[Perspectives of genome editing in otorhinolaryngology]. / Perspektiven der Genomeditierung in der Hals-Nasen-Ohren-Heilkunde.

BACKGROUND: Recent advances in DNA sequencing technology have enabled researchers to identify the genetic background underlying human illness. In addition, the latest genome editing technology, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9), provides great potential to edit genomic DNA sequences precisely with high efficiency. This technology has been evaluated for treatment of genetic diseases in recently published preclinical studies. Since many such genetic disorders can affect functional structures in the head and neck area, the technology bears high therapeutic potential in otorhinolaryngology. OBJECTIVE: In this article, we summarize the concept of CRISPR-Cas9-based therapies, recent achievements in preclinical applications, and future challenges for the implementation of this technology in otolaryngology. MATERIALS AND METHODS: Genetic targeting strategies were analyzed or established using genome sequencing data derived from online databases and literature. RESULTS: Recent research on animal models has shown that genome editing can be used to treat genetic diseases by specifically targeting mutant genomic loci. For example, one preclinical study in the field of otolaryngology has demonstrated that inherited autosomal dominant deafness in mice can be treated using CRISPR-Cas9. Moreover, the same strategies can be used to establish applications for the treatment of head and neck cancer. The greatest challenge appears to be establishment of a system for the safe and efficient delivery of therapeutic nucleotides in clinics. CONCLUSIONS: In theory, genome editing could be used in otolaryngology to target disease-causing genomic loci specifically. However, various challenges have to be overcome until applications can be used clinically.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics.

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Many transcription factors interact synergistically with steroid receptors.

Progesterone (PRE) or glucocorticoid receptor (GRE) DNA binding sites are often found clustered with binding sites for other transcription factors. Individual protein binding sites were tested without the influence of adjacent factors by analyzing isolated combinations of several transcription factor binding sites with PREs or GREs. All show strong synergistic effects on steroid induction. The degree of synergism is inversely related to the strength of the GRE. Thus, a steroid responsive unit can be composed of several modules that, if positioned correctly, act synergistically.

Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activated the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation was not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3.

Glucocorticoid-mediated repression of cytokine gene transcription in human arteritis-SCID chimeras.

Giant cell arteritis (GCA) is a vasculitic syndrome that preferentially affects medium and large-sized arteries. Glucocorticoid therapy resolves clinical symptoms within hours to days, but therapy has to be continued over several years to prevent disease relapses. It is not known whether and how glucocorticoids affect the function of the inflammatory infiltrate or why the disease persists subclinically despite chronic treatment. GCA is self-sustained in temporal arteries engrafted into SCID mice, providing a model in which the mechanisms of action and limitations of glucocorticoid therapy can be examined in vivo. Administration of dexamethasone to temporal artery-SCID chimeras for 1 wk induced a partial suppression of T cell and macrophage function as indicated by the reduced tissue concentrations of IL-2, IL-1beta, and IL-6 mRNA, and by the diminished expression of inducible NO synthase. In contrast, synthesis of IFN-gamma mRNA was only slightly decreased, and expression of TGF-beta1 was unaffected. These findings correlated with activation of the IkappaBalpha gene and blockade of the nuclear translocation of NFkappaB in the xenotransplanted tissue. Dose-response experiments suggested that steroid doses currently used in clinical medicine are suboptimal in repressing NFkappaB-mediated cytokine production in the inflammatory lesions. Chronic steroid therapy was able to deplete the T cell products IL-2 and IFN-gamma, whereas the activation of tissue-infiltrating macrophages was only partially affected. IL-1beta transcription was abrogated; in contrast, TGF-beta1 mRNA synthesis was steroid resistant. The persistence of TGF-beta1-transcribing macrophages, despite paralysis of T cell function, may provide an explanation for the chronicity of the disease, and may identify a novel therapeutic target in this inflammatory vasculopathy.

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Constitutive NF-kappa B activity in neurons.

NF-kappa B is inducible transcription factor present in many cell types in a latent cytoplasmic form. So far, only immune cells including mature B cells, thymocytes, and adherent macrophages have been reported to contain constitutively active forms of NF-kappa B in the nucleus. A recent study showed that the human immunodeficiency virus type 1 (HIV-1) promoter is highly active in several brain regions of transgenic mice (J. R. Corboy, J. M. Buzy, M. C. Zink, and J. E. Clements, Science 258:1804-1807, 1992). Since the activity of this viral enhancer is governed mainly by two binding sites for NF-kappa B, we were prompted to investigate the state of NF-kappa B activity in neurons. Primary neuronal cultures derived from rat hippocampus and cerebral cortex showed a high constitutive expression of an HIV-1 long terminal repeat-driven luciferase reporter gene, which was primarily dependent on intact NF-kappa B binding sites and was abolished upon coexpression of the NF-kappa B-specific inhibitor I kappa B-alpha. Indirect immunofluorescence and confocal laser microscopy showed that the activity of NF-kappa B correlated with the presence of the NF-kappa B subunits p50 and RelA (p65) in nuclei of cultured neurons. NF-kappa B was also constitutively active in neurons in vivo. As investigated by electrophoretic mobility shift assays, constitutive NF-kappa B DNA-binding activity was highly enriched in fractions containing neuronal nuclei prepared from rat cerebral cortex. Nuclear NF-kappa B-specific immunostaining was also seen in cryosections from mouse cerebral cortex and hippocampus. Only a subset of neurons was stained. Activated NF-kappa B in the brain is likely to participate in normal brain function and to reflect a distinct state of neuronal activity or differentiation. Furthermore, it may explain the high level of activity of the HIV-1 enhancer in neurons, an observation potentially relevant for the etiology of the AIDS dementia complex caused by HIV infection of the central nervous system.

Mathematical model for NF-kappaB-driven proliferation of adult neural stem cells.

Neural stem cells (NSCs) are early precursors of neuronal and glial cells. NSCs are capable of generating identical progeny through virtually unlimited numbers of cell divisions (cell proliferation), producing daughter cells committed to differentiation. Nuclear factor kappa B (NF-kappaB) is an inducible, ubiquitous transcription factor also expressed in neurones, glia and neural stem cells. Recently, several pieces of evidence have been provided for a central role of NF-kappaB in NSC proliferation control. Here, we propose a novel mathematical model for NF-kappaB-driven proliferation of NSCs. We have been able to reconstruct the molecular pathway of activation and inactivation of NF-kappaB and its influence on cell proliferation by a system of nonlinear ordinary differential equations. Then we use a combination of analytical and numerical techniques to study the model dynamics. The results obtained are illustrated by computer simulations and are, in general, in accordance with biological findings reported by several independent laboratories. The model is able to both explain and predict experimental data. Understanding of proliferation mechanisms in NSCs may provide a novel outlook in both potential use in therapeutic approaches, and basic research as well.

NF-kappa B: a crucial transcription factor for glial and neuronal cell function.

Transcription factors provide the link between early membrane-proximal signalling events and changes in gene expression. NF-kappa B is one of the best-characterized transcription factors. It is expressed ubiquitously and regulates the expression of many genes, most of which encode proteins that play an important and often determining role in the processes of immunity and inflammation. Apart from its role in these events, evidence has begun to accumulate that NF-kappa B is involved in brain function, particularly following injury and in neurodegenerative conditions such as Alzheimer's disease. NF-kappa B might also be important for viral replication in the CNS. An involvement of NF-kappa B in neuronal development is suggested from studies that demonstrate its activation in neurones in certain regions of the brain during neurogenesis. Brain-specific activators of NF-kappa B include glutamate (via both AMPA/KA and NMDA receptors) and neurotrophins, pointing to an involvement in synaptic plasticity. NF-kappa B can therefore be considered as one of the most important transcription factors characterized in brain to date and it might be as crucial for neuronal and glial cell function as it is for immune cells.

Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro.

The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF-alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF-alpha strongly activates the canonical Nuclear Factor Kappa-B (NF- kappaB) pathway. In order to investigate further functions of TNF in neural stem cells (NSCs) we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h) is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC) or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators.

NF-kappaB is an inducible transcription factor, which is regulated by interaction with inhibitory IkappaB proteins. Previous studies linked the activity of NF-kappaB to the proliferative state of the cell. Here we have analysed the function of NF-kappaB in the cell cycle. Inhibition of NF-kappaB in HeLa cells by stable overexpression of a transdominant negative IkappaB-alpha protein reduced cell growth. A kinetic analysis of the cell cycle revealed a retarded G1/S transition. The IkappaB-alpha overexpressing cell clones showed a decreased percentage of cells in the S phase and an impaired incorporation of bromodeoxyuridine (BrdU). The amounts of cyclins A, B1, D1, D3, and E were unchanged, but the G1-specific proteins cyclin D2 and cdk2 were strongly elevated in the IkappaB-alpha overexpressing cell clones. These cell clones also displayed an increase in cyclin D1-dependent kinase activity, pointing to a cell cycle arrest at the late G1 phase. IkappaB-alpha overexpression crosstalked to cell cycle checkpoints via a reduction of transcription factor p53 and elevation of p21WAF. Surprisingly, the IkappaB-alpha overexpressing cells showed an enrichment of c-Myc in the nucleoli, although the total amount of c-Myc protein was unchanged. These experiments identify an important contribution of the NF-kappaB/IkappaB system for the growth of HeLa cells.

DNA array analysis of the developing rat cerebellum: transforming growth factor-beta2 inhibits constitutively activated NF-kappaB in granule neurons.

The nuclear factor-kappaB (NF-kappaB) pathway is important in neuronal survival and in integration of external signals e.g. cytokines, glutamate, Abeta and nerve growth factor (NGF). During rat cerebellar development NF-kappaB activity is high in granule cells before postnatal day 7 (P7) and declines after P7. Using gene expression profiles, measured by cDNA arrays, up-regulation of transforming growth factor-beta2 (TGF-beta2) was correlated with the developmental down-regulation of NF-kappaB. TGF-beta2 depicted strongest, more than 4-fold, up-regulation in P12 versus P4 cerebella. In situ hybridization and immunohistochemistry confined upregulated TGF-beta2 to granule cells and correlated mRNA and TGF-beta2-protein increase. Finally TGF-beta2 repressed NF-kappaB activity, in an in vitro system resembling migrating cerebellar granule cells. Thus, TGF-beta might fulfill an important role in repressing developmentally activated NF-kappaB in granule neurons.

Brain synapses contain inducible forms of the transcription factor NF-kappa B.

We investigated the rat brain for the presence and activation state of the inducible transcription factor NF-kappa B. Two forms of NF-kappa B containing the transactivating p65 subunit were found in all brain regions investigated. The majority of NF-kappa B was in an inducible cytoplasmic form by virtue of its association with the inhibitory subunit I kappa B. Significant amounts of inducible NF-kappa B forms were present in synaptosomes, as suggested by electrophoretic mobility shift assay and Western blot analysis of subcellular brain fractions. A synaptic localization of NF-kappa B was further evident from immunostaining of inner and outer plexiform layers of the retina with an antibody directed against the p50 subunit of NF-kappa B. In cerebral cortex and striatum, NF-kappa B-specific antibodies showed a punctate immunostaining partially overlapping with that for the synaptic marker protein synaptophysin. NF-kappa B is thus the first transcription factor found in synapses of neurons. With its unusual subneuronal localization, the inducible transcription factor has the potential to function as retrograde messenger mediating stimulus-response coupling and long-term changes in gene expression following presynaptic stimulation.

DNase I hypersensitive sites far upstream of the rat tryptophan oxygenase gene direct developmentally regulated transcription in livers of transgenic mice.

Expression of the gene coding for tryptophan oxygenase (TO) is switched on in rat liver about two weeks after birth. We identified two clusters of DNaseI hypersensitive (HS) sites in the TO gene upstream region; one near the promoter, the other at a distant upstream location (-8.5 kb). Hypersensitivity of upstream sites was present in adult and in 7 day old rat liver, but absent in kidney. To investigate their role in transcriptional regulation, a reporter gene controlled by both HS site regions was used to generate transgenic mice. In these animals the transgene followed the cell specific and developmental regulation of the endogenous gene: inactive after birth and active in adult liver. Transgenes containing only the promoter proximal HS site were non-functional.

Multiple domains of the glucocorticoid receptor involved in synergism with the CACCC box factor(s).

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor-binding sequences with adjacent transcription factor-binding sites. To analyze the mechanism of synergy we tested different human glucocorticoid receptor mutants for synergistic function with another transcription factor in comparison with intrinsic trans-activation obtained with a single receptor binding site (glucocorticoid response element). Multiple domains were found to be involved in synergistic activity of the glucocorticoid receptor with the CACCC box factor. Deletions within the N-terminal receptor half affected simultaneously intrinsic trans-activation and synergism. However, deletion of the hormone-binding domain mainly impaired synergism rather than intrinsic trans-activation, clearly showing that this domain synergizes by a mechanism independent of intrinsic activation. A chimeric protein where the DNA-binding domain of the glucocorticoid receptor was replaced by that of the yeast GAL4 protein also showed functional synergism. These data suggest that some of the receptor domains outside the DNA-binding domain synergize by their intrinsic trans-activating property, but the hormone-binding domain contributes to synergism by a different mechanism.

Transcription factor NF-kappaB and inhibitor I kappaBalpha are localized in macrophages in active multiple sclerosis lesions.

NF-kappaB is a transcription factor family which on translocation to the nucleus regulates gene expression during cell activation. As such, NF-kappaB may play a role in the microglial response to myelin damage in multiple sclerosis (MS) lesions. Here the cellular localization of NF-kappaB and expression of the inhibitory I kappaBalpha were examined by immunocytochemistry on central nervous system (CNS) tissue from MS and control cases. In normal control white matter, the active form of the NF-kappaB subunit RelA (p65) was localized in microglial nuclei, while the c-Rel and p50 subunits and the inhibitory I kappaBalpha were restricted to the cytoplasm. In contrast, in actively demyelinating plaques, the RelA, c-Rel, and p50 subunits of NF-kappaB and I kappaBalpha were all present in macrophage nuclei in both parenchymal and perivascular areas. RelA was also found in the nuclei of a subset of hypertrophic astrocytes. Only c-Rel had a nuclear localization in lymphocytes in perivascular inflammatory cuffs. Our results suggest that constitutive activation of the RelA subunit in the nuclei of resting microglia may facilitate a rapid response to pathological stimuli in the CNS. Activation of the inducible NF-kappaB pool in macrophages in MS lesions could amplify the inflammatory reaction through upregulation of NF-kappaB-controlled adhesion molecules and cytokines.

Cloning of the murine relA (p65 NF-kappa B) gene and comparison to the human gene reveals a distinct first intron.

A murine genomic clone encoding the relA (p65 NF-kappa B) locus was isolated and characterized. The clone spanned about 20 kb and included at least 10 exons and the relA promoter. Sequencing of the promoter region revealed the presence of a murine-specific intron of 546 bp intervening the homologue of human exon 1. The species-specific organisation of relA genes and promoter elements is compared and discussed. Despite the presence of potential binding sites for transcription factors NF-kappa B and AP-1 in the murine relA promoter, the constitutive relA transcript level was not affected by treatment of cells with phorbol ester or pyrrolidinedithiocarbamate, compounds which strongly affect NF-kappa B and AP-1 activities. This provides pharmacological evidence that the relA gene is not inducibly controlled by NF-kappa B or AP-1.

Potential involvement of the transcription factor NF-kappa B in neurological disorders.

Reactive oxygen intermediates (ROIs) are involved in many neurological diseases. Despite the toxic nature of these compounds, low concentrations of ROIs can function as signaling molecules. One target for their signaling function is the inducible transcription factor NF-kappa B. Predominantly in lymphoid cells, induction of NF-kappa B in response to oxidative stress leads to transcriptional activation of many genes which are relevant for pathogen defense. These include the TNF, IL-6, IL-8, GM-CSF, beta-interferon, MHC class I and V-CAM genes. However, NF-kappa B is also abundant in various cell types of the nervous system, including neurons. We propose that NF-kappa B plays a role as a redox-controlled transcriptional activator also in cells of the nervous system and in that property may contribute to neurological disorders. Our finding that some neurons from healthy brain contain constitutively active NF-kappa B suggests a role of NF-kappa B in normal brain function as well.

Activation of NF-kappa B by reactive oxygen intermediates in the nervous system.

Nuclear factor kappa B (NF-kappa B) is a transcription factor crucially involved in glial and neuronal function. NF-kappa B is ubiquitously distributed within the nervous system, and its inducible activity can be discerned from constitutive activity. Prototypic inducible NF-kappa B in the nervous system is composed of the DNA-binding subunits p50 and p65 complexed with an inhibitory I kappa B-alpha molecule. A number of signals from the cell surface can lead to rapid activation of NK-kappa B, thus releasing the inhibition by I kappa B. This activates translocation of NF-kappa B to the nucleus, where it binds to kappa B motifs of target genes and activates transcription. Previous findings have identified reactive oxygen intermediates (ROI) as a common denominator of NF-kappa B activating signals. More specifically, hydrogen peroxide (H2O2) might be used as second messenger in the NF-kappa B system, despite its cytotoxicity. Analysis of pathways leading to NF-kappa B activation in the nervous system has identified a number of ROI-dependent pathways such as cytokine- and neurotrophin-mediated activation, glutamatergic signal transduction, and various diseases with crucial ROI involvement (e.g., Alzheimer's disease, Parkinson's disease, experimental autoimmune encephalomyelitis, multiple sclerosis, amyotrophic lateral sclerosis, and injury). A number of NF-kappa B-specific target genes contribute to the production of ROI or are involved in detoxification of ROIs. In this review, possible mechanisms and regulatory pathways of ROI-mediated NF-kappa B activation are discussed.

Transcription factor NF-kappa B is activated in microglia during experimental autoimmune encephalomyelitis.

NF-kappa B is an inducible transcription factor involved in the induction of multiple genes during inflammatory processes. So far the information pertaining to the role of NF-kappa B in autoimmune processes has been restricted to in vitro analysis. To further characterize the role of NF-kappa B in vivo, the involvement of NF-kappa B has been studied by immunocytochemistry in T cell-mediated autoimmune encephalomyelitis (EAE) of the Lewis rat. In non-diseased animals, immunoreactivity for the DNA-binding subunit p50 and for the DNA-binding and transactivating subunit p65 was low and restricted to the surface of small to medium-sized blood vessels. Strong immunoreactivities for p50 and p65 were detected at the peak of clinical disease. At the recovery stage of EAE, p50 and p65 immunoreactivities had declined to base line levels. Within the resident glial cell population, p50 and p65-immunoreactive cells were identified as OX-42-positive microglia. GFAP-positive astrocytes did not show significant p50 or p65 immunoreactivity. In the core and the vicinity of perivascular inflammatory lesions, both ED-1-positive macrophages and W3/13-positive T lymphocytes and monocytes were strongly immunoreactive for NF-kappa B. Our data suggest a crucial involvement of the transcription factor NF-kappa B in autoimmune diseases of the central nervous system. Furthermore, NF-kappa B appears as a useful marker for inflammatory processes in vivo.