remain recalcitrant to the development of effective vaccination. Recent advances in vaccine technology, immunology, structural biology, and other fields may yet yield insight that will address these diseases; they may also help improve societies' preparedness for future pandemics. On June 1-4, 2022, experts in vaccinology from academia, industry, and government convened for the Keystone symposium "Progress in Vaccine Development for Infectious Diseases" to discuss state-of-the-art technologies, recent advancements in understanding vaccine-mediated immunity, and new aspects of antigen design to aid vaccine effectiveness.

Annals of the New York Academy of Sciences

Cable J
Graham BS
Koup RA
Seder RA
Karikó K
Pardi N
Barouch DH
Sharma B
Rauch S
Nachbagauer R
Forsell MNE
Schotsaert M
Ellebedy AH
Loré K
Irvine DJ
Pilkington E
Tahtinen S
Thompson EA
Feraoun Y
King NP
Saunders K
Alter G
Moin SM
Sliepen K
Karlsson Hedestam GB
Wardemann H
Pulendran B
Doria-Rose NA
He WT
Juno JA
Ataca S
Wheatley AK
McLellan JS
Walker LM
Lederhofer J
Lindesmith LC
Wille H
Hotez PJ
Bekker LG

Humans
Pandemics
COVID-19
Vaccines
Communicable Diseases
Vaccination
Vaccine Development

Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural


Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice

Abstract: As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)-encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8+ T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

Ramos da Silva J
Bitencourt Rodrigues K
Formoso Pelegrin G
Silva Sales N
Muramatsu H
de Oliveira Silva M
Porchia BFMM
Moreno ACR
Aps LRMM
Venceslau-Carvalho AA
Tombácz I
Fotoran WL
Karikó K
Lin PJC
Tam YK
de Oliveira Diniz M
Pardi N
de Souza Ferreira LC

Animals
Female
Mice
Cancer Vaccines
CD8-Positive T-Lymphocytes
Disease Models, Animal
Immunization
Mice, Inbred C57BL
Neoplasms
Papillomavirus E7 Proteins
Papillomavirus Infections
Papillomavirus Vaccines
Recombinant Proteins
RNA, Messenger
Vaccines, DNA

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't


Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

Abstract: Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target. We investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing. We identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2-4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.

Foster JB
Griffin C
Rokita JL
Stern A
Brimley C
Rathi K
Lane MV
Buongervino SN
Smith T
Madsen PJ
Martinez D
Delaidelli A
Sorensen PH
Wechsler-Reya RJ
Karikó K
Storm PB
Barrett DM
Resnick AC
Maris JM
Bosse KR

Brain Neoplasms
Cell Line, Tumor
Cerebellar Neoplasms
Child
Glioma
Glypicans
Humans
Medulloblastoma
Neuroblastoma
Oncogene Proteins
RNA, Messenger
Receptors, Chimeric Antigen
Single-Chain Antibodies
Xenograft Model Antitumor Assays


Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses

Alameh MG
Tombácz I
Bettini E
Lederer K
Sittplangkoon C
Wilmore JR
Gaudette BT
Soliman OY
Pine M
Hicks P
Manzoni TB
Knox JJ
Johnson JL
Laczkó D
Muramatsu H
Davis B
Meng W
Rosenfeld AM
Strohmeier S
Lin PJC
Mui BL
Tam YK
Karikó K
Jacquet A
Krammer F
Bates P
Cancro MP
Weissman D
Luning Prak ET
Allman D
Locci M
Pardi N


A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA

Abstract: RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. It remains unclear whether pseudouridylation of mRNA can be snoRNA-guided, bearing important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at relatively low efficiencies, and is constrained by mRNA localization, snoRNA expression levels and the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously shown to suppress translational termination. However, in this and follow-up experiments in human cells we observe no evidence for significant levels of readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, 'design rules', constraints and consequences of snoRNA-mediated pseudouridylation.

Nir R
Hoernes TP
Muramatsu H
Faserl K
Karikó K
Erlacher MD
Sas-Chen A
Schwartz S

Humans
Protein Biosynthesis
Pseudouridine
RNA Processing, Post-Transcriptional
RNA, Messenger
RNA, Ribosomal
RNA, Small Nucleolar


Developing mRNA for Therapy

Abstract: Messenger RNA was discovered in 1961 and it took 60 years until the first mRNA became FDA-approved product in the form of COVID-19 mRNA vaccine. During those years a lot of progress has been made by hundreds of scientists. It was 1978 when the first-time isolated mRNA delivered into mammalian cells produced the encoded protein. In vitro transcription introduced in 1984 made it possible to generate any desired mRNA from the encoding plasmid using phage RNA polymerases. In the early 90s mRNA was used for therapy as well as vaccine against infectious diseases and cancer. Inflammatory nature of the mRNAs limited their in vivo use. Replacing uridine with pseudouridine made the mRNA non-immunogenic, more stable and highly translatable. Delivery of the lipid nanoparticle-formulated nucleoside-modified mRNA encoding viral antigens became a platform for effective vaccine. Labile nature of the mRNA is ideal for transient production of the viral antigen, to generate effective antibody and cellular immune response. The mRNA platform is revolutionizing the delivery of effective and safe vaccines, therapeutics and gene therapies.

Animals
COVID-19
COVID-19 Vaccines
Humans
Liposomes
Mammals
Nanoparticles
RNA, Messenger
Vaccines, Synthetic
mRNA Vaccines


Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA

Abstract: The presence of the cap structure on the 5'-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5'-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

Vlatkovic I
Ludwig J
Boros G
Szabó GT
Reichert J
Buff M
Baiersdörfer M
Reinholz J
Mahiny AJ
Şahin U
Karikó K


Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine

Abstract: Lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccines have proven to be very successful in the fight against the coronavirus disease 2019 (COVID-19) pandemic. They are effective, safe, and can be produced in large quantities. However, the long-term storage of mRNA-LNP vaccines without freezing is still a challenge. Here, we demonstrate that nucleoside-modified mRNA-LNPs can be lyophilized, and the physicochemical properties of the lyophilized material do not significantly change for 12 weeks after storage at room temperature and for at least 24 weeks after storage at 4°C. Importantly, we show in comparative mouse studies that lyophilized firefly luciferase-encoding mRNA-LNPs maintain their high expression, and no decrease in the immunogenicity of a lyophilized influenza virus hemagglutinin-encoding mRNA-LNP vaccine was observed after 12 weeks of storage at room temperature or for at least 24 weeks after storage at 4°C. Our studies offer a potential solution to overcome the long-term storage-related limitations of nucleoside-modified mRNA-LNP vaccines.

Molecular therapy : the journal of the American Society of Gene Therapy

Muramatsu H
Lam K
Bajusz C
Laczkó D
Karikó K
Schreiner P
Martin A
Lutwyche P
Heyes J
Pardi N

Animals
COVID-19
Freeze Drying
Influenza Vaccines
Liposomes
Mice
Nanoparticles
Nucleosides
RNA, Messenger
Vaccines, Synthetic
mRNA Vaccines


Abstract: Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Adaptor Proteins, Signal Transducing
Adjuvants, Immunologic
Animals
B-Lymphocytes
COVID-19
COVID-19 Vaccines
Germinal Center
HEK293 Cells
Humans
Immunity, Humoral
Interleukin-6
Liposomes
Mice
Mice, Inbred BALB C
Nanoparticles
Protein Subunits
SARS-CoV-2
T-Lymphocytes, Helper-Inducer
mRNA Vaccines


Modified uridines are the key to a successful message


Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models

Abstract: Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

Hotz C
Wagenaar TR
Gieseke F
Bangari DS
Callahan M
Cao H
Diekmann J
Diken M
Grunwitz C
Hebert A
Hsu K
Bernardo M
Karikó K
Kreiter S
Kuhn AN
Levit M
Malkova N
Masciari S
Pollard J
Qu H
Ryan S
Selmi A
Schlereth J
Singh K
Sun F
Tillmann B
Tolstykh T
Weber W
Wicke L
Witzel S
Yu Q
Zhang YA
Zheng G
Lager J
Nabel GJ
Sahin U
Wiederschain D

Cytokines
Humans
Neoplasms
RNA, Messenger

Journal Article
Research Support, Non-U.S. Gov't


What does the success of mRNA vaccines tell us about the future of biological therapeutics?


Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

Abstract: Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.

Szőke D
Kovács G
Kemecsei É
Bálint L
Szoták-Ajtay K
Aradi P
Styevkóné Dinnyés A
Mui BL
Tam YK
Madden TD
Karikó K
Kataru RP
Hope MJ
Weissman D
Mehrara BJ
Pardi N
Jakus Z

Animals
Blood Vessels
Cell Proliferation
Diphtheria Toxin
Disease Models, Animal
HEK293 Cells
Humans
Immunity
Injections, Intradermal
Lipids
Lymphangiogenesis
Lymphatic Vessels
Lymphedema
Mice, Inbred C57BL
Nanoparticles
Nucleosides
Organ Specificity
Poly C
RNA, Messenger
Tamoxifen
Vascular Endothelial Growth Factor C
Mice


BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans

Abstract: BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.

Sahin U
Muik A
Vogler I
Derhovanessian E
Kranz LM
Vormehr M
Quandt J
Bidmon N
Ulges A
Baum A
Pascal KE
Maurus D
Brachtendorf S
Lörks V
Sikorski J
Koch P
Hilker R
Becker D
Eller AK
Grützner J
Tonigold M
Boesler C
Rosenbaum C
Heesen L
Kühnle MC
Poran A
Dong JZ
Luxemburger U
Kemmer-Brück A
Langer D
Bexon M
Bolte S
Palanche T
Schultz A
Baumann S
Mahiny AJ
Boros G
Reinholz J
Szabó GT
Karikó K
Shi PY
Fontes-Garfias C
Perez JL
Cutler M
Cooper D
Kyratsous CA
Dormitzer PR
Jansen KU
Türeci Ö

Adolescent
Adult
Antibodies, Neutralizing
Antibodies, Viral
BNT162 Vaccine
CD8-Positive T-Lymphocytes
COVID-19
COVID-19 Vaccines
Epitopes, T-Lymphocyte
Female
Humans
Immunoglobulin G
Immunologic Memory
Interferon-gamma
Interleukin-2
Male
Middle Aged
SARS-CoV-2
Spike Glycoprotein, Coronavirus
T-Lymphocytes
Th1 Cells
Young Adult

Clinical Trial, Phase I
Clinical Trial, Phase II
Journal Article


Publisher Correction: COVID-19 vaccine BNT162b1 elicits human antibody and T(H)1 T cell responses

Sahin U
Muik A
Derhovanessian E
Vogler I
Kranz LM
Vormehr M
Baum A
Pascal K
Quandt J
Maurus D
Brachtendorf S
Lörks V
Sikorski J
Hilker R
Becker D
Eller AK
Grützner J
Boesler C
Rosenbaum C
Kühnle MC
Luxemburger U
Kemmer-Brück A
Langer D
Bexon M
Bolte S
Karikó K
Palanche T
Fischer B
Schultz A
Shi PY
Fontes-Garfias C
Perez JL
Swanson KA
Loschko J
Scully IL
Cutler M
Kalina W
Kyratsous CA
Cooper D
Dormitzer PR
Jansen KU
Türeci Ö


A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis

Abstract: The ability to control autoreactive T cells without inducing systemic immune suppression is the major goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery of pharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Ψ mRNA) coding for disease-related autoantigens results in antigen presentation on splenic CD11c+ antigen-presenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis, the disease is suppressed by treatment with such m1Ψ mRNA. The treatment effect is associated with a reduction of effector T cells and the development of regulatory T cell (Treg cell) populations. Notably, these Treg cells execute strong bystander immunosuppression and thus improve disease induced by cognate and noncognate autoantigens.

Krienke C
Kolb L
Diken E
Streuber M
Kirchhoff S
Bukur T
Akilli-Öztürk Ö
Kranz LM
Berger H
Petschenka J
Diken M
Kreiter S
Yogev N
Waisman A
Karikó K
Türeci Ö
Sahin U


COVID-19 vaccine BNT162b1 elicits human antibody and T(H)1 T cell responses

Abstract: An effective vaccine is needed to halt the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes the receptor binding domain (RBD) of the SARS-CoV-2 spike protein1. Here we present antibody and T cell responses after vaccination with BNT162b1 from a second, non-randomized open-label phase I/II trial in healthy adults, 18-55 years of age. Two doses of 1-50 μg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those seen in serum from a cohort of individuals who had recovered from COVID-19. Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-μg dose) to 3.5-fold (50-μg dose) those of the recovered individuals. Immune sera broadly neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had T helper type 1 (TH1)-skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to protect against COVID-19 through multiple beneficial mechanisms.

Adult
Antibodies, Neutralizing
Antibodies, Viral
CD8-Positive T-Lymphocytes
COVID-19
COVID-19 Vaccines
Coronavirus Infections
Cytokines
Female
Germany
Humans
Immunoglobulin G
Male
Middle Aged
Pandemics
Pneumonia, Viral
Th1 Cells
Viral Vaccines
Young Adult


A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice

Abstract: SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Laczkó D
Hogan MJ
Toulmin SA
Hicks P
Lederer K
Gaudette BT
Castaño D
Amanat F
Muramatsu H
Oguin TH 3rd
Ojha A
Zhang L
Mu Z
Parks R
Manzoni TB
Roper B
Strohmeier S
Tombácz I
Arwood L
Nachbagauer R
Karikó K
Greenhouse J
Pessaint L
Porto M
Putman-Taylor T
Strasbaugh A
Campbell TA
Lin PJC
Tam YK
Sempowski GD
Farzan M
Choe H
Saunders KO
Haynes BF
Andersen H
Eisenlohr LC
Weissman D
Krammer F
Bates P
Allman D
Locci M
Pardi N

Animals
Antibodies, Neutralizing
Antibodies, Viral
B-Lymphocytes
Betacoronavirus
CD4-Positive T-Lymphocytes
CD8-Positive T-Lymphocytes
COVID-19
COVID-19 Vaccines
Coronavirus Infections
Disease Models, Animal
Furin
Humans
Immunity, Humoral
Immunization
Immunogenicity, Vaccine
Immunologic Memory
Lymphocyte Activation
Mice
Mice, Inbred BALB C
Nanoparticles
Pandemics
Pneumonia, Viral
RNA, Messenger
RNA, Viral
SARS-CoV-2
Spike Glycoprotein, Coronavirus
Vaccines, Synthetic
Viral Vaccines

Journal Article
Research Support, N.I.H., Extramural


Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques

Abstract: Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability.

Pardi N
LaBranche CC
Ferrari G
Cain DW
Tombácz I
Parks RJ
Muramatsu H
Mui BL
Tam YK
Karikó K
Polacino P
Barbosa CJ
Madden TD
Hope MJ
Haynes BF
Montefiori DC
Hu SL
Weissman D


A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA

Abstract: The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications.

Baiersdörfer M
Boros G
Muramatsu H
Mahiny A
Vlatkovic I
Sahin U
Karikó K


In vitro-Transcribed mRNA Therapeutics: Out of the Shadows and Into the Spotlight

Cancer Vaccines
Drug Delivery Systems
Genetic Therapy
Humans
In Vitro Techniques
RNA, Messenger
Transcription, Genetic


The Emerging Role of In Vitro-Transcribed mRNA in Adoptive T Cell Immunotherapy

Abstract: Adoptive T cell therapy is a form of cellular therapy that utilizes human immune cells, often empowered by the expression of recombinant proteins, to attack selected targets present on tumor or infected cells. T cell-based immunotherapy has been progressing over the past several decades, and reached a milestone with the recent US Food and Drug Administration (FDA) approval of chimeric antigen receptor T cell therapy for relapsed and refractory leukemia and lymphoma. Although most studies have used viral vectors, a growing number of researchers have come to appreciate in vitro-transcribed (IVT) mRNA for the development, testing, and application of T cell-based immunotherapeutics. IVT mRNA offers inherent safety features, highly efficient recombinant protein translation, and the ability to control pharmacokinetic properties of the therapy. In this review, we discuss the history of IVT mRNA in adoptive T cell therapy, from tumor-infiltrating lymphocytes and T cell receptor-based therapies to chimeric antigen receptor therapy and gene-editing techniques, as well as prior and ongoing clinical trials.

Animals
Cell- and Tissue-Based Therapy
Gene Editing
Genetic Vectors
Humans
Immunotherapy, Adoptive
In Vitro Techniques
Lymphocytes, Tumor-Infiltrating
Mice
Neoplasms
RNA, Messenger
Receptors, Antigen, T-Cell
Receptors, Chimeric Antigen
T-Lymphocytes
Transcription, Genetic


Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies

Abstract: Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.

Pardi N
Parkhouse K
Kirkpatrick E
McMahon M
Zost SJ
Mui BL
Tam YK
Karikó K
Barbosa CJ
Madden TD
Hope MJ
Krammer F
Hensley SE
Weissman D

Animals
Antibodies, Viral
Cells, Cultured
Dogs
Enzyme-Linked Immunosorbent Assay
Female
Ferrets
Flow Cytometry
Hemagglutinins
Influenza A Virus, H5N1 Subtype
Madin Darby Canine Kidney Cells
Mice
Mice, Inbred BALB C
Orthomyxoviridae
Phylogeny
RNA, Messenger
Rabbits


Purification of mRNA Encoding Chimeric Antigen Receptor Is Critical for Generation of a Robust T-Cell Response

Abstract: T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Foster JB
Choudhari N
Perazzelli J
Storm J
Hofmann TJ
Jain P
Storm PB
Pardi N
Weissman D
Waanders AJ
Grupp SA
Karikó K
Resnick AC
Barrett DM

Adoptive Transfer
Animals
Antigens, CD19
Cell Line, Tumor
Electroporation
Humans
Leukemia
Mice
Mice, Inbred NOD
Mice, Knockout
Mice, SCID
RNA, Messenger
Receptors, Chimeric Antigen
T-Lymphocytes
Xenograft Model Antitumor Assays


Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

Abstract: T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

Pardi N
Hogan MJ
Naradikian MS
Parkhouse K
Cain DW
Jones L
Moody MA
Verkerke HP
Myles A
Willis E
LaBranche CC
Montefiori DC
Lobby JL
Saunders KO
Liao HX
Korber BT
Sutherland LL
Scearce RM
Hraber PT
Tombácz I
Muramatsu H
Ni H
Balikov DA
Li C
Mui BL
Tam YK
Krammer F
Karikó K
Polacino P
Eisenlohr LC
Madden TD
Hope MJ
Lewis MG
Lee KK
Hu SL
Hensley SE
Cancro MP
Haynes BF
Weissman D

Adjuvants, Immunologic
Animals
Antibodies, Neutralizing
Antibody Formation
Antigens
B-Lymphocytes
Germinal Center
Lipids
Macaca mulatta
Nanoparticles
Nucleosides
Protein Subunits
RNA, Messenger
T-Lymphocytes, Helper-Inducer
Time Factors
Vaccination
Vaccines, Subunit

Research Support, U.S. Gov't, Non-P.H.S.


Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

Abstract: The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O3 ), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein-D (SP-D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP-D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate-dependent manner. Combined allergic airway sensitization and O3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S-nitrosylation related de-oligomerisation of SP-D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP-D. Importantly, S-nitrosylation abolished the ability of SP-D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP-D and eosinophils is destroyed by the NO-rich oxidative lung tissue environment in asthma exacerbations.

Yousefi S
Sharma SK
Stojkov D
Germic N
Aeschlimann S
Ge MQ
Flayer CH
Larson ED
Redai IG
Zhang S
Koziol-White CJ
Karikó K
Simon HU
Haczku A

Pulmonary Surfactant-Associated Protein D


Erratum: Elimination of large tumors in mice by mRNA-encoded bispecific antibodies

Abstract: This corrects the article DOI: 10.1038/nm.4356.

Stadler CR
Bähr-Mahmud H
Celik L
Hebich B
Roth AS
Roth RP
Karikó K
Türeci Ö
Sahin U


Elimination of large tumors in mice by mRNA-encoded bispecific antibodies

Abstract: The potential of bispecific T cell-engaging antibodies is hindered by manufacturing challenges and short serum half-life. We circumvented these limitations by treating mice with in vitro-transcribed pharmacologically optimized, nucleoside-modified mRNA encoding the antibody. We achieved sustained endogenous synthesis of the antibody, which eliminated advanced tumors as effectively as the corresponding purified bispecific antibody. Because manufacturing of pharmaceutical mRNA is fast, this approach could accelerate the clinical development of novel bispecific antibodies.

Animals
Antibodies, Bispecific
Cell Line, Tumor
Cytokines
Enzyme-Linked Immunosorbent Assay
Female
Humans
Immunoblotting
Immunohistochemistry
In Vitro Techniques
Luminescent Measurements
Male
Mice
Mice, Inbred BALB C
Mice, Inbred NOD
Neoplasms
RNA, Messenger
T-Lymphocytes
Tumor Burden
Xenograft Model Antitumor Assays


Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

Abstract: Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg-1 of mRNA into mice results in ∼170 μg ml-1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg-1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml-1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases.

Pardi N
Secreto AJ
Shan X
Debonera F
Glover J
Yi Y
Muramatsu H
Ni H
Mui BL
Tam YK
Shaheen F
Collman RG
Karikó K
Danet-Desnoyers GA
Madden TD
Hope MJ
Weissman D

Animals
Antibodies, Monoclonal
Antibodies, Neutralizing
Broadly Neutralizing Antibodies
Drug Administration Schedule
Female
HIV Antibodies
HIV Infections
HIV-1
Humans
Immunization, Passive
Lipids
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Mice, Inbred NOD
Mice, SCID
Mice, Transgenic
Nanoparticles
Nucleosides
RNA, Messenger


Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Abstract: Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Pardi N
Hogan MJ
Pelc RS
Muramatsu H
Andersen H
DeMaso CR
Dowd KA
Sutherland LL
Scearce RM
Parks R
Wagner W
Granados A
Greenhouse J
Walker M
Willis E
Yu JS
McGee CE
Sempowski GD
Mui BL
Tam YK
Huang YJ
Vanlandingham D
Holmes VM
Balachandran H
Sahu S
Lifton M
Higgs S
Hensley SE
Madden TD
Hope MJ
Karikó K
Santra S
Graham BS
Lewis MG
Pierson TC
Haynes BF
Weissman D

Animals
Antibodies, Neutralizing
Antibodies, Viral
Antigens, Viral
Female
Glycoproteins
Injections, Intradermal
Macaca mulatta
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Nanoparticles
RNA Stability
RNA, Messenger
RNA, Viral
Time Factors
Vaccination
Viral Envelope Proteins
Viral Vaccines
Zika Virus
Zika Virus Infection

Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't


Measuring Hematocrit in Mice Injected with In Vitro-Transcribed Erythropoietin mRNA

Abstract: In vitro-transcribed (IVT) mRNA encoding therapeutic protein has the potential to treat a variety of diseases by serving as template for translation in the patient. To optimize conditions for such therapy, reporter protein-encoding mRNAs are usually used. One preferred reporter is erythropoietin (EPO), which stimulates erythropoiesis and leads to an increase in hematocrit. Measurement of hematocrit is a fast and reliable method to determine the potency of the in vitro-transcribed EPO mRNA. However, frequent blood draw from mice can increase hematocrit due to blood loss. Therefore, instead of using conventional hematocrit capillary tubes, we adapted glass microcapillaries for hematocrit measurement. Daily monitoring of mice can be accomplished by drawing less than 20 μL of blood, thus avoiding blood loss-related hematocrit increase. Due to the small volume of the withdrawn blood the hematocrit remains the same for mice injected with control mRNA, whereas significant hematocrit increase is measured between day 4 and 20 postinjection for those injected with pseudouridine-modified EPO mRNA. Following hematocrit measurement the microcapillaries are snapped easily to recover plasma for further analyses, including EPO measurement by ELISA.

Methods in molecular biology (Clifton, N.J.)

Animals
Erythrocytes
Erythropoietin
Hematocrit
Humans
In Vitro Techniques
Mice
Mice, Inbred BALB C
RNA, Messenger


Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage

Abstract: In vitro-synthesized mRNA containing nucleoside modifications has great therapeutical potential to transiently express proteins with physiological importance. One such protein is photolyase which rapidly removes UV-induced DNA damages, but this enzyme is absent in humans. Here, we apply a novel mRNA-based platform to achieve functional nonhuman photolyase production in cultured human keratinocytes. Transfection of nucleoside-modified mRNA encoding photolyase leads to accelerated repair of DNA photolesions in human keratinocytes.

Boros G
Karikó K
Muramatsu H
Miko E
Emri E
Hegedűs C
Emri G
Remenyik É

Cell Line
DNA
DNA Damage
DNA Repair
Deoxyribodipyrimidine Photo-Lyase
Humans
Keratinocytes
Nucleosides
RNA, Messenger
Transfection


mRNA: Fulfilling the Promise of Gene Therapy

Animals
Gene Expression
Genetic Therapy
Humans
RNA, Messenger


Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

Abstract: In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins.

Journal of controlled release : official journal of the Controlled Release Society

Pardi N
Tuyishime S
Muramatsu H
Kariko K
Mui BL
Tam YK
Madden TD
Hope MJ
Weissman D

Animals
Cells, Cultured
Dendritic Cells
Female
HEK293 Cells
Humans
Kinetics
Luciferases, Firefly
Lung
Mice, Inbred BALB C
Nanoparticles
Phosphatidylethanolamines
Pseudouridine
RNA, Messenger
Transfection


Identification of Cyclobutane Pyrimidine Dimer-Responsive Genes Using UVB-Irradiated Human Keratinocytes Transfected with In Vitro-Synthesized Photolyase mRNA

Abstract: Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases.

Boros G
Miko E
Muramatsu H
Weissman D
Emri E
van der Horst GT
Szegedi A
Horkay I
Emri G
Karikó K
Remenyik É

Animals
Cell Line
Cyclin E
Cyclin-Dependent Kinase Inhibitor p15
DNA Repair
Deoxyribodipyrimidine Photo-Lyase
Gene Expression Regulation
Humans
JNK Mitogen-Activated Protein Kinases
Keratinocytes
MAP Kinase Signaling System
Oligonucleotide Array Sequence Analysis
Oncogene Proteins
Potoroidae
Pyrimidine Dimers
RNA, Messenger
Real-Time Polymerase Chain Reaction
Reproducibility of Results
Stress, Physiological
Transcription, Genetic
Transfection
Ultraviolet Rays


mRNA-based therapeutics--developing a new class of drugs

Abstract: In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

Animals
Drug Delivery Systems
Drug Design
Humans
Immunotherapy
Pluripotent Stem Cells
Proteins
RNA, Messenger


Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential

Abstract: UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine.

Journal of photochemistry and photobiology. B, Biology

Boros G
Miko E
Muramatsu H
Weissman D
Emri E
Rózsa D
Nagy G
Juhász A
Juhász I
van der Horst G
Horkay I
Remenyik É
Karikó K
Emri G

Animals
Cell Line
Cell Proliferation
DNA Repair
Deoxyribodipyrimidine Photo-Lyase
Humans
Interleukin-6
Keratinocytes
Light
Lipids
Potoroidae
Pseudouridine
Pyrimidine Dimers
RNA, Messenger
Transfection
Ultraviolet Rays


HPLC purification of in vitro transcribed long RNA

Abstract: In vitro transcription of DNA with phage RNA polymerases is currently the most efficient method to produce long sequence-specific RNA. While the reaction can yield large quantities of RNA, it contains impurities due to various unwanted activities of the polymerases. Here, we described an easily performed HPLC purification that removes multiple contaminants from in vitro transcribed RNA and is scalable. The purified RNA is translated at much greater levels, especially in primary cells and in vivo. HPLC purification of RNA containing modified nucleosides that suppress RNA-mediated activation of innate immune sensors leads to a non-immunogenic RNA with superior translational capacity.

Chromatography, High Pressure Liquid
DNA
DNA-Directed RNA Polymerases
Nucleosides
RNA
Transcription, Genetic


In vitro transcription of long RNA containing modified nucleosides

Abstract: The in vitro synthesis of long RNA can be accomplished using phage RNA polymerase and template DNA. However, the in vitro synthesized RNA, unlike those transcribed in vivo in cells, lacks nucleoside modifications. Introducing modified nucleosides into in vitro transcripts is important because they reduce the potential of RNA to activate RNA sensors and translation of such nucleoside-modified RNA is increased in cell lines, primary cells, and after in vivo delivery. Here, we describe the in vitro synthesis of nucleoside-modified RNA with enhanced translational capacity and reduced ability to activate immune sensors.

Animals
Bacteriophages
Cell Line
DNA-Directed RNA Polymerases
Genetic Engineering
Humans
Nucleosides
RNA
Transfection
Viral Proteins

Journal Article
Research Support, N.I.H., Extramural
Review