RESUMO
Recombinant adeno-associated virus (AAV) vectors are the leading platform for gene delivery for a variety of clinical applications. Patients with preexisting antibodies to AAV are currently excluded from most AAV gene therapy trials to avoid vector neutralization and ensure response to therapy. Anti-AAV neutralizing antibodies (NAbs) are typically assessed by in vitro cell-based transduction inhibition (TI) assays. However, clinical relevance of the determined enrollment cutoff and the inherent variability of a cell-based assay present challenges for use as an enrollment screening test. Here, we describe an enrollment cutoff that was clinically validated and strategies to overcome assay challenges to enable long-term stable performance. A validated anti-AAV6 cell-based TI assay was used to support clinical enrollment across multiple investigational gene therapies and to evaluate AAV6 seroprevalence in healthy and disease populations. The clinical enrollment cutoff was determined statistically using samples collected from healthy donors, applying a 0.1% false error rate with the inclusion of a minimum significant ratio (MSR) metric and in consideration of results from in vivo mouse passive transfer studies. Our strategy for long-term monitoring and control of assay performance employed plate quality control samples flanking the predefined cutoff. An approach using donor samples was implemented to bridge different lots of critical reagents without the need to redefine the cutoff.
Assuntos
Anticorpos Neutralizantes , Vetores Genéticos , Camundongos , Animais , Estudos Soroepidemiológicos , Vetores Genéticos/genética , Terapia Genética/métodos , Transgenes , Dependovirus , Anticorpos AntiviraisRESUMO
Zinc-finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis I (MPS I) (n = 3), MPS II (n = 9), and hemophilia B (n = 1). Treatment was well tolerated with no serious treatment-related adverse events. At the 1e13 vg/kg dose, evidence of genome editing was detected through albumin-transgene fusion transcripts in liver for MPS II (n = 2) and MPS I (n = 1) subjects. The MPS I subject also had a transient increase in leukocyte iduronidase activity to the lower normal range. At the 5e13 vg/kg dose, one MPS II subject had a transient increase in plasma iduronate-2-sulfatase approaching normal levels and one MPS I subject approached mid-normal levels of leukocyte iduronidase activity with no evidence of genome editing. The hemophilia B subject was not able to decrease use of factor IX concentrate; genome editing could not be assessed. Overall, ZFN in vivo editing therapy had a favorable safety profile with evidence of targeted genome editing in liver, but no long-term enzyme expression in blood.
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Nucleases de Dedos de Zinco , HumanosRESUMO
Fabry disease, a lysosomal storage disorder resulting from the deficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted in vivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with <10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated in vivo genome editing has the potential to be an effective one-time therapy for Fabry disease.
Assuntos
Doença de Fabry/genética , Doença de Fabry/terapia , Edição de Genes , Hepatócitos/metabolismo , Nucleases de Dedos de Zinco/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , Animais , Dependovirus/genética , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Técnicas de Transferência de Genes , Engenharia Genética , Terapia Genética , Vetores Genéticos/genética , Humanos , Camundongos , TransgenesRESUMO
Mucopolysaccharidosis type I (MPS I) is a severe disease due to deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA) and the subsequent accumulation of the glycosaminoglycans (GAG), leading to progressive, systemic disease and a shortened lifespan. Current treatment options consist of hematopoietic stem cell transplantation, which carries significant mortality and morbidity risk, and enzyme replacement therapy, which requires lifelong infusions of replacement enzyme; neither provides adequate therapy, even in combination. A novel in vivo genome-editing approach is described in the murine model of Hurler syndrome. A corrective copy of the IDUA gene is inserted at the albumin locus in hepatocytes, leading to sustained enzyme expression, secretion from the liver into circulation, and subsequent uptake systemically at levels sufficient for correction of metabolic disease (GAG substrate accumulation) and prevention of neurobehavioral deficits in MPS I mice. This study serves as a proof-of-concept for this platform-based approach that should be broadly applicable to the treatment of a wide array of monogenic diseases.
Assuntos
Edição de Genes/métodos , Terapia Genética/métodos , Mucopolissacaridose I/terapia , Nucleases de Dedos de Zinco/metabolismo , Animais , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Feminino , Glicosaminoglicanos/metabolismo , Iduronidase/metabolismo , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Masculino , Camundongos , Mucopolissacaridose I/tratamento farmacológico , Mucopolissacaridose I/metabolismo , Nucleases de Dedos de Zinco/genéticaRESUMO
OBJECTIVE: To determine a just and consistent practice for creating nursing assignments. BACKGROUND: Traditional methods of assigning patients to nurses may lead to unbalanced nursing workload. This article describes the ongoing, hospital-wide effort to evaluate and implement a nursing assignment tool based on electronic health record (EHR) functionality and auto-calculated nursing workload scores. METHODS: EHR records of individual patient workload scores from all hospital units were collected from August 2017 to June 2018. A nurse-specific total workload score was summed for each staff. Then, each hospital unit's mean nurse workload score and standard deviation, along with the unit's nurse-to-patient ratio, were used to calculate levels of high, medium, and low nursing workload measurement (NWM). RESULTS: Mean patient-specific workload scores varied greatly across hospital units. Unit-specific nurse-to-patient ratios were factored into NWM scores to create ranges for assignments that were relatively consistent across the institution. CONCLUSION: The use of objective, electronically generated nursing workload scores, combined with traditional nurse-to-patient ratios, provides accurate real-time nurse staffing needs that can inform best practice in staffing. The confirmation of individual patient workload scores and an appreciation for the complexity of EHR vendor rules are necessary for successful implementation. Automation ensures patient safety, staff satisfaction, and optimal resource allocation.
Assuntos
Registros Eletrônicos de Saúde , Recursos Humanos de Enfermagem Hospitalar/provisão & distribuição , Admissão e Escalonamento de Pessoal , Qualidade da Assistência à Saúde/normas , Carga de Trabalho/psicologia , Humanos , Pacientes Internados , Pesquisa em Administração de Enfermagem , Admissão e Escalonamento de Pessoal/organização & administração , Admissão e Escalonamento de Pessoal/normas , Índice de Gravidade de Doença , Inquéritos e QuestionáriosRESUMO
Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by deficiency of iduronate 2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs) in tissues of affected individuals, progressive disease, and shortened lifespan. Currently available enzyme replacement therapy (ERT) requires lifelong infusions and does not provide neurologic benefit. We utilized a zinc finger nuclease (ZFN)-targeting system to mediate genome editing for insertion of the human IDS (hIDS) coding sequence into a "safe harbor" site, intron 1 of the albumin locus in hepatocytes of an MPS II mouse model. Three dose levels of recombinant AAV2/8 vectors encoding a pair of ZFNs and a hIDS cDNA donor were administered systemically in MPS II mice. Supraphysiological, vector dose-dependent levels of IDS enzyme were observed in the circulation and peripheral organs of ZFN+donor-treated mice. GAG contents were markedly reduced in tissues from all ZFN+donor-treated groups. Surprisingly, we also demonstrate that ZFN-mediated genome editing prevented the development of neurocognitive deficit in young MPS II mice (6-9 weeks old) treated at high vector dose levels. We conclude that this ZFN-based platform for expression of therapeutic proteins from the albumin locus is a promising approach for treatment of MPS II and other lysosomal diseases.
Assuntos
Metabolismo Energético , Dosagem de Genes , Edição de Genes , Iduronato Sulfatase/genética , Mucopolissacaridose II/genética , Mucopolissacaridose II/metabolismo , Fenótipo , Animais , Biomarcadores , Modelos Animais de Doenças , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Íntrons , Camundongos , Mucopolissacaridose II/patologia , Mucopolissacaridose II/fisiopatologia , Dedos de Zinco/genéticaRESUMO
Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(Δ69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.
Assuntos
Antígenos Virais de Tumores/metabolismo , Proteínas Culina/antagonistas & inibidores , Proteínas Substratos do Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologia , Vírus 40 dos Símios/química , Análise de Variância , Animais , Proteínas Culina/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Microscopia , Microscopia de Fluorescência , Proteólise , Interferência de RNA , Vírus 40 dos Símios/metabolismo , Ubiquitina/metabolismoRESUMO
Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production systems. We developed a baculovirus-based insect cell production system-the SGMO Helper-with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, in vitro and in vivo, compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and in vitro potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.
RESUMO
Chemotherapy often generates intratumoral senescent cancer cells that strongly modify the tumor microenvironment, favoring immunosuppression and tumor growth. We discovered, through an unbiased proteomics screen, that the immune checkpoint inhibitor programmed cell death 1 ligand 2 (PD-L2) is highly upregulated upon induction of senescence in different types of cancer cells. PD-L2 is not required for cells to undergo senescence, but it is critical for senescent cells to evade the immune system and persist intratumorally. Indeed, after chemotherapy, PD-L2-deficient senescent cancer cells are rapidly eliminated and tumors do not produce the senescence-associated chemokines CXCL1 and CXCL2. Accordingly, PD-L2-deficient pancreatic tumors fail to recruit myeloid-derived suppressor cells and undergo regression driven by CD8 T cells after chemotherapy. Finally, antibody-mediated blockade of PD-L2 strongly synergizes with chemotherapy causing remission of mammary tumors in mice. The combination of chemotherapy with anti-PD-L2 provides a therapeutic strategy that exploits vulnerabilities arising from therapy-induced senescence.
Assuntos
Neoplasias Pancreáticas , Animais , Camundongos , Neoplasias Pancreáticas/metabolismo , Linfócitos T CD8-Positivos/patologia , Tolerância Imunológica , Terapia de Imunossupressão , Senescência Celular , Microambiente TumoralRESUMO
BACKGROUND: Negative feedback regulation of insulin signaling involves ubiquitin-dependent degradation of insulin receptor substrate 1 (IRS1). RESULTS: Cullin-RING E3 ubiquitin ligase 7 (CRL7) mediates the ubiquitination of IRS1 in hyperphosphorylated form. CONCLUSION: Multisite IRS1 phosphorylation triggers interactions with CRL7 for ubiquitin modification. SIGNIFICANCE: Insulin signaling is self-restrained when its downstream effector kinases are hyperactivated to trigger the negative feedback inhibition. Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1-260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.
Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Fosforilação , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Kidney damage generates changes at the phenotypic and genotypic levels that allow its monitoring using different biomarkers in blood, urine or serum. Among these biomarkers, kidney failure causes the urine overrepresentation of the alanine aminopeptidase (APN) enzyme. Here, we describe the design of a molecular probe (NB-ALA) based on the Nile Blue fluorophore (NB), which can detect the APN enzyme in urine by simple fluorometric measurements.
Assuntos
Antígenos CD13 , Corantes Fluorescentes , Biomarcadores , Rim , Sondas Moleculares , Nefropatias/diagnósticoRESUMO
Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity.
RESUMO
Cell senescence has recently emerged as a potentially relevant pathogenic mechanism in fibrosing interstitial lung diseases (f-ILDs), particularly in idiopathic pulmonary fibrosis. We hypothesized that senescent human fibroblasts may suffice to trigger a progressive fibrogenic reaction in the lung. To address this, senescent human lung fibroblasts, or their secretome (SASP), were instilled into the lungs of immunodeficient mice. We found that: (1) human senescent fibroblasts engraft in the lungs of immunodeficient mice and trigger progressive lung fibrosis associated to increasing levels of mouse senescent cells, whereas non-senescent fibroblasts do not trigger fibrosis; (2) the SASP of human senescent fibroblasts is pro-senescence and pro-fibrotic both in vitro when added to mouse recipient cells and in vivo when delivered into the lungs of mice, whereas the conditioned medium (CM) from non-senescent fibroblasts lacks these activities; and, (3) navitoclax, nintedanib and pirfenidone ameliorate lung fibrosis induced by senescent human fibroblasts in mice, albeit only navitoclax displayed senolytic activity. We conclude that human senescent fibroblasts, through their bioactive secretome, trigger a progressive fibrogenic reaction in the lungs of immunodeficient mice that includes the induction of paracrine senescence in the cells of the host, supporting the concept that senescent cells actively contribute to disease progression in patients with f-ILDs.
Assuntos
Fibrose Pulmonar Idiopática , Doenças Pulmonares Intersticiais , Humanos , Animais , Camundongos , Compostos de Anilina , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Senescência Celular , Fibrose , Fibroblastos/patologiaRESUMO
Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.
Assuntos
Senescência Celular , Fenótipo Secretor Associado à Senescência , Humanos , Ferro , Rim , FibroseRESUMO
Procalcitonin (PCT)-a diagnostic serum parameter for bacterial infection and sepsis-is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC(50)) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7 µg L(-1) (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R (2): assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Calcitonina/sangue , Calcitonina/imunologia , Precursores de Proteínas/sangue , Precursores de Proteínas/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Reações Antígeno-Anticorpo , Análise Química do Sangue , Peptídeo Relacionado com Gene de Calcitonina , Ensaio de Imunoadsorção Enzimática , Humanos , Ratos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
The nonclinical safety assessments for gene therapies are evolving, leveraging over 20 years of experimental and clinical experience. Despite the growing experience with these therapeutics, there are no approved harmonized global regulatory documents for developing gene therapies with only the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) S12 guidance on nonclinical biodistribution currently under discussion. Several health authorities have issued guidance over the last 15 years on the nonclinical safety aspects for gene therapy products, but many of the recommendations are limited to high-level concepts on nonclinical safety aspects or altogether silent on key topics. Historically, this approach was appropriately vague given our relatively small dataset of nonclinical experience, where a comprehensive and detailed regulatory guidance approach was unlikely to be appropriate to address all scenarios. However, harmonization of key considerations and assumptions can provide a consistent basis for developing the appropriate nonclinical safety development plans for individual programs, reducing uncertainty across regulatory regions and unnecessary animal use. Several key areas of nonclinical safety testing are nearing maturation for a harmonized approach, including species selection, certain aspects of study design, study duration, and unintended genomic integration risks. Furthermore, several emerging topics are unaddressed in current regulatory guidance for gene therapy products, which will become key areas of differentiation for the next generation of therapeutics. These topics include redosing, juvenile/pediatric safety, and reproductive/developmental safety testing, where relevant experience from other modalities can be applied. The rationale and potential study design considerations for these topics will be proposed, acknowledging that certain aspects of gene therapy development are not considered appropriate for harmonization. This article provides an overview of the current nonclinical safety regulatory landscape, summarizes typical nonclinical safety study designs, highlights areas of uncertainty, and discusses emerging topics that warrant consideration. Specific recommendations and perspectives are provided to inform future regulatory discussions and harmonization efforts.
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Terapia Genética , Humanos , Animais , Criança , Distribuição Tecidual , Terapia Genética/efeitos adversosRESUMO
OBJECTIVE: The purpose of this article is to describe the indications and appropriate imaging studies for various jaw tumors and tumorlike lesions, the imaging findings, the differential diagnosis, and appropriate treatment options. CONCLUSION: It is important for radiologists to recognize the indications and appropriate imaging studies for various jaw lesions. Radiography is typically used for first-line imaging. If necessary, it is followed by CT for evaluation of osseous lesions and MRI for characterization of soft-tissue lesions.
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Doenças Maxilomandibulares/diagnóstico , Diagnóstico Diferencial , Humanos , Neoplasias Maxilomandibulares/diagnóstico , Imageamento por Ressonância Magnética , Radiografia Panorâmica , Cintilografia , Tomografia Computadorizada por Raios XRESUMO
ABSTRACT: The former Apollo facility converted enriched uranium hexafluoride into uranium oxide for shipment to nuclear fuel fabrication plants from 1957 to 1983. This paper describes quantification of the source term from the Apollo facility in terms of quantities of uranium released, particle size, and solubility characteristics. Releases occurred through stacks, rooftop vents, and an incinerator that operated from 1964 to 1969. Incidental and accidental releases are addressed as part of this analysis. Atmospheric releases of uranium from plant operations were estimated from stack sampling and production records. Roof vents, both filtered and unfiltered, were the major emission points from the plant. The total estimated release of uranium activity (including 234U, 235U, and 238U) to the air was 28 GBq. Measurements by others found that the releases were primarily associated with large particles and that their solubility was variable but generally low (Class Y). The release estimates presented here and those findings were incorporated into a sophisticated atmospheric transport model to estimate atmospheric concentrations and soil contamination levels due to the releases and to reconstruct historical doses to individuals that lived in the vicinity of the former Apollo facility.
Assuntos
Poluentes Radioativos do Solo , Urânio , Humanos , Pennsylvania , Poluentes Radioativos do Solo/análise , Urânio/análiseRESUMO
Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.
Assuntos
Apoptose , Cardiomiopatias/enzimologia , Miócitos Cardíacos/enzimologia , Transdução de Sinais , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proteínas Culina , Dependovirus , Fibrose , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução GenéticaRESUMO
The former Apollo facility in western Pennsylvania converted enriched uranium hexafluoride into uranium oxide for shipment to nuclear fuel fabrication plants from 1957 to 1983. Atmospheric releases of uranium from plant operations were estimated from stack sampling and production records. Releases occurred through stacks, rooftop vents, and an incinerator that operated from 1964 to 1969. Roof vents that exhausted workplace air was the major emission source from the plant. Total estimated release of uranium activity (including 234U, 235U, and 238U) to the air was 27.9 GBq. Atmospheric transport modeling was performed using a complex terrain model because the plant was located in an incised river valley. Almost two years of meteorological data were collected from a nearby 10-m tower, along with sounding from a collocated sodar. Light mean wind speed (1.56â¯mâ¯s-1) and predominately stable atmospheric conditions frequently resulted in poor dispersion conditions in the facility environs. Environmental sampling included continuous air monitoring data and depth profiles of uranium in soil that was deposited from airborne releases. Soil measurements exhibited a sharp drop-off in uranium concentrations with distance from the facility, indicating that large non-inhalable particles were emitted to the atmosphere. Large particles (~15-25⯵m aerodynamic equivalent diameter) accounted for 17.5% of the total emissions. Soil measurements were used for model calibration and validation, while air measurements were used to evaluate model performance. Air concentrations were generally over-predicted for locations near the facility but showed only a slight positive bias for locations north of the facility. Predicted uranium activity air concentrations from Apollo sources averaged over 34 years were about three times greater than the background gross alpha activity value of 81 µBq m-3 in a ~0.5â¯km2 region surrounding the Apollo facility. The contribution of Apollo uranium to the gross alpha air concentration would have been negligible several kilometers from the facility.