San Raffaele Telethon Institute for Gene Therapy

Gene transfer technologies and new gene therapy strategies


Head of Unit

Luigi Naldini


Our 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 centers worldwide. To date, several hundred patients suffering from immunodeficiencies, blood or neurodegenerative diseases have markedly benefited from LV-mediated hematopoietic stem cell (HSC) gene therapy and several thousands of cancer patients have benefited  from LV-engineered CAR T cells. 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 exploits an ever-growing and versatile armamentarium of platforms that relies on specific combinations of domains with DNA recognition and DNA modifying functions. We have provided the first proof-of-principle of targeted genome editing in human HSCs, followed by several studies highlighting the major barriers to attain efficient and safe editing by any of these platforms (p53 activation upon editing, cell cycle state, DNA repair proficiency, and preservation of genome integrity). Contextually, we have developed new strategies overcoming them by leveraging on novel delivery modalities, fine-tuning culture conditions, dampening adverse cellular responses, and bypassing genotoxic conditioning regimens.


Associated Research Unit: Targeted Cancer Gene Therapy


Research activity

Our current research encompasses four 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 by homology-directed repair (HDR) 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. 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. In close collaboration with the SR-Tiget Process Development Laboratory, we have established an optimized, proprietary and clinically compliant gene editing platform that is now reaching clinical testing. We are now committed to capitalize on our proprietary HSC gene editing platforms by pursuing its clinical translation for the treatment of other, life-threatening genetic diseases.
  2. Enhancing HDR-mediated Gene Editing. HDR-mediated editing is currently the only strategy allowing targeted integration of large therapeutic DNA sequences. Yet, its widespread adoption in HSPCs is limited by the poor efficiency, the burden of the ex-vivo culture and editing procedure on treated cells, and the safety concerns related to frequent occurrence of unintended, potentially genotoxic, repair outcomes at the nuclease on- and off-target sites. Here, 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. In our vision, coupling selection strategies, optimized culture conditions, and harmless delivery modalities will enable the application of ex-vivo gene editing to a broader range of diseases, which require higher correction thresholds than primary immunodeficiencies.
  3. Exploring Novel Applications of Emerging Editing Systems. We have shown that emerging gene editing technologies, i.e., base and prime editors, can be highly efficient in human HSPCs, albeit not eluding cellular sensing and potentially raising concerns because of residual DNA DSB generation at the target site and guide-RNA independent activity genome wide. While we keep monitoring the precision of these emerging platforms by leveraging on next-generation multiomic technologies, we aim to capitalize on our improved protocols for base and prime editing to model and correct hematologic disorders that are not amenable to nuclease-based gene editing and bear significant unmet medical needs.
  4. Developing Non-genotoxic Conditioning Strategies for Engrafting HSPC. To leverage the full potential of autologous HSPC gene editing, our research is pioneering approaches to circumvent the detrimental off-target effects associated with traditional conditioning regimens. We are exploring innovative strategies that include the selective immunodepletion of resident progenitor cells or their forced mobilization. This toxic-free conditioning is followed by the infusion of edited HSPCs endowed with transient engraftment advantage, thus promoting exchange within the marrow niches and establishing a partial mixed chimerism sufficient to provide therapeutic benefit. Our cutting-edge research extends to large animal models to evaluate the feasibility of translating our mobilization-based conditioning strategy into clinical applications. Furthermore, we are exploiting gain-of-function screenings in human hematochimeric mouse models 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.