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BACKGROUND: Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized. METHODS: We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing to describe the comprehensive transcriptional and phenotypic landscape of 437 126 monocytes. RESULTS: This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi (major histocompatibility complex class II), monocyte-platelet aggregates, as well as nonclassical, and several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- nonclassical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. CONCLUSIONS: This integrative and cross-species comparative analysis provides a new perspective on the comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in cardiovascular disease and the potential for monocyte subpopulation targeted therapies.
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Doenças Cardiovasculares , Monócitos , Análise de Célula Única , Monócitos/metabolismo , Monócitos/imunologia , Animais , Análise de Célula Única/métodos , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Humanos , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Feminino , Transcriptoma , Fatores de Risco de Doenças Cardíacas , Pessoa de Meia-Idade , Perfilação da Expressão Gênica/métodosRESUMO
Kidneys are intricate three-dimensional structures in the body, yet the spatial and molecular principles of kidney health and disease remain inadequately understood. We generated high-quality datasets for 81 samples, including single-cell, single-nuclear, spot-level (Visium) and single-cell resolution (CosMx) spatial-RNA expression and single-nuclear open chromatin, capturing cells from healthy, diabetic and hypertensive diseased human kidneys. Combining these data, we identify cell types and map them to their locations within the tissue. Unbiased deconvolution of the spatial data identifies the following four distinct microenvironments: glomerular, immune, tubule and fibrotic. We describe the complex organization of microenvironments in health and disease and find that the fibrotic microenvironment is able to molecularly classify human kidneys and offers an improved prognosis compared to traditional histopathology. We provide a comprehensive spatially resolved molecular roadmap of the human kidney and the fibrotic process, demonstrating the clinical utility of spatial transcriptomics.
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Microambiente Celular , Progressão da Doença , Fibrose , Nefropatias , Rim , Análise de Célula Única , Humanos , Rim/patologia , Microambiente Celular/genética , Nefropatias/genética , Nefropatias/patologia , Transcriptoma , Perfilação da Expressão Gênica , MultiômicaRESUMO
Human genetic studies have repeatedly associated SNPs near the gene ADAMTS7 with atherosclerotic cardiovascular disease. Subsequent investigations in mice demonstrated that ADAMTS7 is proatherogenic, induced in response to vascular injury, and alters smooth muscle cell function. However, the mechanisms governing this function and its relationship to atherosclerosis remain unclear. Here, we report the first conditional Adamts7 transgenic mouse in which the gene can be conditionally overexpressed in smooth muscle cells, mimicking its induction in atherosclerosis. We observed that smooth muscle cell Adamts7 overexpression results in a 3.5-fold increase in peripheral atherosclerosis, coinciding with an expansion of smooth muscle foam cells. RNA sequencing of Adamts7 overexpressed primary smooth muscle cells revealed an upregulation in the expression of lipid uptake genes. Subsequent experiments in primary smooth muscle cells demonstrated that increased Spi1 and Cd36 expression leads to increased smooth muscle cell oxLDL uptake. To uncover ADAMTS7 expression in human disease, we have interrogated the largest scRNA-seq dataset of human carotid atherosclerosis. This analysis discovered that endothelial cells had the highest expression level of ADAMTS7 with lesser expression in smooth muscle cells, fibroblasts, and mast cells. Subsequent conditional knockout studies in smooth muscle cells surprisingly showed no change in atherosclerosis, suggesting redundant expression of this secreted factor in the vessel wall. Finally, mice overexpressing Adamts7 in endothelial cells also exhibit increased atherosclerosis, suggesting that multiple vascular cell types can contribute to ADAMTS7-mediated foam cell expansion. In summary, Adamts7 is expressed by multiple vascular cell types in atherosclerosis, and ADAMTS7 promotes oxLDL uptake in smooth muscle cells, increasing smooth muscle foam cell formation and peripheral atherosclerosis in mice.
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BACKGROUND: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. METHODS: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. RESULTS: We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. CONCLUSIONS: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.
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Aterosclerose , Doenças das Artérias Carótidas , Placa Aterosclerótica , Humanos , Células Endoteliais/metabolismo , Aterosclerose/patologia , Placa Aterosclerótica/patologia , Doenças das Artérias Carótidas/patologia , Epitopos/metabolismo , Miócitos de Músculo Liso/metabolismoRESUMO
Spatial transcriptomics (ST) has demonstrated enormous potential for generating intricate molecular maps of cells within tissues. Here we present iStar, a method based on hierarchical image feature extraction that integrates ST data and high-resolution histology images to predict spatial gene expression with super-resolution. Our method enhances gene expression resolution to near-single-cell levels in ST and enables gene expression prediction in tissue sections where only histology images are available.
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For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.
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Proteínas do Capsídeo , Capsídeo , Humanos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Células HEK293 , Vetores Genéticos/genética , Dependovirus/genética , Dependovirus/metabolismoRESUMO
Background: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, we have limited understanding of the comprehensive transcriptional and phenotypical landscape of the cells within these lesions. Methods: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. Results: We identified 25 distinct cell populations each having a unique multi-omic signature, including macrophages, T cells, NK cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Within the macrophage populations, we identified 2 proinflammatory subsets that were enriched in IL1B or C1Q expression, 2 distinct TREM2 positive foam cell subsets, one of which also expressed inflammatory genes, as well as subpopulations displaying a proliferative gene expression signature and one expressing SMC-specific genes and upregulation of fibrotic pathways. An in-depth characterization uncovered several subsets of SMCs and fibroblasts, including a SMC-derived foam cell. We localized this foamy SMC to the deep intima of coronary atherosclerotic lesions. Using CITE-seq data, we also developed the first flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Last, we found that the proportion of efferocytotic macrophages, classically activated endothelial cells, contractile and modulated SMC-derived cell types were reduced, and inflammatory SMCs were enriched in plaques of clinically symptomatic vs. asymptomatic patients. Conclusions: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. This facilitates both the mapping of cardiovascular disease susceptibility loci to specific cell types as well as the identification of novel molecular and cellular therapeutic targets for treatment of the disease.
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BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. METHODS: Through RNA-sequencing of human monocyte-derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA (SIMALR). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR. Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques. RESULTS: RNA-sequencing of human monocyte-derived macrophages identified SIMALR, a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ-stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ-stimulated macrophages showed Netrin-1 (NTN1) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ-stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. CONCLUSIONS: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.
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Aterosclerose , Placa Aterosclerótica , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/metabolismo , Placa Aterosclerótica/metabolismo , Lipopolissacarídeos , Netrina-1 , Células HEK293 , Macrófagos/metabolismo , Aterosclerose/metabolismo , Apoptose , Fator 1 Induzível por HipóxiaRESUMO
Transthyretin amyloidosis (ATTR) is a progressive and fatal disease caused by transthyretin (TTR) amyloid fibril accumulation in tissues, which disrupts organ function. As the TTR protein is primarily synthesized by the liver, liver transplantation can cure familial ATTR but is not an option for the predominant age-related wild-type ATTR. Approved treatment approaches include TTR stabilizers and an RNA-interference therapeutic, but these require regular re-administration. Gene editing could represent an effective one-time treatment. We evaluated adeno-associated virus (AAV) vector-delivered, gene-editing meganucleases to reduce TTR levels. We used engineered meganucleases targeting two different sites within the TTR gene. AAV vectors expressing TTR meganuclease transgenes were first tested in immunodeficient mice expressing the human TTR sequence delivered using an AAV vector and then against the endogenous TTR gene in rhesus macaques. Following a dose of 3 × 1013 genome copies per kilogram, we detected on-target editing efficiency of up to 45% insertions and deletions (indels) in the TTR genomic DNA locus and >80% indels in TTR RNA, with a concomitant decrease in serum TTR levels of >95% in macaques. The significant reduction in serum TTR levels following TTR gene editing indicates that this approach could be an effective treatment for ATTR.
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Neuropatias Amiloides Familiares , Dependovirus , Humanos , Camundongos , Animais , Dependovirus/genética , Dependovirus/metabolismo , Macaca mulatta/genética , Macaca mulatta/metabolismo , Neuropatias Amiloides Familiares/terapia , Neuropatias Amiloides Familiares/tratamento farmacológico , Pré-Albumina/genética , Pré-Albumina/metabolismo , Pré-Albumina/uso terapêutico , RNA/uso terapêuticoRESUMO
Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.
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[This corrects the article DOI: 10.1016/j.omtm.2022.09.017.].
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The isolation of adeno-associated virus (AAV) genomes from biomaterials at the molecular level has traditionally relied on polymerase chain reaction-based and cloning-based techniques. However, when applied to samples containing multiple species, traditional techniques for isolating viral genomes can amplify artificial recombinants and introduce polymerase misincorporation errors. In this study, we describe AAV single-genome amplification (AAV-SGA): a powerful technique to isolate, amplify, and sequence single AAV genomes from mammalian genomic DNA, which can then be used to construct vectors for gene therapy. We used AAV-SGA to precisely isolate 15 novel AAV genomes belonging to AAV clades A, D, and E and the Fringe outgroup. This technique also enables investigations of AAV population dynamics and recombination events to provide insights into virus-host interactions and virus biology. Using AAV-SGA, we identified regional heterogeneity within AAV populations from different lobes of the liver of a rhesus macaque and found evidence of frequent genomic recombination between AAV populations. This study highlights the strengths of AAV-SGA and demonstrates its capability to provide valuable insights into the biology and diversity of AAVs.
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Dependovirus , Terapia Genética , Animais , Dependovirus/genética , Macaca mulatta/genética , Terapia Genética/métodos , Genoma Viral , Reação em Cadeia da Polimerase , Vetores Genéticos/genética , Mamíferos/genéticaRESUMO
The depth of anesthesia monitoring is helpful to guide administrations of general anesthetics during surgical procedures,however, the conventional 2-4 channels electroencephalogram (EEG) derived monitors have their limitations in monitoring conscious states due to low spatial resolution and suboptimal algorithm. In this study, 256-channel high-density EEG signals in 24 subjects receiving three types of general anesthetics (propofol, sevoflurane and ketamine) respectively were recorded both before and after anesthesia. The raw EEG signals were preprocessed by EEGLAB 14.0. Functional connectivity (FC) analysis by traditional coherence analysis (CA) method and a novel sparse representation (SR) method. And the network parameters, average clustering coefficient (ACC) and average shortest path length (ASPL) before and after anesthesia were calculated. The results show SR method find more significant FC differences in frontal and occipital cortices, and whole brain network (p<0.05). In contrast, CA can hardly obtain consistent ASPL in the whole brain network (p>0.05). Further, ASPL calculated by SR for whole brain connections in all of three anesthesia groups increased, which can be a unified EEG biomarker of general anesthetics-induced loss of consciousness (LOC). Therefore FC analysis based on SR analysis has better performance in distinguishing anesthetic-induced LOC from awake state.
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Eletroencefalografia , Propofol , Anestesia Geral , Estado de Consciência , Humanos , Propofol/farmacologia , Sevoflurano/farmacologiaRESUMO
Maple syrup urine disease (MSUD) is a rare, inherited metabolic disorder characterized by a dysfunctional mitochondrial enzyme complex, branched-chain alpha-keto acid dehydrogenase (BCKDH), which catabolizes branched-chain amino acids (BCAAs). Without functional BCKDH, BCAAs and their neurotoxic alpha-keto intermediates can accumulate in the blood and tissues. MSUD is currently incurable and treatment is limited to dietary restriction or liver transplantation, meaning there is a great need to develop new treatments for MSUD. We evaluated potential gene therapy applications for MSUD in the intermediate MSUD (iMSUD) mouse model, which harbors a mutation in the dihydrolipoamide branched-chain transacylase E2 (DBT) subunit of BCKDH. Systemic delivery of an adeno-associated virus (AAV) vector expressing DBT under control of the liver-specific TBG promoter to the liver did not sufficiently ameliorate all aspects of the disease phenotype. These findings necessitated an alternative therapeutic strategy. Muscle makes a larger contribution to BCAA metabolism than liver in humans, but a muscle-specific approach involving a muscle-specific promoter for DBT expression delivered via intramuscular (IM) administration only partially rescued the MSUD phenotype in mice. Combining the muscle-tropic AAV9 capsid with the ubiquitous CB7 promoter via IM or IV injection, however, substantially increased survival across all assessed doses. Additionally, near-normal serum BCAA levels were achieved and maintained in the mid- and high-dose cohorts throughout the study; this approach also protected these mice from a lethal high-protein diet challenge. Therefore, administration of a gene therapy vector that expresses in both muscle and liver may represent a viable approach to treating patients with MSUD.
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Dependovirus/genética , Terapia Genética/métodos , Doença da Urina de Xarope de Bordo/genética , Doença da Urina de Xarope de Bordo/terapia , Fenótipo , Administração Intravenosa , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Masculino , Camundongos , MutaçãoRESUMO
SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.
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Enzima de Conversão de Angiotensina 2 , Tratamento Farmacológico da COVID-19 , Dependovirus , Terapia Genética , Vetores Genéticos , SARS-CoV-2 , Administração Intranasal , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/genética , COVID-19/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMO
Gene disruption via programmable, sequence-specific nucleases represents a promising gene therapy strategy in which the reduction of specific protein levels provides a therapeutic benefit. Proprotein convertase subtilisin/kexin type 9 (PCSK9), an antagonist of the low-density lipoprotein (LDL) receptor, is a suitable target for nuclease-mediated gene disruption as an approach to treat hypercholesterolemia. We sought to determine the long-term durability and safety of PCSK9 knockdown in non-human primate (NHP) liver by adeno-associated virus (AAV)-delivered meganuclease following our initial report on the feasibility of this strategy. Six previously treated NHPs and additional NHPs administered AAV-meganuclease in combination with corticosteroid treatment or an alternative AAV serotype were monitored for a period of up to 3 years. The treated NHPs exhibited a sustained reduction in circulating PCSK9 and LDL cholesterol (LDL-c) through the course of the study concomitant with stable gene editing of the PCSK9 locus. Low-frequency off-target editing remained stable, and no obvious adverse changes in histopathology of the liver were detected. We demonstrate similar on-target nuclease activity in primary human hepatocytes using a chimeric liver-humanized mouse model. These studies demonstrate that targeted in vivo gene disruption exerts a lasting therapeutic effect and provide pivotal data for safety considerations, which support clinical translation.
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Edição de Genes , Lipoproteínas LDL/metabolismo , Pró-Proteína Convertase 9/genética , Animais , Sistemas CRISPR-Cas , Dependovirus/genética , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Lipoproteínas LDL/genética , Fígado/metabolismo , Camundongos , Camundongos Knockout , Primatas , Pró-Proteína Convertase 9/metabolismoRESUMO
Our group previously used adeno-associated viral vectors (AAVs) to express an engineered meganuclease specific for a sequence in the PCSK9 gene (M2PCSK9), a clinical target for treating coronary heart disease. Upon testing this nuclease in non-human primates, we observed specific editing characterized by several insertions and deletions (indels) in the target sequence as well as indels in similar genomic sequences. We hypothesized that high nuclease expression increases off-target editing. Here, we reduced nuclease expression using two strategies. The first was a self-targeting strategy that involved inserting the M2PCSK9 target sequence into the AAV genome that expresses the nuclease and/or fusing the nuclease to a specific peptide to promote its degradation. The second strategy used a shortened version of the parental promoter to reduce nuclease expression. Mice administered with these second-generation AAV vectors showed reduced PCSK9 expression due to the nuclease on-target activity and reduced off-target activity. All vectors induced a stable reduction of PCSK9 in primates treated with self-targeting and short-promoter AAVs. Compared to the meganuclease-expressing parental AAV vector, we observed a significant reduction in off-target activity. In conclusion, we increased the in vivo nuclease specificity using a clinically relevant strategy that can be applied to other genome-editing nucleases.
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Dependovirus/genética , Endonucleases/genética , Edição de Genes , Vetores Genéticos/genética , Lipoproteínas LDL/sangue , Inibidores de PCSK9 , Regiões Promotoras Genéticas , Animais , Humanos , Camundongos , Primatas , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismoRESUMO
The administration of adeno-associated virus (AAV) vectors to nonhuman primates (NHP) via the blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (i.e., <14 days), similar from one to 5 months post-injection, and was less severe after 6 months. Vector purification method had no impact, and all capsids and promoters that we tested resulted in some DRG pathology. The data presented here from five different capsids, five different promoters, and 20 different transgenes suggest that DRG pathology is almost universal after AAV gene therapy in nonclinical studies using NHP. None of the animals receiving a therapeutic transgene displayed any clinical signs. Incorporation of sensitive techniques such as nerve-conduction velocity testing can show alterations in a minority of animals that correlate with the severity of peripheral nerve axonopathy. Monitoring sensory neuropathies in human central nervous system and high-dose IV clinical studies seems prudent to determine the functional consequences of DRG pathology.
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Dependovirus/genética , Gânglios Espinais/patologia , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Condução Nervosa , Animais , Feminino , Gânglios Espinais/metabolismo , Macaca fascicularis , Macaca mulatta , Masculino , Transdução GenéticaRESUMO
PURPOSE: The aims of the study were to identify factors related to (1) adolescents and young adults (AYA) desire to discuss health topics; (2) whether discussions occurred at their last medical visit; and (3) the gap (unmet need) between desire and actual discussion. METHODS: We used data from a nationally representative, cross-sectional online survey of AYA aged 13-26 years (n = 1,509) who had had a visit in the past 2 years. Bivariate analyses examined 11 topics. Multivariable regression identified health care factors and demographic factors related to unmet need across four salient topics (HIV/sexually transmitted infections, alcohol and drug use, tobacco, and contraception). RESULTS: Across 11 topics, unmet need averaged 28% and ranged as high as 60%; unmet need generally increased with AYA age. In multivariable analyses, ever having discussed confidentiality with a health care provider was associated with greater desire to discuss three of four salient topics, increased discussions (four of four topics), and reduced unmet need (two topics). Patient use of a clinical checklist/questionnaire at the last medical visit was associated with an increase in discussions (four topics) and reduced unmet need (four topics). Longer office visits were associated with an increase in discussions (three topics) and reduced unmet need (two topics). Older and minority youth had greater desire for discussions and unmet need. CONCLUSIONS: A considerable gap exists between young people's desire to discuss health topics with their health care providers and actual practice.