Dissecting and Overcoming Innate Immune Barriers for Therapeutically Efficient Hematopoietic Stem Cell Gene Engineering (ImmunoStem)
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 819815)
The low gene manipulation efficiency of human hematopoietic stem cells (HSC) remains a major hurdle for sustainable and broad clinical application of innovative therapies for a wide range of disorders. Indeed, high vector doses and prolonged ex vivo culture are still required for clinically relevant levels of gene transfer even with the most established lentiviral vector-based delivery platforms.
Current and emerging gene transfer and editing technologies expose HSC to components potentially recognized by host antiviral factors and nucleic acid sensors that likely restrict their genetic engineering and contribute to broad individual variability in clinical outcomes observed in recent gene therapy trials. Nevertheless, specific effectors are yet to be identified in HSC. We have recently identified an antiviral factor that potently blocks gene transfer in HSC and have discovered small molecules that efficiently counteract it. This is the first example of how manipulating a single host factor can significantly impact gene transfer efficiencies in HSC but likely represents the mere tip of the iceberg of the plethora of innate sensing mechanisms potentially hampering genetic manipulation of this primitive cell compartment.
On these premises, this proposal aims to determine the restriction factors and innate sensing pathways that prevent efficient modification of HSC and to mitigate their effect using methods developed through a thorough understanding of their mechanisms of action. My approach builds on the idea that understanding the crosstalk between HSC and viral vectors will instruct us on which immune sensors and effectors to avoid and how, with direct implications for all gene engineering technologies. Successful completion of this project will deliver novel paradigms of innate pathogen recognition that can be exploited for the development of innovative cell and gene therapies and to fight infectious and autoimmune diseases.
ImmunoStem Aims
AIM 1: Dissecting the determinants of intrinsic antiviral resistance in HSC
We have gained mechanistic insight into how innate immune factors block gene therapy vector entry into HSPC and how our pharmacological strategies counteract them. We have identified the cellular pathway involved in antiviral factor degradation by transduction enhancers (Petrillo et al., Cell Stem Cell 2018) and uncovered novel molecular determinants required for their antiviral activity not only against gene therapy vectors but also other pathogens, including the pandemic causing SARS-CoV2 (Unali et al., EMBO J 2023). In addition, our studies on enhancement of gene therapy in HSC have unravelled additional, potentially beneficial effects our transduction enhancers seem to have on pathways relevant for stem cell biology.
AIM 2: Investigating HSC-specific nucleic acid sensing mechanisms
We have taken advantage of viral vectors to dissect how HSPC detect incoming pathogens leading to activation of antiviral type I IFN responses. Interestingly, we have observed that this activation is independent of the canonical nucleic acid-sensing pathways and seems to occur at the cytoplasmic level through sensors detecting structural rather than genetic components of pathogens in HSPC.
Furthermore, we have investigated vector signalling in the context of diseased HSC and have expanded our investigations to other relevant gene therapy target tissues such as the central nervous system (CNS). We reveal activation of p53-mediated DNA damage responses upon AAV and LV exposure in human iPSC-derived CNS cells, similarly to what we observed for HSC, and of inflammatory and type I IFN responses related to transgene expression (Costa-Verdera et al., Research Square 2024, now in press in Nature Communications). Our work has also contributed to the characterization of a novel STING mutant, involved in detection of cytosolic DNA, causative of the autoimmune interferonopathy SAVI (Valeri et al., J. Exp. Med 2024).
AIM 3: Development of improved HSC gene engineering strategies
To reliably test enhanced transduction and gene editing protocols in pre-clinical in vivo studies, we have set-up and characterized a research-grade LV purification pipeline (Soldi et al.,2020). This effort has allowed us to contribute to a collaboration with the group of Luigi Naldini describing how preventing vector-induced activation of p53 benefits also clonality of gene edited HSPC (Ferrari et al., Nature Biotech, 2020). Moreover, our enhanced LV transduction protocols enable to use of non-integrating LV for gene editing with benefits in terms of cargo capacity and less off-target insertions compared to other donor DNA template delivery platforms (Ferrari et al., Cell Stem Cell, 2022). We have also developed a novel transduction protocol based on the use of CsH and deoxynucleotides (dNs) that enable efficient modification of quiescent unstimulated HSPC with the benefit of achieving higher and more polyclonal engraftment (Valeri et al., Mol Ther 2024).