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Expanding and extending gene therapy of monogenic diseases of the haematopoietic system

Periodic Reporting for period 3 - GENE FOR CURE (Expanding and extending gene therapy of monogenic diseases of the haematopoietic system)

Reporting period: 2019-10-01 to 2021-03-31

Primary immunodeficiencies (PIDs) constitute a heterogeneous group of inherited diseases that mainly affect children. The most severe PIDs are life-threatening conditions, characterized by (i) susceptibility to infections (ranging from a broad spectrum of pathogens to single microbial agents), and (ii) a tendency to develop autoimmune disorders and cancer. The use of supportive treatments and improvements in clinical care have increased the life expectancy of patients with PIDs - some of whom can now expect to reach adulthood. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative option for a growing number of inherited and acquired diseases of the lymphohaematopoietic system. Given that (i) not all patients have an HLA-genoidentical sibling donor and (ii) severe immunological complications worsen the outcome in HLA-partially-matched HSCT, the genetic modification of autologous hematopoietic stem and progenitor cells (HSPCs) has become a powerful curative treatment option for these individuals. The early experiments on primary immune deficiencies (PIDs) have confirmed this hypothesis and constitute the basis for extending gene correction procedures to more prevalent and/or complex genetic disorders of haematopoiesis, such as Wiskott-Aldrich syndrome (WAS) and sickle cell anemia (SCA).
The present project seeks to further consolidate the rationale for replacing HLA-partially-matched HSCT with a gene therapy approach to treat PIDs and to explore new strategies of gene modification by gene-editing technology.
The main goals of this project are as follows:
- WP1: Immunological and haematological reconstitution after gene therapy in WAS patients: implications for the clinical outcome, with a particular focus on autoimmunity and microthrombocytopenia
- WP2: Gene therapy of a SCID caused by an Artemis mutation: implementation of the first clinical trial for this indication
- WP3: Gene therapy for IPEX syndrome
- WP4: Gene therapy of SCA
WP1 : Immunological and haematological reconstitution after gene therapy in WAS patients: implications for the clinical outcome, with a particular focus on autoimmunity and microthrombocytopenia.2028

With this WP1 we aimed at investigating the robustness of our first gene therapy trial for WAS in terms of the correction of all blood lineages, with a particular focus on the long-term cure of autoimmunity and thrombocytopenia.
Since the beginning of this grant we are regularly following 4 patients treated with lentiviral vector based-gene therapy, 5 to 7 years ago. All these patients are alive and well. Clinical and biological data are regularly collected over time, including classical immunological analysis and quantification of WAS gene transduction via quantification of gene marking and WASP expression in myeloid and lymphoid populations, TREC/KREC analyses, TCR repertoire by NGS analyses, as well as integration sites (IS) analysis in subpopulation. The aim of this WP was to focus in particular on a) platelet recovery, b) autoimmunity and B cell function after gene therapy and c) long term hematopoietic reconstitution analysis through IS clonal tracking.

1. Platelet function and characterization after gene therapy
A comprehensive analysis of platelet function and characterization was performed for the first time on our patients. For a better comprehension of results after GT, they were compared not only to healthy donor volunteers (in parallel to each analysis performed), but also to WAS patients after conventional allogeneic hematopoietic stem cell transplantation and WAS untreated patients.
Patients did not develop important hemorrhagic symptoms, despite a persisting micro thrombocytopenia over time.

2. Autoimmunity and B cell function after gene therapy
With the aim at exploring the B cell compartment, a study of B cell function was performed over time. This included the analysis of IgG IgA and IgM production over time, B cell subpopulations phenotype, WASP expression in B cell subsets, quantification of WAS gene transduction in the memory vs the naïve B cell compartments by VCN quantification, autoimmune panel. KREC analyses were also performed at different time points.
Results over time show a global increase in B cell function and KREC analyses. At least two patients could stop Ig replacement and have IgG production in normal ranges.
In order to explore the autoimmunity status of the patients, a panel of autoantibodies before and after gene therapy treatment at different time points was tested. No significant positive autoantibody was detected after gene therapy.

3. Long term hematopoietic reconstitution analysis through IS clonal tracking
We performed an in depth analysis of long term hematopoietic reconstitution aiming at understanding the key parameters of long-term reconstitution, that could impact especially platelet and B cell reconstitution.
Using clonal tracking in cell lineages through integration sites (IS) sequencing, we developed novel statistical methods for the quantitative analysis of HSC. We determined that the minimal estimated number of active HSC, providing hematological reconstitution in these patients, correlating with the number of corrected infused HSPC. We also quantified HSC progeny and highlighted the heterogeneity of human HSC with distinct HSC subsets.

WP2 : Gene therapy of a SCID caused by an Artemis mutation: implementation of the first clinical trial for this indication.

The GMP batch for the Artemis deficiency gene therapy clinical trial was sent by Yposkesi to the Biotherapy Clincial Investigation Center in January 2019. We first checked the efficacy of this GMP batch on bone marrow CD34+ cells from a healthy donor (HD). The cells were transduced with the Artemis vector or an empty vector in serum free conditions supplemented with a cytokine cocktail and growth factors as previously used in other gene therapy trials targeting CD34+ hematopoietic cells. Cells were then cultured in vitro either in the cytokine cocktail to measure cell proliferation or on a feeder-cell-free culture system based on Delta-like ligand-4 (DL4), followed by a co-culture on OP9 stromal cells expressing the Notch ligand Delta1 (DL1) to evaluate T cell differentiation. In this experiment, we observed a low vector copy number (VCN) per cell and a low expression of Artemis transgene, especially during T cell differentiation. These results will be confirmed in the next weeks by testing others Artemis probes.
Based on the results of these two experiments, we will set-up the definitive transduction protocol and validate it on Artemis deficient cells, as required by the French competent authorities before the beginning of each new gene therapy clinical trial. The opening of this phase I/II gene therapy clinical trial is expected for the end of 2019-beginning of 2020.

WP3 : Gene therapy for IPEX syndrome

1. Design of new vector suitable for clinical use
Preclinical and clinical studies suggest that T cell gene therapy approaches designed to selectively restore the repertoire of Treg cells by transfer of wild type FOXP3 gene is a promising potential cure for IPEX. However, there is still a need for a vector that can be used efficiently for the preparation of said Treg cells. We compared 6 different lentiviral constructs according to 4 criteria (vector titers, level of transduction of human CD4+ T cells, level of expression of FOXP3 and tracker genes, degree of correlation between both expression) and selected one construct.

2. Generation of regulatory T cells by lentiviral transfer of FOXP3 into murine CD4+ Tcells
We have already demonstrated that conventional murine CD4 T-cells can be converted to regulatory T cells by gene transfer of human FOXP3, as tested with the in vitro suppression assay. A need to develop an efficient transduction protocol for murine cells with lentiviral vectors has emerged especially in the context of preclinical proof of concept. An optimized protocol in which a nontoxic transduction enhancer called Lentiboost (Sirion Biotech), enables the efficient transduction of primary murine T cells with lentiviral vectors has been set up. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs.

WP4 : Gene therapy of SCA

1. Design of a new LV vector
Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling β-globin variant is a potential treatment for sickle cell anemia (SCA). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling β-globin transgene (β-globin AS3), a minimal β-globin promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCA patients were transduced with LVs containing either HS2 and HS3 (β-AS3) or HS2, HS3, and HS4 (β-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCA bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling β-globin representing up to ∼60% of the total β-globin-like chains. The expression of the anti-sickling β-globin and the reduced incorporation of the sickle βS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling β-globin transgene ameliorating the SCA cell phenotype (Weber et al., Mol Ther Methods Clin Dev., 2018).
The optimization of transduction conditions led to high level of transduction of Plerixafor-mobilized SCA hematopoietic stem cells (HSCs) by β-AS3 LV, as assessed by xenotransplantation in immunodeficient mice: these results have formed the basis to start a gene therapy trial for SCA.

2. Alternative therapeutic strategies based on genome-editing technologies
Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of SCA and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the adult δ- and β-globin genes and a putative 3.5-kb γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in fetal γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCA patient-derived HSPCs, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies (Antoniani, Meneghini et al., Blood, 2018).
WP1 : Immunological and haematological reconstitution after gene therapy in W AS patients: implications for the clinical outcome, with a particular focus on autoimmunity and microthrombocytopenia
WP1 aims at investigating the long term follow up of our first gene therapy trial for WAS in terms of the correction of all blood lineages, with a particular focus on the long-term cure of autoimmunity and thrombocytopenia. The study of platelet function and autoimmunity is ongoing and will be completed by the end of the project.

WP2 : Gene therapy of a SCID caused by an Artemis mutation: implementation of the first clinical trial for this indication
The main objective of this work package is to open a phase I/II clinical trial to treat 5 patients with Artemis immune deficiency. We expect at the end of the grant to have opened and included the 5 patients to be treated.

WP3 : Gene therapy for IPEX syndrome
We shall then assess the ability of CD4SfFOXP3 cells to cure the scurfy mouse’s autoimmune symptoms by adoptive transfer in scurfy male recipients. We shall compare the efficiency of these CD4SfFOXP3 cells with that of eGFP Tregs in controlling the autoimmune manifestation of scurfy mice and define the cell dose required for a sustained effect. We shall analyse the long-term persistence of the corrected CD4SfFOXP3 cells over several months.

The stability of the generated Tregs is a critical parameter for robust suppressor function. Recent studies have also revealed the plasticity of the Treg lineage (Sawant and Vignali, 2014). Therefore, in addition to restoration of FOXP3 expression in IPEX cells, we shall explore their transcriptional signatures and (i) look for expression of other core suppression factors that act in synergy with Foxp3 to lock the regulatory signature (Fu et al., 2012) and (ii) define the whole signature by comparison with healthy Tregs (Ferraro et al., 2014).
Considering our preliminary data showing the feasibility of regulatory T cell engineering though lentiviral transduction of FOXP3 in diseased CD4 T cells, we expect to demonstrate the ability of transduced CD4SfFOXP3 to control the disease as efficiently as nTreg. We expect a reasonable stability of the cells allowing controlling the disease for a couple of months.

WP4 : Gene therapy of SCA
Compared to previously published studies, we have developed a novel high-titer vector expressing an anti-sickling transgene that will be use in a clinical trial expected to start at Necker Hospital in 2019. In parallel, we have developed a novel genome editing strategy to reactivate therapeutic HbF levels in vitro. Future experiments will aim at selecting guide RNAs specifically targeting the β-globin locus that lead to efficient HbF reactivation in the progeny of bona fide HSCs (as assessed by xenotransplantation in immunodeficient mice) without evidence of toxicity.