RESUMO
Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.
Assuntos
HIV-1/imunologia , Latência Viral/imunologia , Latência Viral/fisiologia , Animais , Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Modelos Animais de Doenças , Infecções por HIV/virologia , Soropositividade para HIV/tratamento farmacológico , HIV-1/patogenicidade , Humanos , Camundongos , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/imunologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/imunologia , Ativação Viral , Replicação ViralRESUMO
Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma , Edição de RNA/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA/genética , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Fatores de Transcrição/genéticaRESUMO
Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.
Assuntos
Proteínas de Transporte/genética , Pegada de DNA/métodos , Genômica/métodos , Proteínas Nucleares/genética , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Sítios de Ligação , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Elementos Facilitadores Genéticos , Eritrócitos/fisiologia , Eritropoese , Genoma Humano , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismoRESUMO
Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.
Assuntos
Biotecnologia/métodos , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Marcação de Genes/métodos , Genoma de Planta/genética , Zea mays/genética , Dedos de Zinco , Desoxirribonucleases/genética , Alimentos Geneticamente Modificados , Genes de Plantas/genética , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Hereditariedade , Fosfatos de Inositol/metabolismo , Mutagênese Sítio-Dirigida/métodos , Plantas Geneticamente Modificadas , Recombinação Genética/genética , Reprodutibilidade dos TestesRESUMO
Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/terapia , Fatores de Transcrição/metabolismo , Dedos de Zinco/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Insuficiência Cardíaca/genética , Humanos , Cinética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Dedos de Zinco/genéticaRESUMO
Neuronal tau reduction confers resilience against ß-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.
Assuntos
Doença de Alzheimer , Fatores de Transcrição , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Camundongos , Placa Amiloide/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
INTRODUCTION: Cutaneous body image (CBI) is a construct encompassing how individuals perceive their hair, skin, and nails. Negative CBI has been related to negative psychological outcomes and body image concerns. The first aim of our study was to further validate CBI as a construct. Second, as individuals with dermatologic conditions are at an increased risk for anxiety and depression, the study examined CBI as a mediator of the relationships between having a skin condition and anxiety and depression. METHODS: A convenience sample of clinical participants with dermatologist-validated diagnoses of psoriasis, atopic dermatitis, or acne who were currently taking systemic medication (n = 128) were matched to a sample of comparison participants without skin conditions (n = 128) on self-reported gender, ethnicity, developmental stage, and weight status (body mass index). All participants reported on their CBI, self-esteem (global, appearance-related, and weight-related), body dissatisfaction, drive for thinness, dietary restraint, anxiety, depression, and demographic characteristics. RESULTS: Cutaneous body image was more negative in those respondents with skin conditions (regression analysis B = - 0.61, standard error 0.23, p = 0.008), demonstrating the criterion-related validity of the measure. CBI was significantly correlated with global (r = 0.39, p < 0.001) and appearance-related self-esteem (r = 0.50, p < 0.001), which establishes convergent validity. CBI was not significantly related to a drive for thinness (r = - 0.12, p = 0.06) or to dietary restraint (r = - 0.05, p = 0.39), supporting discriminant validity. CBI mediated the relationships between having a dermatologic condition and anxiety [point estimate of indirect effect 0.07, 95% confidence interval (CI) 0.02, 0.15] and depression (point estimate of indirect effect 0.04, 95% CI 0.01, 0.08). CONCLUSIONS: The measure of CBI has been further validated. Dermatologists must be aware that various dermatoses may impact patient mental health via the mechanism of negative CBI.
RESUMO
Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.
Assuntos
Clivagem do DNA , Edição de Genes/métodos , Linfócitos T , Sequência de Bases , DNA/genética , DNA/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas , Humanos , Células K562 , Domínios Proteicos , RNA MensageiroRESUMO
Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Edição de Genes/métodos , Genoma Humano , Engenharia de Proteínas/métodos , Nucleases de Dedos de Zinco/genética , Pareamento de Bases , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Biblioteca Genômica , Humanos , Mutação INDEL , Células K562 , Biblioteca de Peptídeos , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Nucleases de Dedos de Zinco/metabolismoRESUMO
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
Assuntos
Alelos , Proteína Huntingtina/genética , Doença de Huntington/terapia , Mutação , Transcrição Gênica , Dedos de Zinco , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neuroproteção , Repetições de TrinucleotídeosRESUMO
BACKGROUND: With the estimated increase in the number of Americans with vision-related disabilities on the horizon, the need for optometrists with expertise in low vision rehabilitation services will increase. State optometric certification in low vision rehabilitation is currently only available in 3 states through affiliations between the state optometric associations and other organizations or government entities. METHODS: A mail survey was conducted among Michigan Optometric Association member optometrists designed to address the current percentage of optometrists who practice low vision rehabilitation as well as the percentage who are low vision certified through the Michigan Optometric Association and to establish future need for additional low vision rehabilitation providers in the state. RESULTS: Of the 188 participants, only 26.0% stated that they provide low vision rehabilitation services, the majority of whom provide only primary care low vision rehabilitation. Only 6.4% of respondents are certified low vision rehabilitation specialists through the Michigan Optometric Association. CONCLUSION: The low percentage of optometrists in the state of Michigan who practice low vision rehabilitation may lead to a shortage of providers for the aging and visually impaired Michigan population seeking low vision rehabilitation services. Even fewer are certified low vision providers through the voluntary Michigan Optometric Association certification process.
Assuntos
Certificação/legislação & jurisprudência , Atenção à Saúde/normas , Pessoas com Deficiência/reabilitação , Pessoal de Saúde/legislação & jurisprudência , Optometria , Inquéritos e Questionários , Baixa Visão/reabilitação , Humanos , Michigan , Optometria/legislação & jurisprudência , Recursos HumanosRESUMO
Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
Assuntos
Técnicas de Química Combinatória/métodos , Engenharia Genética , Mutagênese Sítio-Dirigida/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/genética , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Genoma , Humanos , Células K562 , Dados de Sequência Molecular , Receptores CCR5/genética , Fator A de Crescimento do Endotélio Vascular/genética , XanthomonasRESUMO
Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).