Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?

The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system

G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. G4s have been extensively described in the human genome, especially in telomeres and oncogene promoters; in recent years the presence of G4s in viruses has attracted increasing interest. Indeed, G4s have been reported in several viruses, including those involved in recent epidemics, such as the Zika and Ebola viruses. Viral G4s are usually located in regulatory regions of the genome and implicated in the control of key viral processes; in some cases, they have been involved also in viral latency. In this context, G4 ligands have been developed and tested both as tools to study the complexity of G4-mediated mechanisms in the viral life cycle, and as therapeutic agents. In general, G4 ligands showed promising antiviral activity, with G4-mediated mechanisms of action both at the genome and transcript level. This review aims to provide an updated close-up of the literature on G4s in viruses. The current state of the art of G4 ligands in antiviral research is also reported, with particular focus on the structural and physicochemical requirements for optimal biological activity. The achievements and the to-dos in the field are discussed

RNA interference approaches for treatment of HIV-1 infection.

HIV/AIDS is a chronic and debilitating disease that cannot be cured with current antiretroviral drugs. While combinatorial antiretroviral therapy (cART) can potently suppress HIV-1 replication and delay the onset of AIDS, viral mutagenesis often leads to viral escape from multiple drugs. In addition to the pharmacological agents that comprise cART drug cocktails, new biological therapeutics are reaching the clinic. These include gene-based therapies that utilize RNA interference (RNAi) to silence the expression of viral or host mRNA targets that are required for HIV-1 infection and/or replication. RNAi allows sequence-specific design to compensate for viral mutants and natural variants, thereby drastically expanding the number of therapeutic targets beyond the capabilities of cART. Recent advances in clinical and preclinical studies have demonstrated the promise of RNAi therapeutics, reinforcing the concept that RNAi-based agents might offer a safe, effective, and more durable approach for the treatment of HIV/AIDS. Nevertheless, there are challenges that must be overcome in order for RNAi therapeutics to reach their clinical potential. These include the refinement of strategies for delivery and to reduce the risk of mutational escape. In this review, we provide an overview of RNAi-based therapies for HIV-1, examine a variety of combinatorial RNAi strategies, and discuss approaches for ex vivo delivery and in vivo delivery

Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels

Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (Naᵥ1.1), SCN2A (Naᵥ1.2), SCN3A (Naᵥ1.3) and SCN8A (Naᵥ1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge

ModHMM: A Modular Supra-Bayesian Genome Segmentation Method

Genome segmentation methods are powerful tools to obtain cell type or tissue-specific genome-wide annotations and are frequently used to discover regulatory elements. However, traditional segmentation methods show low predictive accuracy and their data-driven annotations have some undesirable properties. As an alternative, we developed ModHMM, a highly modular genome segmentation method. Inspired by the supra-Bayesian approach, it incorporates predictions from a set of classifiers. This allows to compute genome segmentations by utilizing state-of-the-art methodology. We demonstrate the method on ENCODE data and show that it outperforms traditional segmentation methods not only in terms of predictive performance, but also in qualitative aspects. Therefore, ModHMM is a valuable alternative to study the epigenetic and regulatory landscape across and within cell types or tissues

Epigenetics in Friedreich's ataxia: Challenges and opportunities for therapy

Copyright © 2013 Chiranjeevi Sandi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by homozygous expansion of a GAA·TTC trinucleotide repeat within the first intron of the FXN gene, leading to reduced FXN transcription and decreased levels of frataxin protein. Recent advances in FRDA research have revealed the presence of several epigenetic modifications that are either directly or indirectly involved in this FXN gene silencing. Although epigenetic marks may be inherited from one generation to the next, modifications of DNA and histones can be reversed, indicating that they are suitable targets for epigenetic-based therapy. Unlike other trinucleotide repeat disorders, such as Huntington disease, the large expansions of GAA·TTC repeats in FRDA do not produce a change in the frataxin amino acid sequence, but they produce reduced levels of normal frataxin. Therefore, transcriptional reactivation of the FXN gene provides a good therapeutic option. The present paper will initially focus on the epigenetic changes seen in FRDA patients and their role in the silencing of FXN gene and will be concluded by considering the potential epigenetic therapies.This study is supported by funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant agreement no. 242193/EFACTS; and by funding from theWellcome Trust (089757)