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African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.
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Adenoviridae , Vírus da Febre Suína Africana , Febre Suína Africana , Vetores Genéticos , Genótipo , Vacinas Virais , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Febre Suína Africana/virologia , Febre Suína Africana/imunologia , Suínos , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/administração & dosagem , Vetores Genéticos/genética , Adenoviridae/genética , Adenoviridae/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/genética , Antígenos Virais/imunologia , Antígenos Virais/genéticaRESUMO
SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of cell-specific AAV capsids and promotors to specifically target ON-BCs for gene delivery. Subretinal injection of one vector demonstrated safety and efficacy with robust and stable rescue of electroretinography signals and night vision up to 1 y, paving the way for clinical trials in patients.
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Doenças Genéticas Ligadas ao Cromossomo X , Cegueira Noturna , Animais , Dependovirus/genética , Cães , Eletrorretinografia , Oftalmopatias Hereditárias , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Humanos , Proteínas de Membrana/genética , Miopia , Cegueira Noturna/genética , Cegueira Noturna/terapiaRESUMO
BACKGROUND: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. METHODS: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. RESULTS: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of SARS-CoV-2 that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. CONCLUSIONS: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.
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COVID-19 , Vírus da Influenza A Subtipo H1N1 , Vírus de RNA , Genoma Viral , Humanos , SARS-CoV-2/genética , Sequenciamento Completo do GenomaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Single-molecule localization microscopy (SMLM), while well established for cultured cells, is not yet fully compatible with tissue-scale samples. We introduce single-molecule oblique-plane microscopy (obSTORM), which by directly imaging oblique sections of samples with oblique light-sheet illumination offers a deep and volumetric SMLM platform that is convenient for standard tissue samples and small intact animals. We demonstrate super-resolution imaging at depths of up to 66 µm for cells, Caenorhabditis elegans gonads, Drosophila melanogaster larval brain, mouse retina and brain sections, and whole stickleback fish.
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Encéfalo/diagnóstico por imagem , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/metabolismo , Peixes/metabolismo , Microscopia de Fluorescência/métodos , Retina/diagnóstico por imagem , Imagem Individual de Molécula/métodos , Células A549 , Animais , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The recent approval of voretigene neparvovec (Luxturna®) for patients with biallelic RPE65 mutation-associated inherited retinal dystrophy with viable retinal cells represents an important step in the development of ocular gene therapies. Herein, we review studies investigating the episomal persistence of different recombinant adeno-associated virus (rAAV) vector genomes and the pre-clinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies. A targeted review of articles published between 1974 and January 2021 in Medline®, Embase®, and other databases, was conducted, followed by a descriptive longitudinal analysis of the clinical trial outcomes of voretigene neparvovec. Following an initial screening, 14 publications examining the episomal persistence of different rAAV genomes and 71 publications evaluating gene therapies in animal models were included. Viral genomes were found to persist for at least 22 months (longest study follow-up) as transcriptionally active episomes. Treatment effects lasting almost a decade were reported in canine disease models, with more pronounced effects the earlier the intervention. The clinical trial outcomes of voretigene neparvovec are consistent with pre-clinical findings and reveal sustained results for up to 7.5 years for the full-field light sensitivity threshold test and 5 years for the multi-luminance mobility test in the Phase I and Phase III trials, respectively. In conclusion, the therapeutic effect of voretigene neparvovec lasts for at least a decade in animal models and 7.5 years in human subjects. Since retinal cells can retain functionality over their lifetime after transduction, these effects may be expected to last even longer in patients with a sufficient number of outer retinal cells at the time of intervention.
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Müller glial (MG) cells in the zebrafish retina respond to injury by acquiring retinal stem-cell characteristics. Thousands of gene expression changes are associated with this event. Key among these changes is the induction of Ascl1a and Lin28a, two reprogramming factors whose expression is necessary for retina regeneration. Whether these factors are sufficient to drive MG proliferation and subsequent neuronal-fate specification remains unknown. To test this, we conditionally expressed Ascl1a and Lin28a in the uninjured retina of male and female fish. We found that together, their forced expression only stimulates sparse MG proliferation. However, in combination with Notch signaling inhibition, widespread MG proliferation and neuron regeneration ensued. Remarkably, Ascl1 and Lin28a expression in the retina of male and female mice also stimulated sparse MG proliferation, although this was not enhanced when combined with inhibitors of Notch signaling. Lineage tracing in both fish and mice suggested that the proliferating MG generated multipotent progenitors; however, this process was much more efficient in fish than mice. Overall, our studies suggest that the overexpression of Ascl1a and Lin28a in zebrafish, in combination with inhibition of Notch signaling, can phenocopy the effects of retinal injury in Müller glia. Interestingly, Ascl1 and Lin28a seem to have similar effects in fish and mice, whereas Notch signaling may differ. Understanding the different consequences of Notch signaling inhibition in fish and mice, may suggest additional strategies for enhancing retina regeneration in mammals.SIGNIFICANCE STATEMENT Mechanisms underlying retina regeneration in fish may suggest strategies for stimulating this process in mammals. Here we report that forced expression of Ascl1 and Lin28a can stimulate sparse MG proliferation in fish and mice; however, only in fish does Notch signaling inhibition collaborate with Ascl1a and Lin28a to stimulate widespread MG proliferation in the uninjured retina. Discerning differences in Notch signaling between fish and mice MG may reveal strategies for stimulating retina regeneration in mammals.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regeneração Nervosa/fisiologia , Proteínas de Ligação a RNA/metabolismo , Receptores Notch/metabolismo , Retina/fisiologia , Animais , Proliferação de Células/fisiologia , Células Ependimogliais/metabolismo , Feminino , Masculino , Camundongos , Neurogênese/fisiologia , Peixe-ZebraRESUMO
In 2015, a mass die-off of ≈200,000 saiga antelopes in central Kazakhstan was caused by hemorrhagic septicemia attributable to the bacterium Pasteurella multocida serotype B. Previous analyses have indicated that environmental triggers associated with weather conditions, specifically air moisture and temperature in the region of the saiga antelope calving during the 10-day period running up to the event, were critical to the proliferation of latent bacteria and were comparable to conditions accompanying historically similar die-offs in the same areas. We investigated whether additional viral or bacterial pathogens could be detected in samples from affected animals using 3 different high-throughput sequencing approaches. We did not identify pathogens associated with commensal bacterial opportunisms in blood, kidney, or lung samples and thus concluded that P. multocida serotype B was the primary cause of the disease.
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Doenças dos Animais/mortalidade , Antílopes , Doenças dos Animais/epidemiologia , Doenças dos Animais/história , Doenças dos Animais/microbiologia , Animais , Antílopes/microbiologia , Infecções Bacterianas/veterinária , Feminino , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Geografia Médica , História do Século XXI , Cazaquistão/epidemiologia , Masculino , Metagenômica , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: Few resources are available to support caregivers of patients who have survived critical illness; consequently, the caregivers' own health may suffer. We studied caregiver and patient characteristics to determine which characteristics were associated with caregivers' health outcomes during the first year after patient discharge from an intensive care unit (ICU). METHODS: We prospectively enrolled 280 caregivers of patients who had received 7 or more days of mechanical ventilation in an ICU. Using hospital data and self-administered questionnaires, we collected information on caregiver and patient characteristics, including caregiver depressive symptoms, psychological well-being, health-related quality of life, sense of control over life, and effect of providing care on other activities. Assessments occurred 7 days and 3, 6, and 12 months after ICU discharge. RESULTS: The caregivers' mean age was 53 years, 70% were women, and 61% were caring for a spouse. A large percentage of caregivers (67% initially and 43% at 1 year) reported high levels of depressive symptoms. Depressive symptoms decreased at least partially with time in 84% of the caregivers but did not in 16%. Variables that were significantly associated with worse mental health outcomes in caregivers were younger age, greater effect of patient care on other activities, less social support, less sense of control over life, and less personal growth. No patient variables were consistently associated with caregiver outcomes over time. CONCLUSIONS: In this study, most caregivers of critically ill patients reported high levels of depressive symptoms, which commonly persisted up to 1 year and did not decrease in some caregivers. (Funded by the Canadian Institutes of Health Research and others; ClinicalTrials.gov number, NCT00896220.).
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Cuidadores/psicologia , Estado Terminal/enfermagem , Depressão/etiologia , Família/psicologia , Adulto , Idoso , Depressão/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Qualidade de Vida , Estresse PsicológicoRESUMO
Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.
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Córtex Cerebral/metabolismo , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Análise Mutacional de DNA , Dependovirus/genética , Epigênese Genética , Feminino , Biblioteca Gênica , Loci Gênicos , Vetores Genéticos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Retina/metabolismo , Transdução GenéticaRESUMO
Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East, and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa, as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both enzyme-linked immunosorbent assay and virus neutralization, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8+ T cells, S96 induced a higher number of CD4+ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.IMPORTANCE Despite the widespread use of live attenuated PPRV vaccines, this is the first systematic analysis of the immune response elicited in small ruminants. These data will help in the establishment of the immunological determinants of protection, an important step in the development of new vaccines, especially DIVA vaccines using alternative vaccination vectors. This study is also the first controlled test of the ability of the two major vaccines used against virulent PPRV strains from all genetic lineages of the virus, showing conclusively the complete cross-protective ability of these vaccines.
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Anticorpos Antivirais/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Peste dos Pequenos Ruminantes/imunologia , Vírus da Peste dos Pequenos Ruminantes/classificação , Vacinas Virais/imunologia , África , Animais , Ásia , Evolução Molecular , Cabras/imunologia , Índia , Peste dos Pequenos Ruminantes/prevenção & controle , Vírus da Peste dos Pequenos Ruminantes/imunologia , Filogenia , Filogeografia , Ovinos/imunologia , Vacinação/veterinária , Vacinas Atenuadas/classificação , Vacinas Atenuadas/imunologiaRESUMO
We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.
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Modelos Animais de Doenças , Células Fotorreceptoras de Vertebrados/patologia , Mutação Puntual , Retina/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/genética , Animais , Eletrorretinografia , Microscopia , Microscopia Eletrônica , Fenótipo , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Degeneração Retiniana/fisiopatologiaRESUMO
Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes and channelrhodopsin-2 (ChR2)-mediated optical activation, we found that increased spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.
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Interneurônios/fisiologia , Córtex Visual/citologia , Córtex Visual/fisiologia , Percepção Visual/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Channelrhodopsins , Aprendizagem por Discriminação , Camundongos , Modelos Neurológicos , Inibição Neural/fisiologia , Parvalbuminas/metabolismo , Rodopsinas Microbianas/metabolismo , Vigília/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The clinical success of gene replacement therapies in recent years has served as a proof of concept for the treatment of inherited retinal degenerations using adeno-associated virus (AAV) as viral vector. However, inherited retinal degenerative diseases showcase a broad genetic and mechanistic heterogeneity, challenging the development of mutation-specific therapies for each specific mutation. Mutation-independent approaches must be developed to slow down retinal degeneration regardless of the underlying genetic mutation and onset of the disease. New understanding of cell death mechanisms in rod-cone dystrophies have led to promising rescue of photoreceptor cell death by virally mediating expression of anti-apoptotic factors and secretion of retinal neurotrophic factors. Optogenetic therapies are also able to restore light sensitivities in blind retinas.
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Distrofias de Cones e Bastonetes/terapia , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Optogenética/métodos , Morte Celular , Channelrhodopsins/genética , Channelrhodopsins/uso terapêutico , Distrofias de Cones e Bastonetes/genética , Dependovirus/genética , Progressão da Doença , Células Ependimogliais/metabolismo , Humanos , Mutação , Fatores de Crescimento Neural/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Transdução GenéticaRESUMO
Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.
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Células Ependimogliais/fisiologia , Proteínas do Tecido Nervoso/genética , Retinose Pigmentar/genética , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Terapia Genética , Células HEK293 , Humanos , Injeções Intravítreas , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Retina/patologia , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/terapiaRESUMO
RATIONALE: Disability risk groups and 1-year outcome after greater than or equal to 7 days of mechanical ventilation (MV) in medical/surgical intensive care unit (ICU) patients are unknown and may inform education, prognostication, rehabilitation, and study design. OBJECTIVES: To stratify patients for post-ICU disability and recovery to 1 year after critical illness. METHODS: We evaluated a multicenter cohort of 391 medical/surgical ICU patients who received greater than or equal to 1 week of MV at 7 days and 3, 6, and 12 months after ICU discharge. Disability risk groups were identified using recursive partitioning modeling. MEASUREMENTS AND MAIN RESULTS: The 7-day post-ICU Functional Independence Measure (FIM) determined the recovery trajectory to 1-year after ICU discharge and was an independent risk factor for 1-year mortality. The 7-day post-ICU FIM was predicted by age and ICU length of stay. By 2 weeks of MV, ICU patients could be stratified into four disability groups characterized by increasing risk for post ICU disability, ICU and post-ICU healthcare use, and disposition. Patients less than 42 years with ICU length of stay less than 2 weeks had the best function and fewest deaths at 1 year compared with patients greater than 66 years with ICU length of stay greater than 2 weeks who sustained the worst disability and 40% 1-year mortality. Depressive symptoms (17%) and post-traumatic stress disorder (18%) persisted at 1 year. CONCLUSIONS: ICU survivors of greater than or equal to 1 week of MV may be stratified into four disability groups based on age and ICU length of stay. These groups determine 1-year recovery and healthcare use and are independent of admitting diagnosis and illness severity. Clinical trial registered with www.clinicaltrials.gov (NCT 00896220).
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Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.
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Ativação do Canal Iônico , Canais Iônicos/efeitos da radiação , Luz , Células Ganglionares da Retina/efeitos da radiação , Visão Ocular , Animais , Cegueira/fisiopatologia , Canais Iônicos/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Células Ganglionares da Retina/fisiologiaRESUMO
In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.
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Proteínas de Membrana/metabolismo , Células Fotorreceptoras/metabolismo , Retinose Pigmentar/etiologia , Retinose Pigmentar/metabolismo , Animais , Células Ependimogliais/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retinose Pigmentar/genéticaRESUMO
Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination.
Assuntos
Optogenética/métodos , Rodopsina/genética , Visão Ocular/genética , Animais , Dependovirus/genética , Expressão Ectópica do Gene , Potenciais Evocados Visuais/genética , Potenciais Evocados Visuais/efeitos da radiação , Terapia Genética , Vetores Genéticos/genética , Luz , Camundongos , Estimulação Luminosa , Retina/citologia , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução Genética , Percepção VisualRESUMO
Systemic delivery of AAV9 offers the potential for widespread and efficient gene delivery to the retina, and may thus be a useful approach for treatment of disease where intraocular injections are not possible, for syndromes affecting multiple organs, or where early intervention is required. The expression resulting from intravenous injection of AAV9 is more efficient in neonates than adults, and here we characterize the effect of age on retinal transduction of AAV9 in the mouse retina. We find that the pattern of expression in neonatal mice is correlated to the development of the retinal vasculature, and that the area of the retinal transduction as well as the cell types infected vary depending on the age at injection. Furthermore, we demonstrate that sequential injections of AAV9 vectors carrying two different transgenes infect adjacent areas of the retina, providing a larger area of coverage. Lastly, we show that the retina's endogenous spatiotemporal expression pattern of Mfsd2a, a protein associated with the maturation of a functional blood-brain barrier, coincides with suppression of retinal transduction by intravenously-delivered AAV9, suggesting that AAV9 crosses the blood-retina barrier through transcytosis.