9^th International Conference and Exhibition on Advanced Cell and Gene Therapy March 21-22, 2019 Hotel: Holiday Inn Rome Aurelia, Rome, Italy

Biography:

Abstract:

Efficient intracellular delivery of exogenous materials remains a critical issue in fundamental biological researches and clinical applications. Here, we developed a novel chemical-free method for intracellular delivery enhancement using a designed gigahertz ultrasonic electromechanical resonator. When excited by a sinusoidal electric signal, the propagation and attenuation of acoustic wave in liquid will generate high-speed acoustic streaming. The liquid above the device working area will be accelerated and strike the substrate surface, thus generates pressure on cells, induces deformation and membrane poration, and finally realizes delivery of exogenous materials. To verify the intracellular delivery ability, DOX was selected as an example, and an enhanced fluorescence of DOX in cells exposed to resonator stimulation can be seen. We also realized the delivery of fluorescent-labeled DNA strains and plasmids. Besides, different power applied to the resonator can induce different fluid velocity, thus generate different force intensity and control the deliver efficiency. Pores on membranes induced by acoustic streaming treatment were observed by SEM. Disrupted cell membranes and porous structures can be seen after treatment, and resealed after 10 min recovery, indicating a strong fluid force exerted on cells and the influence is temporary and reversible.

Biography:

Ingegnere T has conducted his studies needed for both the first grade degree (laurea triennale) and second grade degree (laurea specialistica) at Sapienza University of Rome. After his graduation he did his PhD at CNR in Rome. From 2014 to 2016 he spent his first Postdoc in the Immunology Lab of Patrizio Giacomini at IFO-Regina Elena Cancer Institute. Since April 2016 he is in the lab of Prof. Moretta as Postdoc in the Immunology Unit of Pediatric Hospital Bambino Gesù.

Abstract:

Cell-mediated immune responses play a central role in the control of infections and tumor growth. In particular, cytolytic T lymphocytes (CTL) and natural killer (NK) cells are fundamental effectors against virus-infected, tumor and leukemia cells. Both T and NK cells are particularly efficient also in allogeneic settings such as the allogeneic haemopoietic stem cell transplantation (HSCT) to cure hematologic malignancies. Another particularly promising approach of cellular therapy is the use of genetically-engineered autologous T cells with chimeric antigen receptors (CAR) conferring specificity for antigens expressed by tumor cells. Also NK cells can be genetically engineered with CAR. Different from CAR-T, NK cells, equipped with an array of receptors involved in tumor cell recognition and killing, retain their ability to target neoplastic cells through such receptors, possibly making tumor escape mechanisms less effective. In addition, they may be complementary to CAR-T cells. However, NK cell transfection resulted quite challenging. Thus, viral transduction display to have variable levels of transgene expression and may compromise NK cell viability. Moreover, viral transduction requires dedicated facilities, high costs and lengthy preparation. Recently, electroporation of mRNA has been proposed as alternative of viral methods. Although the mRNA electroporation has a very low effect on the vitality and good efficacy, a relevant drawback are represented by the short-time expression of the transgene. Here we show a new procedure for NK cells transfection with plasmid DNA. With an efficiency of up to 50% and viability up to 65% it is the most efficient, non-viral, methodology existing so far to deliver exogenous DNA into NK cells. By applying this method, we transfected exogenous CCR7 chemokine receptor conferring to the NK cells the ability to efficiently migrate in response to the chemokines. Moreover, the introduction of an anti-CD19 CAR confer to transfected NK cells a specific and powerful cytotoxicity against CD19+ leukemic cells. These results illustrate some of potential important applications of this novel transfection approach. Notably, the electroporation of DNA may allow to a non-integrating gene transfer with episomal vectors.

Biography:

Sharmila Fagoonee did her PhD thesis on heme and hemoglobin metabolism at the University of Turin. During her Post-doc studies, she started working in the field of Regenerative Medicine after a training at BIDMC, Harvard, Boston. As a CNR Researcher, she is currently studying new therapies and biomarkers for liver diseases. She is Co-Investigator in several research projects (Telethon, PRIN, Regional grants). She has published more than 142 scientific papers. She has served as Reviewer for several scientific journals, and is an Editorial Board Member of Journal of Clinical Medicine and Minerva Biotecnologica.

Abstract:

The Crigler Najjar syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of a population of stem cells isolated from cryopreserved hepatocytes known as human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1^-/- mice, which closely mimic the pathological manifestations in CNSI patients. In order to assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture in this 3D setting ex vivo, HLSC differentiated into hepatocyte-like cells expressing markers such as albumin and cytochrome 1a1. For the in vivo human cell engraftment and recovery experiments in the Crigler-Najjar mouse model, NSG/Ugt1^-/- mice were generated. A single dose of HLSC was injected in the liver parenchyma of 5 days old phototherapy-treated NSG/Ugt1^-/- pups and HLSC functionality and phenotype rescue were assessed in vivo at post-natal Day 21. HLSC expressed UGT1A1 in vivo, induced a decrease in serum unconjugated bilirubin, and improved phenotype and survival compared to untreated controls. A significant reduction in eosinophilic neurons was also observed in HLSC-injected mutant mice hippocampus and cerebellum reflecting recovery from brain damage versus controls. Our results show that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1^-/- mice, and show promises for the treatment of CNSI.

Biography:

Farzin Farzaneh holds the Chair of Molecular Medicine at King’s College London. He has published over 250 research articles with an average citation of over 30, and an Impact Factor of 47. He has run a licensed GMP facility at King’s College London, since 2001, for the production of cell and gene therapy based investigational medicinal products. Farzin has extensive industrial and academic collaborations, including research council, charitable and pharmaceutical sponsorships of £25M. He has initiated a number of clinical trials in novel applications of gene therapy and holds MHRA licences (IMPs and “Specials"). He is a Qualified Person (QP) for release of cell and gene therapy products in UK and EU and an Individual Designate under a Human Tissue Authority licence that allows procurement, testing, processing, distribution and/or import/export of tissues and or cells intended for human applications. He is also appointed by the Commission on Human Medicines, as a member of the Clinical Trials, Biologicals & Vaccines Expert Advisory Group since 2016.

Abstract:

The therapeutic potential of cell and gene therapies has created a great deal of excitement in a range of biomedical fields. The path from fundamental research, through to licensing applications involves several important components. These include, studies assessing the validity of critical concepts, pre-clinical potential safety and efficacy studies, the design of clinical trials, production of products that are suitable for clinical investigations. Similarly important is the process leading to regulatory authorisation of clinical studies, the efficient conduct of meaningful clinical investigations, proof of concept and pivotal trials through to licensing applications. These processes constitute a complex path, with multiple critical steps and potential bottlenecks, involving important design and implementation challenges.

In an open discussion forum, some of the most prominent of these challenges will be identified and strategies to address them will be examined.