X-linked Chronic Granulomatous Disease Research Articles

Very Early-Onset Inflammatory Manifestations of X-Linked Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare primary immune deficiency caused by mutations in genes coding for components of the nicotinamide adenine dinucleotide phosphate oxidase, characterized by severe and recurrent bacterial and fungal infections, together with inflammatory complications. Dysregulation of inflammatory responses are often present in this disease and may lead to granulomatous lesions, most often affecting the gastrointestinal (GI) and urinary tracts. Treatment of inflammatory complications usually includes corticosteroids, whereas antimicrobial prophylaxis is used for infection prevention. Curative treatment of both infectious susceptibility and inflammatory disease can be achieved by hematopoietic stem cell transplantation. We report herein three patients with the same mutation of the CYBB gene who presented with very early-onset and severe GI manifestations of X-linked CGD. The most severely affected patient had evidence of antenatal inflammatory involvement of the GI and urinary tracts. Extreme hyperleukocytosis with eosinophilia and high inflammatory markers were observed in all three patients. A Mycobacterium avium lung infection and an unidentified fungal lung infection occurred in two patients both during their first year of life, which is indicative of the severity of the disease. All three patients underwent bone marrow transplantation and recovered fully from their initial symptoms. To our knowledge, these are the first reports of patients with such an early-onset and severe inflammatory manifestations of CGD.

Mutation in an exonic splicing enhancer site causing chronic granulomatous disease

In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.

Chronic Granulomatous Disease: An Unreported Mutation

Chronic Granulomatous Disease (CGD) is caused by defects in the phagocyte NADPH oxidase and occurs in approximately 1:200,000 births worldwide. It presents with early onset of severe recurrent bacterial and fungal infections. This is a case of a 9-year old male with severe, recurrent bacterial infections since 3 weeks of age. Initial Nitroblue tetrazolium (NBT) reduction tests were normal but a DNA analysis revealed a previously unreported homozygous mutation in CYBB, p.S418Y. Dihydrorhodamine (DHR) test showed poor neutrophil oxidation consistent with X-linked CGD. Definitive microbiologic diagnosis is essential for directing therapy for recurrent bacterial and fungal infections. Treatment of infections should be aggressive. Lifelong bacterial and fungal prophylaxis is necessary for prolonged survival. We report a case of confirmed CGD with the previously unreported mutation.

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

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. We sought to understand the clinical manifestations associated with the X-linked CGD carrier state. 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. 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. A low %DHR+ value strongly predicts infection risk in X-linked CGD carriers, and the carrier state itself is associated with autoimmunity.

Major CD4 T-Cell Depletion and Immune Senescence in a Patient with Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) results from primary defects in phagocytic reactive oxygen species (ROS) production. T-cell evaluation is usually neglected during patients’ follow-up, although T-cell depletion has been reported in CGD through unknown mechanisms. We describe here a 36-year-old patient with X-linked CGD with severe CD4 T-cell depletion <200 CD4 T-cells/μl, providing insights into the mechanisms that underlie T-cell loss in the context of oxidative burst defects. In addition to the typical infections, the patient featured a progressive T-cell loss associated with persistent lymphocyte activation, expansion of interleukin (IL)-17-producing CD4 T-cells, and impaired thymic activity, leading to a reduced replenishment of the T-cell pool. A relative CD4 depletion was also found at the gut mucosal level, although no bias to IL-17-production was documented. This immunological pattern of exhaustion of immune resources favors prompt, potentially curative, therapeutic interventions in CGD patients, namely, stem-cell transplantation or gene therapy. Moreover, this clinical case raises new research questions on the interplay of ROS production and T-cell homeostasis and immune senescence.

Infection Profile in Chronic Granulomatous Disease: a 23-Year Experience from a Tertiary Care Center in North India.

Chronic granulomatous disease (CGD) is an inherited phagocytic disorder characterized by recurrent infections with usually catalase-positive organisms. Infections in CGD from developing countries are expected to be different from those in the Western countries. We report the profile of infections in children diagnosed with CGD from a tertiary care center in North India. Case records of children diagnosed with CGD at Pediatric Immunodeficiency Clinic, Advanced Pediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India, from August 1993 to April 2016 (23years) were analyzed. Thirty-eight children were diagnosed to have CGD. Median follow-up of patients was 2years (interquartile range 0.75, 6.0). Staphylococcus aureus and Pseudomonas spp. were the two most common causative bacteria isolated. Aspergillus was the most common fungus isolated. The most common organ involved was the lung (94.7%). Liver abscesses were identified in 5 patients (13.2%), and 20 (52.6%) patients had lymphadenitis. Infections with Pseudomonas spp. were high in our cohort (15.7%) compared to the other studies. Infections with some unusual organisms (e.g., Fusarium dimerium and Chryseobacterium gleum) were also seen in our cohort. Children with X-linked CGD presented earlier and also had a greater number of infections as compared to autosomal recessive CGD. Various socioeconomic factors coupled with the lack of awareness and paucity of readily available diagnostic facilities for primary immunodeficiencies accounted for a late clinical presentation with severe infections and increased mortality (28.9%) in our cohort. However, mortality was similar in X-linked and autosomal recessive CGD as was the number of fungal infections. The incidence of infections and mortality was significantly lower after initiation of antibacterial and antifungal prophylaxis.

CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants.

Chronic granulomatous disease (CGD) is characterized by defects in the production of microbicidal reactive oxygen species (ROS) by phagocytes. Testing of gene and cell therapies for the treatment of CGD in human hematopoietic cells requires preclinical transplant models. The use of the lymphocyte-deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse strain for human hematopoietic cell xenografts to test CGD therapies is complicated by the presence of functional mouse granulocytes capable of producing ROS for subsequent bacterial and fungal killing. To establish a phagocyte-defective mouse model of X-linked CGD (X-CGD) in NSG mice, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was utilized for targeted knockout of mouse Cybb on the X-chromosome by microinjection of NSG mouse zygotes with Cas9 mRNA and CRISPR single-guide RNA targeting Cybb exon 1 or exon 3. This resulted in a high incidence of indel formation at the CRISPR target site, with all mice exhibiting deletions in at least one Cybb allele based on sequence analysis of tail snip DNA. A female mouse heterozygous for a 235-bp deletion in Cybb exon 1 was bred to an NSG male to establish the X-CGD NSG mouse strain, NSG.Cybb[KO]. Resulting male offspring with the 235 bp deletion were found to be defective for production of ROS by neutrophils and other phagocytes, and demonstrated increased susceptibility to spontaneous bacterial and fungal infections with granulomatous inflammation. The establishment of the phagocyte-defective NSG.Cybb[KO] mouse model enables the in vivo assessment of gene and cell therapy strategies for treating CGD in human hematopoietic cell transplants without obfuscation by functional mouse phagocytes, and may also be useful for modeling other phagocyte disorders in humanized NSG mouse xenografts.

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochrome b558 composed of NOX2 and p22phox, the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22phox liposomes was estimated to be around 700 nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47phox, p67phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22phox liposomes for 8 h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments.

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13minigene into CYBB exon 1 resulted in no detectable gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13minigene into exon 2 restored both gp91phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.

NADPH Oxidase-2 and Atherothrombosis: Insight From Chronic Granulomatous Disease.

The phagocytic cell enzyme NADPH oxidase-2 (Nox2) is critical for killing micro-organisms via production of reactive oxygen species and thus is a key element of the innate immune system. Nox2 is also detectable in endothelial cells and platelets where it has vasoconstrictive and aggregating properties, respectively. Patients with X-linked chronic granulomatous disease with hereditary Nox2 deficiency not only have impaired bacterial killing but, in association with loss of Nox2 function, also have enhanced carotid artery dilation, impaired platelet-related thrombosis, and reduced carotid atherosclerotic burden. Experimental studies corroborated these reports in chronic granulomatous disease by demonstrating (1) Nox2 is upregulated in atherosclerotic plaque, and this upregulation significantly correlates with oxidative stress and (2) pharmacological inhibition of Nox2 is associated with a delayed atherosclerotic progression in animal models. Furthermore, the role of Nox2 in platelet-associated thrombosis was substantiated by experiments showing impaired platelet activation in animals treated with a Nox2 inhibitor or impaired platelet aggregation along with reduced platelet-related thrombosis in the mouse knockout model of Nox2. Interestingly, in chronic granulomatous disease patients and in the mouse knockout model of Nox2, no defects of primary hemostasis were detected. This review analyses experimental and clinical data suggesting Nox2 is a potential target for counteracting the atherothrombotic process.

A New Chapter on Targeted Gene Insertion for X-CGD: Do Not Skip the Intro(n)

X-linked chronic granulomatous disease (X-CGD) is a rare, devastating primary immunodeficiency disorder (PID) characterized by a defective host defense against certain bacteria and fungi.1 It is caused by mutations in the CYBB gene, encoding the gp91phox subunit of NADPH oxidase, an enzyme complex responsible for pathogen clearance in professional phagocytic cells. Like many monogenic disorders of the immune system, X-CGD is amenable to gene therapy through gene addition. More recently, scientists have also started to explore nuclease-mediated targeted gene insertion as a strategy to restore gp91phox expression in reprogrammed induced pluripotent stem cells (iPSCs)2–4 and hematopoietic stem and progenitor cells (HSPCs)5 from CGD patients.

CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease.

Gene repair of CD34+ hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34+ HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc-/- mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

High Efficiency Gene Correction in Hematopoietic Cells By Template-Free Crispr/Cas9 Genome Editing

IntroductionA significant fraction of inherited monogenic disorders is caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical gene therapy trials. Most of these trials employed retroviral vectors, which by inserting randomly throughout the genome deprived the transduced genes of their endogenous control and caused insertional mutations leading to secondary disease. The development of genome editing tools capable of modifying any prespecified genomic sequence with unprecedented accuracy opened up a wide range of new possibilities in gene manipulation including targeted gene repair. In particular, CRISPR/Cas9, a prokaryotic adaptive immune system, and its swift repurposing for genome editing was widely adopted as the hitherto simplest genome editing tool. CRISPR/Cas9 is an RNA guided endonuclease that uses RNA-DNA base pairing to target genomic DNA. Bound to its target via the guide (g)RNA, Cas9 induces DNA double strand breaks (DBS) at prespecified genomic sites that promptly activate the endogenous DNA repair machinery. DSB repair is accomplished by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Thus far, correction of human mutations in hematopoietic cells relied entirely on HDR requiring gene specific donor templates in addition to the RNA guided endonucleases (RGNs). However, although the HDR offers precision, its efficiency is low and requires positive selection to enrich for gene corrected cell. Because in mammalian cells DSB repair by NHEJ significantly exceeds HDR and even more importantly, is the dominant DSB repair pathway in hematopoietic stem- and progenitor cells (HSPC), we thought to exploit NHEJ for gene therapy because in theory, approximately one third of the indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation. This would lead to a significant number of ORF reconstitutions of which some, depending on the position and type of the original mutation, should either completely or partially recover protein function.ResultsTo test gene repair efficiency by NHEJ in human hematopoietic cells, we generated PLB-985 (PLB) reporter cells expressing mutationally inactivated EGFP (mEGFP). Transduction of mEGFP expressing PLBs (mEGFP-PLB) with integrase-deficient lentiviral (IDLV) particles encoding for RGNs targeting the EGFP mutation reconstituted EGFP expression in up to 27% of the mEGFP PLB cells. Indel analysis revealed that 13 out of 28 (46%) restored the EGFP-open reading frame of which 7 (25%) reconstituted EGFP activity. To test whether the donor-template free IDLV strategy would also effectively correct bone fide disease mutations, we performed similar experiments with X-CGD PLB cells expressing transgenes encoding patient-specific frameshift, missense and nonsense CYBB mutations causing X-linked chronic granulomatous disease (X-CGD) which is an inherited, life threatening immunodeficiency disorder. Transduction of the cells with IDLVs carrying RGNs directed against each of these mutations restored CYBB function in up to 10% of cells harboring frameshift mutations which is sufficient to protect X-CGD patients from microbial infections. However, RGNs directed against the nonsense or missense mutations restored CYBB function in only 1-2% of the cells, suggesting that these mutations are less amenable to CRISPR/Cas9 mediated repair. As Cas9 frequently tolerates single nucleotide mismatches, selection against solitary nucleotide substitutions may explain this low efficiency of gene repair.ConclusionsFrameshift mutations can be effectively repaired by NHEJ in hematopoietic cells by CRISPR/Cas9 transducing IDLVs. As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that about a quarter of patients suffering from monogenic blood disorders could benefit from personalized, template free CRISPR/Cas9 gene therapy. DisclosuresNo relevant conflicts of interest to declare.

The NADPH Oxidase Catalytic Subunit Nox2 Displays Differential Roles in Arterial Vs. Venous Thrombosis

NADPH oxidase is a major superoxide generating enzyme in vascular tissues. Deficiency of the Nox2 (gp91phox) catalytic subunit of NADPH oxidase is a genetic cause of X-linked chronic granulomatous disease. These patients are prone to infection due to loss of oxidant production by neutrophils, and recent evidence suggests they also have a defect in platelet adhesion to collagen. However, the role of Nox2 in thrombosis is controversial, with one study demonstrating no effect of murine Nox2 deficiency on platelet activation and another study implicating Nox2 in platelet aggregation as well as thrombosis in small vessels. Given that Nox2 may be important for both platelet activation as well as release of neutrophil extracellular traps (NETs, a mediator of venous thrombosis), we hypothesized that genetic deficiency in murine Nox2 leads to decreased susceptibility to experimental thrombosis in both arterial and venous systems. We studied male Nox2 deficient (Cybb-/y) mice and wild type (Cybb +/y) littermates at 10-14 weeks of age. There were no differences in platelet count (921.9±33.6 x103/µl vs. 978±85.2 x103/µl), hematocrit (36.5±0.6% vs. 38.7±0.8% ) or white blood cell count (5.5±0.3 x103/µl vs. 6.1±0.6 x103/µl) between Cybb+/y and Cybb-/y mice. We next, examined platelet activation in response to thrombin (0.05 and 0.2 U/ml) or collagen (80 and 320 ng/ml) using flow cytometry. We observed similar levels of integrin α2bβ3 activation, fibrinogen binding, and intra-platelet levels of H2O2 in platelets from Cybb+/y and Cybb-/y mice after activation with either agonist, which suggests no alteration in platelet inside-out signaling due to loss of Nox2. No significant difference in susceptibility to carotid artery thrombosis in a photochemical injury model was observed between Cybb+/y and Cybb-/- mice (time to stable occlusion 21.97±4.7 vs 27.6±4.2, P>0.4). In contrast, Cybb-/y mice demonstrated significant decreases in the weight and length of venous thrombi in the inferior vena cava after 48 hours of stenosis (P<0.05). Our findings suggest that Nox2 is not a major mediator of platelet activation or arterial thrombosis but contributes to the development of venous thrombi. Future studies are warranted to examine the role of NETs and the prothrombotic effects of Nox2 in association with other cardiovascular risk factors. DisclosuresLentz:Novo Nordisk: Consultancy, Research Funding; Celgene: Equity Ownership; Opko: Membership on an entity's Board of Directors or advisory committees.

P206 Cutaneous manifestations among a female infant carrier of X-linked CGD

P206 Cutaneous manifestations among a female infant carrier of X-linked CGD

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy.

Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

A novel missense mutation in the NADPH binding domain of CYBB abolishes the NADPH oxidase activity in a male patient with increased susceptibility to infections

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X-linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-γ). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91+CGD patient by rhIFN-γ.

Successful immune reconstitution by means of hematopoietic stem cell transplantation in a Colombian patient with chronic granulomatous disease.

Chronic granulomatous disease is a primary immunodeficiency that results from mutations in proteins of the NADPH oxidase system that affect the microbicidal activity of phagocytes. Immune reconstitution by hematopoietic stem cell transplantation is currently the only curative therapy for this disease. To describe the clinical and molecular characterization of a patient with X-linked chronic granulomatous disease and the successful immune reconstitution by means of a hematopoietic stem cell transplantation. The respiratory burst was measured by flow cytometry using the dihydrorodamine 123 (DHR) oxidation test in neutrophils of peripheral blood. Mutational analysis of CYBB was performed by PCR amplification in complementary DNA, as well as sequencing and comparative genomic hybridization in genomic DNA. HLA-identical stem cells from the patient's younger brother were used for the transplantation and reduced intensity pre-transplantation conditioning was administered. Post-transplantation immune reconstitution was evaluated periodically by serial complete blood counts and DHR 123 in peripheral blood neutrophils. The diagnosis of X-linked chronic granulomatous disease resulted from a hemizygous deletion affecting Xp21.1 that included the entire CYBB. Post-transplantation engraftment was documented in platelets and peripheral blood neutrophils at days 10 and 11, respectively. Total hematological reconstitution was achieved by day 30 post-transplantation and no complications or infections have been observed in the three years since the transplantation. Hemopoietic stem cell transplantation allows for total reconstitution of the immune function related to microbicidal activity of phagocytic cells from patients with X-linked chronic granulomatous disease.

557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30-kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPR-mediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB2-13 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.

224. A Gene Therapy Clinical Study of a Patient with X-Linked Chronic Granulomatous Disease

BACKGROUND: X-linked Chronic granulomatous disease (X-CGD) is a primary immunodeficiency disease characterized by failure of the nicotinamide adenine dinucleotide phosphate enzyme system in phagocytes to produce reactive oxygen species (ROS). This disorder derives from defects in the CYBB gene encoding gp91phox and leads to recurrent infection and hyper-inflammation, occasionally represented by CGD-associated colitis (CGD colitis). Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for X-CGD, and in Europe and America, gene therapy clinical trials have been performed to patients who have no HLA-matched donor. To assess the safety and efficacy of hematopoietic stem cell gene therapy, we commenced a retroviral gene therapy clinical study using a busulfan-based preconditioning treatment regimen for CGD patients. METHODS AND PATIENT: The patient was a 27-year- old man with X-CGD. His condition was complicated by refractory subcutaneous abscess, fungal lung disease, lymphadenitis and CGD colitis, and he did not have an appropriate HLA-matched donor for HSCT. Granulocyte colony-stimulating factor -mobilized CD34+ stem cells were transduced with an MFGS-based retrovirus vector encoding the therapeutic gene of CYBB ex vivo, and infused back to the patient after busulfan-based preconditioning. Clinical and biochemical assessments were performed to evaluate the safety and efficacy of retrovirus gene therapy. RESULTS: After engraftment of 6.5×106/kg of CD34+ cells including 76% of gp91phox expressing cells, the gene-modified phagocytes appeared in peripheral blood after day +14. Dihydrorhodamine (DHR) flow cytometry assay demonstrated that the 2.3% of neutrophils in peripheral blood produced ROS, and a vector insert copy number was 2.63 per DHR+ neutrophil at day +95. His clinical symptoms of subcutaneous abscess and CGD colitis improved at almost the same time. The retrovirus insertion site of gene-modified peripheral blood cells revealed that there was no evidence of abnormal growth of clones with MDS-EVI1, SETBP1 or PRDM16 integrants at day +63. At day +182, gene marking of peripheral blood decreased to about 0.1% of neutrophils. PCR quantification of vector gene marking showed a vector insert copy number of 0.17 and 0.04 per neutrophil at day +95 and day +182, respectively. A vector insertion copy of bone marrow cells was 0.08 per marrow cell at day +183, suggesting that a reduction of gene-modified CD34+ stem cells would cause a decrease of gp91phox expressing phagocytes in peripheral blood. CONCLUSION: The therapeutic gene itself does not confer a growth advantage on the transduced cells in CGD, which would impact on the long-term gene marking and the withdrawal of clonal expansion. As other studies have reported that residual ROS production could contribute to the survival of CGD patients, the presence of gene-modified ROS-producing phagocytes led to the improvement of severe infection in our patient.