Autoimmunity in thymic epithelial tumors: a not yet clarified pathologic paradigm associated with several unmet clinical needs.

Thymic epithelial tumors (TETs) are rare mediastinal cancers originating from the thymus, classified in two main histotypes: thymoma and thymic carcinoma (TC). TETs affect a primary lymphoid organ playing a critical role in keeping T-cell homeostasis and ensuring an adequate immunological tolerance against "self". In particular, thymomas and not TC are frequently associated with autoimmune diseases (ADs), with Myasthenia Gravis being the most common AD present in 30% of patients with thymoma. This comorbidity, in addition to negatively affecting the quality and duration of patients' life, reduces the spectrum of the available therapeutic options. Indeed, the presence of autoimmunity represents an exclusion criteria for the administration of the newest immunotherapeutic treatments with checkpoint inhibitors. The pathophysiological correlation between TETs and autoimmunity remains a mystery. Several studies have demonstrated the presence of a residual and active thymopoiesis in adult patients affected by thymomas, especially in mixed and lymphocytic-rich thymomas, currently known as type AB and B thymomas. The aim of this review is to provide the state of art in regard to the histological features of the different TET histotype, to the role of the different immune cells infiltrating tumor microenvironments and their impact in the break of central immunologic thymic tolerance in thymomas. We discuss here both cellular and molecular immunologic mechanisms inducing the onset of autoimmunity in TETs, limiting the portfolio of therapeutic strategies against TETs and greatly impacting the prognosis of associated autoimmune diseases.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Transplantation after CD45-ADC corrects Rag1 immunodeficiency in congenic and haploidentical settings.

BACKGROUND: Mutations in the recombinase-activating genes 1 and 2 (RAG1, RAG2) cause a spectrum of phenotypes, ranging from severe combined immune deficiency to combined immune deficiency with immune dysregulation (CID-ID). Hematopoietic cell transplantation is a curative option. Use of conditioning facilitates robust and durable stem cell engraftment and immune reconstitution but may cause toxicity. Transplantation from haploidentical donors is associated with poor outcome in patients with CID-ID. OBJECTIVES: We sought to evaluate multilineage engraftment and immune reconstitution after conditioning with CD45-antibody drug conjugate (CD45-ADC) as a single agent in hypomorphic mice with Rag1 mutation treated with congenic and haploidentical hematopoietic cell transplantation. METHODS: Rag1-F971L mice, a model of CID-ID, were conditioned with various doses of CD45-ADC, total body irradiation, or isotype-ADC, and then given transplants of total bone marrow cells from congenic or haploidentical donors. Flow cytometry was used to assess chimerism and immune reconstitution. Histology was used to document reconstitution of thymic architecture. RESULTS: Conditioning with CD45-ADC as a single agent allowed robust engraftment and immune reconstitution, with restoration of thymus, bone marrow, and peripheral compartments. The optimal doses of CD45-ADC were 1.5 mg/kg and 5 mg/kg for congenic and haploidentical transplantation, respectively. No graft-versus-host disease was observed. CONCLUSIONS: Conditioning with CD45-ADC alone allows full donor chimerism and immune reconstitution in Rag1 hypomorphic mice even following haploidentical transplantation, opening the way for the implementation of similar approaches in humans.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

A novel peptide 'T14' reflects age and photo-aging in human skin.

T14 is a 14mer peptide derived from the C-terminus of acetylcholinesterase (AChE). Once cleaved, it is independently bioactive of the parent molecule and enhances calcium influx in different cell types, in a range of scenarios: it binds to an allosteric site selectively on the alpha-7 receptor, where it modulates calcium influx and is thus a potential trophic agent, as already reported in a range of normal developmental scenarios. However, if inappropriately activated, this erstwhile beneficial effect converts to a toxic one, resulting in pathologies as disparate as Alzheimer's and various metastatic cancers. Given that epidermal keratinocyte cells have the same ectodermal origin as brain cells, as well as expressing AChE and the alpha-7 receptor, we have explored whether T14 plays a comparable role. Here we report that the T14 immunoreactivity is detectable in human keratinocytes with levels inversely related to age: this decrease is even more apparent with chronic photo-exposure and thus accelerated skin aging. We conclude that T14, an agent promoting cell growth and renewal in other parts of the body, also operates in skin, Moreover, monitoring of keratinocyte T14 levels might offer further insights into the now well reported link between degenerative diseases and epidermal cell profile.

Correcting inborn errors of immunity: From viral mediated gene addition to gene editing.

Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

Osteoclast rich osteopetrosis due to defects in the TCIRG1 gene.

Discovery that mutations in TCIRG1 (also known as Atp6i) gene are responsible for most instances of autosomal recessive osteopetrosis (ARO) heralded a new era for comprehension and treatment of this phenotypically heterogeneous rare bone disease. TCIRG1 encodes the a3 subunit, an essential isoform of the vacuolar ATPase proton pump involved in acidification of the osteoclast resorption lacuna and in secretory lysosome trafficking. TCIRG1 defects lead to inefficient bone resorption by nonfunctional osteoclasts seen in abundance on bone marrow biopsy, delineating this ARO as 'osteoclast-rich'. Presentation is usually in early childhood and features of extramedullary haematopoiesis (hepatosplenomegaly, anaemia, thrombocytopenia) due to bone marrow fibrosis, and cranial nerve impingement (blindness in particular). Impaired dietary calcium uptake due to high pH causes the co-occurrence of rickets, described as "osteopetrorickets". Osteoclast dysfunction leads to early death if untreated, and allogeneic haematopoietic stem cell transplantation is currently the treatment of choice. Studies of patients as well as of mouse models carrying spontaneous (the oc/oc mouse) or targeted disruption of Atp6i (TCIRG1) gene have been instrumental providing insight into disease pathogenesis and development of novel cellular therapies that exploit gene correction.

PBX1-directed stem cell transcriptional program drives tumor progression in myeloproliferative neoplasm.

PBX1 regulates the balance between self-renewal and differentiation of hematopoietic stem cells and maintains proto-oncogenic transcriptional pathways in early progenitors. Its increased expression was found in myeloproliferative neoplasm (MPN) patients bearing the JAK2V617F mutation. To investigate if PBX1 contributes to MPN, and to explore its potential as therapeutic target, we generated the JP mouse strain, in which the human JAK2 mutation is induced in the absence of PBX1. Typical MPN features, such as thrombocythemia and granulocytosis, did not develop without PBX1, while erythrocytosis, initially displayed by JP mice, gradually resolved over time; splenic myeloid metaplasia and in vitro cytokine independent growth were absent upon PBX1 inactivation. The aberrant transcriptome in stem/progenitor cells from the MPN model was reverted by the absence of PBX1, demonstrating that PBX1 controls part of the molecular pathways deregulated by the JAK2V617F mutation. Modulation of the PBX1-driven transcriptional program might represent a novel therapeutic approach.

Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria.

In this paper, we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 µM CdCl2 for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two foci was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of the Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

NKG2A expression identifies a subset of human Vδ2 T cells exerting the highest antitumor effector functions.

Human Vδ2 cells are innate-like γδ T effectors performing potent immune surveillance against tumors. The constitutive expression of NKG2A identifies a subset of Vδ2 T cells licensed with an intrinsic hyper-responsiveness against cancer. Indeed, the transcriptomic profiles of NKG2A+ and NKG2A- cells characterize two distinct "intralineages" of Vδ2 T lymphocytes that appear early during development, keep their phenotypes, and show self-renewal capabilities in adult life. The hyper-responsiveness of NKG2A+ Vδ2 T cells is counterbalanced by the inhibitory signaling delivered by human leukocyte antigen E (HLA-E) expressed on malignant cells as a tumor-escape mechanism. However, either masking or knocking out NKG2A restores the capacity of Vδ2 T cells to exert the highest effector functions even against HLA-E+ tumors. This is highly relevant in the clinic, as the different degrees of engagement of the NKG2A-HLA-E checkpoint in hepatocellular carcinoma, glioblastoma, and non-small cell lung cancer directly impact patients' overall survival. These findings open avenues for developing combined cellular and immunologic anticancer therapies.

A novel intronic variant in PIGB in Acrofrontofacionasal dysostosis type 1 patients expands the spectrum of phenotypes associated with GPI biosynthesis defects.

Acrofrontofacionasal dysostosis type 1 (AFFND1) is an extremely rare disorder characterized by several dysmorphic features, skeletal abnormalities and intellectual disability, and described only in seven patients in the literature. A biallelic variant in the Neuroblastoma Amplified Sequence (NBAS) gene was recently identified in two Indian patients with AFFND1. Here we report genetic investigation of AFFND1 in the originally described Brazilian families and the identification of an extremely rare, recessively-inherited, intronic variant in the Phosphatidylinositol Glycan class B (PIGB) gene NC_000015.10 (NM_004855.4): c.795-19T > G) in the affected individuals. The PIGB gene encodes an enzyme involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is required for the post-translational modification of a large variety of proteins, enabling their correct cellular localization and function. Recessive variants in PIGB have previously been reported in individuals with a neurodevelopmental syndrome having partial overlap with AFFND1. In vitro assays demonstrated that the intronic variant leads to exon skipping, suggesting the Brazilian AFFND1 patients may be null for PIGB, in agreement with their severe clinical phenotype. These data increase the number of pathogenic variants in the PIGB gene, place AFFND1 among GPI deficiencies and extend the spectrum of phenotypes associated with GPI biosynthesis defects.

Thymic Epithelial Cell Alterations and Defective Thymopoiesis Lead to Central and Peripheral Tolerance Perturbation in MHCII Deficiency.

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII-/- mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII-/- mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.

Premature Senescence and Increased Oxidative Stress in the Thymus of Down Syndrome Patients.

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues. Based on previous studies suggesting immune senescence in DS, we hypothesized that induction of cellular senescence may contribute to early thymic involution and immune dysregulation. Immunohistochemical analysis of thymic tissue showed signs of accelerated thymic aging in DS patients, normally seen in older healthy subjects. Moreover, our whole transcriptomic analysis on human Epcam-enriched thymic epithelial cells (hTEC), isolated from three DS children, which revealed disease-specific transcriptomic alterations. Gene set enrichment analysis (GSEA) of DS TEC revealed an enrichment in genes involved in cellular response to stress, epigenetic histone DNA modifications and senescence. Analysis of senescent markers and oxidative stress in hTEC and thymocytes confirmed these findings. We detected senescence features in DS TEC, thymocytes and peripheral T cells, such as increased ß-galactosidase activity, increased levels of the cell cycle inhibitor p16, telomere length and integrity markers and increased levels of reactive oxygen species (ROS), all factors contributing to cellular damage. In conclusion, our findings support the key role of cellular senescence in the pathogenesis of immune defect in DS while adding new players, such as epigenetic regulation and increased oxidative stress, to the pathogenesis of immune dysregulation.

Autosomal recessive osteopetrosis: mechanisms and treatments.

Autosomal recessive osteopetrosis (ARO) is a severe inherited bone disease characterized by defective osteoclast resorption or differentiation. Clinical manifestations include dense and brittle bones, anemia and progressive nerve compression, which hamper the quality of patients' lives and cause death in the first 10 years of age. This Review describes the pathogenesis of ARO and highlights the strengths and weaknesses of the current standard of care, namely hematopoietic stem cell transplantation (HSCT). Despite an improvement in the overall survival and outcomes of HSCT, transplant-related morbidity and the pre-existence of neurological symptoms significantly limit the success of HSCT, while the availability of human leukocyte antigen (HLA)-matched donors still remains an open issue. Novel therapeutic approaches are needed for ARO patients, especially for those that cannot benefit from HSCT. Here, we review preclinical and proof-of-concept studies, such as gene therapy, systematic administration of deficient protein, in utero HSCT and gene editing.

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency.

BACKGROUND: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. OBJECTIVES: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). METHODS: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. RESULTS: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. CONCLUSIONS: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.

Innovative Cell-Based Therapies and Conditioning to Cure RAG Deficiency.

Genetic defects in recombination activating genes (RAG) 1 and 2 cause a broad spectrum of severe immune defects ranging from early severe and repeated infections to inflammation and autoimmune manifestations. A correlation between in vitro recombination activity and immune phenotype has been described. Hematopoietic cell transplantation is the treatment of care; however, the availability of next generation sequencing and whole genome sequencing has allowed the identification of novel genetic RAG variants in immunodeficient patients at various ages, raising therapeutic questions. This review addresses the recent advances of novel therapeutic approaches for RAG deficiency. As conventional myeloablative conditioning regimens are associated with acute toxicities and transplanted-related mortality, innovative minimal conditioning regimens based on the use of monoclonal antibodies are now emerging and show promising results. To overcome shortage of compatible donors, gene therapy has been developed in various RAG preclinical models. Overall, the transplantation of autologous gene corrected hematopoietic precursors and the use of non-genotoxic conditioning will open a new era, offering a cure to an increasing number of RAG patients regardless of donor availability and severity of clinical conditions.