Lysosomal acid lipase (LAL) deficiency is a rare, autosomal recessive condition caused by mutations in the gene encoding for LAL. Wolman disease (WD) is the most severe form with an incidence of 1 in 300.000 livebirth, resulting in failure to thrive, hepatomegaly, and hepatic failure, and a life expectancy of 3-8 months. Enzyme replacement therapy using Sebelipase ?, a recombinant human LAL, is a very promising treatment, but access, frequent infusion and cost represent clear impediments to this life-long treatment. Allogenic hematopoietic stem cells (HSC) transplantation might be curative; however, it suffers of several post-transplantation complications. To minimize these issues, we proposed to transplant gene corrected autologous HSC. For this we will take advantage of a new lal-/- mouse model. Mouse HSC will be collected and ex vivo modified to express supraphysiological levels of the missing enzyme, which are required to correct the pathology when HSC are reintroduced into the patient. To stably insert the LAL transgene we will take advantage of the CRISPR/Cas9 system to achieve integration in the a-globin locus, which guarantees safe and effective transgene expression in the erythroid lineage. To easily follow in time disease progression and treatment, we will establish a pipeline to analyze lipids and lysosomes accumulation in blood cells using imaging flow cytometry. Finally, for clinical translation, we will optimize donor DNA delivery in human HSC by replacing AAV vector with a novel integrase defective lentiviral vector. Briefly, the proposal has 4 aims: 1) to find an effective treatment for WD; 2) to establish a novel analysis pipeline to follow WD treatment; 3) to develop novel donor DNA vector template; 4) to establish an HSC based GT platform for protein replacement therapy. This research will pave the way for the development of a novel and urgently needed gene therapy approach for WD, as well as for other lysosomal storage disorders.

B cells produce antibodies (Ab), play an essential role in the immune system and are important therapeutic targets. As hybridomas, B cells are also key for biotechnological production of recombinant monoclonal immunoglobulin (Ig) reagents. The possibility of engineering the B-cell genome for Ig production has potentially far-reaching interests in human health through vaccines, cancer, auto-immunity, infectious or genetic diseases and biomedical diagnosis applications. However, primary B-cells remain difficult to modify genetically. Gene editing technology is not commonly used in B cells to induce the production of a desired Ab instead of the cells’s own Ig. We have recently discovered a new system for efficient gene delivery to human and murine B-cells, which we propose to exploit for CRISPR/cas9 genome editing. Three laboratories will collaborate to develop tools and therapeutically-relevant applications in human and murine models. The efforts will initially be focused on editing the Ig heavy chain gene locus. One goal is to precisely redirect antibody specificity by induced antigenic-specificity replacement (iASR). Well-described Ab will be used to test iASR strategies in vitro and in vivo. Another goal is to modify IgH constant region to force a specific class switch recombination (iCSR). This strategy will be developed to generate IgM Abs for diagnostics and blood cell typing. Overall this project’s ambition is to overcome technological limitations to obtain an efficient platform for B-cell enginering and vectored antibody therapy.

Genome editing (GE) technologies based on CRISPR/Cas systems allow targeted genomic modification and have emerged as powerful alternatives to conventional gene addition for various human diseases, with a series of clinical trials in progress. However, some challenges remain to be addressed to obtain more effective precise sequence changes. Novel approaches, such as prime editors (PEs) have raised exciting possibilities, allowing double-strands break-free GE. However, a major limitation of PEs is highly variable efficiency both from one target to another and between cell types. In addition, PEs can only make small sequence changes. Our objectives are to develop (i) alternative, more efficient and safer GE tools based on the PE strategy We will design and evaluate novel PE tools in order to both increase activity per se and overcome cell-specific limitations. We will translate the novel PE tools in an in vivo vertebrate system, the zebrafish, chosen for its amenability to high throughput screening and ease of manipulation. (ii) universal gene therapy strategies for Duchenne Muscular Dystrophy (DMD) and ?-hemoglobinopathies taking advantage of the power of the novel GE tools DMD is the most common form of muscular dystrophy and is caused by mutations in the Dystrophin gene. There is still no effective treatment and a promising universal therapeutic strategy consists in upregulating expression of utrophin in skeletal muscle. We will use PE to upregulate Utrophin by disrupting repressor binding sites. ?-thalassemia and Sickle Cell Disease are frequent genetic diseases caused by mutations in the ?-globin locus. Importantly, clinical severity is alleviated in patients with genetic variants causing hereditary persistence of fetal hemoglobin (HPFH). A universal therapeutic strategy thus aims at generating HPFH mutations in hematopoietic stem/progenitor cells. We propose to use PE to introduce multiple mutations in one step to better rescue the ?-hemoglobinopathy phenotype.

Sickle cell disease (SCD) is a genetic recessive inherited disorder caused by a Glu-to-Val substitution in the β globin protein resulting in abnormal hemoglobin (HbS) that polymerizes under hypoxia driving red cell sickling and reduced half-life. SCD is a severe multisystem disease characterized by hemolytic anemia, high susceptibility to infections, inflammation, recurrent painful vaso-occlusive crises and organ failure. SCD is also characterized by stress erythropoiesis, with abnormalities during terminal erythroid differentiation, suggesting that anemia could also be impacted by defects of central origin. We recently demonstrated the occurrence of ineffective erythropoiesis in the bone marrow of SCD patients and characterized the molecular mechanism as involving the cytoplasmic trapping of HSP70 chaperon protein by HbS polymers under partial hypoxia. Although SCD has been investigated for decades, there is still an urgent need for more studies to understand the complexity of its molecular and cellular defects and to develop new treatments in a personalized medicine perspective. The main treatments in SCD target the causative defect, i.e. HbS, either by adding normal hemoglobin to the circulation, through chronic blood transfusion or allogeneic hematopoietic stem cell transplantation, or by inducing endogenous fetal hemoglobin (HbF) using hydroxycarbamide (HC). Surprisingly, although HC was first tested in SCD patients more than 35 years ago, the molecular mechanisms underlying its mediated induction of HbF are still poorly understood. HbF expression is a known modulator of disease severity in SCD as it inhibits HbS polymerization, prolonging the lifespan of the red cells in the circulation. Importantly, we have recently revealed a new anti-apoptotic role for HbF during terminal erythroid differentiation in SCD by showing that it rescues erythroblasts from cell death at the polychromatic and orthochromatic stages. We have designed an ambitious project to address the unknown molecular mechanisms involved in ineffective erythropoiesis in SCD and to test the effect of known and novel therapeutic strategies on erythroid differentiation. In particular, our IRIS project will investigate the (i) molecular bases of ineffective erythropoiesis in SCD related to the auto-oxidation of HbS and to the impaired α and sickle β chain coupling, and (ii) the impact of erythroblasts’ death on the erythroid niche, particularly on the central macrophage of the erythroblastic island. We will also develop innovative therapeutic strategies based on gene and base editing and assess their effect on ineffective erythropoiesis, together with the effect of other therapeutical molecules such as HC. Our study will be conducted in vitro and in vivo, using a wide panel of tools and human material comprising patient hematopoietic primary cells and cell lines engineered for the project purposes, as well as the humanized Townes SCD mouse model. The IRIS proposal tackles precedingly unexplored areas of the SCD pathophysiology, both on the fundamental and translational research levels. It will reveal new and important biological aspects of SCD that may explain the large variability in disease severity and degree of anemia. It will also offer new molecular therapeutical strategies that can improve patient life expectancy and quality of life and likely reduce the treatment costs.

Histidine-rich peptides that were designed and investigated in our laboratories exhibit a large variety of biological functions that can be tuned by subtle sequence variations, pH, salt and other environmental conditions. These LAH4 peptides exhibit potent antimicrobial and pore forming activities, which are more pronounced when the histidines are charged at low pH than under neutral conditions. Furthermore, self-assembling globular complexes with nucleic acids, are very potent transfection agents when the same peptide has high cell penetrating capabilities also for other cargo. Finally, other derivatives in this peptide family exhibit excellent transduction enhancement in experiments with lenti- and adeno-associated viruses. In this project we will investigate why such subtle variations in sequence can have such profound effects on biological activities. Based on preliminary data we hypothesize that supramolecular architecture enhances small differences in sequence and ultimately determines biological functionality. Such information may be very important to determine the best usage of these adjuvants of transduction (in vitro and in vivo). Although the peptides are closely related and all made up from the same set of amino acids they can occur in mono- or small oligomeric states (antimicrobial), globular complexes in the nano- to micrometer range (transfection) or as fibrous aggregates forming hydrogels (transduction enhancement). Therefore, we propose to investigate in a systematic manner how the aggregation states in solution and in membranes correlates with their activities in antimicrobial, nucleic acid transfection and lentiviral transduction assays. Furthermore, the aggregates will be investigated by a number of highly complementary biophysical approaches including state-of-the art EM, atomic force microscopy, X-ray diffraction and solid-state NMR spectroscopy. The high resolution structural models thus developed will allow for a molecular understanding of the interactions between peptide units, between peptides and nucleic acids and between peptide complexes and membranes. In this manner it can be rationalized which amino acid residues are essential for supramolecular self-assembly, opening the path to the design of new sequences with improved characteristics for a given application. As the supramolecular assembly in aqueous environments is reversible as well as dependent on peptide concentration and chemical environment particular emphasis will be given to mimic as closely as possible the relevant conditions also in biophysical and structural investigations. Therefore, the advancement of novel solid-state NMR technologies is of utmost importance to permit the study of biomacromolecules also at low concentrations or under a restricted range of chemical environments. This problem can only be tackled through a network of 3 partners with experts in biophysics, solid-state NMR spectroscopy, cell biology and biomedical applications. In addition, through an industry-academia partnership Dynamic Nuclear Polarisation and ultra-fast MAS solid-state NMR spectroscopy will be made available, combined and further developed for this project and for biomolecular applications in general. Both technologies have been developed to boost the sensitivity of solid-state NMR experiments and to make the direct observation of 1H nuclei possible also in solid or semi solid samples. Proton-detection is of particular importance when intermolecular contact sites need to be determined, as these nuclei constitute the outermost shell of the molecules.