San Raffaele Telethon Institute for Gene Therapy

Gene transfer technologies and new gene therapy strategies


Head of Unit

Luigi Naldini


Group's early work laid the foundation for the current broad use of lentiviral vectors (LV), both in biomedical research and as advanced therapy medicinal products in several gene therapy clinical trials, carried out in our own Institute and several other centres worldwide. To date, several hundred patients suffering from immunodeficiencies, blood or neurodegenerative diseases, and some types of cancer, have markedly benefited from HSC or T-cell gene therapy. As our early research has now reached the clinic, the unit is back at the bench working on the next generation genetic engineering.


Since the first report of programmable nucleases to enable targeted genome editing, our lab has been at the forefront of exploiting new powerful tools for precise genetic engineering of hematopoietic cells, with the ultimate goal of achieving in situ gene correction or targeted transgene integration with limited risk of genome-wide mutagenesis. Ex vivo gene editing is mostly performed by exploiting: (i) programmable endonucleases that induce a DNA double strand break (DSB) in the genomic site of interest, and (ii) DNA vectors that transfer the template for homology-directed repair (HDR) of the DSB. We have provided the first proof-of-principle of targeted genome editing in human hematopoietic stem cells (HSC) followed by several studies highlighting the major barriers to attain efficient editing (p53 activation upon editing, cell cycle state and HDR proficiency) and demonstrated new strategies to overcome them.


Associated Research Unit: Targeted Cancer Gene Therapy


Research activity

Our current research encompasses two major areas:

  1. Clinical Translation of ex vivo Gene Editing of Hematopoietic Cells. We are working to advance ex vivo gene editing of hematopoietic cells towards clinical testing for the treatment of life-threatening diseases that provide a suitable risk-benefit ratio for a first-in-human and first-of-its-kind clinical trial. We have prioritized the translation of targeted gene editing in human T cells for the treatment of Hyper-IgM1 syndrome, a life-threatening primary immunodeficiency caused by mutations in the CD40L gene and consequently impaired T cell function. We selected this application because of the potential disease rescue by a limited input of corrected cells and the possibility of directly correcting T cells, providing for easier translation, lower safety concerns and potentially comparable clinical benefits than editing hematopoietic stem and progenitor cells (HSPC). This will be the first indication pursued by SR-Tiget in the gene editing space and may well become one of the first HDR-based editing strategy clinically developed worldwide. We will take advantage of a close collaboration with our spinoff company Genespire and the SR-Tiget Process Development Laboratory, with the final goal of establishing an optimized and proprietary HDR-based gene editing platform, which will then be adapted also to HSPCs and more broadly applied to the treatment of other genetic diseases.
  2. Enhancing HSC Gene Editing and Developing Non-genotoxic Conditioning Strategies for Engrafting HSPC. Widespread adoption of HSPC gene editing for the treatment of genetic diseases is still limited by the efficiency of HDR-mediated integration of the donor cassette and the burden of the procedure on treated cells, which ultimately shrink the yield and the clonal complexity of the edited cell population. Furthermore, the induction of a DSB may cause, in some cells, adverse events at the genomic target site, such as large deletions and structural rearrangements (e.g., chromotripsis). Here, we investigate the major biological constraints to HSPC gene therapy and develop strategies to overcome them. We aim to improve the efficiency of HDR in the more primitive progenitors and purge unwanted genomic events by selecting cells with the desired editing outcome. These studies will enable application of gene editing to a broader range of diseases, which require higher correction thresholds than primary immunodeficiencies.
    In parallel, we are exploring the applicability of novel DNA double-strand break (DSB)-free editors as potential alternatives to HDR-mediated gene editing for the site-specific correction of disease-causing mutations.
    Furthermore, to leverage the full potential of autologous HSPC gene editing, we are investigating strategies to bypass the off-target toxicity of current conditioning regimens by exploiting selective immunodepletion of resident progenitors or forcing their mobilization, followed by the infusion of edited HSPCs endowed with transient engraftment advantage, thus promoting exchange within the marrow niches and establishment of partial mixed chimerism with engineered cells sufficient to provide therapeutic benefit. We are exploiting gain-of-function screenings in human hematochimeric mouse model to identify key regulators of human HSPC engraftment and integrate them in the gene editing pipeline. If successful, our studies will establish a new golden standard for precision engineering of hematopoiesis, providing for robust polyclonal engraftment of HSPCs carrying the desired gene edit while minimizing the acute morbidity and long-term safety concerns of the procedure.