RESUMO
Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.
Assuntos
Sistemas CRISPR-Cas/genética , Testes Genéticos/métodos , Genoma/genética , Células-Tronco Pluripotentes/metabolismo , HumanosRESUMO
CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Engenharia Genética , Células-Tronco Pluripotentes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Deleção de Genes , Humanos , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Verubecestat is a potent BACE1 enzyme inhibitor currently being investigated in Phase III trials for the treatment of mild-to-moderate and prodromal Alzheimer's disease. Multiple anti-amyloid immunotherapies have been dose-limited by adverse amyloid related imaging abnormalities such as vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H) observed in human trials and mice. Verubecestat was tested in a 12-week nonclinical study for the potential to exacerbate microhemorrhage (ARIA-H) profiles in 18-22-month-old post-plaque Tg2576-AßPPswe mice. Animals were treated with verubecestat or controls including the anti-Aß antibody analog of bapineuzumab (3D6) as a positive control for ARIA induction. ARIA-H was measured using in-life longitudinal T2*-MRI and Prussian blue histochemistry at study end. Verubecestat reduced plasma and cerebrospinal fluid Aß40 and Aß42 byâ>90% and 62% to 68%, respectively. The ARIA-H profile of verubecestat-treated mice was not significantly different than controls. Anti-Aß treatment significantly increased ARIA-H detected by Prussian blue staining; however, anti-Aß antibody treatment did not impact plaque status. Verubecestat treatment significantly suppressed the accumulation of total levels of brain Aß40 and Aß42 and Thioflavin S positive plaque load. Stereological analysis of cortex and hippocampus plaque load similarly revealed significantly reduced area of Aß immunoreactivity and reduced plaque number in verubecestat-treated animals compared to controls. The absence of elevated ARIA events in verubecestat-treated mice was associated with a significant reduction in the level of accumulated CNS amyloid pathology and brain Aß peptides; effects consistent with the desired therapeutic mechanism of verubecestat in AD patients. These data will be compared with longitudinal MRI profiles from ongoing clinical trials.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Antipsicóticos/uso terapêutico , Óxidos S-Cíclicos/uso terapêutico , Placa Amiloide/patologia , Tiadiazinas/uso terapêutico , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Colágeno Tipo IV/metabolismo , Óxidos S-Cíclicos/sangue , Óxidos S-Cíclicos/líquido cefalorraquidiano , Modelos Animais de Doenças , Humanos , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/etiologia , Estudos Longitudinais , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Mutação/genética , Placa Amiloide/tratamento farmacológico , Presenilina-1/genética , Tiadiazinas/sangue , Tiadiazinas/líquido cefalorraquidianoRESUMO
BACKGROUND: Relapsing fever (RF) spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. An additional role in tissue localization is suggested by the finding that isogenic serotypes 1 (Bt1) and 2 (Bt2) of the RF spirochete Borrelia turicatae, which differ only in the Vsp they express, exhibit marked differences in clinical disease severity and tissue localization during infection. METHODOLOGY/PRINCIPAL FINDINGS: Here we used known vsp DNA sequences encoding for B. turicatae and Borrelia hermsii Vsp proteins with variable regions and then studied whether there are differences in disease expression and tissue localization of their corresponding serotypes during mouse infection. For sequence and structural comparisons we focused exclusively on amino acid residues predicted to project away from the spirochetes surface, referred to as the Vsp dome. Disease severity and tissue localization were studied during persistent infection with individual or mixed serotypes in SCID mice. The results showed that all Vsp domes clustered into 3 main trunks, with the domes for B. turicatae Vsp1 (BtVsp1) and BtVsp2 clustering into separate ones. B. hermsii serotypes whose Vsp domes clustered with the BtVsp1 dome were less virulent but localized to the brain more. The BtVsp2 dome was the oddball among all and Bt2 was the only serotype that caused severe arthritis. CONCLUSION/SIGNIFICANCE: These findings indicate that there is significant variability in Vsp dome structure, disease severity, and tissue localization among serotypes of B. hermsii.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Borrelia/metabolismo , Spirochaetales/metabolismo , Animais , Análise por Conglomerados , Primers do DNA/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Conformação Molecular , Filogenia , Reação em Cadeia da Polimerase , Spirochaetales/genética , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
BACKGROUND: Neurological involvement is common in the spirochetal infection relapsing fever (RF) in both humans and experimental animals. RF is best known for antigenic variation caused by the sequential expression of variable outer membrane lipoproteins of 2 sizes, variable small (Vsp) and variable large (Vlp) proteins. Less understood is the persistence of RF borreliae in the brain after they are cleared from the blood, referred to as residual brain infection (RBI). Our goal was to investigate the phenomenon of RBI in RF. METHODS: We studied RBI in immunocompetent mice by culturing blood and perfused brain samples 1 month after intraperitoneal inoculation with Borrelia turicatae serotype 1 (Bt1). Mice deficient in Toll-like receptor 2 (TLR2-/-) or in B and T cells (scid) were included for comparison. RESULTS: All scid mice had persistent infection in blood and brain. RBI was found in 3 (19%) of 16 immunocompetent and TLR2-/- mice. RBI was caused by either persistence of the original serotype (Bt1) or newly emerged Vsp (n=1, renamed Bt3) or Vlp serotypes. The Vsp of Bt1 (Vsp1) and Bt3 (Vsp3) were 75% identical. CONCLUSIONS: RBI in RF is relatively frequent and can occur by persistence of the original or newly emerged serotypes.
Assuntos
Borrelia/classificação , Infecções Bacterianas do Sistema Nervoso Central/microbiologia , Febre Recorrente/microbiologia , Animais , Borrelia/genética , Borrelia/patogenicidade , Encéfalo/microbiologia , Encéfalo/patologia , Infecções Bacterianas do Sistema Nervoso Central/sangue , DNA Bacteriano/análise , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Reação em Cadeia da Polimerase , Febre Recorrente/sangue , SorotipagemRESUMO
Relapsing fever (RF) is a spirochetal infection characterized by relapses of a febrile illness and spirochetemia due to the sequential appearance and disappearance of isogenic serotypes in the blood. The only difference between isogenic serotypes is the variable major outer membrane lipoprotein. In the absence of specific antibody, established serotypes cause persistent infection. Studies in our laboratory indicate that another consequence of serotype switching in RF is a change in neuroinvasiveness. As the next step to elucidate this phenomenon, we studied the interaction of the neurotropic Oz1 strain of the RF agent Borrelia turicatae with the cerebral microcirculation. During persistent infection of antibody-deficient mice, we found that serotype 1 entered the brain in larger numbers and caused more severe cerebral microgliosis than isogenic serotype 2. Microscopic examination revealed binding of B. turicatae to brain microvascular endothelial cells in vivo. In vitro we found that B. turicatae associated with brain microvascular endothelial cells (BMEC) significantly more than with fibroblasts or arachnoidal cells. The binding was completely eliminated by pretreatment of BMEC with proteinase K. Using transwell chambers with BMEC barriers, we found that serotype 1 crossed into the lower compartment significantly better than serotype 2. Heat killing significantly reduced BMEC crossing but not binding. We concluded that the interaction of B. turicatae with the cerebral microcirculation involves both binding and crossing brain microvascular endothelial cells, with significant differences among isogenic serotypes.
Assuntos
Borrelia/patogenicidade , Encéfalo/irrigação sanguínea , Circulação Cerebrovascular , Animais , Barreira Hematoencefálica/microbiologia , Borrelia/classificação , Borrelia/fisiologia , Encéfalo/microbiologia , Encéfalo/patologia , Linhagem Celular , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Feminino , Humanos , Neuroborreliose de Lyme/microbiologia , Neuroborreliose de Lyme/patologia , Neuroborreliose de Lyme/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microcirculação/microbiologia , Microcirculação/patologia , Febre Recorrente/microbiologia , Febre Recorrente/patologia , SorotipagemRESUMO
Lyme borreliosis is a multisystemic disease caused by infection with various genospecies of the spirochete Borrelia burgdorferi. The organs most often affected are the skin, joints, the heart, and the central and peripheral nervous systems. Multiple neurological complications can occur, including aseptic meningitis, encephalopathy, facial nerve palsy, radiculitis, myelitis, and peripheral neuropathy. To investigate spinal cord involvement in the nonhuman primate (NHP) model of Lyme borreliosis, we inoculated 25 adult Macaca mulatta with B. burgdorferi sensu strictu strains N40 by needle (N=9) or by tick (N=4) or 297 by needle (N=2), or with B. burgdorferi genospecies garinii strains Pbi (N=4), 793 (N=2), or Pli (N=4) by needle. Immunosuppression either transiently (TISP) or permanently (IS) was used to facilitate establishment of infection. Tissues and fluids were collected at necropsy 7-24 weeks later. Hematoxylin and eosin staining was used to study inflammation, and immunohistochemistry and digital image analysis to measure inflammation and localize spirochetes. The spirochetal load and C1q expression were measured by TaqMan RT-PCR. The results showed meningoradiculitis developed in only one of the 25 NHP's examined, TISP NHP 321 inoculated with B. garinii strain Pbi. Inflammation was localized to nerve roots, dorsal root ganglia, and leptomeninges but rarely to the spinal cord parenchyma itself. T cells and plasma cells were the predominant inflammatory cells. Significantly increased amounts of IgG, IgM, and C1q were found in inflamed spinal cord. Taqman RT-PCR found spirochetes in the spinal cord only in IS-NHP's, mostly in nerve roots and ganglia rather than in the cord parenchyma. C1q mRNA expression was significantly increased in inflamed spinal cord. This is the first comprehensive study of spinal cord involvement in Lyme borreliosis.
Assuntos
Borrelia burgdorferi/fisiologia , Neuroborreliose de Lyme/patologia , Macaca mulatta , Medula Espinal/patologia , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Antibacterianos/análise , Mordeduras e Picadas , Borrelia burgdorferi/isolamento & purificação , Borrelia burgdorferi/patogenicidade , Complemento C1q/metabolismo , Dexametasona/administração & dosagem , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Processamento de Imagem Assistida por Computador , Hospedeiro Imunocomprometido , Técnicas Imunoenzimáticas , Terapia de Imunossupressão , Ixodes/microbiologia , Neuroborreliose de Lyme/imunologia , Neuroborreliose de Lyme/metabolismo , RNA Bacteriano/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/microbiologiaRESUMO
Borrelia burgdorferi sensu lato, the causative organism of Lyme borreliosis, is a heterogeneous group of spirochetes, consisting of at least three pathogenic species. To test the hypothesis that the genetic heterogeneity is the reason for the clinical differences, we investigated whether the experimental disease induced by European isolates is different from that induced by American isolates. Two American isolates of species B. burgdorferi sensu stricto were compared with three European isolates, two of species B. garinii, and one of species B. afzelii. The patterns of infection, immunity, and inflammation induced by the different species was distinctive. Inflammatory cells and levels of antibody in B. garinii- and B. afzelii-infected animals were lower than in B. burgdorferi s.s.-infected animals, whereas levels of spirochetal infection in the skin and nervous system were higher in the former group of animals. These data demonstrate that B. burgdorferi s.s. strains are more infective and inflammatory, whereas B. garinii and B. afzelii strains can survive the adaptive immune response to a greater degree and persist at greater numbers in the skin and nervous system. The results explain to a large extent the disparities between LNB in humans in the United States and Europe.