42 correction of scid x1 by targeted genome editing of hematopoietic stem progenitor cells hspc in the mouse model

Targeted genome editing by engineered nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy trials with integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of leukemias due to insertional mutagenesis and unregulated transgene expression. To model SCID-X1 gene correction in preclinical studies, we developed a mouse model carrying the IL2RG human gene harboring a common disease-causing mutation in place of the murine Il2rg, allowing to use of the same reagents developed for gene correction of human cells. These mice have impaired lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute in part the T and B cell compartments. We then tested gene correction of the murine Lin- HSPC by the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol yielded high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR but also high cytotoxicity (65% cell loss) under the conditions we used. The surviving cells remained capable of expansion in culture and maintained their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over the un-corrected SCID cells. These data indicate functional correction of the defective IL2RG gene by targeted editing. Furthermore, upon challenging the mice with a murine pathogen we observed viral-specific γIFN production by CD8+ gene corrected cells, proving their in vivo functionality. Yet, measuring the percentage of edited cells (either by NHEJ or HDR) within the HSC compartment long-term, we found that it was nearly undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells persisted in the mice up to 7 months post transplantation within all the hematopoietic organs, indicating successful editing of at least 1% progenitors able to sustain long-term lymphopoiesis and partially correct the disease phenotype. We then developed a new protocol exploiting CRISPR/Cas9 technology that enabled to achieve substantial levels of targeted DNA repair by NHEJ (up to 70%) and HDR (up to 25%) with minimal cytotoxicity and provided stable engraftment of the edited cells in transplanted mice. By this strategy we are now assessing the impact of HSC vs. progenitor targeted editing and conditioning regimen for the extent and stability of disease correction. These studies will help establish the key factors underlying safe and effective rescue of the disease by HSPC gene editing and assist in the design of the protocol for its first clinical testing

481. Targeted Genome Editing in Mouse Hematopoietic Stem/Progenitor Cells (HSPC) To Model Gene Correction of SCID-X1

Targeted genome editing by artificial nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy with early generation integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of adverse events due to insertional mutagenesis and unregulated transgene expression. We recently reported a strategy that enabled targeted integration of a corrective cDNA into the IL2RG gene in 6% of human HSPC with high specificity. Gene corrected HSPC generated polyclonal lymphoid cells that express the IL2RG protein and have a selective growth advantage over those carrying disruptive IL2RG mutations (Genovese, Nature, 2014). Here, to model SCID-X1 disease correction, we developed a mouse model carrying the IL2RG human gene including a common disease-causing mutation in place of the murine Il2rg gene, allowing to use the same reagents utilized for gene correction of human cells. These mice have impairment in lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we first performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute at least in part the T and B cell compartments. We then developed a protocol to obtain gene correction in murine Lin- HSPC based on the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol was associated with high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR, but also with high levels of acute cytotoxicity (65% cell loss). The surviving cells remained capable of expansion in culture and preserved their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over un-corrected cells. These data indicate functional correction of the IL2RG gene by our strategy. Yet, measuring percentage of correction within myeloid cells at long-term we found that it was almost undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells stably persisted in the mice up to 7 months post transplantation within all the hematopoietic organs. Furthermore, upon challenging the transplanted mice with a murine pathogen (LCMV Arm.) we observed viral-specific γIFN production by CD8+ gene corrected cells at a similar extent as for WT mice, proving in vivo the functionality of corrected T cells. These results suggest that our protocol achieves biologically relevant levels of gene correction in progenitors that sustain long-term lymphopoiesis but is limited in multipotent HSC. Ongoing studies aim to improve murine HSC gene targeting and to compare safety and efficacy of gene correction vs gene replacement in our disease model

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Artt. 2519-2521

Commento ad articoli del codice civile relativi alla normativa applicabile ed alla costituzione delle società cooperativ

Fallimento e impresa

Lo studio analizza la disciplina dei presupposti del fallimento nel quadro della disciplina di riforma della legge fallimentare introdotta nel triennio 2005-2007, nella prospettiva della definizione dell'ambito applicativo della stessa disciplina dal punto di vista dei soggetti cui essa si riferisce e delle condizioni oggettive che ne consentono l'operatività