Interleukin-2 Receptor Gamma Research Articles

IL-2RG as a possible immunotherapeutic target in CRC predicting poor prognosis and regulated by miR-7-5p and miR-26b-5p

Despite advances in treatment strategies, colorectal cancer (CRC) continues to cause significant morbidity and mortality, with mounting evidence a close link between immune system dysfunctions issued. Interleukin-2 receptor gamma (IL-2RG) plays a pivotal role as a common subunit receptor in the IL-2 family cytokines and activates the JAK-STAT pathway. This study delves into the role of Interleukin-2 receptor gamma (IL-2RG) within the tumor microenvironment and investigates potential microRNAs (miRNAs) that directly inhibit IL-2RG, aiming to discern their impact on CRC clinical outcomes. Bioinformatics analysis revealed a significant upregulation of IL-2RG mRNA in TCGA-COAD samples and showed strong correlations with the infiltration of various lymphocytes. Single-cell analysis corroborated these findings, highlighting IL-2RG expression in critical immune cell subsets. To explore miRNA involvement in IL-2RG dysregulation, mRNA was isolated from the tumor tissues and lymphocytes of 258 CRC patients and 30 healthy controls, and IL-2RG was cloned into the pcDNA3.1/CT-GFP-TOPO vector. Human embryonic kidney cell lines (HEK-293T) were transfected with this construct. Our research involved a comprehensive analysis of miRPathDB, miRWalk, and Targetscan databases to identify the miRNAs associated with the 3′ UTR of human IL-2RG. The human microRNA (miRNA) molecules, hsa-miR-7-5p and hsa-miR-26b-5p, have been identified as potent suppressors of IL-2RG expression in CRC patients. Specifically, the downregulation of hsa-miR-7-5p and hsa-miR-26b-5p has been shown to result in the upregulation of IL-2RG mRNA expression in these patients. Prognostic evaluation of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p, using TCGA-COAD data and patient samples, established that higher IL-2RG expression and lower expression of both miRNAs were associated with poorer outcomes. Additionally, this study identified several long non-coding RNAs (LncRNAs), such as ZFAS1, SOX21-AS1, SNHG11, SNHG16, SNHG1, DLX6-AS1, GAS5, SNHG6, and MALAT1, which may act as competing endogenous RNA molecules for IL2RG by sequestering shared hsa-miR-7-5p and hsa-miR-26b-5p. In summary, this investigation underscores the potential utility of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p as serum and tissue biomarkers for predicting CRC patient prognosis while also offering promise as targets for immunotherapy in CRC management.Graphical

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, in Combination with Costimulatory Blockers Delayed Skin Graft Rejection in Mice

There is a current medical need to identify innovative maintenance therapies to prevent allograft rejection in solid organ transplantation. During transplant rejection, recipient immune cells identify a grafted organ as foreign and attack it: CD4 + T cells orchestrate transplant rejection by recruiting a range of effector cells responsible for the damage of rejection including CD8 + T cells, NK cells, B cells and macrophages. These pathogenic immune cell responses can be driven by cytokines of the common gamma chain (γ c) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γ c cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the role of γ c cytokines in driving transplant rejection, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γ c cytokine-induced signaling, and we tested its ability to suppress allogeneic immune cell responses in a mouse model of skin graft rejection when delivered as a monotherapy and in combination with mycophenolate mofetil (MMF) or T cell costimulatory pathway blockers. In this model, recipient B6/129 (mixed background) Il2rg hu/hu mice were transplanted with tail skin from BALB/cJ donor mice. Graft survival was analyzed using a log rank Mantel-Cox test to detect statistically significant differences across groups (*=p<0.033; **= p<0.002; ***=p<0.0002; ****=p<0.0001). In a mouse model of skin graft rejection, γ c cytokine signaling blockade with REGN7257 given as maintenance immunosuppression effectively improved overall graft survival (Median Survival Time [MST] IL2RG=13) compared to mice that received a control antibody (MST CTRL=9 [****vs IL2RG Ab]), and with similar efficacy to that observed with the calcineurin inhibitor tacrolimus (MST TAC=12.5 [**vs control, n.s. vs IL2RG Ab]). In treatment combination experiments, while monotherapy treatment with the antiproliferative agent MMF was not as efficient as REGN7257 at delaying skin graft rejection (MST MMF=10 [*vs control] and MST IL2RG=12 [*vs MMF]), simultaneous blockade of cell proliferation and γ c cytokine signaling conferred an advantage over monotherapies (MST MMF+IL2RG=15.5 [****vs control, **vs IL2RG Ab]). Similarly, induction therapy consisting of γ c cytokine signaling blockade followed by maintenance immunosuppression using the CTLA4-Ig fusion protein abatacept, significantly improved overall skin graft survival (MST IL2RG+CTLA4=19.5 [****vs control]) compared to REGN7257 induction only (MST IL2RG=11 [****vs control and combo]) or abatacept maintenance only (MST CTLA4=10 [*vs control, ****vs combo]). Finally, maintenance immunosuppression combining blocking of γc cytokine signaling together with blocking of both CD28 and CD40L costimulatory signals prolonged skin graft survival better than any combination therapies of only 2 pathway blockers (MST TRIPLE=26 [**vs control] and MST DOUBLE=16-21 [n.s.-** vs triple]). Overall, our study showed that γ c cytokine blockade could potentially be an alternative to calcineurin inhibitor-based regimens to prevent rejection by specifically targeting immune cells. Our data also highlighted the inherent complexity of effector mechanisms and pathways involved in allograft rejection and showed that prolonged allograft survival requires simultaneous blockade of multiple T cell activating signals, including inhibition of cytokine signaling and costimulatory signals. These data provide evidence of γ c cytokines as key drivers of allogeneic immune cell responses during acute transplant rejection, offering a potentially novel strategy for the management of transplant patients.

Abstract P3073: Interleukin-2 Receptor Gamma Signaling Is Required For The Regenerative Adaptive Immune Response In The Adult Zebrafish Heart

Ischemic heart disease remains a leading cause of death worldwide. Unlike adult mammals, zebrafish have the ability to fully regenerate their heart after injury through processes including epicardium and endocardium activation, coronary revascularization, cardiomyocyte dedifferentiation and proliferation, and scar resolution. Immune cells are critical to remove damaged tissue, sustain repair and initiate regeneration after injury. Our previous data show that blocking antigen presentation impairs cardiac regeneration, pointing at a role for the adaptive immune system during this process. However, the molecular mechanisms underlying a pro-regenerative adaptive immune response remain largely unknown. Based on bulk transcriptomics, single-cell transcriptomics, and qPCR data, we have identified il2rga ( interleukin-2 receptor gamma a ) and jak3 ( janus kinase 3) as potential regulators of the adaptive immune response during cardiac regeneration. We indeed found that il2rga mutants exhibit a reduced expression of T-cell markers, as well as impaired cardiac regeneration, as observed by decreased cardiomyocyte dedifferentiation and proliferation, and defective scar resolution. In addition, il2rga mutants display decreased expression of pro-regenerative factors (e.g., igf1, ngfb, nrg1, pdgfaa, tgfb2, vegfc ). Similarly, loss of Jak3, which functions downstream of Il2rga, also leads to reduced cardiomyocyte dedifferentiation and proliferation, as well as impaired endocardium activation. Bulk transcriptomics further shows the downregulation of key genes involved in the adaptive immune response in il2rga mutants. Specifically, we found that il13 , a recently described cardiomyocyte cell cycle regulator and T helper 2 (Th2) cytokine, is strongly downregulated upon the loss of il2rga . Altogether, our findings reveal the crucial role of Il2rga/Jak3 signaling during cardiac regeneration, thereby opening new avenues to identify and understand the adaptive immune mechanisms capable of sustaining regenerative responses post-cardiac injury.

Multi-omics study of key genes, metabolites, and pathways of periodontitis

ObjectiveThis study aimed to explore the key genes, metabolites, and pathways that influence periodontitis pathogenesis by integrating transcriptomic and metabolomic studies. DesignGingival crevicular fluid samples from periodontitis patients and healthy controls were collected for liquid chromatography/tandem mass-based metabolomics. RNA-seq data for periodontitis and control samples were obtained from the GSE16134 dataset. Differential metabolites and differentially expressed genes (DEGs) between the two groups were then compared. Based on the protein-protein interaction (PPI) network module analysis, key module genes were selected from immune-related DEGs. Correlation and pathway enrichment analyses were performed for differential metabolites and key module genes. A multi-omics integrative analysis was performed using bioinformatic methods to construct a gene-metabolite-pathway network. ResultsFrom the metabolomics study, 146 differential metabolites were identified, which were mainly enriched in the pathways of purine metabolism and Adenosine triphosphate binding cassette transporters (ABC transporters). The GSE16134 dataset revealed 102 immune-related DEGs (458 upregulated and 264 downregulated genes), 33 of which may play core roles in the key modules of the PPI network and are involved in cytokine-related regulatory pathways. Through a multi-omics integrative analysis, a gene-metabolite-pathway network was constructed, including 28 genes (such as platelet derived growth factor D (PDGFD), neurturin (NRTN), and interleukin 2 receptor, gamma (IL2RG)); 47 metabolites (such as deoxyinosine); and 8 pathways (such as ABC transporters). ConclusionPDGFD, NRTN, and IL2RG may be potential biomarkers of periodontitis and may affect disease progression by regulating deoxyinosine to participate in the ABC transporter pathway.

BSA-AIE Nanoparticles with Boosted ROS Generation for Immunogenic Cell Death Immunotherapy of Multiple Myeloma.

The main obstacle of multiple myeloma (MM) therapy is the compromised immune microenvironment, which leads to MM relapses and extramedullary disease progression. In this study, a novel strategy is reported of enhanced immunogenic cell death (ICD) immunotherapy with aggregation-induced emission (AIE) photosensitizer-loaded bovine serum albumin (BSA) nanoparticles (referred as BSA/TPA-Erdn), which can activate T cells, convert the cold tumor to hot, and reverse T cell senescence to restore the immune microenvironment for MM treatment. Loading AIE photosensitizer into the hydrophobic domain of BSA proteins significantly immobilizes the molecular geometry, which massively increases reactive oxygen species (ROS) generation and elicits a promising ICD immune response. Employing a NOD-SCID IL-2receptor gamma null mice model with MM patients' monocytes, it is shown that BSA/TPA-Erdn can simulate human dentric cell maturation, activate functional T lymphocytes, and increase additional polarization and differentiation signals to deliver a promising immunotherapy performance. Intriguingly, for the first time, it is shown that BSA/TPA-Erdn can greatly reverse T cell senescence, a main challenge in treating MM. Additionally, BSA/TPA-Erdn can effectively recruit more functional T lymphocytes into MM tumor. As a consequence, BSA/TPA-Erdn restores MM immune microenvironment and shows the best MM tumor eradication performance, which shall pave new insights for MM treatment in clinical practices.

Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing.

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.

Evaluation of Common Gamma Chain Cytokine Signaling Blockade with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, on Immune Cell Populations in Monkey and Human

The common γ chain cytokine receptor (γc; IL2RG) family of cytokines includes interleukin 2 (IL2), IL4, IL7, IL9, IL15, and IL21. This set of cytokines exhibits broad pleiotropic actions on both the innate and adaptive immune system with each cytokine sharing the IL2RG chain as part of its signaling receptor complex. Mutations in the IL2RG gene result in X-linked severe combined immunodeficiency (XSCID) in humans, whereby patients present with dramatically diminished numbers of T-cells and NK-cells, and dysfunctional B-cells. Given the crucial role for γc cytokines in the development and function of lymphocytes, modulating their activities may offer therapeutic potential in a range of immune-mediated diseases. To understand the effects of γc cytokine blockade on immune cells including T-cell subsets, we utilized REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and tested its ability to suppress immune cell populations and their functions in cynomolgus monkey and human. Mixed lymphocyte reaction assays were performed to look at the impact of γc cytokine signaling blockade on activation/proliferation of human T-cells. To further analyze the contribution of γc cytokines to T-cell differentiation and function, we performed in vitro transcriptomic studies on human peripheral blood mononuclear cells stimulated with anti-CD3/CD28 beads. In addition, we assessed the effects of γc cytokine signaling blockade on both human T-cell and NK-cell effector functions in target cell killing assays. Finally, we evaluated the effects of γc cytokine signaling blockade with REGN7257 on immune cell populations and their activation/proliferation status in cynomolgus monkeys by flow cytometry. γc cytokine signaling blockade with REGN7257 demonstrated potent inhibition of allogeneic responses in mixed human lymphocyte reaction assays, by preventing T-cell activation and proliferation. Two-tailed gene set enrichment analysis identified 4 distinct patterns of significantly enriched gene sets that were impacted by γc cytokine signaling upon CD3/CD28-induced activation of human T-cells: gene sets involved in inflammatory responses, differentiation and proliferation, activation and immune responses, as well as those related to adhesion and migration. These signatures were blocked with REGN7257 treatment. In addition, REGN7257 potently blocked activation (i.e. IFNG production) and cytotoxic activity of both human T-cells and NK-cells in in vitro target cell killing assays. Finally, the impact of γc cytokine signaling blockade on lymphocyte populations in vivo were investigated in cynomolgus monkeys in a single dose pharmacokinetic/pharmacodynamic study and a repeat-dose toxicology study. Inhibition of γc cytokine signaling with REGN7257 in cynomolgus monkeys reduced circulating T-cells and NK-cells, without impacting B-cells, granulocytes, platelets or red blood cells. γc cytokine signaling blockade led to a reduction in both peripheral CD4+ and CD8+ T-cell counts with effector memory T-cells being the most impacted T-cell population studied. Similarly to that observed in human T-cell MLR assays, both activated and proliferating T-cells were reduced in monkeys dosed with REGN7257. Taken together, these pharmacological observations highlight the major role for γc cytokine signaling in maintenance of lymphocyte populations (i.e. NK-cells and T-cells), but not other immune cell populations, such as granulocytes, platelets and red blood cells. Furthermore, our data highlight the importance of γc cytokines in driving functions of cynomolgus monkeys and human effector NK and T-cells, opening a potential new route for the management of immune-mediated diseases.

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, Protected Mice from Inflammatory and Autoimmune Diseases

Pathogenic immune cell responses in inflammatory and autoimmune diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the roles of γc cytokines in driving inflammatory and autoimmune diseases, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic immune cell responses in mouse models of graft-versus-host disease (GVHD), immune aplastic anemia and multiple sclerosis (MS). We first evaluated the efficacy of REGN7257 in xenogeneic and allogeneic mouse models of GVHD. In the xenogeneic model, immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). In the allogeneic GVHD model, irradiated BALB/c mice were engrafted with bone marrow cells and splenocytes from Il2rghu/hu mice. In the xenogeneic GVHD mouse model, we also analyzed the effects of γc cytokine signaling blockade with REGN7257 on inflammation and immune cell infiltration into tissues (e.g. liver, bone marrow), as well as liver fibrosis. Finally, we assessed the effects of IL2RG blockade on pathogenic immune cell responses in an experimental autoimmune encephalomyelitis (EAE) model of MS, looking at immune cell infiltration into the central nervous system (CNS) and associated demyelination. In both xenogeneic and allogeneic models of GVHD, prophylactic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Similarly, in the xenogeneic GVHD model, dosing with REGN7257 starting 3 weeks after huPBMC administration effectively protected mice from weight loss and death. Consistent with the classic pathology of GVHD, tissues of control mice were highly infiltrated by immune cells, including CD4+ and CD8+ T-cells, while γc cytokine signaling blockade strongly reduced immune cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Finally, in an EAE mouse model of MS, inhibition of γc cytokine signaling with REGN7257 alleviated disease progression and significantly prolonged survival. This was associated with reduced inflammation and infiltration by immune cells (CD4+ T-cells, CD8+ T-cells, B-cells and monocytic cells) in the CNS, as well as decreases in myelin oligodendrocyte glycoprotein-specific antibody production. We showed that REGN7257 treatment efficiently protected the spinal cord from demyelination, which was associated with decreased oligodendrocyte injury and neuron protection. Blockade of γc cytokine signaling with REGN7257 protected mice against immune cell-mediated pathology in multiple disease models: an EAE model of MS, an allogeneic GVHD model, and a xenogeneic GVHD model that uniquely presents hallmarks of both acute and chronic GVHD as well as immune aplastic anemia. These data provide evidence of γc cytokines as key drivers of pathogenic immune cell responses, offering a potentially novel strategy for the management of inflammatory and autoimmune diseases, such as GVHD, immune aplastic anemia and MS.

Pre-clinical efficacy of CD20-targeted chimeric antigen receptor T cells for non-Hodgkin's lymphoma

BackgroundA 4-1BB/CD3-ζ-costimulated CAR-T against CD20 (CAR-T20) was subjected to a systemic efficacy evaluation in a cell co-culture model, and NOD-SCID IL-2 receptor gamma null mice (short for NSG mice) were xenografted with human Burkitt's lymphoma Raji cells.MethodsCAR-T20 cells were incubated with target cells (K562, K562 CD20 or Raji cells) at ratios of 10:1 and 5:1 for 24 h, and the killing rate was estimated by an LDH cytotoxicity assay. To evaluate the effect of CAR-T20 on the survival time of tumor-bearing animals, 30 NSG mice were employed, and Raji-Luc cells (5 × 105 cells per mouse) were administered prior to CAR-T20 administration. The survival time, optical intensity of Raji-Luc cells, clinical symptoms, and body mass of the animals were observed. Another 144 male NSG mice were employed to investigate the proliferation and antitumor effects of CAR-T20. Human cytokine and murine cytokines were detected at 1, 7, 14, 21, 28, 42, 56 and 90 days post-CAR-T administration, while biochemistry index analysis, T-cell and CAR-T-cell detection in peripheral blood, and histopathological examination were performed at 14, 28, 56 and 90 days post-administration.ResultsCAR-T20 cells had a specific killing effect on CD20-expressing cells in vitro. At a dose of 1 × 106 per mouse or above, CAR-T20 prolonged the median survival time from 14 days to more than 3 months, inhibited the proliferation of Raji cells in mice, and alleviated the clinical manifestations and weight loss caused by the Raji-Luc cell load. CAR-T20 at a dose of 2 × 106 per mouse or above inhibited the proliferation of Raji cells in mice for up to 111 days post-administration without recurrence. The numbers of T cells and CAR-T cells in the animals administered CAR-T20 increased significantly when Raji cells were markedly proliferated and subsequently decreased when Raji cells were predominantly inhibited. CAR-T20 increased human IFN-γ, murine TNF and murine IL-6 levels and decreased human IL-10 levels in tumor-bearing mice. The incidences of xenografted tumors in organs/tissues were also reduced effectively by CAR-T20.ConclusionThe effective dose of CAR-T20 in mice starts from 1 × 106 per mouse, equivalent to a clinical dose of 5 × 106/kg. Together, our data support the clinical translation of CAR-T20 for R/R B-cell NHL patients.

ATYPICAL PRESENTATION OF X-LINKED IL-2 RECEPTOR GAMMA MUTATION CHARACTERIZED BY RECURRENT CUTANEOUS AND GENITAL WARTS

<h3>Introduction</h3> Combined immunodeficiency syndromes constitute a group of inherited immune disorders characterized by diminished lymphocyte number and function. Late onset combined immunodeficiency can be difficult to diagnose as patients initially have a less severe phenotype compared to their SCID counterparts. Here we describe a case of an atypical presentation of late onset combined immunodeficiency due to a hemizygous IL-2 receptor gamma (IL-2Rg) mutation. <h3>Case</h3> The patient is a 41-year-old male with a 25-year history of recurrent cutaneous warts of the hands and feet. At the age of 36 he developed perianal condylomata acuminata that later developed into squamous cell carcinoma. He reported no history of recurrent sinopulmonary infection, atopic disease, or autoimmunity. Immune phenotyping demonstrated normal B- and NK cell counts but CD4+ T-cell lymphopenia with poor proliferation to antigenic stimulation. Total immunoglobulins were normal but without vaccine responses. Genetic testing revealed a hemizygous variant of unknown significance in IL2Rg gene. NK cell cytotoxicity assay showed suboptimal cytotoxicity and CD8+ T-cells exhibited impaired pSTAT5 confirming the likely pathogenicity of this variant. <h3>Discussion</h3> IL-2 plays a critical role in the activation among a host of T-cells and NK cells. Deleterious mutations of the IL-2Rg gene are associated with X-linked SCID displaying a T−B+NK− phenotype. Milder phenotypes have been reported with hypomorphic mutations of IL-2Rg, with a recent report describing recurrent cutaneous warts and respiratory infections in two brothers with splice site mutations. This patient's chronic HPV infection resulted in malignant sequalae, highlighting the importance for awareness of the phenotypic heterogeneity that can occur in X-linked SCID.

Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model

Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.

A high-quality severe combined immunodeficiency (SCID) rat bioresource.

Immunodeficient animals are valuable models for the engraftment of exogenous tissues; they are widely used in many fields, including the creation of humanized animal models, as well as regenerative medicine and oncology. Compared with mice, laboratory rats have a larger body size and can more easily undergo transplantation of various tissues and organs. Considering the absence of high-quality resources of immunodeficient rats, we used the CRISPR/Cas9 genome editing system to knock out the interleukin-2 receptor gamma chain gene (Il2rg) in F344/Jcl rats-alone or together with recombination activating gene 2 (Rag2)-to create a high-quality bioresource that researchers can freely use: severe combined immunodeficiency (SCID) rats. We selected one founder rat with frame-shift mutations in both Il2rg (5-bp del) and Rag2 ([1-bp del+2-bp ins]/[7-bp del+2-bp ins]), then conducted mating to establish a line of immunodeficient rats. The immunodeficiency phenotype was preliminarily confirmed by the presence of severe thymic hypoplasia in Il2rg-single knockout (sKO) and Il2rg/Rag2-double knockout (dKO) rats. Assessment of blood cell counts in peripheral blood showed that the white blood cell count was significantly decreased in sKO and dKO rats, while the red blood cell count was unaffected. The decrease in white blood cell count was mainly caused by a decrease in lymphocytes. Furthermore, analyses of lymphocyte populations via flow cytometry showed that the numbers of B cells (CD3- CD45+) and natural killer cells (CD3- CD161+) were markedly reduced in both knockout rats. In contrast, T cells were markedly reduced but showed slightly different results between sKO and dKO rats. Notably, our immunodeficient rats do not exhibit growth retardation or gametogenesis defects. This high-quality SCID rat resource is now managed by the National BioResource Project in Japan. Our SCID rat model has been used in various research fields, demonstrating its importance as a bioresource.

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions.

IntroductionPreviously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model.MethodsThe IL2RG XKOY pigs used in this study were obtained by mating IL2RG XKOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity.ResultsTumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed.ConclusionsIL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Generation and Characterization of a Zebrafish IL-2Rγc SCID Model.

The IL-2 family of cytokines act via receptor complexes that share the interleukin-2 receptor gamma common (IL-2Rγc) chain to play key roles in lymphopoiesis. Inactivating IL-2Rγc mutations results in severe combined immunodeficiency (SCID) in humans and other species. This study sought to generate an equivalent zebrafish SCID model. The zebrafish il2rga gene was targeted for genome editing using TALENs and presumed loss-of-function alleles analyzed with respect to immune cell development and impacts on intestinal microbiota and tumor immunity. Knockout of zebrafish Il-2rγc.a resulted in a SCID phenotype, including a significant reduction in T cells, with NK cells also impacted. This resulted in dysregulated intestinal microbiota and defective immunity to tumor xenotransplants. Collectively, this establishes a useful zebrafish SCID model.

Interleukin-15 receptor subunit alpha regulates interleukin-15 localization and protein expression in skeletal muscle cells.

What is the central question of this study? How are the dynamics of interleukin (IL)-15 and its receptors altered during the differentiation of myoblasts into myotubes, and how is IL-15 regulated? What is the main finding and its importance? The mRNA levels of IL-15 and interleukin-2 receptor subunits beta and gamma increase during skeletal muscle differentiation, whereas interleukin-15 receptor subunit alpha (IL-15RA) exhibits different kinetics. IL-15RA regulates the localization and expression of IL-15 at the protein level. Interleukin-15 (IL-15) is a myokine in the interleukin-2 (IL-2) family that is generated in the skeletal muscle during exercise. The functional effect of IL-15 involves muscle regeneration and metabolic regulation in skeletal muscle. Reports have indicated that interleukin-15 receptor subunit alpha (IL-15RA) acts by regulating IL-15 localization in immune cells. However, the dynamics of IL-15 and its receptors, which regulate the IL-15 pathway in skeletal muscle differentiation, have not yet been clarified. In this study, we investigated the mechanism of IL-15 regulation using a mouse skeletal muscle cell line, C2C12 cells. We found that the mRNA expression of IL-15, interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) increased, but that IL-15RA exhibited different kinetics as differentiation progressed. We also found that IL-15, mainly present in the cytosol, pre-assembled with IL-15RA in the cytosol and fused to the plasma membrane. Moreover, IL-15RA increased IL-15 protein levels. Our findings suggest that genes involved in the IL-15 signalling complex are enhanced with the differentiation of myotubes and that IL-15RA regulates the protein kinetics of IL-15 signalling in skeletal muscle.

Hematopoietic cell transplantation in severe combined immunodeficiency: The SCETIDE 2006-2014 European cohort

Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for patients with severe combined immunodeficiency (SCID), a group of monogenic immune disorders with an otherwise fatal outcome. We performed a comprehensive multicenter analysis of genotype-specific HSCT outcome, including detailed analysis of immune reconstitution (IR) and the predictive value for clinical outcome. HSCT outcome was studied in 338 patients with genetically confirmed SCID who underwent transplantation in 2006-2014 and who were registered in the SCETIDE registry. In a representative subgroup of 152 patients, data on IR and long-term clinical outcome were analyzed. Two-year OS was similar with matched family and unrelated donors and better than mismatched donor HSCT (P<.001). The 2-year event-free survival (EFS) was similar in matched and mismatched unrelated donor and less favorable in mismatched related donor (MMRD) HSCT (P< .001). Genetic subgroups did not differ in 2-year OS (P= .1) and EFS (P=.073). In multivariate analysis, pretransplantation infections and use of MMRDs were associated with less favorable OS and EFS. With a median follow-up of 6.2 years (range, 2.0-11.8 years), 73 of 152 patients in the IR cohort were alive and well without Ig dependency. IL-2 receptor gamma chain/Janus kinase 3/IL-7 receptor-deficient SCID, myeloablative conditioning, matched donor HSCT, and naive CD4 T lymphocytes >0.5×10e3/μL at+1 year were identified as independent predictors of favorable clinical and immunologic outcome. Recent advances in HSCT in SCID patients have resulted in improved OS and EFS in all genotypes and donor types. To achieve a favorable long-term outcome, treatment strategies should aim for optimal naive CD4 T lymphocyte regeneration.

Immune Reconstitution and Preliminary Safety Analysis Of 9 Patients Treated With Somatic Gene Therapy For X-Linked Severe Combined Immunodeficiency (SCID-X1) With a Self-Inactivating Gammaretroviral Vector

▪X-linked severe combined immunodeficiency (SCID-X1) is a fatal genetic disease currently treated with hematopoietic stem cell transplantation. Previous gene therapy trials using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (γc) controlled by the viral long-terminal repeat (LTR) induced T cell reconstitution in 18 of 20 boys with SCID-X1, but also resulted in insertional oncogenesis in 5 of 20, leading to leukemia, with 4 of the 5 demonstrating insertion at the LMO2 locus (Trial 1). We tested whether a self-inactivating (SIN) gammaretroviral vector, pSRS11.EFS.IL2RG.pre*, derived by removing the MLV LTR U3 (enhancer/promoter) region and driving expression of γc by an intronless elongation factor 1α (EF1-α) promoter, would promote T cell reconstitution in boys with X-SCID with an improved safety profile (Trial 2). Pre-clinical studies showed a lack of clonal skewing in primary and secondary transplant recipient mice in vivo and lack of insertions in either Lmo2 or Evi1 in these mice. The primary endpoints of the study are: 1. immune recovery defined as CD3+ T cell count >300/microliter and restoration of proliferation to phytohemagglutinin (PHA); and 2. Lack of insertional oncogenesis.To date 9 boys with SCID-X1 age 3.9 to 10.5 months were enrolled with 5-32 months follow-up on the current trial. All received bone marrow derived CD34+ cells transduced with the pSRS11.EFS.IL2RG.pre* vector, infused without conditioning. Transduction was performed at each site with one of two common protocols and the same GMP-produced vector supernatant. Transduction efficiency of infused CD34+ cells ranged from 0.25-2.92 vector copy number (VCN). One patient with a low initial transduction underwent a repeat procedure 1 month after infusion of the first product. Overall survival is excellent with 8 out of 9 patients alive and well; 1 patient died of overwhelming adenoviral infection, present at study entry, at 4 months prior to full reconstitution with genetically modified T cells. Excluding 1 patient with high level maternal engraftment who is unevaluable with regard to T cell number, 5 of 7 (71%) achieved CD3+ T cell count >300/microliter. The 2 patients who did not achieve T cell count >300/microliter received CD34+ cells with the lowest vector copy number (VCN). An early rise in CD16/56+ NK cells was also associated with T cell recovery. Of 8 surviving patients, 7 of 8 (87.5%) had PHA stimulation index >15 at or before 6 months (n=8; median 85.5; range 0.2-224); the patient who had no functional recovery received mismatched allogeneic cord blood transplant. At last follow-up, the remaining 7 patients have evidence of γc transgene expression in T cells, naïve T cell generation, and normal diversity. They are free of SCID related infections and humoral immune evaluation is ongoing.We compared the early immune recovery and peripheral blood insertion site analysis between 6 evaluable subjects on Trial 2 with the 20 subjects enrolled on the previous trials of MLV-based γc vector. The CD3+ T cell count at 6 months for Trial 2 (n=6; median 548/microliter; range 87-3960) does not appear to be lower than for Trial 1 (n=19; median 1755/microliter; range 200-4759; p=0.14 in 2-sided Wilcoxon rank sum test). However, we recognize that this test is underpowered, and results are preliminary. Because the latency time to leukemic events in the previous trial was long (33-60 months), we compared insertion sites as a surrogate measure of safety. Insertional activation of growth controlling genes has been demonstrated to be associated in some murine models with clonal skewing during in vitro expansion and in vivo reconstitution. Therefore, we hypothesized that removal of the transcription factor binding site-rich LTR U3 enhancer regions would change the insertion pattern detected in peripheral blood after transplantation away from the 5' ends of genes important for cell proliferation or persistence. Whereas in Trial 1, LMO2, EVI1 and other lymphoid proto-oncogenes showed prominent clusters of integration sites post-transplantation, these locations were not significant clusters in the second trial (p<10-4; comparison of n=19 Trial 1 versus n=6 Trial 2). Thus, we show promising early data that a modified gammaretroviral vector retains efficacy in treatment of SCID-X1 and is likely to have an improved safety profile. Disclosures:Off Label Use: Off-label use of CliniMACS device for purification of CD34+ bone marrow cells. Baum:Transatlantic Gene Therapy Consortium: Patents & Royalties.

Warfarin Impairs the Bone Marrow Microenvironment and Hematopoiesis Via the Periostin/Integrin β3 Pathway

The coagulation system is a complex system comprised of various proteases and other factors which result in the formation of a blood clot, and proteases in general are known to regulate the mobilization of normal hematopoietic stem cells (HSC) from the bone marrow microenvironment (BMM). However, whether the proteases of the coagulation system are involved in the mobilization or maintenance of hematopoietic stem and progenitor cells (HSPC) is unknown.Hypothesizing that the proteases of the coagulation system play a role in the normal physiology of the BMM and that modulation of coagulation factors may have an effect on HSPC, we tested the effects of the anticoagulant warfarin, used in 1% of people worldwide for prophylactic or therapeutic anticoagulation. Indeed, treatment of wildtype mice with warfarin led to a decrease in myeloid cells in peripheral blood, decreased HSC function and mobilization from the BMM and an almost 8 fold reduction of functional HSC. Intrafemoral co-transplantation of warfarin-treated, ex-vivo expanded primary murine bone marrow stromal cells with untreated HSPC led to decreased engraftment of HSPC compared to co-transplantation of vehicle-treated stroma cells suggesting that warfarin may compromise bone marrow stroma. We have implicated periostin, a vitamin K dependent factor produced by bone marrow stroma - binding to integrin β3 and signaling via AKT in HSC - as being the cause of impairment of hematopoiesis during treatment with warfarin.In addition, warfarin treatment of NOD SCID interleukin-2 receptor gamma knockout (NSG) mice or intrafemoral cotransplantation of human warfarin-treated stroma cells and untreated human CD34+ HSC led to reduced engraftment of human CD45+ leukocytes. In corroboration of our murine data we showed that the total leukocyte and monocyte count, as well as the percentage of basophils and eosinophils were significantly reduced in 282 patients receiving warfarin and 89 patients receiving another vitamin K antagonist, fluindione, for anticoagulation compared to untreated healthy controls.In summary, warfarin or vitamin K antagonism treatment impairs HSC function in humans and mice via modification of the BMM and, specifically, by the impairment of the periostin/integrin β3 axis, which we, thereby, implicate also for the maintenance of normal HSC. [Display omitted] DisclosuresFontenay:Celgene: Research Funding.

Development of human hepatocellular carcinoma in X-linked severe combined immunodeficient pigs: An orthotopic xenograft model.

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6-15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC.

Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.