690. Permanent Epigenetic Silencing of Human Genes With Artificial Transcriptional Repressors

There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship

729 inheritable silencing of endogenous gene by hit and run targeted epigenetic editing

Gene silencing holds great promise for the treatment of several diseases and can be exploited to investigate gene function and activity of the regulatory genome. Here, we develop a novel modality of gene silencing that exploits epigenetics to achieve stable and highly efficient repression of target genes. To this end, we generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of chromatin-modifying enzymes involved in silencing process of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains (namely KRAB, DNMT3A and DNMT3L) that, when transiently co-assembled on the promoter of the reporter cassette, recreate a powerful embryonic-specific repressive complex capable of inducing full and long-term (>150 days) silencing of transgene expression in up to 90% of the cells. The ATR-induced silencing was cell type and locus independent, and resistant to metabolic activation of the cells. Importantly, these findings were holding true also for endogenous genes embedded in their natural chromatin context, as shown for the highly and ubiquitously expressed B2M gene. Here, transient co-delivery of TALE-based ATRs resulted in loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of the cells. This phenotype was associated with a drastic switch in the epigenetic and transcriptional state of the constitutively active B2M promoter, which become highly decorated with de novo DNA methylation and deprived of RNAP II. Importantly, silencing was sharply confined to the targeted gene and resistant to INF-γ, a potent natural activator of B2M. We further extended these studies by showing that our silencing approach is portable to the CRISPR/dCas9 DNA binding technology. In this setting, comparable levels of B2M silencing (up to 80%) were achieved using either pools or even individual sgRNAs coupled to dCas9-based ATRs. Yet, adoption of this technology allowed performing simultaneous, highly efficient multiplex gene silencing within the same cell, as shown for B2M, IFNAR1 and VEGFA. Finally, we assessed resistance of the silenced gene to activity of potent artificial transcription activators and chromatin remodelers, and found that only targeted DNA demethylation was able to reawaken the silent gene. This allowed performing iterative cycles of silencing and reactivation of the same gene in the same cell population. Overall, these data provide the first demonstration of efficient and stable epigenetic silencing of endogenous genes upon transient delivery of ATRs. This was accomplished by repurposing the ERVs silencing machinery, which instructs self-sustaining repressive epigenetic states to the target gene. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for both basic and translational research

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS

Effect of the mandatory vaccination law on measles and rubella incidence and vaccination coverage in Italy (2013-2019)

Despite the introduction of the trivalent vaccine (measles, mumps, rubella) more than 20 years ago, measles outbreaks have occurred in Europe, including Italy, due to its underutilization. In Italy mandatory vaccination was established in 2017 (Decree Law 119/2017). This study aimed at evaluating the impact of mandatory vaccination and determining the trend in vaccination coverage for measles and rubella in Italy. We retrieved data from the Annual Status Update, a form sent annually by the Italian National Verification Committee to the Regional Verification Committee for Europe, from 1st January 2013 to 31st December 2019. Since the beginning of 2013, 14,788 cases of measles have been reported, ranging from 256 (3.9 x 1,000,000) to 5,397 (88.4 x 1,000,000) compared to 259 rubella cases for the same period. From 2013 to 2015, vaccination coverage decreased for the first dose of measles (90.4% to 85.3%) and rubella vaccine (90.3% to 85.2%), but then it increased significantly, reinforced by the Italian Decree Law, reaching 94.4% in 2019. The trend for the second dose showed a decrease from 2013 to 2016 (84.1% to 82.2% for measles and 83.7% to 82.0% for rubella), but then increased significantly and reached 90.2% in 2019 for measles and 90.0% for rubella. The mandatory vaccination law has resulted in a significant increase in vaccination coverage for measles and rubella in Italy, and demonstrates encouraging progress toward the 95% target and the restriction of measles transmission. Special attention should be paid to maintaining and further improving vaccination coverage

High Incidence of Candidemia in Critically Ill COVID-19 Patients Supported by Veno-Venous Extracorporeal Membrane Oxygenation: A Retrospective Study

Background: The incidence of candidemia in severe COVID-19 patients (0.8–14%) is two- to ten-fold higher than in non-COVID-19 patients. Methods: This retrospective analysis aimed to analyse the incidence of bloodstream infections (BSI) due to Candida in a cohort of COVID-19 patients supported with ECMO. Results: Among 138 intubated and ventilated patients hospitalized for ≥10 days in the intensive care unit of a teaching hospital, 45 (32.6%) patients received ECMO support, while 93 patients (67.4%) did not meet ECMO criteria and were considered the control group. In the ECMO group, 16 episodes of candidaemia were observed, while only 13 in patients of the control group (36.0% vs. 14.0%, p-value 0.004). It was confirmed at the survival analysis (SHR: 2.86, 95% CI: 1.39–5.88) and at the multivariable analyses (aSHR: 3.91, 95% CI: 1.73–8.86). A higher candida score seemed to increase the hazard for candidemia occurrence (aSHR: 3.04, 95% CI: 2.09–4.42), while vasopressor therapy was negatively associated with the outcome (aSHR: 0.15, 95% CI: 0.05–0.43). Conclusions: This study confirms that the incidence of candidemia was significantly higher in critically ill COVID-19 patients supported with VV-ECMO than in critically ill COVID patients who did not meet criteria for VV-ECMO

Precision Medicine and Public Health: New Challenges for Effective and Sustainable Health

The development of high-throughput omics technologies represents an unmissable opportunity for evidence-based prevention of adverse effects on human health. However, the applicability and access to multi-omics tests are limited. In Italy, this is due to the rapid increase of knowledge and the high levels of skill and economic investment initially necessary. The fields of human genetics and public health have highlighted the relevance of an implementation strategy at a national level in Italy, including integration in sanitary regulations and governance instruments. In this review, the emerging field of public health genomics is discussed, including the polygenic scores approach, epigenetic modulation, nutrigenomics, and microbiomes implications. Moreover, the Italian state of implementation is presented. The omics sciences have important implications for the prevention of both communicable and noncommunicable diseases, especially because they can be used to assess the health status during the whole course of life. An effective population health gain is possible if omics tools are implemented for each person after a preliminary assessment of effectiveness in the medium to long term

Comparing the Occurrence of Healthcare-Associated Infections in Patients with and without COVID-19 Hospitalized during the Pandemic: A 16-Month Retrospective Cohort Study in a Hospital Intensive Care Unit

The COVID-19 pandemic has increased the healthcare-associated infection (HAI) risk in intensive care unit (ICU) patients. However, a comparison between patients with and without COVID-19 in terms of HAI incidence has been rarely explored. In this study, we characterized the occurrence of HAI among patients with and without COVID-19 admitted to the ICU of the Umberto I hospital of Rome during the first 16 months of the pandemic and also identified risk factors for HAI acquisition. Patients were divided into four groups according to their ICU admission date. A multivariable conditional risk set regression model for multiple events was constructed for each admission period. Adjusted hazard ratios and 95% confidence intervals were calculated. Overall, 352 COVID-19 and 130 non-COVID-19 patients were included, and a total of 361 HAIs were recorded. We found small differences between patients with and without COVID-19 in the occurrence and type of HAI, but the infections in the two cohorts mostly involved different microorganisms. The results indicate that patient management was likely an important factor influencing the HAI occurrence during the pandemic. Effective prevention and control strategies to reduce HAI rates should be implemented

Mid-term results on a new self-expandable covered stent combined with branched stent-grafts: insights from a multicenter Italian registry

Objective: To investigate the technical periprocedural and mid-term outcomes of endovascular repairs with multibranched (BEVAR) or iliac-branch devices (IBD) combined with a new self-expanding covered stent. Methods: The COBRA (COvera in BRAnch) registry is a physician-initiated, multicenter, ambispective, observational registry (ClinicalTrials.gov Identifier: NCT04598802) enrolling patients receiving a BEVAR or IBD procedure mated with Bard Covera Plus (Tempe, Arizona, USA) covered stent, designed to evaluate the outcomes of the covered stent mated with patient-specific and off-the-shelf branched stent-graft. Primary endpoints were technical success, branch instability, and freedom from aortic and branch-related reintervention within 30 days and at follow-up. Preoperative characteristics, comorbidities, and outcomes definition were graded according to the Society for Vascular Surgery reporting standards. Results: Two hundred eighty-four patients [76 years (70-80); 79% males] in 24 centers were enrolled for a total of 708 target vessels treated. The CSs were mated with an off-the-shelf graft in 556 vessels (79%) and a custom-made graft in 152 (21%). Three-hundred-seven adjunctive relining stents in 277 vessels (39%) were deployed, of which 116 (38%) proximal, 66 (21%) intrastent and 125 (41%) distal. Adjunctive relining stent placement was more frequent when landing in a vessel branch instead of the main trunk (59%vs39%, p=.031), performing a percutaneous access (49%vs35%, p<.001), using a stent with a diameter ≥8mm (44%vs36%, p=.032) and a length ≥80mm (65%vs55%, p=.005), when a post-dilatation was not performed (45%vs29%, p<.001) and when an inner-branch configuration was used (55%vs35%, p<.001). Perioperative technical bridging success was 98%. Eight patients (3%) died in the perioperative period. Two deaths (1%) were associated with renal branch occlusion followed by acute kidney injury and paraplegia. Follow-up data were available for 638 vessels (90%) at a median of 32 months (Q1, Q3: 21,46). A branch instability was reported in 1% of branches. Forty-six (17%) patients died during follow-up, nine (3%) of them due to aortic-related causes. Primary patency rates at one year, two and three years were 99% (581/587), 99% (404/411), and 97% (272/279) respectively. Branch instability was associated with patient-specific devices (9% vs 4%, p=.014) and intrastent adjunctive stent placement (12% vs 2%, p=.003), especially when a bare metal balloon-expandable stent was used (25%vs3%, p<.001). Conclusions: The use of this new self-expanding covered stent mated with branched endografts proved to be safe and feasible with high technical procedural success rates. Low rates of branch instability were observed at mid-term follow-up. Comparative studies with other commercially available covered stents are warranted