In his early career, L. Naldini identified the ligand for the Met receptor with Hepatocyte Growth Factor (HGF), proved its identity with Scatter Factor and elucidated its mechanism of regulation and function in triggering motility and invasion of epithelial cells. MET has since been one of the most investigated oncogene in epithelial cancer and metastasis. During his stay within Inder Verma and Didier Trono laboratories at the Salk Institute for Biological Studies, La Jolla (1994-96), he first described the use of HIV-derived hybrid lentiviral vectors for gene transfer into non-dividing cells. The original paper reporting this work is one of top-cited articles in the journal Science (more than 3,430 citations). He then developed the technology for safe and efficient use working as a senior scientist at Cell Genesys, Foster City, CA. He discussed with the RAC, FDA and EMA the requirements and implications of lentiviral vector administration to humans. Overall, this work laid the foundation for the currently broad use of lentiviral vectors; what was initially received as an elegant proof-of-principle of an unlikely and fearsome technology, has become one of the most widely used tool in biomedical research. At the end of 1998, L. Naldini returned to academia as professor at the University of Torino and in 2003 moved to the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) in Milan, initially as co-director with Maria Grazia Roncarolo and since 2008 as director of the Institute. Throughout this time, he has continued to investigate new strategies to overcome the major hurdles to safe and effective gene transfer, bringing about innovative solutions that not only were translated into new therapeutic strategies for genetic disease and cancer but also allowed gaining novel insights into fundamental biological processes such as hematopoietic stem cell function, induction of immunological tolerance and tumor angiogenesis. Concerning vector development, L. Naldini’s work led to improved gene transfer into relevant cell types such as hematopoietic stem cells (HSC). By reaching exhaustive cell marking with minimal interference with cell function, individual HSC activity can now be monitored in vivo to unprecedented levels. A boost towards the broad use of lentiviral vectors came from studies primarily conducted in Naldini’s laboratory showing that the advanced design of lentiviral vectors is associated with much lower genotoxicity than conventional gamma-retroviral vectors, thus providing for a safer gene transfer platform despite the original concerns raised by the nature of the parental virus. The demonstration of high gene transfer efficiency coupled with improved safety provided by these pre-clinical studies was crucial for moving lentiviral vectors to clinical testing. Gratifyingly, these predictions have now been verified in a growing number of clinical studies, as mentioned below. By tracking the hematopoietic cell contribution to angiogenesis, Naldini’s work established a novel paradigm in which the bone marrow contributes essential paracrine regulators to the newly formed vessels. These studies helped defining a subset of proangiogenic monocytes, which selectively engage in tissue remodeling and regeneration and can be distinguished from conventional monocytes by gene expression, surface markers and functional properties. Naldini and his collaborators are now exploiting these findings to develop a new therapeutic strategy by which the progeny of transplanted hematopoietic progenitors is engineered to selectively target gene therapy to tumors, thus enhancing therapeutic efficacy and avoiding systemic toxicity. A clinical trial for the first-in-human testing of this strategy opened at the beginning of 2019 for the treatment of glioblastoma multiforme sponsored by a biotech company spin-off co-founded by Luigi Naldini and the San Raffaele Scientific Institute. In another development, Naldini’s research applied microRNA regulation to vector design and provided the prototype for making transgenes and medically used viruses stringently responsive to cell type- and differentiation-specific cues. By using this innovative approach, Naldini’s team could overcome the immunological barrier to stable gene transfer, one of the major hurdles to successful gene therapy, establish long-term correction of hemophilia in small and large animal models and induce active tolerance to the transgene. Ongoing work performed in collaboration with Biogen/Bioverativ (recently transferred to Sanofi Aventis) is progressing these studies towards clinical translation of a new gene therapy treatment for hemophilia. The strategy of microRNA regulation is now widely exploited to develop safer vectors, oncolytic viruses and viral vaccines. In collaboration with John Dick’s group, L. Naldini identified microRNAs with specific activity in HSC, showed that miR-126 sets a threshold for HSC activation and governs HSC pool size, and contribute to key pathogenic features of leukemia initiating cells when aberrantly expressed. The expression pattern of miR-126 was then exploited to design vectors transcriptionally silent in HSC but active in their mature progeny. Naldini’s laboratory also pioneered the use of engineered Zinc-finger nucleases to edit the human genome in relevant cells for therapeutic applications. These studies opened the way to correct, rather than replace genes, a potentially revolutionary approach that may substantially expand the scope and power of genetic manipulation. Together with Chiara Bonini’s group, L. Naldini provided the first proof-of-principle of T-cell receptor genetic editing as a novel means of T-cell therapy, in which a new biological function is instructed to an immune effector cell by genetically re-writing its endogenous antigen specificity. L. Naldini’s group also reported the first evidence of successful targeted genome editing in human HSC and its application to correct mutations causing some primary immunodeficiencies in patients’ cells and in mouse disease models. Recently, optimization of the editing procedure, also using CRISPR/Cas technology, has allowed achieving substantial levels of targeted gene editing in human long-term repopulating HSC to support further development towards clinical testing. In 2015-2017, L. Naldini was appointed member of an international study committee on Human Gene Editing by the National Academy of Sciences, USA, which issued widely received recommendations for the development of this technology in view of its scientific potential medical and ethical implications. Throughout the years, L. Naldini’s efforts towards improving gene therapy have always been pursued with the clear goal in mind of therapeutic translation. Work from his laboratory showed that the post-transplant recruitment of hematopoietic cells to the resident microglia pool could be exploited to deliver gene therapy to the central and peripheral nervous system and treat leukodystrophies in the mouse model. Successful clinical testing of lentiviral vectors in HSC gene therapy was first reported in 2009 by a French team led by Patrick Aubourg to treat adrenoleukodystrophy (ALD), using the vector platform previously developed by Naldini and collaborators. Shortly thereafter, a lentiviral vector-based HSC gene therapy trial was launched at SR-Tiget for metachromatic leukodystrophy (MLD), which is invariably lethal and without any effective conventional treatment. Children treated before or early after symptoms onset are reported at the latest follow-up, reaching up to 10 years, in good conditions and leading a normal or near normal life, whereas they would have already succumbed to the disease if left untreated. Application of lentiviral vector HSC gene therapy continues to expand, at SR-Tiget and elsewhere in the world, to treat patients with immunodeficiencies, storage diseases and hemoglobinopathies like thalassemia, again showing excellent safety and clinical improvements reaching up to full transfusion independence. Overall, >80 patients have been treated at SR-Tiget in Milan and >200 worldwide, with all studies reporting excellent safety and efficacy. Molecular monitoring of the patients in these trials show extensive genetic engineering of human hematopoiesis, with highly polyclonal reconstitution and no evidence of genotoxicity, consistently with the advanced engineering of the vector design and the preclinical predictions from experimental models. Moreover, these studies allow unprecedented insights into the clonal dynamics of human hematopoiesis, providing the first glimpses of HSC activity in living humans. Based on the pioneering work of SR-Tiget in the clinical development of early generation HSC gene therapy for Adenosine Deaminase Deficiency (ADA-SCID) and the leadership provided in pursuing a new generation of vectors based on lentiviruses, SR-Tiget entered in 2010 in a strategic alliance with GlaxoSmithKline (recently transferred to Orchard Therapeutics) to support further clinical development and market access of these therapies and make HSC gene therapy a clinical reality. This first-of-its-kind agreement between a major pharmaceutical company and an academic center engaged in gene therapy highlighted a road map for many more such alliances to come in recent years and was credited in 2016 by the successful registration in EU of the first ex vivo gene therapy product worldwide, Strimvelis, and to market entry of lentiviral MLD gene therapy in 2020.