Noncoding RNAs in Hepatocellular Carcinoma: Potential Applications in Combined Therapeutic Strategies and Promising Candidates of Treatment Response.

The incidence of hepatocellular carcinoma (HCC) is increasing, and 40% of patients are diagnosed at advanced stages. Over the past 5 years, the number of clinically available treatments has dramatically increased for HCC, making patient management particularly complex. Immune checkpoint inhibitors (ICIs) have improved the overall survival of patients, showing a durable treatment benefit over time and a different response pattern with respect to tyrosine kinase inhibitors (TKIs). Although there is improved survival in responder cases, a sizeable group of patients are primary progressors or are ineligible for immunotherapy. Indeed, patients with nonviral etiologies, such as nonalcoholic steatohepatitis (NASH), and alterations in specific driver genes might be less responsive to immunotherapy. Therefore, improving the comprehension of mechanisms of drug resistance and identifying biomarkers that are informative of the best treatment approach are required actions to improve patient survival. Abundant evidence indicates that noncoding RNAs (ncRNAs) are pivotal players in cancer. Molecular mechanisms through which ncRNAs exert their effects in cancer progression and drug resistance have been widely investigated. Nevertheless, there are no studies summarizing the synergistic effect between ncRNA-based strategies and TKIs or ICIs in the preclinical setting. This review aims to provide up-to-date information regarding the possible use of ncRNAs as therapeutic targets in association with molecular-targeted agents and immunotherapies and as predictive tools for the selection of optimized treatment options in advanced HCCs.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma.

BACKGROUND: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker. METHODS: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats. RESULTS: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells. CONCLUSIONS: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder.

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

Luteolin Treatment Ameliorates Brain Development and Behavioral Performance in a Mouse Model of CDKL5 Deficiency Disorder.

CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene, is characterized by early-onset epilepsy, intellectual disability, and autistic features. Although pharmacotherapy has shown promise in the CDD mouse model, safe and effective clinical treatments are still far off. Recently, we found increased microglial activation in the brain of a mouse model of CDD, the Cdkl5 KO mouse, suggesting that a neuroinflammatory state, known to be involved in brain maturation and neuronal dysfunctions, may contribute to the pathophysiology of CDD. The present study aims to evaluate the possible beneficial effect of treatment with luteolin, a natural flavonoid known to have anti-inflammatory and neuroprotective activities, on brain development and behavior in a heterozygous Cdkl5 (+/-) female mouse, the mouse model of CDD that best resembles the genetic clinical condition. We found that inhibition of neuroinflammation by chronic luteolin treatment ameliorates motor stereotypies, hyperactive profile and memory ability in Cdkl5 +/- mice. Luteolin treatment also increases hippocampal neurogenesis and improves dendritic spine maturation and dendritic arborization of hippocampal and cortical neurons. These findings show that microglia overactivation exerts a harmful action in the Cdkl5 +/- brain, suggesting that treatments aimed at counteracting the neuroinflammatory process should be considered as a promising adjuvant therapy for CDD.

Reduced Follicular Regulatory T Cells in Spleen and Pancreatic Lymph Nodes of Patients With Type 1 Diabetes.

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at risk for type 1 diabetes (T1D), multiple studies have analyzed and reported alterations in T follicular helper (Tfh) cells in presymptomatic AAb+ subjects and patients with T1D. Yet, whether the regulatory counterpart of Tfh cells, represented by T follicular regulatory (Tfr) cells, is similarly altered is still unclear. To address this question, we performed analyses in peripheral blood, spleen, and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb- and AAb+ subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed among T1D, AAb-, and AAb+ adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared with nondiabetic control subjects. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D, suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.

Inhibition of microglia overactivation restores neuronal survival in a mouse model of CDKL5 deficiency disorder.

BACKGROUND: CDKL5 deficiency disorder (CDD), a severe neurodevelopmental disorder characterized by early onset epilepsy, intellectual disability, and autistic features, is caused by mutations in the CDKL5 gene. Evidence in animal models of CDD showed that absence of CDKL5 negatively affects neuronal survival, as well as neuronal maturation and dendritic outgrowth; however, knowledge of the substrates underlying these alterations is still limited. Neuroinflammatory processes are known to contribute to neuronal dysfunction and death. Recent evidence shows a subclinical chronic inflammatory status in plasma from CDD patients. However, to date, it is unknown whether a similar inflammatory status is present in the brain of CDD patients and, if so, whether this plays a causative or exacerbating role in the pathophysiology of CDD. METHODS: We evaluated microglia activation using AIF-1 immunofluorescence, proinflammatory cytokine expression, and signaling in the brain of a mouse model of CDD, the Cdkl5 KO mouse, which is characterized by an impaired survival of hippocampal neurons that worsens with age. Hippocampal neuron survival was determined by DCX, NeuN, and cleaved caspase-3 immunostaining in Cdkl5 KO mice treated with luteolin (10 mg/kg), a natural anti-inflammatory flavonoid. Since hippocampal neurons of Cdkl5 KO mice exhibit increased susceptibility to excitotoxic stress, we evaluated neuronal survival in Cdkl5 KO mice injected with NMDA (60 mg/kg) after a 7-day treatment with luteolin. RESULTS: We found increased microglial activation in the brain of the Cdkl5 KO mouse. We found alterations in microglial cell morphology and number, increased levels of AIF-1 and proinflammatory cytokines, and activation of STAT3 signaling. Remarkably, treatment with luteolin recovers microglia alterations as well as neuronal survival and maturation in Cdkl5 KO mice, and prevents the increase in NMDA-induced cell death in the hippocampus. CONCLUSIONS: Our results suggest that neuroinflammatory processes contribute to the pathogenesis of CDD and imply the potential usefulness of luteolin as a treatment option in CDD patients.

Treatment with a GSK-3ß/HDAC Dual Inhibitor Restores Neuronal Survival and Maturation in an In Vitro and In Vivo Model of CDKL5 Deficiency Disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a rare neurodevelopmental disorder characterized by early-onset seizures and severe cognitive, motor, and visual impairments. To date there are no therapies for CDKL5 deficiency disorder (CDD). In view of the severity of the neurological phenotype of CDD patients it is widely assumed that CDKL5 may influence the activity of a variety of cellular pathways, suggesting that an approach aimed at targeting multiple cellular pathways simultaneously might be more effective for CDD. Previous findings showed that a single-target therapy aimed at normalizing impaired GSK-3ß or histone deacetylase (HDAC) activity improved neurodevelopmental and cognitive alterations in a mouse model of CDD. Here we tested the ability of a first-in-class GSK-3ß/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3ß and HDAC6 activity, not only restored maturation, but also significantly improved survival of both human CDKL5-deficient cells and hippocampal neurons from Cdkl5 KO mice. Importantly, in vivo treatment with C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice, suggesting that dual GSK-3ß/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.

Age-Related Cognitive and Motor Decline in a Mouse Model of CDKL5 Deficiency Disorder is Associated with Increased Neuronal Senescence and Death.

CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene. Children affected by CDD display a clinical phenotype characterized by early-onset epilepsy, intellectual disability, motor impairment, and autistic-like features. Although the clinical aspects associated with CDKL5 mutations are well described in children, adults with CDD are still under-characterized. Similarly, most animal research has been carried out on young adult Cdkl5 knockout (KO) mice only. Since age represents a risk factor for the worsening of symptoms in many neurodevelopmental disorders, understanding age differences in the development of behavioral deficits is crucial in order to optimize the impact of therapeutic interventions. Here, we compared young adult Cdkl5 KO mice with middle-aged Cdkl5 KO mice, at a behavioral, neuroanatomical, and molecular level. We found an age-dependent decline in motor, cognitive, and social behaviors in Cdkl5 KO mice, as well as in breathing and sleep patterns. The behavioral decline in older Cdkl5 KO mice was not associated with a worsening of neuroanatomical alterations, such as decreased dendritic arborization or spine density, but was paralleled by decreased neuronal survival in different brain regions such as the hippocampus, cortex, and basal ganglia. Interestingly, we found increased ß-galactosidase activity and DNA repair protein levels, γH2AX and XRCC5, in the brains of older Cdkl5 KO mice, which suggests that an absence of Cdkl5 accelerates neuronal senescence/death by triggering irreparable DNA damage. In summary, this work provides evidence that CDKL5 may play a fundamental role in neuronal survival during brain aging and suggests a possible worsening with age of the clinical picture in CDD patients.

A GABAB receptor antagonist rescues functional and structural impairments in the perirhinal cortex of a mouse model of CDKL5 deficiency disorder.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early-onset epilepsy and intellectual disability. Studies in mouse models have linked CDKL5 deficiency to defects in neuronal maturation and synaptic plasticity, and disruption of the excitatory/inhibitory balance. Interestingly, increased density of both GABAergic synaptic terminals and parvalbumin inhibitory interneurons was recently observed in the primary visual cortex of Cdkl5 knockout (KO) mice, suggesting that excessive GABAergic transmission might contribute to the visual deficits characteristic of CDD. However, the functional relevance of cortical GABAergic circuits abnormalities in these mutant mice has not been investigated so far. Here we examined GABAergic circuits in the perirhinal cortex (PRC) of Cdkl5 KO mice, where we previously observed impaired long-term potentiation (LTP) associated with deficits in novel object recognition (NOR) memory. We found a higher number of GABAergic (VGAT)-immunopositive terminals in the PRC of Cdkl5 KO compared to wild-type mice, suggesting that increased inhibitory transmission might contribute to LTP impairment. Interestingly, while exposure of PRC slices to the GABAA receptor antagonist picrotoxin had no positive effects on LTP in Cdkl5 KO mice, the selective GABAB receptor antagonist CGP55845 restored LTP magnitude, suggesting that exaggerated GABAB receptor-mediated inhibition contributes to LTP impairment in mutants. Moreover, acute in vivo treatment with CGP55845 increased the number of PSD95 positive puncta as well as density and maturation of dendritic spines in PRC, and restored NOR memory in Cdkl5 KO mice. The present data show the efficacy of limiting excessive GABAB receptor-mediated signaling in improving synaptic plasticity and cognition in CDD mice.

Increased DNA Damage and Apoptosis in CDKL5-Deficient Neurons.

Mutations in the CDKL5 gene, which encodes a serine/threonine kinase, causes a rare encephalopathy, characterized by early-onset epilepsy and severe intellectual disability, named CDKL5 deficiency disorder (CDD). In vitro and in vivo studies in mouse models of Cdkl5 deficiency have highlighted the role of CDKL5 in brain development and, in particular, in the morphogenesis and synaptic connectivity of hippocampal and cortical neurons. Interestingly, Cdkl5 deficiency in mice increases vulnerability to excitotoxic stress in hippocampal neurons. However, the mechanism by which CDKL5 controls neuronal survival is far from being understood. To investigate further the function of CDKL5 and dissect the molecular mechanisms underlying neuronal survival, we generated a human neuronal model of CDKL5 deficiency, using CRISPR/Cas9-mediated genome editing. We demonstrated that CDKL5 deletion in human neuroblastoma SH-SY5Y cells not only impairs neuronal maturation but also reduces cell proliferation and survival, with alterations in the AKT and ERK signaling pathways and an increase in the proapoptotic BAX protein and in DNA damage-associated biomarkers (i.e., γH2AX, RAD50, and PARP1). Furthermore, CDKL5-deficient cells were hypersensitive to DNA damage-associated stress, accumulated more DNA damage foci (γH2AX positive) and were more prone to cell death than the controls. Importantly, increased kainic acid-induced cell death of hippocampal neurons of Cdkl5 KO mice correlated with an increased γH2AX immunostaining. The results suggest a previously unknown role for CDKL5 in DNA damage response that could underlie the pro-survival function of CDKL5.

Correction to: Increased DNA Damage and Apoptosis in CDKL5-Deficient Neurons.

The original version of this article unfortunately contained error in Fig. 5a to where a panel is missing.

Pharmacotherapy with sertraline rescues brain development and behavior in a mouse model of CDKL5 deficiency disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a severe neurodevelopmental disorder, CDKL5 deficiency disorder (CDD). CDKL5 is fundamental for correct brain development and function, but the molecular mechanisms underlying aberrant neurologic dysfunction in CDD are incompletely understood. Here we show a dysregulation of hippocampal and cortical serotonergic (5-HT) receptor expression in heterozygous Cdkl5 knockout (KO) female mice, suggesting that impaired 5-HT neurotransmission contributes to CDD. We demonstrate that targeting impaired 5-HT signaling via the selective serotonin reuptake inhibitor (SSRI) sertraline rescues CDD-related neurodevelopmental and behavioral defects in heterozygous Cdkl5 KO female mice. In particular, chronic treatment with sertraline normalized locomotion, stereotypic and autistic-like features, and spatial memory in Cdkl5 KO mice. These positive behavioral effects were accompanied by restored neuronal survival, dendritic development and synaptic connectivity. At a molecular level, sertraline increased brain-derived neurotrophic factor (BDNF) expression and restored abnormal phosphorylation levels of tyrosine kinase B (TrkB) and its downstream target the extracellular signal-regulated kinase (ERK1/2). Since sertraline is an FDA-approved drug with an extensive safety and tolerability data package, even for children, our findings suggest that sertraline may improve neurodevelopment in children with CDD. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Reduced PD-1 expression on circulating follicular and conventional FOXP3+ Treg cells in children with new onset type 1 diabetes and autoantibody-positive at-risk children.

Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 diabetes (T1D), but the contribution of follicular regulatory T (Treg) cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5+) and conventional (CXCR5-) Treg cells in the blood of children with new-onset T1D, and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood follicular and conventional Treg cells were higher in frequency in children with new onset T1D, but expressed reduced amounts of PD-1 as compared to AAb-negative children. Interestingly, the proportion of circulating FOXP3+ Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to AAb-negative controls, suggesting its potential use as a biomarker of disease progression. Follicular Treg cells were reduced in frequency in the spleens of prediabetic NOD mice as they became older and turned diabetic. Interestingly, PD-1 expression declined also on circulating follicular and conventional Treg cells in prediabetic NOD mice as they aged. Together, these findings show that the frequency of circulating follicular and conventional Treg cells and their levels of PD-1 change with disease progression in children at-risk for developing T1D and in NOD mice.

Functional and Structural Impairments in the Perirhinal Cortex of a Mouse Model of CDKL5 Deficiency Disorder Are Rescued by a TrkB Agonist.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental encephalopathy caused by mutations in the CDKL5 gene and characterized by early-onset epilepsy and intellectual and motor impairments. No cure is currently available for CDD patients, as limited knowledge of the pathology has hindered the development of therapeutics. Cdkl5 knockout (KO) mouse models, recently created to investigate the role of CDKL5 in the etiology of CDD, recapitulate various features of the disorder. Previous studies have shown alterations in synaptic plasticity and dendritic pattern in the cerebral cortex and in the hippocampus, but the knowledge of the molecular substrates underlying these alterations is still limited. Here, we have examined for the first time synaptic function and plasticity, dendritic morphology, and signal transduction pathways in the perirhinal cortex (PRC) of this mouse model. Being interconnected with a wide range of cortical and subcortical structures and involved in various cognitive processes, PRC provides a very interesting framework for examining how CDKL5 mutation leads to deficits at the synapse, circuit, and behavioral level. We found that long-term potentiation (LTP) was impaired, and that the TrkB/PLCγ1 pathway could be mechanistically involved in this alteration. PRC neurons in mutant mice showed a reduction in dendritic length, dendritic branches, PSD-95-positive puncta, GluA2-AMPA receptor levels, and spine density and maturation. These functional and structural deficits were associated with impairment in visual recognition memory. Interestingly, an in vivo treatment with a TrkB agonist (the 7,8-DHF prodrug R13) to trigger the TrkB/PLCγ1 pathway rescued defective LTP, dendritic pattern, PSD-95 and GluA2-AMPA receptor levels, and restored visual recognition memory in Cdkl5 KO mice. Present findings demonstrate a critical role of TrkB signaling in the synaptic development alterations due to CDKL5 mutation, and suggest the possibility of TrkB-targeted pharmacological interventions.

CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling.

CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene, a member of a highly conserved family of serine-threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF-ß action, is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF-ß1 treatment normalized defective neuronal survival and maturation in Cdkl5 knockout (KO) neurons. Moreover, we demonstrate that Cdkl5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF-ß1 prevents increased NMDA-induced cell death in hippocampal neurons from Cdkl5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of Cdkl5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.

Tr1 cell immunotherapy promotes transplant tolerance via de novo Tr1 cell induction in mice and is safe and effective during acute viral infection.

Tr1 cell therapy is considered an emerging approach to improve transplant tolerance and enhance allogeneic graft survival. However, it remains unclear how Tr1 cells promote transplant tolerance and whether they will be safe and stable in the face of an acute viral infection. By employing a mouse model of pancreatic islet transplantation, we report that Tr1 cell therapy promoted transplant tolerance via de novo induction of Tr1 cells in the recipients. Acute viral infection with lymphocytic choriomeningitis virus (LCMV) had no impact on Tr1 cell number and function, neither on the Tr1 cells infused nor on the ones induced, and that was reflected in the robust maintenance of the graft. Moreover, Tr1 cell immunotherapy had no detrimental effect on CD8 and CD4 anti-LCMV effector T-cell responses and viral control. Together, these data suggest that Tr1 cells did not convert to effector cells during acute infection with LCMV, maintained transplant tolerance and did not inhibit antiviral immunity.

Comparison of Zinc Finger Nucleases Versus CRISPR-Specific Nucleases for Genome Editing of the Wiskott-Aldrich Syndrome Locus.

Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in γ-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.

Murine Pancreatic Islets Transplantation under the Kidney Capsule.

Type 1 diabetes (T1D) is an autoimmune disease caused by the lack of insulin-producing pancreatic beta cells leading to systemic hyperglycemia. Pancreatic islet transplantation is a valid therapeutic approach to restore insulin loss and to promote adequate glycemic control. Pancreatic islet transplantation in mice is an optimal preclinical model to identify new therapeutic strategies aiming at preventing rejection and optimizing post-transplant immuno-suppressive/-tolerogenic therapies. Islet transplantation in preclinical animal models can be performed in different sites such the kidney capsule, spleen, bone marrow and pancreas. This protocol describes murine islet transplantation under the kidney capsule. This is a widely accepted procedure for research purposes. Stress caused in the animals is minimal and it leads to reliable and reproducible results.

PTPN22 and islet-specific autoimmunity: What have the mouse models taught us?

An allelic variant of the protein tyrosin phosphatase non-receptor 22 (PTPN22) gene, PTPN22 R620W, constitutes the strongest non-HLA genetic risk factor for the development of type 1 diabetes (T1D). A number of studies using mouse models have addressed how PTPN22 predisposes to T1D. PTPN22 downmodulation, overexpression or expression of the variant gene in genetically manipulated mice has generated controversial results. These discrepancies probably derive from the fact that PTPN22 has differential effects on innate and adaptive immune responses. Moreover, the effects of PTPN22 are dependent on other genetic variables. Here we discuss these findings and try to explain the discrepancies. Exploring the mechanism by which PTPN22 contributes to islet-specific autoimmunity could help us understand its role in T1D pathogenesis and exploit it as a potential therapeutic target to prevent the disease.

Extrinsic Protein Tyrosine Phosphatase Non-Receptor 22 Signals Contribute to CD8 T Cell Exhaustion and Promote Persistence of Chronic Lymphocytic Choriomeningitis Virus Infection.

A genetic variant of the protein tyrosine phosphatase non-receptor 22 (PTPN22) is associated with a wide range of autoimmune diseases; however, the reasons behind its prevalence in the general population remain not completely understood. Recent evidence highlights an important role of autoimmune susceptibility genetic variants in conferring resistance against certain pathogens. In this study, we examined the role of PTPN22 in persistent infection in mice lacking PTPN22 infected with lymphocytic choriomeningitis virus clone 13. We found that lack of PTPN22 in mice resulted in viral clearance 30 days after infection, which was reflected in their reduced weight loss and overall improved health. PTPN22-/- mice exhibited enhanced virus-specific CD8 and CD4 T cell numbers and functionality and reduced exhausted phenotype. Moreover, mixed bone marrow chimera studies demonstrated no differences in virus-specific CD8 T cell accumulation and function between the PTPN22+/+ and PTPN22-/- compartments, showing that the effects of PTPN22 on CD8 T cells are T cell-extrinsic. Together, these findings identify a CD8 T cell-extrinsic role for PTPN22 in weakening early CD8 T cell responses to collectively promote persistence of a chronic viral infection.