Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. OBJECTIVE: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. METHODS: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. RESULTS: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. CONCLUSION: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.

Case report: Persistent shedding of a live vaccine-derived rubella virus in a young man with severe combined immunodeficiency and cutaneous granuloma.

A young man with X-linked severe combined immunodeficiency developed a persistent vaccine-derived rubella virus (VDRV) infection, with the emergence of cutaneous granulomas more than fifteen years after receipt of two doses of measles-mumps-rubella (MMR) vaccine. Following nasopharyngeal swab (NP) collection, VDRV was detected by real-time polymerase chain reaction (RT-qPCR) and sequencing, and live, replication-competent VDRV was isolated in cell culture. To assess duration and intensity of viral shedding, sequential respiratory samples, one cerebrospinal fluid sample, and two urine samples were collected over 15 months, and VDRV RNA was detected in all samples by RT-qPCR. Live VDRV was cultured from nine of the eleven respiratory specimens and from one urine specimen. To our knowledge, this was the first reported instance of VDRV cultured from respiratory specimens or from urine. To assess potential transmission to close contacts, NP specimens and sera were collected from all household contacts, all of whom were immunocompetent and previously vaccinated with MMR. VDRV RNA was not detected in any NP swabs from the contacts, nor did serologic investigations suggest VDRV transmission to any contacts. This report highlights the need to understand the prevalence and duration of VDRV shedding in granuloma patients and to estimate the risk of VDRV transmission to immune and non-immune contacts.

Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System.

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

Artificial thymic organoids represent a reliable tool to study T-cell differentiation in patients with severe T-cell lymphopenia.

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαß+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gene correction for SCID-X1 in long-term hematopoietic stem cells.

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

The original version of this Article omitted the following from the Acknowledgements: "G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.

Aberrant Clonal Hematopoiesis following Lentiviral Vector Transduction of HSPCs in a Rhesus Macaque.

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.

Disseminated and Congenital Toxoplasmosis in a Mother and Child With Activated PI3-Kinase δ Syndrome Type 2 (APDS2): Case Report and a Literature Review of Toxoplasma Infections in Primary Immunodeficiencies.

Phosphoinositide 3-kinase (PI3K) plays an integral role in lymphocyte function. Mutations in PIK3CD and PIK3R1, encoding the PI3K p110δ and p85α subunits, respectively, cause increased PI3K activity and result in immunodeficiency with immune dysregulation. We describe here the first cases of disseminated and congenital toxoplasmosis in a mother and child who share a pathogenic mutation in PIK3R1 and review the mechanisms underlying susceptibility to severe Toxoplasma gondii infection in activated PI3Kδ syndrome (APDS) and in other forms of primary immunodeficiency.

X-linked carriers of chronic granulomatous disease: Illness, lyonization, and stability.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by recurrent life-threatening bacterial and fungal infections and aberrant inflammation. Mutations in CYBB cause X-linked CGD and account for 65% to 70% of cases in Western countries. OBJECTIVE: We sought to understand the clinical manifestations associated with the X-linked CGD carrier state. METHODS: We undertook a comprehensive retrospective study of 162 affected female subjects. We examined dihydrorhodamine 123 (DHR) oxidation data for percentage of X-chromosome inactivation. We correlated lyonization (%DHR+) with clinical features. Where possible, we followed %DHR+ values over time. RESULTS: Clinical data were available for 93 female subjects: %DHR+ values were 46% (mean) and 47% (median; SD, 24). Using the %DHR+ value as the criterion for X inactivation, 78% of patients had levels of inactivation of 20% to 80%, suggesting random inactivation that was independent of age. In contrast, carriers with CGD-type infections had median %DHR+ values of 8% (n = 14; range, 0.06% to 48%), and those with only autoimmune or inflammatory manifestations had median %DHR+ values of 39% (n = 31; range, 7.4% to 74%). Those with both infections and autoimmunity had low %DHR+ values (n = 6; range, 3% to 14%). A %DHR+ value of less than 10% was strongly associated with infections (odds ratio, 99). Strong association persisted when %DHR+ values were less than 20% (odds ratio, 12). Autoimmunity was not associated with %DHR+ values. In 2 sets of identical twins, the %DHR+ populations tracked closely over time. Although the %DHR+ populations were very similar between sisters, those between mothers and daughters were unrelated. CONCLUSIONS: A low %DHR+ value strongly predicts infection risk in X-linked CGD carriers, and the carrier state itself is associated with autoimmunity.

The Changing Paradigm of Management of Liver Abscesses in Chronic Granulomatous Disease.

Background: Chronic granulomatous disease (CGD) is a rare genetic disorder causing recurrent infections. More than one-quarter of patients develop hepatic abscesses and liver dysfunction. Recent reports suggest that disease-modifying treatment with corticosteroids is effective for these abscesses. Comparison of corticosteroid therapy to traditional invasive treatments has not been performed. Methods: Records of 268 patients with CGD treated at the National Institutes of Health from 1980 to 2014 were reviewed. Patients with liver involvement and complete records were included. We recorded residual reactive oxygen intermediate (ROI) production by neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase germline mutation status, laboratory values, imaging characteristics, time to repeat hepatic interventions, and overall survival among 3 treatment cohorts: open liver surgery (OS), percutaneous liver-directed interventional radiology therapy (IR), and high-dose corticosteroid management (CM). Results: Eighty-eight of 268 patients with CGD suffered liver involvement. Twenty-six patients with a median follow-up of 15.5 years (8.5-32.9 years of follow-up) had complete records and underwent 100 standard interventions (42 IR and 58 OS). Eight patients received a treatment with high-dose corticosteroids only. There were no differences in NADPH genotype, size, or number of abscesses between patients treated with OS, IR, or CM. Time to repeat intervention was extended in OS compared with IR (18.8 vs 9.5 months, P = .04) and further increased in CM alone (median time to recurrence not met). Impaired macrophage and neutrophil function measured by ROI production correlated with shorter time to repeat intervention (r = 0.6, P = .0019). Conclusions: Treatment of CGD-associated liver abscesses with corticosteroids was associated with fewer subsequent hepatic interventions and improved outcome compared to invasive treatments.

Interleukin-27 Enhances the Potential of Reactive Oxygen Species Generation from Monocyte-derived Macrophages and Dendritic cells by Induction of p47phox.

Interleukin (IL)-27, a member of the IL-12 cytokine family, plays an important and diverse role in the function of the immune system. We have previously demonstrated that IL-27 is an anti-viral cytokine which inhibits HIV-1, HIV-2, Influenza virus and herpes simplex virus infection, and enhances the potential of reactive oxygen species (ROS) generating activity during differentiation of monocytes to macrophages. In this study, we further investigated the mechanism of the enhanced potential for ROS generation by IL-27. Real time PCR, western blot and knock down assays demonstrate that IL-27 is able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells (iDC) in association with the induction of p47phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase, and the increase in amounts of phosphorylated p47phox upon stimulation. We also demonstrate that IL-27 is able to induce extracellular superoxide dismutase during differentiation of monocytes but not in terminal differentiated macrophages. Since ROS plays an important role in a variety of inflammation, our data demonstrate that IL-27 is a potent regulator of ROS induction and may be a novel therapeutic target.

Gastrointestinal Features of Chronic Granulomatous Disease Found During Endoscopy.

BACKGROUND & AIMS: Chronic granulomatous disease (CGD) is an inherited disorder of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex within phagocytic cells that predisposes people to bacterial and fungal infections. Approximately 40% of patients with CGD have gastrointestinal involvement. We aimed to characterize the endoscopic features of gastrointestinal CGD and define the role of endoscopy in patients. METHODS: We created a database of all patients with CGD seen at the National Institutes of Health from 1990 through 2010. We identified patients who had an endoscopy, and collected information from those with CGD-associated inflammatory bowel disease. We analyzed clinical data (demographic information and symptoms), endoscopic data (indication, preparation quality, degree of inflammation, mucosal findings, and complications), and pathologic data. RESULTS: A total of 211 endoscopies (96 esophagogastroduodenoscopies, 82 colonoscopies, and 33 flexible sigmoidoscopies) were performed at the National Institutes of Health on 78 patients with CGD. Esophageal, gastric, and duodenal inflammation were detected in 21%, 74%, and 37% of patients, respectively. Esophageal dysmotility and structural abnormalities were noted in 26%. Of the patients who had colonic CGD-inflammatory bowel disease, 74% had skip lesions and 93% had anorectal disease. Enteric fistulae were found in 18% of patients; 73% of these were perianal. Colonic strictures were observed in 24% of patients; 80% were in the anorectal area. CONCLUSIONS: Based on an analysis of clinical and endoscopic data from 78 patients, CGD-inflammatory bowel disease is a distinct entity, primarily involving the anus and rectum, with skip lesions in the remaining bowel. Bowel strictures and fistulae are present in a significant number of patients. Upper gastrointestinal tract inflammatory disease is common, although typically not as severe as colonic disease. Upper and lower endoscopies are important in characterizing the gastrointestinal features of CGD.

Common severe infections in chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is due to defective nicotinamide adenine dinucleotide phosphate oxidase activity and characterized by recurrent infections with a limited spectrum of bacteria and fungi as well as inflammatory complications. To understand the impact of common severe infections in CGD, we examined the records of 268 patients followed at a single center over 4 decades. METHODS: All patients had confirmed diagnoses of CGD, and genotype was determined where possible. Medical records were excerpted into a standard format. Microbiologic analyses were restricted to Staphylococcus, Burkholderia, Serratia, Nocardia, and Aspergillus. RESULTS: Aspergillus incidence was estimated at 2.6 cases per 100 patient-years; Burkholderia, 1.06 per 100 patient-years; Nocardia, 0.81 per 100 patient-years; Serratia, 0.98 per 100 patient-years, and severe Staphylococcus infection, 1.44 per 100 patient-years. Lung infection occurred in 87% of patients, whereas liver abscess occurred in 32%. Aspergillus incidence was 55% in the lower superoxide-producing quartiles (quartiles 1 and 2) but only 41% in the higher quartiles (rate ratio, <0.0001). Aspergillus and Serratia were somewhat more common in lower superoxide producing gp91phox deficiency. The median age at death has increased from 15.53 years before 1990 to 28.12 years in the last decade. Fungal infection carried a higher risk of mortality than bacterial infection and was the most common cause of death (55%). Gastrointestinal complications were not associated with either infection or mortality. CONCLUSIONS: Fungal infections remain a major determinant of survival in CGD. X-linked patients generally had more severe disease, and this was generally in those with lower residual superoxide production. Survival in CGD has increased over the years, but infections are still major causes of morbidity and mortality.

Human syndromes of immunodeficiency and dysregulation are characterized by distinct defects in T-cell receptor repertoire development.

BACKGROUND: Human immunodeficiencies characterized by hypomorphic mutations in critical developmental and signaling pathway genes allow for the dissection of the role of these genes in the development of the T-cell receptor (TCR) repertoire and the correlation of alterations of the TCR repertoire with diverse clinical phenotypes. OBJECTIVE: The presence of T cells in patients with Omenn syndrome (OS) and patients with atypical presentations of severe combined immunodeficiency gene mutations presents an opportunity to study the effects of the causal genes on TCR repertoires and provides a window into the clinical heterogeneity observed. METHODS: We performed deep sequencing of TCRß complementarity-determining region 3 (CDR3) regions in subjects with a series of immune dysregulatory conditions caused by mutations in recombination activating gene 1/2 (RAG 1/2), IL-2 receptor γ (IL2RG), and ζ chain-associated protein kinase 70 (ZAP70); a patient with atypical DiGeorge syndrome; and healthy control subjects. RESULTS: We found that patients with OS had marked reductions in TCRß diversity compared with control subjects, as expected. Patients with atypical presentations of RAG or IL2RG mutations associated with autoimmunity and granulomatous disease did not have altered overall diversity but instead had skewed V-J pairing and skewed CDR3 amino acid use. Although germline TCRs were more abundant and clonally expanded in patients with OS, nongermline sequences were expanded as well. TCRß from patients with RAG mutations had less junctional diversity and smaller CDR3s than patients with OS caused by other gene mutations and healthy control subjects but relatively similar CDR3 amino acid use. CONCLUSIONS: High-throughput TCR sequencing of rare immune disorders has demonstrated that quantitative TCR diversity can appear normal despite qualitative changes in repertoire and strongly suggests that in human subjects RAG enzymatic function might be necessary for normal CDR3 junctional diversity.

Diabetes, renal and cardiovascular disease in p47 phox-/- chronic granulomatous disease.

Chronic granulomatous disease is a rare immunodeficiency due to defects in the phagocyte NADPH oxidase. The X-linked form (gp91 (phox) deficiency) accounts for about 70 % of cases; autosomal recessive p47 (phox) deficiency accounts for about 25 % of cases. We identified a 10 % incidence of diabetes in p47 (phox) deficient CGD, but none in X-linked CGD. Renal and cardiovascular diseases were also higher in p47 (phox) deficiency. p47 (phox) deficient CGD has non-infectious morbidities distinct from those in X-linked CGD.