484. Preclinical Proof of Concept of Transcriptional Silencing and Replacement Strategy for Treatment of Retinitis Pigmentosa Due To RHODOPSIN Mutations

Silencing and replacement strategy is a promising approach to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) due to rhodopsin gene (RHO) approximately 200 different mutations have been described, posing a challenge for the design of effective therapeutics.We designed a silencing and replacement strategy based on transcriptional silencing through an artificial zinc finger DNA-binding protein lacking effector domains (ZF6DBD), and tested both efficacy and safety in two animal models.In a murine model of adRP, we show that AAV-mediate retinal delivery (AAV2/8-CMV-ZF6-DBD) is associated with selective transcriptional silencing of the human mutated allele resulting in morphological and functional (Electroretinography, ERG a-wave and b-wave responses) rescue. We then tested the effect of transcriptional silencing in the porcine large pre-clinical model. Delivery of a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) of the ZF6 transcriptional silencer to the porcine retina resulted in robust transcriptional silencing of the endogenous porcine RHO transcript. Cell sorting of transduced photoreceptors showed an almost complete RHO transcriptional silencing effect (90% RHO transcriptional repression), underscoring the potency of the system. To determine the safety of the zinc-finger silencer we performed extensive RNA-seq analysis on treated and control retinae. The data sets generated demonstrate selective RHO gene transcriptional repression and a remarkably low number of differential expressed genes (DEGs), supporting specificity and thus, safety. The co-administration to the porcine retina of the AAV-ZF6 silencer (AAV2/8-CMV-ZF6-DBD) and the AAV-RHO replacement (5×10e11 vg, AAV2/8-GNAT1-HumanRHO) constructs resulted in a balanced silencing and replacement effect. This data support the use of zinc-finger based RHO transcriptional silencing for the development of a clinical trial for adRP patients

320 transcriptional silencing via synthetic dna binding protein lacking canonical repressor domains as a potent tool to generate therapeutics

Transcription factors (TFs) function by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs). Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain entails complete and gene-specific transcriptional silencing. To silence RHODOPSIN (RHO) gain-of-function mutations, we engineered a synthetic DNA-binding protein lacking canonical repressor domains and targeted to the regulatory region of the RHO gene. AAV-mediate retinal delivery at a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) in the porcine retina resulted in selective transcriptional silencing of RHO expression. The rod photoreceptors (the RHO expressing cells) transduced cells when isolated by FACS-sorting showed the remarkable 90% RHO transcriptional repression. To evaluate genome-wide transcriptional specificity, we analyzed the porcine retina transcriptome by RNA sequencing (RNA-Seq). The differentially expressed genes (DEGs) analysis showed that only 19 genes were perturbed. In this study, we describe a system based on a synthetic DNA binding protein enabling targeted transcriptional silencing of the RHO gene by in vivo gene transfer. The high rate of transcriptional silencing occurring in transduced cells supports applications of this regulatory genomic interference with a synthetic trans-acting factor for diseases requiring gene silencing in a large number of affected cells, including for instance a number of neurodegeneration disorders. The result support a novel mode of gene targeted silencing with a DNA-binding protein lacking intrinsic activity

Clinical outcome with different doses of low-molecular-weight heparin in patients hospitalized for COVID-19

A pro-thrombotic milieu and a higher risk of thrombotic events were observed in patients with CoronaVirus disease-19 (COVID-19). Accordingly, recent data suggested a beneficial role of low molecular weight heparin (LMWH), but the optimal dosage of this treatment is unknown. We evaluated the association between prophylactic vs. intermediate-to-fully anticoagulant doses of enoxaparin and in-hospital adverse events in patients with COVID-19. We retrospectively included 436 consecutive patients admitted in three Italian hospitals. Outcome according to the use of prophylactic (4000IU) vs. higher (>4000IU) daily dosage of enoxaparin was evaluated. The primary end-point was in-hospital death. Secondary outcome measures were in-hospital cardiovascular death, venous thromboembolism, new-onset acute respiratory distress syndrome (ARDS) and mechanical ventilation. A total of 287 patients (65.8%) were treated with the prophylactic enoxaparin regimen and 149 (34.2%) with a higher dosing regimen. The use of prophylactic enoxaparin dose was associated with a similar incidence of all-cause mortality (25.4% vs. 26.9% with the higher dose; OR at multivariable analysis, including the propensity score: 0.847, 95% CI 0.400-0.1.792; p=0.664). In the prophylactic dose group, a significantly lower incidence of cardiovascular death (OR 0.165), venous thromboembolism (OR 0.067), new-onset ARDS (OR 0.454) and mechanical intubation (OR 0.150) was observed. In patients hospitalized for COVID-19, the use of a prophylactic dosage of enoxaparin appears to be associated with similar in-hospital overall mortality compared to higher doses. These findings require confirmation in a randomized, controlled study

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector in vivo and in vitro only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR

Conformation Regulation of the X Chromosome Inactivation Center: A Model

X-Chromosome Inactivation (XCI) is the process whereby one, randomly chosen X becomes transcriptionally silenced in female cells. XCI is governed by the Xic, a locus on the X encompassing an array of genes which interact with each other and with key molecular factors. The mechanism, though, establishing the fate of the X's, and the corresponding alternative modifications of the Xic architecture, is still mysterious. In this study, by use of computer simulations, we explore the scenario where chromatin conformations emerge from its interaction with diffusing molecular factors. Our aim is to understand the physical mechanisms whereby stable, non-random conformations are established on the Xic's, how complex architectural changes are reliably regulated, and how they lead to opposite structures on the two alleles. In particular, comparison against current experimental data indicates that a few key cis-regulatory regions orchestrate the organization of the Xic, and that two major molecular regulators are involved

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells

Gene expression states influence the three-dimensional conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features, which define promoter activation states or CTCF binding sites. Single cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fibre fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions

Short-term triple therapy with azithromycin for Helicobacter pylori eradication: Low cost, high compliance, but low efficacy

<p>Abstract</p> <p>Background</p> <p>The Brazilian consensus recommends a short-term treatment course with clarithromycin, amoxicillin and proton-pump inhibitor for the eradication of <it>Helicobacter pylori </it>(<it>H. pylori)</it>. This treatment course has good efficacy, but cannot be afforded by a large part of the population. Azithromycin, amoxicillin and omeprazole are subsidized, for several aims, by the Brazilian federal government. Therefore, a short-term treatment course that uses these drugs is a low-cost one, but its efficacy regarding the bacterium eradication is yet to be demonstrated. The study's purpose was to verify the efficacy of <it>H. pylori </it>eradication in infected patients who presented peptic ulcer disease, using the association of azithromycin, amoxicillin and omeprazole.</p> <p>Methods</p> <p>Sixty patients with peptic ulcer diagnosed by upper digestive endoscopy and <it>H. pylori </it>infection documented by rapid urease test, histological analysis and urea breath test were treated for six days with a combination of azithromycin 500 mg and omeprazole 20 mg, in a single daily dose, associated with amoxicillin 500 mg 3 times a day. The eradication control was carried out 12 weeks after the treatment by means of the same diagnostic tests. The eradication rates were calculated with 95% confidence interval.</p> <p>Results</p> <p>The eradication rate was 38% per intention to treat and 41% per protocol. Few adverse effects were observed and treatment compliance was high.</p> <p>Conclusion</p> <p>Despite its low cost and high compliance, the low eradication rate does not allow the recommendation of the triple therapy with azithromycin as an adequate treatment for <it>H. pylori </it>infection.</p