Guest Editors
Daniela Benati
Department of Life Sciences, Centre for Regenerative Medicine University of Modena and Reggio Emilia via Gottardi 100 Modena , Italy
Dr. Daniela Benati received her M. Sc. in Pharmaceutical biotechnology from the University of Bologna. She earned her PhD in Evolutionary Biology from the University of Siena in 2009, working in the laboratory of Prof. Cosima Tatiana Baldari, where she described the immunomodulatory activities of statins on antigen presenting cells. In 2010, she joined the group of Dr. Lisa Chakrabarti at the Pasteur Institute as a postdoctoral fellow. During her 4-year postdoc she identified high avidity HIV-specific TCRs, which she transferred, via lentiviral vectors, to healthy donor T cells to induce efficient anti-HIV responses, thus providing important tools for the development of immunotherapeutic approaches for a functional cure of HIV infection. Since 2015 she has been senior postdoc at the University of Modena and Reggio Emilia working in the laboratory of Dr. Alessandra Recchia. Her research interests cover the development of gene therapy strategies for the correction of genetic mutations causing monogenic disorders. She focuses on the employment of viral and non-viral methods to optimize the delivery of gene editing tools, such as CRISPR/Cas9, into different cell types both in vitro and in vivo.
Alessandra Recchia
University of Modena and Reggio Emilia, Department of Life Sciences
Dr. Alessandra Recchia received her M.Sc. in Biology from II University of Rome “Tor Vergata” in 1994. She obtained her Ph.D. in Experimental Medicine from University of L’Aquila in 1999, working at the Research Institute of Molecular Biology (IRBM) in Rome. In 1997 she was visiting scientist at McMaster University (Ontario, CANADA) under the supervision of Prof. F. Graham who pioneered the Adenoviral vectors. Thanks to a fruitful collaboration between IRBM and Prof. Graham, she developed the first helper dependent Adenoviral vector (HDAd) carrying the integration machinery of adeno-associated virus (AAV) able to integrate in the AAVS1 region of chromosome 19. Then (2000-2002), as post doc in Prof. F. Mavilio’s lab at TIGET Institute (Milan), she generated an improved HDAd/AAV vector demonstrating its application in human primary cells and in transgenic mice carrying the human AAVS1 site. For this study she received in 2004 the Young Investigation Award by the European Society of Gene Therapy. In 2003, as senior post doc in DIBIT Institute (Milan), she participated to the clinical translation of retroviral vector (RV) technology in milestone clinical trials for gene therapy of Junctional Epidermolysis Bullosa (JEB) and she was involved in the molecular follow up of clinical trials for genetic and acquired disorders (adenosine deaminase deficiency, JEB, graft-versus-host disease). In 2005 she joined the Department of Biomedical Science at University of Modena and Reggio Emilia, as Assistant Professor in Molecular Biology. Since 2008 she established her independent group devoted to gene therapy for monogenic disorders. She contributed to unravel the integration profile of RV and lentiviral vectors employed in many clinical trials. In 2011-2012 she began studying gene editing and Sleeping Beauty (SB) system. She employed these systems to correct monogenic disorders (adRP, JEB and RDEB) in vitro and in mouse models. Due to the heterogeneity of diseases and target genes/tissues, she employed and optimized different viral (Ad, AAV, lentiviral vector) and non-viral (electroporation, chemical transfection) systems to achieve efficient and safe delivery of the gene editing toolboxes or SB components into clinically relevant target cells that, in most of the cases, are refractory to canonical chemical transfection.
Collection Overview
CRISPR/Cas genome editing systems represent one of the most robust platforms in basic biomedical research and therapeutic applications. To date, the strategies of CRISPR/Cas9 delivery are primarily physical/chemical approaches such as transfection, electroporation, hydrodynamic injection or viral vectors such as lentivirus, adenovirus, or adeno-associated virus. Despite the several available molecular formats of Cas9/gRNA and molecular components includeing plasmid, mRNA, and ribonucleoprotein for physical/chemical delivery, they often are more successful in vitro than in vivo. To deliver Cas9/gRNA components in vivo, various viral and non-viral methods have been developed. Viral vectors carrying the Cas9/gRNA are used extensively because of their high efficiency of gene delivery and long-term transgene expression. However, their intrinsic drawbacks including risk of carcinogenesis, limitation of insertion size and immune responses severely limit their application. As a low toxicity alternative, non-viral methods including lipid nanoparticles, gold nanoparticles, and nanoparticles conjugated to penetrating peptide (CPP) offer appealing possibilities in CRISPR/Cas9 delivery. Current CRISPR/Cas9 delivery systems have shown relatively low gene delivery efficiency and transgene expression. However, with the rapid development of novel biomaterials in recent years, efficient delivery of gene payloads to pass the multiple barriers present under physiological conditions and promote transgene expression can be achieved. The goal of this Methods Collection is to showcase standardized protocols for delivering Cas9/sgRNA components to target cells both in vitro and in vivo.