RESUMO
We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed 'plasmid-chromatin' deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (â¼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.
Assuntos
Anticorpos Biespecíficos/metabolismo , Sistemas CRISPR-Cas , Cromatina/metabolismo , Edição de Genes/métodos , Plasmídeos/genética , Anticorpos Biespecíficos/imunologia , Cromatina/genética , DNA/genética , DNA/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Transgenes/genéticaRESUMO
Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that â¼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.
Assuntos
Técnicas de Silenciamento de Genes , Oligonucleotídeos/análise , Núcleo Celular/genética , Recuperação de Fluorescência Após Fotodegradação , Humanos , Células MCF-7 , Microinjeções , Microscopia de Fluorescência , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/análiseRESUMO
Etiology and pharmacotherapy of stress-related psychiatric conditions and somatoform disorders are areas of high unmet medical need. Stressors holding chronic plus psychosocial components thereby bear the highest health risk. Although the metabotropic glutamate receptor subtype 5 (mGlu5) is well studied in the context of acute stress-induced behaviors and physiology, virtually nothing is known about its potential involvement in chronic psychosocial stress. Using the mGlu5 negative allosteric modulator CTEP (2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4yl]ethynyl]pyridine), a close analogue of the clinically active drug basimglurant - but optimized for rodent studies, as well as mGlu5-deficient mice in combination with a mouse model of male subordination (termed CSC, chronic subordinate colony housing), we demonstrate that mGlu5 mediates multiple physiological, immunological, and behavioral consequences of chronic psychosocial stressor exposure. For instance, CTEP dose-dependently relieved hypothalamo-pituitary-adrenal axis dysfunctions, colonic inflammation as well as the CSC-induced increase in innate anxiety; genetic ablation of mGlu5 in mice largely reproduced the stress-protective effects of CTEP and additionally ameliorated CSC-induced physiological anxiety. Interestingly, CSC also induced an upregulation of mGlu5 in the hippocampus, a stress-regulating brain area. Taken together, our findings provide evidence that mGlu5 is an important mediator for a wide range of chronic psychosocial stress-induced alterations and a potentially valuable drug target for the treatment of chronic stress-related pathologies in man.
Assuntos
Imidazóis/uso terapêutico , Piridinas/uso terapêutico , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Estresse Psicológico/psicologia , Hormônio Adrenocorticotrópico/sangue , Animais , Ansiedade/etiologia , Ansiedade/psicologia , Doença Crônica , Dominação-Subordinação , Relação Dose-Resposta a Droga , Febre/etiologia , Febre/fisiopatologia , Hidrocortisona/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Glutamato Metabotrópico 5/genética , Meio Social , Regulação para CimaRESUMO
TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with "knob"-mutations, and the variable region of the light chain (VL) fused to CH3 with matching "holes". The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent "in between" these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads.
Assuntos
Anticorpos Biespecíficos , Fragmentos Fab das Imunoglobulinas , Imunoglobulina G , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos/imunologia , Sítios de Ligação , Dissulfetos/química , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Epitopos/imunologia , Engenharia Genética , Humanos , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , TemperaturaRESUMO
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Antígenos de Histocompatibilidade Menor/genética , Proteínas/genética , Proteínas Supressoras de Tumor/genética , Alelos , Toxina Diftérica/administração & dosagem , Técnicas de Inativação de Genes/métodos , Vetores Genéticos/genética , Histidina/análogos & derivados , Histidina/biossíntese , Humanos , Células MCF-7 , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas/metabolismo , Puromicina/administração & dosagem , Transfecção/métodos , Transgenes/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1â¶50. Reproducibility assays showed a variation below 25% at dilutions less than 1â¶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.