Gene transfer technologies and new gene therapy strategies

SR-Tiget Unit - ERC Advanced Grant
Luigi Naldini, Head of Unit

We first described the use of hybrid lentiviral vectors for gene transfer into non-dividing cells in 1996 (the original paper reporting this work is one of top-cited articles in Science). Since then, we have been improving the technology for safe and efficient application by introducing new generations of vector design and establishing methods for manufacturing and qualification. Overall, this early work laid the foundation for the currently broad use of lentiviral vectors, one of the most widely used tools in biomedical research. Throughout this time we have continued to investigate new gene transfer approaches and exploit them to gain insights into fundamental biological processes of high relevance for medicine, such as stem cell activity and tumor angiogenesis, and to develop new therapeutic strategies for treating genetic disease and cancer.
We have shown the proficiency of lentiviral vectors at gene transfer into hematopoietic stem cells in stringent experimental models. By reaching exhaustive cell marking with minimal interference with cell function, individual stem cell activity can now be monitored in vivo to unprecedented levels. An unexpected boost towards the broad use of lentiviral vectors came from studies showing that the advanced design of lentiviral vectors is associated with lower genotoxicity than conventional gamma-retroviral vectors, thus providing 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 studies has been crucial for moving lentiviral vectors to the clinic.
Our efforts towards improving gene transfer have always been pursued with the goal of therapeutic translation. We originally selected lysosomal storage diseases as paradigmatic for testing the therapeutic potential offered by lentiviral vectors. We showed that the post-transplant recruitment of hematopoietic cells to the resident macrophage/microglia pool can be exploited to deliver gene therapy to the central and peripheral nervous system and treat metachromatic leukodystrophy in the mouse model. Based on these preclinical studies, in 2009 we started a clinical trial of hematopoietic stem cell gene therapy for the human disease, which is invariably lethal and currently without any effective treatment. The results up to now show that the treatment is safe and provides clear therapeutic benefit (see clinical trial update in SR-Tiget Clinical Research Unit). These encouraging data are fueling our strategic alliance with GlaxoSmithKline to eventually make hematopoietic stem cell gene therapy a broad clinical reality.
As we test late generation lentiviral vectors in the clinic, we keep us busy in the laboratory to improve the robustness and advance the scope of stem cell gene therapy and to create the next generation vectors, in order to overcome the known risks and limitations of the platforms currently being tested.
We continue to investigate approaches to better preserve and expand hematopoietic stem cells in culture to support their genetic manipulation and improve the safety and efficacy of ex vivo gene therapy. We have also improved vectors to achieve stringently regulated transgene expression. We first applied microRNA regulation to vector design in 2006 and provided the prototype for making transgenes and medically used viruses stringently responsive to cell type and differentiation specific cues. This strategy proved highly robust and versatile and is undergoing an increasingly wider exploitation by the biomedical community. More recently, we have identified microRNAs with specific activity in distinct hematopoietic subpopulations and are exploiting them to negatively regulate transgene expression and achieve targeted expression profiles of unprecedented specificity in hematopoietic stem cell therapy (Project 1).
Concurrently, we have developed powerful vector tools to study microRNA function and have identified a key role of some microRNAs in hematopoietic stem cell quiescence, expansion and myeloid differentiation. Ongoing studies exploit microRNA gain and loss of function and unbiased proteomic and transcriptomic approaches to identify relevant targets of these microRNAs and decipher the molecular network they act on. These data are providing novel insights into how hematopoietic stem cell quiescence, commitment and differentiation are regulated (Project 2).
We have pioneered and continue to develop gene targeting approaches based on engineered Zinc Finger Nucleases to edit the genomic sequence. These strategies bring the possibilities of site-specific integration and correction of defective genes within the reach of gene therapy and offer radical new solutions to overcome the major hurdles that have long hindered progress of the gene therapy field. Gene correction, as opposed to gene replacement, not only restores the function of a diseased gene but also its physiological expression control, while avoiding the risks of insertional mutagenesis. We are optimizing and exploiting this approach in lymphocytes, hematopoietic stem cells and induced pluripotent stem cells to correct mutations causing X-linked primary immunodeficiency and other genetic diseases (Project 3).
Furthermore, we are exploiting artificial DNA-binding proteins to stably modulate epigenetic features at pre-selected loci and specifically silence dominant mutations (Project 4).
We continue to investigate the potential of in vivo gene delivery to treat systemic diseases such as hemophilia, capitalizing on the intriguing properties of a lentiviral vector platform stringently targeted to hepatocytes by a combination of transcriptional and post-transcriptional, microRNA-regulated control. By using this strategy, we 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 affected mice and dogs and induce active tolerance to the transgene. Further studies aim towards clinical translation of this strategy and its broader exploitation to treat a range of metabolic and autoimmune diseases (Project 5).
By tracking the hematopoietic cell contribution to angiogenesis, we have established a novel paradigm in which the bone marrow contributes a myeloid cell population required for promoting new vessel formation. These studies helped defining a lineage of pro-angiogenic monocytes, which selectively engage in tissue remodeling and regeneration and can be distinguished from conventional pro-inflammatory monocytes by gene expression, surface markers and functional properties. We continue to investigate the origin and biological function of these cells and exploit them to develop new therapeutic strategies. In one such strategy, we engineer the progeny of transplanted hematopoietic progenitors to target gene therapy to tumors, enhancing therapeutic efficacy and avoiding systemic toxicity (Project 6).

Versione italiana