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Pathogenesis and therapy of primary immunodeficiencies

SR-Tiget Unit
Alessandro Aiuti, Deputy Director, Clinical Research Coordinator and Head of Unit

alessandro.aiuti@hsr.it

Primary Immunodeficiencies (PIDs) are a group of rare genetic diseases characterized by an altered innate and adaptive immune system, leading to increased susceptibility to infections, risk of autoimmunity and cancer. Without treatment, many of these conditions are fatal and require early intervention. SR-TIGET was one of the pioneer institute bringing hematopoietic stem / progenitor cell (HSPC) gene therapy (GT) from preclinical studies to successful clinical applications in adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) and Wiskott-Aldrich syndrome (WAS). Our group is working towards the identification of the genetic bases and the pathophysiological processes underlying PIDs and autoimmune/autoinflammatory diseases with the goal of developing novel ex vivo gene and cell therapy strategies for such conditions. In addition to ADA-SCID and WAS, we are currently investigating the molecular and cellular mechanisms linked to mutations in genes encoding the gp91phox NADPH oxidase (Chronic Granulomatous Disease), ARPC1B and ADA2/CECR1 with the final goal to develop a gene therapy approach for these diseases. Moreover, we are characterizing the biological properties of HSPCs and mesenchymal stromal cells (MSCs) and improving the procedures for their ex vivo isolation, genetic modification and transplantation. Specifically, we are conducting vector integration studies using HSPCs isolated from PID patients undergoing gene therapy. These studies will provide crucial information on vector biology, dynamics of gene-corrected HSPCs and safety of retroviral/lentiviral-mediated gene therapy. We are also exploring the possibility to use HSPCs in combination with MSC to improve the outcome of HSPC-gene therapy strategies. Collectively, these studies will contribute to advance our knowledge on PIDs and improve patients’ care.

Research activities (updated December 2017)

HSPC biology: in vivo clonal tracking and cell lineage modeling
Human hematopoietic Stem and Progenitor Cells (HSPC) are an heterogeneous population with different degree of self-renewing and differentiation capacity. Despite their wide clinical exploitation, there remains limited understanding on how distinct human HSPC contribute to hematopoiesis at steady state or after stem cell transplantation, interact with bone marrow (BM) stromal niche cells and recirculate. Integration site (IS) clonal tracking represents a powerful tool to unveil population dynamics and hierarchical relationships in vivo in humans, since upon transduction each cell and its progeny become univocally marked by a distinct IS.
The main goals of this project are: 1) to dissect the role of HSPC subsets in hematopoietic reconstitution through combined clonal tracking with phenotypic, transcriptome profiling and functional studies, during early and late phases after gene therapy (GT); 2) to investigate the interaction between HSPC and mesenchymal stromal cell niche and the ability of these cells to re-circulate; 3) to model hierarchical relationships of HSPC and specific mature lineages.
Applying longitudinally high-throughput IS retrieval to our lentiviral-based HSPC-GT clinical trial for Wiskott-Aldrich Syndrome (WAS) we traced >89.000 clones from 15 distinct BM and peripheral blood (PB) lineages, including BM CD34+ cells isolated from four WAS-GT patients. The progressive increase of shared IS among BM and PB lineages and the presence of stable HSPC multipotent output one year after GT, led us to hypothesize two distinct phases of hematopoietic reconstitution after GT: a first phase (0-12 months after GT) sustained by short-term unipotent HSPC followed by a second phase (>18 months post-GT) sustained by long-living multilineage hematopoietic stem cell (HSPC) (Biasco L., et al., 2016). We are currently extending the resolution of our studies isolating 7 distinct HSPC subtypes at early and steady-state reconstitution phases post-GT. IS shared among HSPC subtypes were also highly represented in mature lineages, indicating our capability to track in real-time active progenitors in vivo. In order to monitor hematopoietic reconstitution after GT, we have also developed a multi-parametric 17-color flow cytometry-based assay, named “Whole Blood Dissection”, able to unambiguously identify up to 23 different blood cell types, including HSPC subtypes and all major mature cell lineages, in BM and PB samples in a single test-tube. These studies will allow us to definitively unveil information on the HSPC clonal behaviors in vivo in humans.
We also exploited a new strategy to characterize in vivo individual mature T-cell subtypes, including the recently defined T Memory Stem Cells (TSCM), a population described to couple memory long-term survival with naïve differentiation potential. Our data confirm the long-term survival potential of memory T cells at clonal resolution level, and the persistence of gene-corrected lymphocytes in patients (Biasco* L, Scala* S, et al., 2015). We provided the first evidence that TSCM actively participate in the long-term graft of infused lymphocytes in humans and constitute a long-term reservoir for the in vivo generation of T-cell memory subsets.

Functional characterization of the bone marrow mesenchymal stromal compartment
Bone marrow (BM) mesenchymal stromal cells (MSCs) are a rare population of multipotent progenitors capable of supporting hematopoiesis and control inflammation. MSCs are isolated thanks to their ability to adhere to tissue culture plastic and expanded in vitro as fibroblast-like cells. They are characterized by the expression of specific markers (CD105, CD73 and CD90) and they can differentiate into osteocytes, adipocytes and chondrocytes. MSCs have been shown to control hematopoietic stem cells (HSCs) homeostasis, self-renewal and differentiation. Indeed, HSCs co-cultured with MSCs are enriched for highly clonogenic CD34+ cells, indicating that MSCs are able to maintain HSCs quiescent in a primitive state. Moreover, MSCs ameliorate the outcome of BM transplantation (BMT), especially when low numbers of cells are available for transplantation, by secreting specific cytokines that prevent graft failure and facilitate HSCs engraftment. The main aim of our study is to develop an MSC-based approach to ameliorate the outcome of HSC-gene therapy (HSC-GT) strategies ongoing at SR-TIGET. We developed a method to isolate and expand ex vivo MSCs from the CD34 negative fraction (CD34 MSCs) of human BM as autologous source of MSCs for future GT and BMT clinical applications. Using this isolation method we aim to:
1) Characterize the mesenchymal compartment of patients pre-GT to identify alterations in the biological and functional properties of MSCs. This is particularly important considering that a functional micro-environment is likely to better sustain gene-corrected HSC engraftment and that functional MSCs should efficiently support maintenance and expansion of primitive HSCs.
2) Set 2D and 3D co-culture methods to use autologous or third-party MSCs as feeder layer to maintain primitive HSCs during gene transfer in vitro.
3) Develop co-infusion protocols of MSCs and HSCs to facilitate the engraftment of gene corrected HSCs.
4) Set 2D and 3D ex-vivo models combining HSCs, MSCs and endothelial cells, to investigate the interactions among the different cellular components of the BM niche. Similarly, we are working on in vivo humanized ossicle models to study BM niche physiology in health and disease.
5) Characterize functional and phenotypic properties of different MSCs subpopulation in vitro and in vivo by genome wide expression analysis.

Gene therapy for Chronic Granulomatous Disease
Chronic Granulomatous Disease (CGD) is a rare genetic condition that affects the immune system caused by loss-of-function mutations in genes encoding the NOX family of NADPH oxidases. NADPH oxidases are key producers of reactive oxygen species (ROS) in immune phagocytes (macrophages, dendritic cells, monocytes), which play a pivotal role in killing of intracellular bacteria and fungi. Therefore, CGD patients recurrently suffer severe infections with bacterial and fungal pathogens, as well as the formation of granulomas in various tissues. Preclinical and preliminary clinical studies have indicated that gene therapy (GT) with hematopoietic stem cells (HSC) may represent an alternative to conventional bone marrow (BM) transplantation. Thus, to this aim we have developed a GT approach mediated by lentiviral vectors (LVs) to target gp91phox expression in immune phagocytes while sparing primitive HSC, thereby reducing the risk of genotoxicity. In preclinical studies, we showed that LVs developed at SR-TIGET efficiently restored gp91phox expression and function in human CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. Moreover, in a mouse model of acute Staphylococcus aureus infection, untreated CGD mice showed an increased production of pro-inflammatory cytokines and chemokine, whereas CGD mice transplanted with gp91phox-corrected HSC showed infiltration of inflammatory cells, cytokine production, bacterial burden and residual lung damage comparable to wild type mice. In addition, LV transduced BM CD34+ cells from CGD patients efficiently engrafted in immune-deficient mice showing restoration of gp91phox expression in vivo. The gp91phox LVs developed at SR-TIGET represents a promising tool for GT in CGD and thus will be further investigated for clinical development.

Next-Generation Sequencing based platform for clinical molecular diagnosis and gene discovery of Primary Immunodeficiencies
The incidence of Primary Immunodeficiencies (PIDs) varies in different countries with a range of 1 in 700 to 1 in 19,000, but increasing evidence has suggested that PIDs are not rare disorders, and are more common than generally thought. More than 260 disorders have been identified, resulting from mutations in over 300 genes. However, with the advent of Next Generation Sequencing (NGS) their number is rapidly increasing. Our laboratory is currently undergoing a NGS-based genetic screening of patients exhibiting symptoms of Combined Immunodeficiencies (CIDs) and Common variable immune deficiency (CVID) with autoimmune features and complex clinical phenotypes characterized by a familiar pattern. We identified several novel mutations in genes responsible for combined immune deficiencies, allowing the identification of suitable specific treatment to patients (i.e. bone marrow transplantation, gene therapy, pharmacological treatment). We identified patients carrying mutations in the gene encoding Actin Related Protein 2/3 Complex Subunit 1B (ARPC1B), one of seven subunits of the human Arp2/3 protein complex implicated in the control of actin polymerization in cells. We are currently investigating cytoskeletal defects in patient cells by phenotypical and functional tests. Moreover, we also identified mutations in adenosine deaminase 2 (ADA2, also known as Cat Eye Syndrome Critical Region Protein 1, CECR1) encoding a member of a subfamily of the adenosine deaminase protein family regulating levels of the signaling molecule adenosine, in patients with systemic vascular inflammatory disorder, stroke and clinical immunodeficiency. We are currently developing lentiviral vector-mediated approaches, in vitro and in vivo models for these diseases. Collectively, our NGS studies will contribute to provide tremendous insights into the molecular basis of PIDs and will set the basis for novel therapeutic strategies for those patients with unmet medical need.

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