Research activity

Our laboratory has long been at the forefront of the development of gene transfer strategies and has exploited the new technologies to gain novel insights into fundamental biological processes of high relevance for molecular medicine, such as stem cell activity and angiogenesis, and to provide proof-of-principle of new strategies for treating genetic disease and cancer.

I first described the use of HIV-derived hybrid lentiviral vectors for gene transfer into non-dividing cells in 1996, together with Inder Verma and Didier Trono at the Salk Institute. The original paper reporting this work is one of the top-cited articles in the journal Science (>1850 citations). Over the following years my laboratory has advanced the technology to allow more efficient gene transfer and safe use. Today, lentiviral vectors represent one of the most broadly used gene transfer tool in experimental biology and biotechnology.

Clinical testing of lentiviral vectors has started few years ago in lymphocytes for the gene therapy of AIDS and has proved safety and possibly some efficacy. Paradoxically, we have recently shown that the integration profile and the advanced design of lentiviral vectors is associated with a much lower risk of insertional mutagenesis and genotoxicity than conventional gamma-retroviral vectors, thus providing for a safer gene transfer platform despite the virus of origin. The high efficiency of gene transfer and the improved safety profile of lentiviral vectors have prompted their test for hematopoietic stem cell (HSC) gene transfer. We and others have shown their proficiency at stably and effectively marking long-term repopulating HSC of mice and humans. We are now applying deep sequencing and bioinformatics to map lentiviral vector integration sites in hematochimeric mouse models of human hematopoiesis. These studies allows monitoring HSC clonal activity and evolution to unprecedented sophistication gaining novel insight into their repopulation/differentiation potential in vivo and validating novel features of vector design improving its safety (Research Area: Safety of Vector Integration, Project leader: Eugenio Montini, Ph.D.)

The encouraging results obtained in pre-clinical models provide a strong rationale for clinical application of lentiviral vectors in HSC gene therapy. Our Institute is a prime actor in these developments and two clinical trials of HSC gene therapy for Wiskott - Aldrich syndrome (WAS) and Metachromatic Leukodystrophy (MLD) are planned to start in 2009. The trials will give us a first in-depth glimpse at the clonal activity of HSC in living humans.

The recent application of microRNA regulation to vector design has provided a new experimental strategy in which transgene expression can be made specifically responsive to the cell lineage and differentiation stage of the target cell. Using this innovative approach, we could overcome the immunological barrier to stable gene transfer, one of the major hurdles to successful gene therapy, and have recently established long-term correction of hemophilia in mouse models. Follow-up studies will determine the efficacy and safety of this strategy to cure the disease in the dog model, a more challenging setting which is more predictive of the outcome in humans. Concurrently, we are exploiting microRNA regulated lentiviral vectors to explore the biology of microRNAs, to monitor their activity in stem cells and their progeny in vivo at the single cell level, and even to knock-them down functionally, phenocopying genetic knock-outs.

We have also explored the use of engineered Zinc-finger nucleases to target vector integration and edit the genome with unprecedented efficiency in primary human cells. These studies open the way to correct inherited mutations, rather than replacing genes, a potentially revolutionary approach as it can restore both the function and the endogenous regulation of the defective gene, without the risks associated with random insertional mutagenesis.

By tracking the hematopoietic cell contribution to angiogenesis, our work has provided formal evidence for a novel paradigm in which bone marrow-derived myeloid cells contribute an essential supporting role to blood vessel formation. We have identified a subset of blood monocytes (TEMs, Tie2-Expressing Monocytes) which are specifically recruited to tumors where they promote angiogenesis and tumor growth. Ongoing studies are investigating the origin and lineage relationship of TEMs with other myeloid lineage cells, their gene expression profile and the mechanisms by which they promote tumor growth. Moroever, our recent studies have provided the proof-of-principle of a promising new strategy by which the TEM progeny of transplanted hematopoietic progenitors can selectively target gene therapy to tumors. (Research Area: Angiogenesis and Tumor Targeting, Project leader: Michele De Palma, Ph.D.)

In the field of neurodegenerative disorders, our work has shown that the recruitment of hematopoietic cells to the resident microglia pool can be exploited to deliver gene therapy to the central and peripheral nervous system, and has reported the first successful cure of MLD in the mouse model. As mentioned above, a lentiviral vector based clinical trial for the human disease, which is lethal and currently without any effective treatment, is planned to start in the San Raffaele Institute next year. We are now further investigating the mechanisms of disease correction by HSC gene therapy and applying this approach to the treatment of other storage disorders, including globoid cell dystrophy and mucopolysaccaridoses, in relevant animal models (Research Area: Gene Therapy of Leukodystrophies, Project leaders: Alessandra Biffi, M.D.)

We are also exploring the biology and transplantation of neural stem cells (NSC) and testing novel combined gene- and NSC-based approaches to correct the nervous system damage in storage disorders (Research Area: Gene/neural stem cell therapy for lysosomal storage diseases, Group leader: Angela Gritti, Ph.D.)

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