RESUMO
Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.
Assuntos
Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Loci Gênicos , Camundongos , Fatores de Transcrição/administração & dosagem , Dedos de ZincoRESUMO
Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , Transativadores/química , Transativadores/metabolismo , DNA/metabolismo , Ligação Proteica , Sequências Repetitivas de Aminoácidos , Ativação TranscricionalRESUMO
BACKGROUND: To adapt to threats in the environment, animals must predict them and engage in defensive behavior. While the representation of a prediction error signal for reward has been linked to dopamine, a neuromodulatory prediction error for aversive learning has not been identified. METHODS: We measured and manipulated norepinephrine release during threat learning using optogenetics and a novel fluorescent norepinephrine sensor. RESULTS: We found that norepinephrine response to conditioned stimuli reflects aversive memory strength. When delays between auditory stimuli and footshock are introduced, norepinephrine acts as a prediction error signal. However, temporal difference prediction errors do not fully explain norepinephrine dynamics. To explain noradrenergic signaling, we used an updated reinforcement learning model with uncertainty about time and found that it explained norepinephrine dynamics across learning and variations in temporal and auditory task structure. CONCLUSIONS: Norepinephrine thus combines cognitive and affective information into a predictive signal and links time with the anticipation of danger.
Assuntos
Norepinefrina , Norepinefrina/metabolismo , Incerteza , Animais , Masculino , Optogenética , Medo/fisiologia , Condicionamento Clássico/fisiologia , Reforço Psicológico , Antecipação Psicológica/fisiologia , Lobo Frontal/metabolismo , Lobo Frontal/fisiologiaRESUMO
Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.
Assuntos
Aminoácidos , Escherichia coli , Humanos , Aminoácidos/genética , Escherichia coli/genética , Fragmentos de Imunoglobulinas , Anticorpos/genéticaRESUMO
The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.
RESUMO
Overexpression of CD74, a type II transmembrane glycoprotein involved in MHC class II antigen presentation, has been reported in many B-cell non-Hodgkin lymphomas (NHLs) and in multiple myeloma (MM). STRO-001 is a site-specific, predominantly single-species antibody-drug conjugate (ADC) that targets CD74 and has demonstrated efficacy in xenograft models of MM and tolerability in non-human primates. Here we report results of preclinical studies designed to elucidate the potential role of STRO-001 in B-cell NHL. STRO-001 displayed nanomolar and sub-nanomolar cytotoxicity in 88% (15/17) of cancer cell lines tested. STRO-001 showed potent cytotoxicity on proliferating B cells while limited cytotoxicity was observed on naïve human B cells. A linear dose-response relationship was demonstrated in vivo for DLBCL models SU-DHL-6 and U2932. Tumor regression was induced at doses less than 5 mg/kg, while maximal activity with complete cures were observed starting at 10 mg/kg. In MCL Mino and Jeko-1 xenografts, STRO-001 starting at 3 mg/kg significantly prolonged survival or induced tumor regression, respectively, leading to tumor eradication in both models. In summary, high CD74 expression levels in tumors, nanomolar cellular potency, and significant anti-tumor in DLBCL and MCL xenograft models support the ongoing clinical study of STRO-001 in patients with B-cell NHL.
Assuntos
Antineoplásicos , Imunoconjugados , Linfoma não Hodgkin , Mieloma Múltiplo , Animais , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/patologia , Linfoma não Hodgkin/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing ß/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.
Assuntos
DNA/química , Imidazóis/química , Nylons/química , Pirróis/química , DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
CD74 is a type II transmembrane glycoprotein that functions as an MHC class II chaperone and displays diverse roles in immune responses. Recently, anti-CD74 immunotherapy has shown promise as an effective treatment strategy for lymphoid neoplasms in preclinical models. Using a human anti-CD74 antibody (SP7219), we defined the expression of CD74 protein in both normal and over 790 neoplastic hematolymphoid tissue samples. We found that CD74 is expressed broadly in normal B-cell compartments including primary and secondary lymphoid follicles and in the thymic medulla. The vast majority of lymphomas expressed CD74, including Hodgkin lymphomas (98%), B-cell lymphomas (96%), extranodal NK/T-cell lymphomas (88%), mature T-cell lymphomas (80%), and plasma cell myeloma (75%). Our findings confirm and expand previous observations regarding the expression of CD74 and suggest that CD74 expression on tumor cells may be directly targeted for immunomodulatory therapy for lymphoid and plasma cell malignancies.
Assuntos
Antígenos CD/imunologia , Imunoconjugados/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Linfoma Extranodal de Células T-NK/patologia , Mieloma Múltiplo/tratamento farmacológico , Sialiltransferases/imunologia , Antígenos CD/metabolismo , Genes MHC da Classe II/genética , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/patologia , Humanos , Linfoma de Células B/patologia , Linfoma Extranodal de Células T-NK/tratamento farmacológico , Mieloma Múltiplo/patologia , Sialiltransferases/metabolismoRESUMO
STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.
RESUMO
INTRODUCTION: Brain-derived neurotrophic factor (BDNF) has been implicated in wide range of neurological diseases and injury. This neurotrophic factor is vital for neuronal health, survival, and synaptic connectivity. Many therapies focus on the restoration or enhancement of BDNF following injury or disease progression. AREAS COVERED: The present review will focus on the mechanisms in which BDNF exerts its beneficial functioning, current BDNF therapies, issues and potential solutions for delivery of neurotrophic factors to the central nervous system, and other disease indications that may benefit from overexpression or restoration of BDNF. EXPERT OPINION: Due to the role of BDNF in neuronal development, maturation, and health, BDNF is implicated in numerous neurological diseases making it a prime therapeutic agent. Numerous studies have shown the therapeutic potential of BDNF in a number of neurodegenerative disease models and in acute CNS injury, however clinical translation has fallen short due to issues in delivering this molecule. The use of MSC as a delivery platform for BDNF holds great promise for clinical advancement of neurotrophic factor restoration. The ease with which MSC can be engineered opens the door to the possibility of using this cell-based delivery system to advance a BDNF therapy to the clinic.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Doenças do Sistema Nervoso/tratamento farmacológico , Animais , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Proteínas Recombinantes/uso terapêuticoRESUMO
The specificity of RNA-guided nucleases has gathered considerable interest as they become broadly applied to basic research and therapeutic development. Reports of the simple generation of animal models and genome engineering of cells raised questions about targeting precision. Conflicting early reports led the field to believe that CRISPR/Cas9 system was promiscuous, leading to a variety of strategies for improving specificity and increasingly sensitive methods to detect off-target events. However, other studies have suggested that CRISPR/Cas9 is a highly specific genome-editing tool. This review will focus on deciphering and interpreting these seemingly opposing claims.
Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma , Genômica/métodos , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Primary dysmenorrhea is the most common gynecologic complaint among adolescents. Conventional treatments include nonsteroidal anti-inflammatory drugs and hormonal contraceptives, but complementary and alternative medicine is a growing area of interest. As patients seek such treatments, pediatric nurse practitioners should be aware of these options to offer the best advice to patients.
Assuntos
Terapias Complementares , Dismenorreia/terapia , Adolescente , Terapias Complementares/enfermagem , Dismenorreia/enfermagem , Dismenorreia/fisiopatologia , Feminino , Humanos , Profissionais de Enfermagem Pediátrica , Fatores de RiscoRESUMO
The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2)--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.
Assuntos
Proteínas de Arabidopsis/fisiologia , DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Arabidopsis/isolamento & purificação , Sítios de Ligação , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Humanos , Luciferases/genéticaRESUMO
Variants that alter the DNA-binding specificity of transcription factors could affect the specificity for and expression of potentially many target genes, as has been observed in several tumor-derived mutations. Here we examined if such trans expression quantitative trait loci (trans-eQTLs) could similarly result from common genetic variants. We chose to focus on the Cys2-His2 class of zinc finger transcription factors because they are the most abundant superfamily of transcription factors in human and have well-characterized DNA binding interactions. We identified 430 SNPs that cause missense substitutions in the DNA-contacting residues. Fewer common missense SNPs were found at DNA-contacting residues compared with non-DNA-contacting residues (P = 0.00006), consistent with possible functional selection against SNPs at DNA-contacting positions. Functional predictions based on zinc finger transcription factor (ZNF) DNA binding preferences also suggested that many common substitutions could potentially alter binding specificity. However, Hardy-Weinberg Equilibrium analysis and examination of seven orthologs within the primate lineage failed to find evidence of trans-eQTLs associated with the DNA-contacting positions or evidence of a different selection pressure on a contemporary and evolutionary timescales. The overall conclusion was that common SNPs that alter the DNA-contacting residues of these factors are unlikely to produce strong trans-eQTLs, consistent with the observations by others that trans-eQTLs in humans tend to be few and weak. Some rare SNPs might alter specificity and remained rare due to purifying selection. The study also underscores the need for large-scale eQTLs mapping efforts that might provide experimental evidence for SNPs that alter the choice of transcription factor binding sites.