DNA Damage: From Threat to Treatment Carusillo, Antonio; Mussolino, Claudio
Cells (Basel, Switzerland),
07/2020, Letnik:
9, Številka:
7
Journal Article
Recenzirano
Odprti dostop
DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic ...sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.
Recent progress in developing programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic ...repeat (CRISPR)-Cas nucleases, have paved the way for gene editing to enter clinical practice. This translation is a result of combining high nuclease activity with high specificity and successfully applying this technology in various preclinical disease models, including infectious disease, primary immunodeficiencies, hemoglobinopathies, hemophilia and muscular dystrophy. Several clinical gene-editing trials, both ex vivo and in vivo, have been initiated in the past 2 years, including studies that aim to knockout genes as well as to add therapeutic transgenes. Here we discuss the advances made in the gene-editing field in recent years, and specify priorities that need to be addressed to expand therapeutic genome editing to further disease entities.
In the last decades, a better understanding of human pathologies has revealed that genetic alterations as well as epigenetic aberrations can be drivers of a disease or exacerbate its manifestation. ...The availability of customizable platforms that allow precise genomic targeting has opened the possibility to cure genetic disorders by tackling directly the origin of the disease. Indeed, tethering of different effectors to a DNA-binding moiety grants precise alterations of the genome, transcriptome, or epigenome with the aim of normalizing disease-causing aberrations. The use of designer nucleases for therapeutic genome editing is currently approaching the clinics, and safety concerns arise with respect to off-target effects. Epigenome editing might be a valuable alternative, as it does not rely on DNA double-strand breaks, one of the most deleterious form of DNA damage, to exert its function. We have recently described designer epigenome modifier (DEM), a novel platform for achieving precise epigenome editing in clinically relevant primary human cells. We discuss the efficiency of DEM and highlight their remarkable safety profile, which certainly makes this platform a valuable candidate for future clinical translation.
Designer effectors based on the DNA binding domain (DBD) of
transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in ...editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive structure of the TALE arrays composing the DBD impedes their generation as gene synthesis product and prevents the delivery of TALE-based genes using lentiviral vectors (LVs), a widely used system for human gene therapy. To overcome these limitations, we aimed at chimerizing the DNA sequence encoding for the TALE-DBDs by introducing sufficient diversity to facilitate both their gene synthesis and enable their lentiviral delivery. To this end, we replaced three out of 17
TALE repeats with TALE-like units from the bacterium
. This was combined with extensive codon variation and specific amino acid substitutions throughout the DBD in order to maximize intra- and inter-repeat sequence variability. We demonstrate that chimerized TALEs can be easily generated using conventional Golden Gate cloning strategy or gene synthesis. Moreover, chimerization enabled the delivery of TALE-based designer nucleases, transcriptome and epigenome editors using lentiviral vectors. When delivered as plasmid DNA, chimerized TALEs targeting the
and
loci showed comparable activities in human cells. However, lentiviral delivery of TALE-based transcriptional activators was only successful in the chimerized form. Similarly, delivery of a chimerized
-specific epigenome editor resulted in rapid silencing of endogenous
expression. In conclusion, extensive codon variation and chimerization of TALE-based DBDs enables both the simplified generation and the lentiviral delivery of designer TALEs, and therefore facilitates the clinical application of these tools to precisely edit the genome, transcriptome and epigenome.
Tissue oxygenation throughout life depends on the activity of hemoglobin (Hb) one of the hemeproteins that binds oxygen in the lungs and secures its delivery throughout the body. Hb is composed of ...four monomers encoded by eight different genes the expression of which is tightly regulated during development, resulting in the formation of distinct hemoglobin tetramers in each developmental stage. Mutations that alter hemoglobin structure or its regulated expression result in a large group of diseases typically referred to as hemoglobinopathies that are amongst the most common genetic defects worldwide. Unprecedented efforts in the last decades have partially unraveled the complex mechanisms that control globin gene expression throughout development. In addition, genome wide association studies have revealed protective genetic traits capable of ameliorating the clinical manifestations of severe hemoglobinopathies. This knowledge has fueled the exploration of innovative therapeutic approaches aimed at modifying the genome or the epigenome of the affected cells to either restore hemoglobin function or to mimic the effect of protective traits. Here we describe the key steps that control the switch in gene expression that concerns the different globin genes during development and highlight the latest efforts in altering globin regulation for therapeutic purposes.
The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained ...in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.
Human leucocyte antigen class I (HLA-I) molecules play a central role for both NK and T-cell responses that prevent serious human cytomegalovirus (HCMV) disease. To create opportunities for viral ...spread, several HCMV-encoded immunoevasins employ diverse strategies to target HLA-I. Among these, the glycoprotein US10 is so far insufficiently studied. While it was reported that US10 interferes with HLA-G expression, its ability to manipulate classical HLA-I antigen presentation remains unknown. In this study, we demonstrate that US10 recognizes and binds to all HLA-I (HLA-A, -B, -C, -E, -G) heavy chains. Additionally, impaired recruitment of HLA-I to the peptide loading complex was observed. Notably, the associated effects varied significantly dependending on HLA-I genotype and allotype: (i) HLA-A molecules evaded downregulation by US10, (ii) tapasin-dependent HLA-B molecules showed impaired maturation and cell surface expression, and (iii) β 2 m-assembled HLA-C, in particular HLA-C*05:01 and -C*12:03, and HLA-G were strongly retained in complex with US10 in the endoplasmic reticulum. These genotype-specific effects on HLA-I were confirmed through unbiased HLA-I ligandome analyses. Furthermore, in HCMV-infected fibroblasts inhibition of overlapping US10 and US11 transcription had little effect on HLA-A, but induced HLA-B antigen presentation. Thus, the US10-mediated impact on HLA-I results in multiple geno- and allotypic effects in a so far unparalleled and multimodal manner.
During a viral infection, the immune system must discriminate between healthy and infected cells to selectively kill infected cells. Healthy cells have different types of molecules known collectively as HLA-I on their surface. These molecules present small fragments of proteins from the cell, called antigens, to patrolling immune cells, known as CTLs or natural killer cells. While CTLs ignore antigens from human proteins (which indicate the cell is healthy), they can bind to and recognize antigens from viral proteins, which triggers them to activate immune responses that kill the infected cell. However, some viruses can prevent infected cells from presenting HLA-I molecules on their surfaces as a strategy to evade the immune system. Natural killer cells have evolved to overcome this challenge. They bind to the HLA-I molecules themselves, which causes them to remain inactive. However, if the HLA-I molecules are missing, the NK cells can more easily switch on and kill the target cell. The human cytomegalovirus is a common virus that causes lifelong infection in humans. Although it rarely causes illness in healthy individuals, it can be life-threatening to newborn babies and for individuals with weakened immune systems. One human cytomegalovirus protein known as US10 was previously found to bind to HLA-I without reducing the levels of these molecules on the surface of the cell. However, its precise role remained unclear. Gerke et al. used several biochemical and cell biology approaches to investigate whether US10 manipulates the quality of the three types of HLA-I, which could impact both CTL and NK cell recognition. The experiments showed that US10 acted differently on the various kinds of HLA-I. To one type, it bound strongly within the cell and prevented it from reaching the surface. US10 also prevented another type of HLA-I from maturing properly and presenting antigens but did not affect the third type of HLA-I. These findings suggest that US10 interferes with the ability of different HLA-I types to present antigens in specific ways. Further research is needed to measure how US10 activity affects immune cells, which may ultimately aid the development of new therapies against human cytomegalovirus and other similar viruses.
Hereditary pulmonary alveolar proteinosis (herPAP) constitutes a rare, life threatening lung disease characterized by the inability of alveolar macrophages to clear the alveolar airspaces from ...surfactant phospholipids. On a molecular level, the disorder is defined by a defect in the CSF2RA gene coding for the GM-CSF receptor alpha-chain (CD116). As therapeutic options are limited, we currently pursue a cell and gene therapy approach aiming for the intrapulmonary transplantation of gene-corrected macrophages derived from herPAP-specific induced pluripotent stem cells (herPAP-iPSC) employing transcriptional activator-like effector nucleases (TALENs). Targeted insertion of a codon-optimized CSF2RA-cDNA driven by the hybrid cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter into the AAVS1 locus resulted in robust expression of the CSF2RA gene in gene-edited herPAP-iPSCs as well as thereof derived macrophages. These macrophages displayed typical morphology, surface phenotype, phagocytic and secretory activity, as well as functional CSF2RA expression verified by STAT5 phosphorylation and GM-CSF uptake studies. Thus, our study provides a proof-of-concept, that TALEN-mediated integration of the CSF2RA gene into the AAVS1 safe harbor locus in patient-specific iPSCs represents an efficient strategy to generate functionally corrected monocytes/macrophages, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of herPAP.
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