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Upcoming Seminars

Alessandra Recchia, PhD - "Modelling the CRISPR/Cas9 system to correct monogenic disorders"

Assistant Professor of Molecular Biology, Department of Life Sciences, University of Modena and Reggio Emilia, Italy
When Nov 18, 2019
from 12:00 PM to 01:00 PM
Where Tigem, Vesuvius Auditorium
Contact Name
Contact Phone 081-19230659
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Abstract
The CRISPR/Cas9 system is an effective genomic scissor guided by a short guide RNA to cleave specific DNA target sequences. The Cas9 introduces double strand breaks into the target gene, stimulating the cell’s DNA repair mechanisms of homology-directed repair and non-homologous end joining. Triggering these cellular mechanisms to purposely edit the gene of interest, CRISPR/Cas9 technology has earned a tremendous impact on genetic manipulation. For its simplicity, CRISPR/Cas9 has been studied and applied the most by scientists and clinicians and also proposed as a gene therapy tool for monogenic disorder. In this seminar I will present you our experience on CRISPR/Cas9 applications to tackle monogenic disorders. Employing different delivery systems and different SpCas9 variants, I will report correction of genetic defects ex vivo, in patients’ cell, and in vivo in preclinical model of dominant retinitis pigmentosa.

Francesca Carlomagno, MD, PhD - "Links Between Cell Cycle Control and Iron Metabolism"

Full Professor, IEOS c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita' degli Studi di Napoli Federico II
When Dec 10, 2019
from 12:00 PM to 01:00 PM
Where Tigem, Vesuvius Auditorium
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Contact Phone 081-19230659
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Iron is crucial for many enzymes involved in DNA replication and for dNTPs synthesis. Indeed, limited iron availability affects cell proliferation inducing G1 arrest in order to avoid replication stress and DNA damage. NCOA4 is an iron-sensing protein that is stabilized in low iron conditions to promote ferritin degradation. Interestingly, NCOA4 exerts also a negative control on DNA replication origin activation in iron deficiency conditions in order to maintain genome stability. Thus, in DFO treated cells we observed increased binding of NCOA4 to MCM2-7 and to chromatin, and in particular to canonical DNA replication origins.  Indeed, in low iron conditions NCOA4-depleted HeLa cells (shNCOA4) activated more DNA replication origins than control cells showing reduced inter-origin distance and decreased fork rate with signs of replication stress.  Unscheduled DNA replication with associated signs of replication stress observed in HeLa-shNCOA4 cells lead to activation of a DNA Damage Response with phosphorylation of ATR, CHK1 and gamma-H2AX, permanent cell cycle arrest and reduced cell survival. Consistently, NCOA4 null mice displayed a decreased tolerance to tissue insults such as Dextran Sulfate-induced acute colitis with decreased proliferating crypts (eg pH3 positive cells) and increased DNA damage (eg pCHK2 positive cells) and apoptotic cells (eg Caspase 3 positive cells), reflecting an overall impairment of intestinal cell renewal. Collectively, our data describe a new cellular response activated in iron deficiency that exploit NCOA4 protein as a cell proliferation brake, to align DNA replication origin activation and iron fluctuations in order to avoid DNA replication stress.

Francesca Fallarino, PhD - "Endogenous produced tryptophan metabolites prevent the development of anti-FVIII antibodies in hemophilia A"

Associate Professor, Department of Experimental Medicine, University of Perugia, ITALY
When Dec 17, 2019
from 12:00 PM to 01:00 PM
Where Tigem, Vesuvius Auditorium
Contact Name
Contact Phone 081-19230659
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Abstract
Hemophilia A is a genetic disorder that manifests itself through an inability to form blood clots within the body. Since this is due to the absence of a clotting protein (factor VIII), the gold-standard treatment is to inject the protein that is missing into the patient’s circulation to make up for the deficiency. Unfortunately, about 30% of hemophilia A patients develop inhibitors against this infused protein and render the treatment ineffective. The interaction between factor VIII and the body’s white blood cells are important for inhibitor generation as well as the tolerance to factor VIII, which is the absence of inhibitor generation to the protein. We reported that the inhibitor-positive status was associated with reduced activity of the immune-regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in dendritic cells, that promotes regulatory effects via the production of tryptophan catabolites, known as kynurenines. Some of those tryptophan derivatives are endogenous ligands for the Aryl hydrocarbon receptor (AhR).
In this study, we tested the potential of tryptophan-related AhR ligands for inhibiting the development of anti-FVIII antibodies in hemophilic (F8 KO) mice. To this aim, F8 KO mice hemophilic mice were treated with recombinant human FVIII (rhFVIII) alone or in combination with selected AhR ligands once weekly for four weeks. All mice treated with rhFVIII developed high-titer anti-FVIII antibodies after 4 weeks of treatment. Administration of specific tryptophan metabolites prevented the generation of anti-FVIII antibodies in almost 80% of F8 KO mice. The protective effect of these AhR ligands was negated by co-administration of the AhR antagonist CH-223191 or in AhR KO mice. Similar results were obtained by administration of engineered gold nanoparticles loaded with the same tryptophan metabolite and rhFVIII.  In addition, in the same model we found that treatment with AhR ligands not only suppressed FVIII-specific antibody titers but, resulted in increased protection against specific  bacteria and fungi infection.  
Thus, these results suggest that the engagement of AhR, by specific tryptophan derivatives, may be a possible new strategy to control the immune response to rhFVIII, while protecting against specific infections.