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Introduction: Fibroblast growth factor 20 (Fgf20), a member of the Fgf9 subfamily, was identified as an important regulator of bone differentiation and homeostasis processes. However, the role of Fgf20 in bone physiology has not been approached yet. Here we present a comprehensive bone phenotype analysis of mice with functional ablation of Fgf20. Methods: The study conducts an extensive analysis of Fgf20 knockout mice compared to controls, incorporating microCT scanning, volumetric analysis, Fgf9 subfamily expression and stimulation experiment and histological evaluation. Results: The bone phenotype could be detected especially in the area ofâ the lumbar and caudal part of the spine and in fingers. Regarding the spine, Fgf20-/- mice exhibited adhesions of the transverse process of the sixth lumbar vertebra to the pelvis as well as malformations in the distal part of their tails. Preaxial polydactyly and polysyndactyly in varying degrees of severity were also detected. High resolution microCT analysis of distal femurs and the fourth lumbar vertebra showed significant differences in structure and mineralization in both cortical and trabecular bone. These findings were histologically validated and may be associated with the expression of Fgf20 in chondrocytes and their progenitors. Moreover, histological sections demonstrated increased bone tissue formation, disruption of Fgf20-/- femur cartilage, and cellular-level alterations, particularly in osteoclasts. We also observed molar dysmorphology, including root taurodontism, and described variations in mineralization and dentin thickness. Discussion: Our analysis provides evidence that Fgf20, together with other members of the Fgf9 subfamily, plays a crucial regulatory role in skeletal development and bone homeostasis.
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Factores de Crecimiento de Fibroblastos , Ratones Noqueados , Animales , Ratones , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Microtomografía por Rayos X , Huesos/metabolismo , Huesos/patología , Huesos/diagnóstico por imagen , Huesos/anomalías , Calcificación Fisiológica , Masculino , Osteogénesis , Femenino , Ratones Endogámicos C57BL , FenotipoRESUMEN
T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas). Clinical-grade lentiviral vector was used to transduce cells with a bivalent CD19/CD22 CAR, and cell counts and flow cytometry were used to monitor the cells throughout the culture. We observed differences in CD4/CD8 ratio when stimulating with the Cloudz activator, where there was a significant skewing toward CD8 T cells. The naive T cell subset expressing CD62L+CCR7+CD45RA+ was the highest in all donors when stimulating with Dynabeads, whereas effector/effector memory cells were highest when using the Cloudz. Functional assays demonstrated differences in killing of target cells and proinflammatory cytokine secretion, with the highest killing from the Cloudz-stimulated cells among all donors. This study demonstrates that the means by which these stimulatory Abs are presented to T cells contribute to the activation, resulting in differing effects on CAR T cell function. These studies highlight important differences in the final product that should be considered when manufacturing CAR T cells for patients in the clinic.
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Activación de Linfocitos , Receptores Quiméricos de Antígenos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Humanos , Activación de Linfocitos/inmunología , Inmunoterapia Adoptiva/métodos , Linfocitos T CD8-positivos/inmunología , Linfocitos T/inmunología , Fenotipo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Antígenos CD19/inmunología , Antígenos CD19/metabolismoRESUMEN
With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (Tn/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O2 from day 5 onward, we could generate >60% Tn/scm-like cells, with longer time in hypoxia correlating with a higher percentage of Tn/scm-like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells.
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Diferenciación Celular , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Proliferación Celular , Linfocitos T/metabolismo , Linfocitos T/citología , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Linfocitos T CD8-positivos/inmunologíaRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.
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Lectina 2 Similar a Ig de Unión al Ácido Siálico , Linfocitos T , Humanos , Concentración de Iones de Hidrógeno , Linfocitos T/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Proliferación Celular , Técnicas de Cultivo de CélulaRESUMEN
Chimeric antigen receptor T cells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, T cell selection, removes contaminating cell types that can potentially suppress T cell expansion and transduction. While positive selection of CD4/CD8 T cells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients. While negative selection may avoid this drawback, it is virtually absent from good manufacturing practices. Here, we performed both CD4/CD8-positive and -negative clinical scale selections of mononuclear cell apheresis products and generated CD22 CARTs per our ongoing clinical trial (NCT02315612NCT02315612). While the selection process did not yield differences in CART expansion or transduction, positively selected CART exhibited a significantly higher in vitro interferon-γ and IL-2 secretion but a lower in vitro tumor killing rate. Notably, though, CD22 CART generated from both selection protocols efficiently eradicated leukemia in NSG mice, with negatively selected cells exhibiting a significant enrichment in γδ CD22 CART. Thus, our study demonstrates the importance of the initial T cell selection process in clinical CART manufacturing.
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The skeletal system mirrors several processes in the vertebrate body that impact developmental malfunctions, hormonal disbalance, malfunction of calcium metabolism and turn over, and inflammation processes such as arthrosis. X-ray micro computed tomography is a useful tool for 3D in situ evaluation of the skeletal system in a time-related manner, but results depend highly on resolution. Here, we provide the methodological background for a graduated evaluation from whole-body analysis of skeletal morphology and mineralization to high-resolution analysis of femoral and vertebral microstructure. We combine an expert-based evaluation with a machine-learning-based computational approach, including pre-setup analytical task lists. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo microCT scanning and skeletal analysis in mice Basic Protocol 2: Ex vivo high-resolution microCT scanning and microstructural analysis of the femur and L4 vertebra.
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Calcinosis , Animales , Ratones , Microtomografía por Rayos X , Modelos Animales de Enfermedad , Fémur/diagnóstico por imagen , Vértebras LumbaresRESUMEN
BACKGROUND AIMS: Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. METHODS: Viability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. RESULTS: All methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze-thaw process, showing decreased viability. CONCLUSIONS: The study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.
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Leucocitos Mononucleares , Azul de Tripano , Reproducibilidad de los Resultados , Supervivencia Celular , Criopreservación/métodos , Citometría de Flujo/métodosRESUMEN
Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.
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Proteínas del Esmalte Dental , Animales , Ratones , Amelogenina/metabolismo , Proteínas del Esmalte Dental/genéticaRESUMEN
BACKGROUND AIMS: Hematopoietic stem cell transplantation using bone marrow as the graft source is a common treatment for hematopoietic malignancies and disorders. For allogeneic transplants, processing of bone marrow requires the depletion of ABO-mismatched red blood cells (RBCs) to avoid transfusion reactions. Here the authors tested the use of an automated closed system for depleting RBCs from bone marrow and compared the results to a semi-automated platform that is more commonly used in transplant centers today. The authors found that fully automated processing using the Sepax instrument (Cytiva, Marlborough, MA, USA) resulted in depletion of RBCs and total mononuclear cell recovery that were comparable to that achieved with the COBE 2991 (Terumo BCT, Lakewood, CO, USA) semi-automated process. METHODS: The authors optimized the fully automated and closed Sepax SmartRedux (Cytiva) protocol. Three reduction folds (10×, 12× and 15×) were tested on the Sepax. Each run was compared with the standard processing performed in the authors' center on the COBE 2991. Given that bone marrow is difficult to acquire for these purposes, the authors opted to create a surrogate that is more easily obtainable, which consisted of cryopreserved peripheral blood stem cells that were thawed and mixed with RBCs and supplemented with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Baxalta, Westlake Village, CA, USA). This "bone marrow-like" product was split into two starting products of approximately 600 mL, and these were loaded onto the COBE and Sepax for direct comparison testing. Samples were taken from the final products for cell counts and flow cytometry. The authors also tested a 10× Sepax reduction using human bone marrow supplemented with human liquid plasma and RBCs. RESULTS: RBC reduction increased as the Sepax reduction rate increased, with an average of 86.06% (range of 70.85-96.39%) in the 10×, 98.80% (range of 98.1-99.5%) in the 12× and 98.89% (range of 98.80-98.89%) in the 15×. The reduction rate on the COBE ranged an average of 69.0-93.15%. However, white blood cell (WBC) recovery decreased as the Sepax reduction rate increased, with an average of 47.65% (range of 38.9-62.35%) in the 10×, 14.56% (range of 14.34-14.78%) in the 12× and 27.97% (range of 24.7-31.23%) in the 15×. COBE WBC recovery ranged an average of 53.17-76.12%. Testing a supplemented human bone marrow sample using a 10× Sepax reduction resulted in an average RBC reduction of 84.22% (range of 84.0-84.36%) and WBC recovery of 43.37% (range of 37.48-49.26%). Flow cytometry analysis also showed that 10× Sepax reduction resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells compared with 12× and 15× reduction. Therefore, a 10× reduction rate was selected for the Sepax process. CONCLUSIONS: The fully automated and closed SmartRedux program on the Sepax was shown to be effective at reducing RBCs from "bone marrow-like" products and a supplemented bone marrow product using a 10× reduction rate.
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Médula Ósea , Trasplante de Células Madre Hematopoyéticas , Humanos , Eritrocitos , Trasplante de Células Madre Hematopoyéticas/métodos , Trasplante de Médula Ósea , Citometría de FlujoRESUMEN
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicerâ¢-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
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MicroARNs , Ribonucleasa III , Ratones , Animales , Ribonucleasa III/metabolismo , Interferencia de ARN , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Portadoras/metabolismo , Mamíferos/metabolismoRESUMEN
The presence of Leishmania RNA virus 1 (LRV1) enables Leishmania protozoan parasites to cause more severe disease than the virus-free strains. The structure of LRV1 virus-like particles has been determined previously, however, the structure of the LRV1 virion has not been characterized. Here we used cryo-electron microscopy and single-particle reconstruction to determine the structures of the LRV1 virion and empty particle isolated from Leishmania guyanensis to resolutions of 4.0 Å and 3.6 Å, respectively. The capsid of LRV1 is built from sixty dimers of capsid proteins organized with icosahedral symmetry. RNA genomes of totiviruses are replicated inside the virions by RNA polymerases expressed as C-terminal extensions of a sub-population of capsid proteins. Most of the virions probably contain one or two copies of the RNA polymerase, however, the location of the polymerase domains in LRV1 capsid could not be identified, indicating that it varies among particles. Importance. Every year over 200 000 people contract leishmaniasis and more than five hundred people die of the disease. The mucocutaneous form of leishmaniasis produces lesions that can destroy the mucous membranes of the nose, mouth, and throat. Leishmania parasites carrying Leishmania RNA virus 1 (LRV1) are predisposed to cause aggravated symptoms in the mucocutaneous form of leishmaniasis. Here, we present the structure of the LRV1 virion determined using cryo-electron microscopy.
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Escherichia coli phage SU10 belongs to the genus Kuravirus from the class Caudoviricetes of phages with short non-contractile tails. In contrast to other short-tailed phages, the tails of Kuraviruses elongate upon cell attachment. Here we show that the virion of SU10 has a prolate head, containing genome and ejection proteins, and a tail, which is formed of portal, adaptor, nozzle, and tail needle proteins and decorated with long and short fibers. The binding of the long tail fibers to the receptors in the outer bacterial membrane induces the straightening of nozzle proteins and rotation of short tail fibers. After the re-arrangement, the nozzle proteins and short tail fibers alternate to form a nozzle that extends the tail by 28 nm. Subsequently, the tail needle detaches from the nozzle proteins and five types of ejection proteins are released from the SU10 head. The nozzle with the putative extension formed by the ejection proteins enables the delivery of the SU10 genome into the bacterial cytoplasm. It is likely that this mechanism of genome delivery, involving the formation of the tail nozzle, is employed by all Kuraviruses.
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Bacteriófagos , Fosmet , Podoviridae , Bacteriófagos/genética , Bacteriófagos/metabolismo , ADN Viral/genética , Genoma Viral/genética , Podoviridae/genéticaRESUMEN
L-BC virus persists in the budding yeast Saccharomyces cerevisiae, whereas other viruses from the family Totiviridae infect a diverse group of organisms including protists, fungi, arthropods, and vertebrates. The presence of totiviruses alters the fitness of the host organisms, for example, by maintaining the killer system in yeast or increasing the virulence of Leishmania guyanensis. Despite the importance of totiviruses for their host survival, there is limited information about Totivirus structure and assembly. Here we used cryo-electron microscopy to determine the structure of L-BC virus to a resolution of 2.9 Å. The L-BC capsid is organized with icosahedral symmetry, with each asymmetric unit composed of two copies of the capsid protein. Decamers of capsid proteins are stabilized by domain swapping of the C-termini of subunits located around icosahedral fivefold axes. We show that capsids of 9% of particles in a purified L-BC sample were open and lacked one decamer of capsid proteins. The existence of the open particles together with domain swapping within a decamer provides evidence that Totiviridae capsids assemble from the decamers of capsid proteins. Furthermore, the open particles may be assembly intermediates that are prepared for the incorporation of the virus (+) strand RNA.
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Totivirus , Virus , Animales , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Totivirus/química , Totivirus/genéticaRESUMEN
Objects: Health Behaviours in School-aged Children (HBSC) is an international survey programme aiming to investigate adolescents' health behaviours, subjective perception of health status, wellbeing, and the related contextual information. Our scoping review aimed to synthesise the evidence from HBSC about the relationship between family environmental contributors and adolescents' health-related outcomes. Methods: We searched previous studies from six electronic databases. Two researchers identified the qualified publications independently by abstract and full-text screening with the assistance of an NLP-based AI instrument, ASReview. Publications were included if they were based on HBSC data and investigated the effects of family environment on adolescents' health outcomes. Researches addressed family-related factors as mediators or moderators were also included. Results: A total of 241 articles were included. Family environmental contributors could be mapped into six categories: (1) Demographic backgrounds (N = 177); (2) General family's psycho-socio functions (N = 44); (3) Parenting behaviours (N = 100); (4) Parental health behaviours (N = 7); (5) Family activities (N = 24); and (6) Siblings (N = 7). Except for 75 papers that assessed family variables as moderators (N = 70) and mediators (N = 7), the others suggested family environment was an independent variable. Only five studies employed the data-driven approach. Conclusion: Our results suggest most research studies focussed on the influences of family demographic backgrounds on adolescents' health. The researches related to parental health behaviours and siblings are most inadequate. Besides, we recommend further research studies to focus on the mediator/moderator roles of the family, for exploring the deep mechanism of the family's impacts. Also, it would be valuable to consider data-driven analysis more in the future, as HBSC has mass variables and data.
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BACKGROUND: Cytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS. METHODS: Forty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes. RESULTS: We found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE). CONCLUSION: Our data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.
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COVID-19 , Gripe Humana , COVID-19/terapia , Síndrome de Liberación de Citoquinas , Citocinas/metabolismo , Genómica , Humanos , Inmunoterapia , Inmunoterapia Adoptiva/métodos , Fosfofructoquinasa-2 , Receptores Quiméricos de AntígenosRESUMEN
The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC-MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6 -methyladenosine and 2'-O-methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1-methyladenosine (m1 A) and 5-methylcytidine (m5 C). Mapping of m1 A and m5 C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3' and 5' fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.
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Nucleósidos , ARN de Transferencia , Animales , Abejas/genética , ARN , ARN Mensajero , ARN de Transferencia/genética , Virión/genéticaRESUMEN
Virus-like nanoparticles are protein shells similar to wild-type viruses, and both aim to deliver their content into a cell. Unfortunately, the release mechanism of their cargo/genome remains elusive. Pores on the symmetry axes were proposed to enable the slow release of the viral genome. In contrast, cryo-EM images showed that capsids of nonenveloped RNA viruses can crack open and rapidly release the genome. We combined in vitro cryo-EM observations of the genome release of three viruses with coarse-grained simulations of generic virus-like nanoparticles to investigate the cargo/genome release pathways. Simulations provided details on both slow and rapid release pathways, including the success rates of individual releases. Moreover, the simulated structures from the rapid release pathway were in agreement with the experiment. Slow release occurred when interactions between capsid subunits were long-ranged, and the cargo/genome was noncompact. In contrast, rapid release was preferred when the interaction range was short and/or the cargo/genome was compact. These findings indicate a design strategy of virus-like nanoparticles for drug delivery.
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Nanopartículas , Virus , Cápside , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Genoma ViralRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. METHODS: Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. RESULTS: After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. CONCLUSION: We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.
Asunto(s)
Receptores Quiméricos de Antígenos , Criopreservación/métodos , Citometría de Flujo/métodos , Inmunoterapia Adoptiva/métodos , Control de Calidad , Receptores de Antígenos de Linfocitos T , Reproducibilidad de los Resultados , Linfocitos TRESUMEN
BACKGROUND: Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. METHODS: Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. RESULTS: The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. CONCLUSIONS: Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.