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Chronic granulomatous disease (CGD) is an inherited blood disorder of phagocytic cells that renders patients susceptible to infections and inflammation. A recent clinical trial of lentiviral gene therapy for the most frequent form of CGD, X-linked, has demonstrated stable correction over time, with no adverse events related to the gene therapy procedure. We have recently developed a parallel lentiviral vector for p47phox-deficient CGD (p47phoxCGD), the second most common form of this disease. Using this vector, we have observed biochemical correction of CGD in a mouse model of the disease. In preparation for clinical trial approval, we have performed standardized preclinical studies following Good Laboratory Practice (GLP) principles, to assess the safety of the gene therapy procedure. We report no evidence of adverse events, including mutagenesis and tumorigenesis, in human hematopoietic stem cells transduced with the lentiviral vector. Biodistribution studies of transduced human CD34+ cells indicate that the homing properties or engraftment ability of the stem cells is not negatively affected. CD34+ cells derived from a p47phoxCGD patient were subjected to an optimized transduction protocol and transplanted into immunocompromised mice. After the procedure, patient-derived neutrophils resumed their function, suggesting that gene correction was successful. These studies pave the way to a first-in-man clinical trial of lentiviral gene therapy for the treatment of p47phoxCGD.

Plasma gelsolin (pGSN) is the secreted isoform of an intracellular actin remodeling protein found in high concentrations in human plasma. Clinical studies demonstrate reduced pGSN concentrations in several disease states, including severe trauma, burns, and sepsis. Markedly decreased pGSN concentrations in these conditions precede and predict adverse clinical outcomes. In this study, we measured pGSN in patients with chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and dysregulated inflammation. pGSN was quantified using a sandwich ELISA in plasma from healthy volunteers, clinically stable CGD patients, and X-linked CGD carriers and in sera from 12 CGD patients undergoing bone marrow transplantation. pGSN was also quantified in healthy volunteers challenged with intravenous endotoxin. pGSN concentrations were lower in CGD patients without active infection or systemic inflammation compared with healthy control subjects. In CGD patients undergoing bone marrow transplantation, pGSN concentrations increased significantly following successful transplant. X-linked carriers of CGD had normal pGSN. Despite reduction of pGSN in CGD patients, we did not detect significant changes in pGSN over 24 h following challenge of healthy volunteers with intravenous endotoxin (4 ng/kg) that elicited a febrile response. We describe, for the first time, significantly lower pGSN in clinically stable patients with CGD compared with age- and sex-matched healthy volunteers. Low pGSN levels in CGD patients significantly increased following bone marrow transplantation. X-linked carriers of CGD had normal pGSN. In healthy volunteers challenged with intravenous endotoxin, pGSN is not an acute phase reactant.

PURPOSE: Haploidentical related donor (HRD) transplantation was performed in 7 recipients with chronic granulomatous disease (CGD) who had no matched-related or unrelated donor. METHODS: Peripheral blood cell (PBC) products were used with a conditioning regimen consisting of low-dose cyclophosphamide, fludarabine, total body irradiation, and busulfan. Graft-versus-host disease (GVHD) prophylaxis consisted of high-dose post-transplant cyclophosphamide and sirolimus. Recipients were ages 14–26 years, and 3 had severe infections active at transplant. RESULTS: All 7 recipients achieved full engraftment with complete donor chimerism early in the post-transplant period. Acute GVHD occurred in all cases and was grade 3 or steroid refractory in 3. Two patients with steroid-refractory GVHD died. Three patients with severe infectious complications active at transplant, 1 Nocardia pneumonia and 2 extensive invasive fungal infections), survived and were cured of their infection at last follow-up. Bacterial disease occurred post-transplant in all recipients, and viral infections/reactivation were common, including 4 cases of BK virus–associated hemorrhagic cystitis. CONCLUSIONS: Seven patients with CGD achieved rapid and full-donor engraftment from HRDs utilizing PBCs and a conditioning regimen with PTCy and sirolimus GVHD prophylaxis. However, the incidence of grade 3 and steroid-refractory GVHD was high and led to 2 deaths. Patients with active infections at transplant had successful transplant courses and were cured of their disease. Although there was an initial success with this regimen, the cumulative experience does not support its use in CGD due to an unacceptable rate of severe GVHD.

We analyzed events and therapies related to febrile neutropenia in patients receiving hematopoietic cell transplantation (HCT) for chronic granulomatous disease (CGD).Three protocols for HCT were used to extract the relation between conditioning and infectious complications during transplantation for CGD, especially the relation of fever and neutropenia to microbiological events and antibiotic therapy.Sixty-nine recipients received either reduced intensity conditioning with matched related or unrelated donors or conditioning specific to haploidentical-related donors utilizing posttransplant cyclophosphamide. Fever prior to neutropenia was common (52) and in eight recipients, Gram negative bacterial infection occurred prior to neutropenia, and in nine during neutropenia. Alemtuzumab as conditioning was associated with preneutropenic infection. Empiric therapy (noncarbapenem) by institutional guideline was given in 40. Carbapenems were given before neutropenia (8) or as empiric therapy in neutropenia (18), or a switch to a carbapenem (n = 22) occurred in 48 cases. No deaths related to infection associated with neutropenia occurred.The management of febrile neutropenia in HCT for CGD led to no deaths related to infection associated with neutropenia. Bacteremias occurred both prior to neutropenia and during neutropenia. Bacteria isolated may have represented the recrudescence of prior infection, representing the population transplanted and the platform for HCT. The treatment of prior infections may have had an influence on the necessity of carbapenem use as either empiric or directed therapy for bacterial infections.

Granulocyte transfusions are sometimes used as adjunctive therapy for the treatment of infection in patients with chronic granulomatous disease (CGD). However, granulocyte transfusions can be associated with a high rate of alloimmunization, and their role in CGD patients undergoing hematopoietic cell transplantation (HCT) or gene therapy (GT) is unknown. We identified 27 patients with CGD who received granulocyte transfusions pre- (within 6months) and/or post-HCT or GT in a retrospective survey. Twelve patients received granulocyte transfusions as a bridge to cellular therapy. Six (50%) of these patients had a complete or partial response. However, six of 10 (60%) patients for whom testing was performed developed anti-HLA antibodies, and three of the patients also had severe immune-mediated cytopenia within the first 100days post-HCT or GT. Fifteen patients received granulocyte transfusions post-HCT only. HLA antibodies were not checked for any of these 15 patients, but there were no cases of early immune-mediated cytopenia. Out of 25 patients who underwent HCT, there were 5 (20%) cases of primary graft failure. Three of the patients with primary graft failure had received granulocyte transfusions pre-HCT and were subsequently found to have anti-HLA antibodies. In this small cohort of patients with CGD, granulocyte transfusions pre-HCT or GT were associated with high rates of alloimmunization, primary graft failure, and early severe immune-mediated cytopenia post-HCT or GT. Granulocyte transfusions post-HCT do not appear to confer an increased risk of graft failure.

INTRODUCTION: Patients with primary immunodeficiencies (PIDs) are potentially cured by allogeneic hematopoietic cell transplantation (HCT). The spectrum of PIDs has expanded greatly beyond those that present in infancy or are diagnosed on newborn screening and require urgent, preemptive HCT. Many PID diagnoses are now made later in life and the role of HCT is only considered upon severe disease manifestations; in these cases, the kinetics and goals of a donor search may be different than for severe combined immunodeficiency. Across all PIDs, related donor searches have the additional selection factor of the inherited disease, such searches may yield more limited options than searches for patients with hematologic malignancies; thus, unrelated donor options often become more critical in these patients. We retrospectively evaluated the outcomes of donor searches among PID patients referred for HCT at the National Institutes of Health (NIH), where the minimum patient age for evaluation is 3 years old and where donor options included matched sibling or matched related (MSD/MRD), HLA-haploidentical (haplo), or 7–8/8 HLA-matched unrelated (mMUD/MUD) donors. METHODS: Patient (n=161) and donor demographics, MUD search results, HLA typing, pedigrees, mutation testing, and donor selection data were collected. The National Marrow Donor Program HapLogic 8/8 HLA-match algorithm was used to predict the likelihood of a successful MUD search and categorized as very good, good, fair, poor, very poor, or futile per the Memorial Sloan Kettering Cancer Center (MSKCC) Search Prognosis method. RESULTS: There were significant differences by PID mode of inheritance in patient age, disposition (receipt of HCT or not), donor source, and donor relatedness. A related or unrelated donor option could be identified for 94% of patients. Of living 1(st) degree relatives (median 3 (range 0–12) per patient), a median of 1 donor remained for autosomal dominant and X-linked (XL) diseases after HLA typing, mutation testing, and other exclusions, and a median of 2 donors remained for autosomal recessive (AR) diseases. Among patients with a PID of known mode of inheritance (n=142), the best related donor was haplo for 99 (70%) patients, with 56 (39%) haplos age 40 years or older and 5 (4%) 2(nd) degree haplos; 13 (9%) had no family donor options. The best related donor was a heterozygote/asymptomatic carrier of the PID mutation in 36 (49%) patients with AR or XL disease (n=73). Among patients with MUD search performed (n=139), 53 (38%) had very poor/futile 8/8 MUD searches, including 6 (32%) of those with unknown PID mutation and therefore no family donor options. The MSKCC Search Prognosis was less favorable for those of non-European ancestry compared to European ancestry, p=0.002. The majority of patients of Hispanic or African ancestry had very poor/futile MUD searches, 71% and 63%, respectively. No HCT recipients with very poor/futile MUD searches (n=38) received 8/8 MUD grafts. DISCUSSION: Alternative donor options, including haplo and unrelated donors, are critical to enable HCT for patients with PID. MUD search success remains low for those of non-European ancestry, and this is of particular concern for patients with PID due to an unknown genetic defect. Among PID patients, related donor options are reduced and haplos age 40 years+ and/or mutation carriers are often the best family option.

Acute graft-versus-host disease (aGVHD) is an immune-mediated reaction that can occur after hematopoietic stem cell transplantation in which donor T cells recognize the host antigens as foreign, destroying host tissues. Establishment of a tolerogenic immune environment while preserving the immune response to infectious agents is required for successful bone marrow transplantation. Pregnancy-specific glycoprotein 1 (PSG1), which is secreted by the human placenta into the maternal circulation throughout pregnancy, likely plays a role in maintaining immunotolerance to prevent rejection of the fetus by the maternal immune system. We have previously shown that PSG1 activates the latent form of transforming growth factor β1 (TGF-β), a cytokine essential for the differentiation of tolerance-inducing CD4+FoxP3+ regulatory T cells (Tregs). Consistent with this observation, treatment of naive murine T cells with PSG1 resulted in a significant increase in FoxP3+ cells that was blocked by a TGF-β receptor I inhibitor. We also show here that PSG1 can increase the availability of active TGF-β in vivo. As the role of CD4+FoxP3+ cells in the prevention of aGVHD is well established, we tested whether PSG1 has beneficial effects in a murine aGHVD transplantation model. PSG1-treated mice had reduced numbers of tissue-infiltrating inflammatory CD3+ T cells and had increased expression of FoxP3 in T cells compared with vehicle-treated mice. In addition, administration of PSG1 significantly inhibited aGVHD-associated weight loss and mortality. On the other hand, administration of PSG1 was less effective in managing aGVHD in the presence of an alloimmune reaction against a malignancy in a graft-versus-leukemia experimental model. Combined, this data strongly suggests that PSG1 could be a promising treatment option for patients with aGVHD following bone marrow transplantation for a nonmalignant condition, such as an autoimmune disorder or a genetic immunodeficiency.

X-linked Chronic Granulomatous Disease (CGD) is a rare inherited immunodeficiency characterized by early life-threatening infections from bacteria and fungi in male children. Female carriers of X-linked CGD usually do not develop any manifestations of the disease, yet in rare cases they may present with CGD-related manifestations due to skewed X chromosome inactivation, even in advanced age. Here, we report the case of a 49-year-old woman with no history of previous frequent or severe infections, who presented acutely with life-threatening bilateral pneumonia caused by Nocardia asteroides and was eventually diagnosed with late-onset X-linked CGD due to skewed X chromosome inactivation in white blood cells. Treatment with interferon-γ as a rescue therapy resulted in normalization of the intensity of the oxidative burst in the residual positive cells and resolution of the infection, which was otherwise resistant to conventional treatments. After discharge, however, recurrent severe pulmonary infections despite prophylactic treatments as well as appearance of granulomatous colitis led to considering definitive treatment. Hematopoietic stem cell transplantation from unaffected HLA-identical brother using a non-myeloablative conditioning protocol with intravenous busulfan followed by high-dose peripheral blood stem cell graft and post-transplant cyclophosphamide was successfully performed. After three years of follow-up, white blood cell chimerism remained stable with about 60% donor cells in the myeloid lineage, with no further infections and no recurrence of inflammatory bowel disease.

PURPOSE: Up to date review on various types of immunodeficiencies with a significant myeloid component including some more recently described congenital disorders. SUMMARY: Neutrophils (or granulocytes) are a major contributor to infection surveillance and clearance and defective neutrophils characteristically lead to pyogenic infections. Deficiency in numbers, either iatrogenic or congenital, functional defects, and/or inability to target to the sites of infection can all lead to serious morbidity and mortality; however, myeloid based immunodeficiencies are not all the same. Having absent neutrophils, that is neutropenia, has implications different to those of having dysfunctional neutrophils as will become evident as the various disorders are reviewed. RECENT FINDINGS: While a number of disorders have been described in the past, genetic sequencing has led to the identification of the specific disorders and clarified their pathophysiology. Advances in genetic therapies including genetic editing should provide future treatments beyond hematopoieitic stem cell transplant for patients with these rare disorders

Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4–1.8 copies per neutrophil) and the persistence of 16–46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients. Initial results from phase I/II lentiviral gene therapy trials provide early evidence supporting its safety and efficacy in treating patients with X-linked chronic granulomatous disease.

Background Patients undergoing allogeneic hematopoietic stem cell transplant require variable, often extensive transfusion support. Identification of factors that predict urgent, intensive, or special needs should improve management of these patients. Study design and methods This is a retrospective study of red blood cell (RBC) and platelet transfusion support provided for sequential matched sibling donor allogeneic transplants conducted at the Clinical Center, National Institutes of Health, from 1993 through 2010. Factors potentially important for predicting quantity of RBC and platelet transfusions, and time to transfusion independence through Day 200 following hematopoietic stem cell transplantation were evaluated. Results Subjects (n = 800) received 10,591 RBC and 10,199 platelet transfusions. Multivariable analysis demonstrated that the need for RBC pretransplant, CD34+ dose, transplant year, diagnostic category, and ABO match were significantly independently associated with quantity of RBC transfusions during Days 0 through 30. Only pretransplant need for RBCs, CD34+ dose, and transplant year had significance during Days 0 through 100. Similar analyses for quantity of platelet transfusions demonstrated that for both Days 0 through 30 and 0 through 100 significant factors were need for platelet support before transplant, CD34+ dose, transplant year, and transplant regimen. Of note, long term, during Days 101 through 200, only CD34+ dose remained significant for quantity of RBC and platelet transfusions. Analysis of time to transfusion independence demonstrated that patients with ABO major mismatches required longer to achieve freedom from RBC transfusion support compared to identical matches or those with minor mismatches. Conclusion Patient-specific factors including CD34+ dose and ABO match of the graft should be given particular consideration by transfusion services when planning support of patients receiving allogeneic hematopoietic stem cell transplant.

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4(+) and CD4(+)CD45RA(+) cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4(+) cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

The purpose of this study was to evaluate engraftment and adverse events with a conditioning and prophylactic regimen intended to achieve high rates of engraftment with minimal graft-versus-host disease (GVHD) in allogeneic transplantation for chronic granulomatous disease in a single center. Forty patients, 37 male, with chronic granulomatous disease were transplanted. Transplant products were matched sibling peripheral blood stem cells (PBSCs) in four and matched unrelated donor (MUD) bone marrow in three, and one patient received mismatched unrelated PBSCs. Thirty-two patients received MUD PBSCs. All patients received a conditioning regimen of busulfan/alemtuzumab (with low-dose total body irradiation for MUD recipients) with sirolimus graft-versus-host disease prophylaxis. Engraftment occured in 38/40 recipients (95%). Acute or chronic GVHD occurred in 18 (45%) and 5 (12.5%), respectively, with 6 episodes of grades III–IV and/or steroid refractory GVHD. Overall survival was 33/40 (82.5%) and event-free survival was 30/40 (80%). Successful engraftment was associated with myeloid and NK cell, but not CD3+ chimerism. Myeloid engraftment was greater than 70% in 30/32 recipients at mean follow-up of 3.4 years. Evidence of persistent immunodeficiency was not seen in successful transplants. Attempts to rescue failed or poorly functioning grafts were associated with unacceptable morbidity and mortality. A reduced-intensity allogeneic transplant protocol based on alemtuzumab and busulfan with sirolimus GVHD prophylaxis produced high rates of successful engraftment and minimal regimen-related toxicity. Prolonged clinical follow-up has confirmed its efficacy in ameliorating CGD-related disease. Outcomes were not acceptable with donor cell infusion rescue of cause with poor graft function.

Esther H Zhou,1 Sally Seymour,2 Margie R Goulding,1 Elizabeth M Kang,1 Jacqueline M Major,1 Solomon Iyasu1 1Division of Epidemiology, Office of Surveillance and Epidemiology, 2Office of New Drugs, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA Background: Emerging safety issues associated with long-acting beta2-agonist (LABA) have led to multiple regulatory activities by the US Food and Drug Administration (FDA) since 2003, including Drug Safety Communications (DSCs) in 2010. These DSCs had three specific recommendations for the safe use of LABA products in adult asthma treatment. Methods: We examined the initiation of LABA-containing products for adult asthma treatment using an intermittent time series approach in a claims database from 2003 to 2012. We assessed the alignment of dispensing patterns with the following 2010 FDA recommendations: 1) contraindicated use of single-ingredient (SI)-LABA without an asthma controller medication (ACM); 2) a LABA should only be used when asthma is not adequately controlled on inhaled corticosteroids (ICSs) or ACM; and 3) step-down asthma therapy (e.g., discontinue LABA) when asthma control is achieved. Results: There were 477,922 adults (18–64years old) dispensed a new LABA during 2003–2012. Among LABA initiators, patients who initiated an SI-LABA and who did “not” have an ACM dispensed on the same date decreased from >9% in 2003 (the initial labeling change) to

IntroductionInflammatory bowel disease (IBD) affects approximately 1/3 of patients with chronic granulomatous disease (CGD). Comprehensive investigation of the effect of allogeneic hematopoietic cell transplantation (HCT) on CGD IBD and the impact of IBD on transplant outcomes is lacking.MethodsWe collected data retrospectively from 145 patients with CGD who had received allogeneic HCT at 26 Primary Immune Deficiency Treatment Consortium (PIDTC) centers between January 1, 2005 and June 30, 2016.ResultsForty-nine CGD patients with IBD and 96 patients without IBD underwent allogeneic HCT. Eighty-nine percent of patients with IBD and 93% of patients without IBD engrafted (p = 0.476). Upper gastrointestinal acute GVHD occurred in 8.5% of patients with IBD and 3.5% of patients without IBD (p = 0.246). Lower gastrointestinal acute GVHD occurred in 10.6% of patients with IBD and 11.8% of patients without IBD (p = 0.845). The cumulative incidence of acute GVHD grades II-IV was 30% (CI 17-43%) in patients with IBD and 20% (CI 12-29%) in patients without IBD (p = 0.09). Five-year overall survival was equivalent for patients with and without IBD: 80% [CI 66-89%] and 83% [CI 72-90%], respectively (p = 0.689). All 33 surviving evaluable patients with a history of IBD experienced resolution of IBD by 2years following allogeneic HCT.ConclusionsIn this cohort, allogeneic HCT was curative for CGD-associated IBD. IBD should not contraindicate HCT, as it does not lead to an increased risk of mortality. This study is registered at clinicaltrials.gov NCT02082353.

Pseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in NCF1, a gene with 2 pseudogenes, NCF1B and NCF1C. The most common NCF1 mutation, a GT deletion (ΔGT) at the start of exon 2 (>90% of alleles), is constitutive to NCF1B and NCF1C. NCF1 ΔGT results in premature termination, undetectable protein expression, and defective production of antimicrobial superoxide in neutrophils. We examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases targeting the exon 2 ΔGT in induced pluripotent stem cells or CD34+ hematopoietic stem cells derived from p47-CGD patients. Correction of ΔGT in NCF1 pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.

The use of HLA-identical hematopoietic stem cell transplantation (HSCT) demonstrates overall survival rates greater than 75% for T-B-NK+ severe combined immunodeficiency secondary to pathogenic mutation of recombinase activating genes 1 and 2 (RAG1/2). Limited data exist regarding the use of HSCT in patients with hypomorphic RAG variants marked by greater preservation of RAG activity and associated phenotypes such as granulomatous disease in combination with autoimmunity. We describe a 17-year-old with combined immunodeficiency and immune dysregulation characterized by granulomatous lung disease and autoimmunity secondary to compound heterozygous RAG mutations. A myeloablative reduced toxicity HSCT was completed using an unrelated bone marrow donor. With the increasing cases of immune dysregulation being discovered with hypomorphic RAG variants, the use of HSCT may advance to the forefront of treatment. This case serves to discuss indications of HSCT, approaches to preparative therapy, and the potential complications in this growing cohort of patients with immune dysregulation and RAG deficiency.

Human cytomegalovirus (CMV) infection and disease remains a significant cause of morbidity and mortality for hematopoietic cell transplantation (HCT) recipients. Disruption of or weak reconstitution of virus-specific cellular immune function, such as with certain HCT approaches, poses significant risk for CMV-related complications. The incidence of and risk factors for CMV infection and the nature of CMV disease were evaluated retrospectively among 356 consecutive HCT recipients transplanted at the National Institutes of Health using all graft sources, including bone marrow, peripheral blood stem cell (PBSC), and umbilical cord blood (UCB), and a range of in vivo and ex vivo approaches for graft-versus-host disease (GVHD) prophylaxis. The cumulative incidence of CMV infection was higher for CMV-seropositive recipients at 33%, regardless of donor CMV serostatus. Patients transplanted with CMV-seropositive donors had a significantly shorter duration of antiviral therapy. Among graft sources UCB was associated with the highest cumulative incidence of CMV infection at 65% and significantly longer treatment duration at a median of 36days, whereas PBSC HCT was associated with the lowest incidence at 26% and the shortest CMV treatment duration at a median of 21days. There were significant differences in the cumulative incidence of CMV infection by T cell manipulation strategy when systemic steroids were included as a risk-modifying event. Over one-third of CMV infections occurred in the setting of systemic steroid administration. CMV disease occurred in 5% of HCT recipients, with 70% of cases in the setting of treatment for GVHD. Although factors related to serostatus, graft source, and GVHD prophylaxis were associated with varied CMV infection incidence, unplanned post-HCT corticosteroid therapy contributed greatly to the incidence of both CMV infection and disease across HCT approaches, highlighting this post-HCT intervention as a key time to potentially tailor the approach to monitoring, preemptive therapy, and even prophylaxis.

Latent EBV infection can lead to EBV-PTLD when proliferating, EBV+ B cells are insufficiently controlled by cellular immunity. At the National Institutes of Health (NIH), all HCT recipients are monitored weekly from day 0-100 with whole blood EBV qPCR. We evaluated the cumulative incidence (CI) of EBV-related events across 4 T-cell manipulation strategies (posttransplantation cyclophosphamide (PTCy), proximal serotherapy, ex vivo T cell depletion (TCD), and calcineurin/mTOR inhibitor (CNI/mTORi)-based), across all graft sources (marrow (BM), peripheral blood (PBSC), and umbilical cord blood (UCB)), and across regimens with and without mTORi drugs. The CI of EBV events (detection of any EBV in blood (Any-EBV), detection of EBV > 3 log10 IU/mL (>3-EBV), pre-emptive therapy for EBV (EBV-Tx), and EBV-PTLD) were determined, with death as a competing risk. EBV-tx included rituximab and/or EBV-specific T-cells. Any-EBV and >3-EBV events were captured through day +100 and EBV-Tx and EBV-PTLD events were captured through 1-year post-HCT. Included are 356 consecutive patients receiving 1st HCT at NIH from 2011-2017 with sufficient weekly day 0-100 EBV qPCRs (> 63% of specimens) and 1 year of follow-up of survivors. Fig 1 shows cohort characteristics. Among T-cell manipulation strategies, the CI of Any-EBV and >3-EBV was lowest for serotherapy, p=0.0007 and p=0.0006, with no difference among strategies in the CI of EBV-Tx or EBV-PTLD. By graft source, UCB had the highest CI of Any-EBV at day +100, p=0.001, with no difference in the CI of >3-EBV owing to higher CI of EBV-Tx among UCB recipients compared to BM and PBSC grafts, p 3-EBV, but no difference in the CI of EBV-Tx or EBV-PTLD. Within CNI/mTORi-based approaches, the CI of Any-EBV and >3-EBV was lower for CNI+mTORi vs CNI without mTORi, p=0.004 and p=0.002, and the CI of EBV-Tx was 3% for CNI+mTORi vs 14% for CNI without mTORi, p=0.009, with no difference in the CI of EBV-PTLD. Among PTCy-containing approaches, with or without serotherapy, there were no differences in the CI of Any-EBV, >3-EBV, EBV-Tx, or EBV-PTLD. The CI of EBV events are summarized in Fig 2. While EBV detection in blood was common post-HCT, the clinical significance of detection and the role of pre-emptive therapy seem to vary by T-cell manipulation strategy, graft source, and use of mTORi. The finding of less EBV detection with mTORi-containing regimens across approaches warrants further investigation into the clinical significance and underlying mechanisms.

Intravenous (IV) busulfan test dose pharmacokinetics (PK) has been shown to accurately predict once-daily dose requirements and improve outcomes in adult transplant patients, but there are limited data to support this approach in children. Test doses of busulfan ∼0.8 mg/kg were infused over 2 to 3 hours, followed by serial sampling to 4-6 hours postinfusion in pediatric hematopoietic stem cell transplant recipients (n = 5). Once-daily busulfan doses were calculated based on a myelosuppressive area under the concentration-time curve (AUC) target of ∼3700 to 4000 μmol·min/L and assumed dose-proportionality to the test dose. PK analysis was then repeated at full daily doses within 6-8 days of test dose administration. Plasma PK samples collected under test and full-dose conditions were analyzed using validated commercial assays and noncompartmental methods. In 4 out of 5 patients, PK estimates after once-daily IV busulfan administration differed in comparison to test dose estimates (AUC range -38.2% to +49.7%, clearance range -34.3% to +61.8%). Patients 1, 2, and 3 required increases in remaining daily busulfan doses to achieve AUC targets, and no adjustment was required in patient 4. Patient 5's AUC was 49.7% higher than expected, and he subsequently developed fatal sinusoidal obstruction syndrome. In our experience with pediatric patients, test dose PK failed to reliably predict daily dosing requirements with large discrepancies from predicted AUC targets. This article highlights the necessity for therapeutic drug monitoring of IV busulfan and inadvisability of relying solely on test-dose busulfan PK in pediatric patients. Furthermore, clinicians should consider strategies to expedite dose adjustments in real time.

In a retrospective cohort study of patients enrolled in the UK Clinical Practice Research Datalink during 2000-2013, we evaluated long-term risks of death, stroke, and acute myocardial infarction (AMI) in adults prescribed clarithromycin. Patients were outpatients aged 40-85 years, who were prescribed clarithromycin (n = 287,748), doxycycline (n = 267,729), or erythromycin (n = 442,999), or Helicobacter pylori eradication therapy with a proton pump inhibitor, amoxicillin, and either clarithromycin (n = 27,639) or metronidazole (n = 14,863). We analyzed time to death, stroke, or AMI with Cox proportional hazards regression. The long-term hazard ratio for death following 1 clarithromycin versus 1 doxycycline prescription was 1.29 (95% confidence interval (CI): 1.21, 1.25), increasing to 1.62 (95% CI: 1.43, 1.84) for ≥5 prescriptions of clarithromycin versus ≥5 prescriptions for doxycycline. Erythromycin showed smaller risks in comparison with doxycycline. Stroke and AMI incidences were also increased after clarithromycin but with smaller hazard ratios than for mortality. For H. pylori eradication, the hazard ratio for mortality following clarithromycin versus metronidazole regimens was 1.09 (95% CI: 1.00, 1.18) overall, and it was higher (hazard ratio = 1.65, 95% CI: 0.88, 3.08) following ≥2 prescriptions in subjects not on statins at baseline. Outpatient clarithromycin use was associated with long-term mortality increases, with evidence for a similar, smaller increase with erythromycin.

Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.

Purpose The aim of this study is to determine (i) the positive predictive value (PPV) of an algorithm using clinical codes to identify incident glaucoma and cataract events in the Clinical Practice Research Datalink (CPRD) and (ii) the ability to capture the correct timing of these clinical events. Methods A total of 21 339 and 5349 potential cataract and glaucoma cases, respectively, were identified in CPRD between 1 January 1990 and 31 December 2010. Questionnaires were sent to the general practitioners (GP) of 1169 (5.5%) cataract and 1163 (21.7%) glaucoma cases for validation. GPs were asked to verify the diagnosis and the timing of the diagnosis and to provide other supporting information. Results A total of 986 (84.3%) valid cataract questionnaires and 863 (74.2%) glaucoma questionnaires were completed. 92.1% and 92.4% of these used information beyond EMR to verify the diagnosis. Cataract and glaucoma diagnoses were confirmed in the large majority of the cases. The PPV (95% CI) of the cataract and glaucoma Read code algorithm were 92.0% (90.3–93.7%) and 84.1% (81.7–86.6%), respectively. However, timing of diagnosis was incorrect for a substantial proportion of the cases (20.3% and 32.8% of the cataract and glaucoma cases, respectively) among whom 30.4% and 49.2% had discrepancies in diagnosis timing greater than 1 year. Conclusions High PPV suggests that the algorithms based on the clinical Read codes are sufficient to identify the cataract and glaucoma cases in CPRD. However, these codes alone may not be able to accurately identify the timing of the diagnosis of these eye disorders. Copyright © 2014 John Wiley & Sons, Ltd.

Background Granulocyte–colony-stimulating factor (G-CSF)-mobilized autologous hematopoietic progenitor cells (HPCs) may be collected by apheresis of patients with chronic granulomatous disease (CGD) and severe combined immunodeficiency (SCID) for use in gene therapy trials. CD34+ cell mobilization has not been well characterized in such patients. Study Design and Methods We retrospectively evaluated CD34+ cell mobilization and collection in 73 consecutive CGD and SCID patients and in 99 age-, weight-, and G-CSF dose–matched healthy allogeneic controls. Results In subjects aged not more than 20 years, Day 5 preapheresis circulating CD34+ counts were significantly lower in CGD and SCID patients than in controls; mean peak CD34+ cell counts were 58 × 106, 64 × 106, and 87 × 106/L, respectively (p = 0.01). The SCIDs had lower CD34+ collection efficiency than CGDs and controls; mean efficiencies were 40, 63, and 57%, respectively (p = 0.003). In subjects aged more than 20 years, the CGDs had significantly lower CD34+ cell mobilization than controls; mean peak CD34+ cell counts were 41 × 106 and 113 × 106/L, respectively (p

To evaluate changes in the dispensing patterns of long-acting beta2-adrenergic agonist (LABA) in pediatric and adolescent asthma patients in relation to multiple Food and Drug Administration (FDA) regulatory activities from 2003 to 2011.We estimated LABA dispensing to pediatric asthma patients across three periods: 2003-2004 (after the first labeling change), 2005-2009 (after regulatory activities in 2005 and before 2010 LABA labeling change) and 2010-2011 (after 2010 LABA labeling change), using the IMS Health Plan Claims database. We estimated dispensing patterns over time for single-ingredient (SI) LABA and fixed-dose combination (FDC) of inhaled corticosteroid (ICS) and LABA (FDC-ICS/LABA). We also evaluated prior use of non-LABA asthma-control medication (ACM) before LABA initiation.Of the 147 862 pediatric and adolescent asthma patients who initiated a LABA during the entire study period, the majority (96%) were FDC-ICS/LABA initiators. The proportion of SI-LABA among any LABA initiators was small and declined (9%, 4% and 2%, trend test p 0.001) for the three periods. Among the patients who initiated, the proportions with prior use of an ACM (1-90 days prior) were 35%, 36% and 39% for the three periods.The significant decline in the proportion of SI-LABA initiation over these years is consistent with FDA's recommendations. However, the favorable trend cannot be solely attributed to FDA activities as changes to clinical practice guidelines, and media publicity may have played a role. Investigating the reasons for the low ACM use before LABA initiation may inform approaches to further improve appropriate use of LABA in young asthma patients.

The mortality rate of alveolar hemorrhage (AH) after allogeneic hematopoietic stem cell transplantation is greater than 60% with supportive care and high-dose steroid therapy. We performed a retrospective cohort analysis to assess the benefits and risks of recombinant human factor VIIa (rFVIIa) as a therapeutic adjunct for AH. Between 2005 and 2012, 57 episodes of AH occurred in 37 patients. Fourteen episodes (in 14 patients) were treated with steroids alone, and 43 episodes (in 23 patients) were treated with steroids and rFVIIa. The median steroid dose was 1.9 mg/kg/d (interquartile range [IQR], 0.8 to 3.5 mg/kg/d; methylprednisolone equivalents) and did not differ statistically between the 2 groups. The median rFVIIa dose was 41 μg/kg (IQR, 39 to 62 μg/kg), and a median of 3 doses (IQR, 2 to 17) was administered per episode. Concurrent infection was diagnosed in 65% of the episodes. Patients had moderately severe hypoxia (median PaO2/FiO2, 193 [IQR, 141 to 262]); 72% required mechanical ventilation, and 42% survived to extubation. The addition of rFVIIa did not alter time to resolution of AH (P = .50), duration of mechanical ventilation (P = .89), duration of oxygen supplementation (P = .55), or hospital mortality (P = .27). Four possible thrombotic events (9% of 43 episodes) occurred with rFVIIa. rFVIIa in combination with corticosteroids did not confer clear clinical advantages compared with corticosteroids alone. In patients with AH following hematopoietic stem cell transplantation, clinical factors (ie, worsening infection, multiple organ failure, or recrudescence of primary disease) may be more important than the benefit of enhanced hemostasis from rFVIIa.

Lentivector mediated gene transfer into hematopoietic stem/progenitor cells and T cells has resulted in long-term stable integration of transgenes and significant clinical benefits in many diseases. We previously reported (De Ravin et al Sci Transl Med. 2016) early outcome data for 5 patients with X-linked severe combined immunodeficiency (X-SCID) enrolled in a first-in-human lentivector gene therapy clinical trial (NCT03315078) as salvage therapy for older children and young adults who had received prior haplo-identical hematopoietic stem cell transplantation (HSCT) as infants without chemotherapy-based conditioning. Lymphocyte-depleted haplo-identical stem cell transplant in X-SCID infants without conditioning generally results in only a T-cell engraftment that may also decline over time which, combined with B- and NK-cell dysfunction, may result in the progression of multi-system clinical problems (bronchiectasis, infections, gastrointestinal malabsorption, failure to grow, immune dysregulation). By 2016 three additional patients were treated and the cohort of 8 patients have now been followed for 3 to 7 years (Cohort A), where we observed gradual clinical benefit in the clearance of chronic norovirus and associated improved abdominal complaints, malabsorption, and growth and IgG production. Four patients were able to cease immunoglobulin replacement therapy. Despite these encouraging results, the relatively inefficient transduction of hematopoietic stem/progenitor cells (HSPCs) required large quantities of vector, and resulted in relatively low vector copy number in myeloid cells in some patients, with delayed immune cell recovery and persistent clinical disease especially in the last patient treated, P8. To address this, we developed a refined enhanced transduction (ET) procedure consisting of a single overnight transduction after 48hours pre-stimulation in cytokines (Stem cell factor, Thrombopoietin, Flt3-ligand; 100ng/mL) and incorporated transduction enhancers LentiBoost 1:100 and dimethyl prostaglandin 2 (dmPGE2; 1mM) and recently treat 6 patients, including re-treatment of P8 (Cohort B). Here we compared the early outcomes from Cohort B patients who received autologous CD34+ HSPCs processed with the enhanced transduction (ET) procedure. These patients (aged 12 to 36 yo) had significant problems with donor T cell infiltration of liver, bone marrow and kidneys, and near absent B and NK cells. Cryopreserved G-CSF/plerixafor mobilized peripheral blood CD34+ HSPCs (5x106/kg to 28x106/kg) were transduced by the ET process at 5% vector concentration, compared to previous method without transduction enhancers of two daily transductions at 20-30% final vector concentration, representing a 10-12 fold reduction in vector used. Colony forming unit assays showed 78%-92% versus 17%-58% vector-positive clones in Cohort B compared with Cohort A, with VCN of 1.5-12 copies (Cohort B), compared with 0.06 to 0.5 copies in Cohort A. At one month following infusion of gene corrected cell product, we isolated peripheral blood CD14+ myeloid cells as indication of early marrow output from engrafted HSPCs, and observed VCN of 1 to 4 copies (P9-12 and re-treat P8), a ≥10x increase from Cohort A (0.04 to 0.23) at same early time point after infusion (Fig 1). In addition to earlier clinical improvement (abdominal complaints, appetite, growth), there was also an early appearance of B and NK cells (Fig. 2) at much higher levels in Cohort B than previously observed even at years after treatment in Cohort A. In conclusion, we observed significantly improved measures of early clinical outcomes from lentivector gene therapy of older children and young adults with X-SCID using enhanced transduction procedure with addition of LentiBoost and dmPGE2, that achieves much greater transduction efficiencies with >10 fold less vector, and results in faster immune reconstitution and more significant clinical benefit by 3 months. The patients are monitored closely for potential risks from higher VCNs that may be balanced by reduced selection pressure due to the greater numbers of gene corrected clones. Figure Disclosures Puck: NIAID: Research Funding; Pfeizer: Other: spouse serves on Rare Disease Advisory Board; Invitae: Other: spouse employment. Cowan:bluebird bio: Consultancy; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; California Institute Of Regenerative Medicine: Research Funding; NIH NIAD: Research Funding; Rocket Pharma: Consultancy; Leadiant: Consultancy. Mamcarz:ASSISI Foundation of Memphis: Research Funding; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; California Institute of Regenerative Medicine: Research Funding; NHLBI: Research Funding; UpToDate: Honoraria; American Lebanese Syrian Associated Charities: Research Funding. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; California Institute for Regenerative Medicine: Research Funding; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; EMD Serono: Honoraria; ASSISI fundation of Memphis: Research Funding; Inmatics: Membership on an entity's Board of Directors or advisory committees. Meagher:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy.

PURPOSE: The major concern associated with isotretinoin treatment is its high teratogenic potential. Therefore ensuring use of contraception while on therapy is an important strategy for at-risk patients and has been emphasized in all risk management programs. iPledge the latest and most rigorous isotretinoin program requires among other stipulations monthly assessments of contraceptive use for patients undergoing isotretinoin treatment. The purpose of this study is to evaluate isotretinoin usage patterns and assess concomitant use of isotretinoin and contraceptives before and after iPledge. METHODS: Female patients aged 13-45 years with a new prescription for isotretinoin products during 2004-2008 were identified in the IMS Health longitudinal prescription claims database. Monthly concomitant use of isotretinoin and contraceptives was estimated. Segmented regression analysis of interrupted time series data was used to assess changes in monthly proportion of concomitant use in the 24 months preceding versus following iPledge implementation. RESULTS: The number of isotretinoin prescriptions decreased after iPledge implementation. A small but significant increase in monthly proportion of patients concomitantly using isotretinoin and contraceptive therapies was observed immediately after iPledge implementation (1.3% p-value = 0.02) particularly among younger patients (2.5% p-value < 0.01). No changes in the proportion of concomitancy over time (i.e. slope) between the periods before and after iPledge implementation were observed. CONCLUSION: The findings of this pharmacy prescription claims-based study suggest a small increase in concomitant use of isotretinoin and contraceptives coincident with the time of implementation of iPledge particularly among younger women. Published 2013. This article is a U. S. Government work and is in the public domain in the USA.

Graft-versus-host disease (GVHD) remains a significant complication of allogeneic transplantation. We previously reported that the adenosine A2A receptor (A2AR) specific agonist, ATL146e, decreases the incidence and severity of GVHD in a mouse transplant model. There is increasing interest in treatments that increase CD4+CD25highFoxp3+ regulatory T cells (Tregs) to suppress GVHD. Our current study found in vitro that A2AR selective agonists enhanced TGF-β–induced generation of mouse Tregs 2.3- to 3-fold. We demonstrated in vivo suppression of GVHD with specific A2AR agonists in two different murine GVHD transplant models associated with profound increases in both circulating and target tissue Tregs of donor origin. Three different A2AR agonists of differing potency, ATL146e, ATL370, and ATL1223, all significantly inhibited GVHD-associated weight loss and mortality. At the same time, Tregs shown to be of donor origin increased 5.1- to 7.4-fold in spleen, 2.7- to 4.6-fold in peripheral blood, 2.3- to 4.7-fold in colon, and 3.8- to 4.6-fold in skin. We conclude that specific activation of A2AR inhibits acute GVHD through an increase of donor-derived Tregs. Furthermore, the increased presence of Tregs in target tissues (colon and skin) of A2AR-specific agonist-treated mice is likely the mechanistic basis for the anti-inflammatory effect preventing acute GVHD.

Gene-editing correction of the GT deletion in exon 2 of NCF1 pseudogenes corrects p47phox-deficient chronic granulomatous disease.The nonfunctional pseudogenes NCF1B and NCF1C can be resurrected to produce functional p47phox protein by gene editing.

The pregnancy-specific glycoproteins (PSGs) are a family of proteins secreted by the syncytiotrophoblast of the placenta and are the most abundant trophoblastic proteins in maternal blood during the third trimester. The human PSG family consists of 10 protein-coding genes, some of which have a possible role in maintaining maternal immune tolerance to the fetus. PSG9 was reported as a potential predictive biomarker of pre-eclampsia, a serious complication of pregnancy that has been related to immunological dysfunction at the fetal-maternal interface. Therefore, we hypothesized that PSG9 may have an immunoregulatory role during pregnancy. We found that PSG9 binds to LAP and activates the latent form of TGF-β1. In addition, PSG9 induces the secretion of TGF-β1 from macrophages but not from CD4+ T-cells. TGF-β1 is required for the ex vivo differentiation of regulatory T-cells and, consistent with the ability of PSG9 to activate this cytokine, we observed that PSG9 induces the differentiation of FoxP3+ regulatory T-cells from naïve murine and human T-cells. Cytokines that are associated with inflammatory responses were also reduced in the supernatants of T-cells treated with PSG9, suggesting that PSG9, through its activation of TGFβ-1, could be a potent inducer of immune tolerance.

linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations in IL2RG encoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1.

Chronic granulomatous disease (CGD) still causes significant morbidity and mortality. The difficulty in considering high-risk yet curative treatments, such as allogeneic bone marrow transplantation, lies in the unpredictable courses of both CGD and bone marrow transplantation in different patients. Some patients with CGD can have frequent infections, granulomatous or autoimmune disorders necessitating immunosuppressive therapy, or both but also experience long periods of relative good health. However, the risk of death is clearly higher in patients with CGD of all types, and the complications of CGD short of death can still cause significant morbidity. Therefore, with recent developments and improvements, bone marrow transplantation, previously considered an experimental or high-risk procedure, has emerged as an important option for patients with CGD. We will discuss the complications of CGD that result in significant morbidity and mortality, particularly the most common infections and autoimmune/inflammatory complications, as well as their typical management. We will then discuss the status of bone marrow transplantation.

Chronic granulomatous disease (CGD) is a rare inherited disorder involving defective nicotinamide adenine dinucleotide phosphate oxidase function, which leads to defective production of antimicrobial superoxide anion and related oxygen intermediates critical for host defense [1]. Clinically, CGD patients develop recurrent life-threatening infections and extensive tissue granuloma formation, autoimmune diseases, and inflammatory bowel disease, which may require immunosuppressive or immunomodulatory therapy. The pathogens that commonly cause infection in CGD include the bacteria Staphylococcus aureus, Serratia marcescens, Burkholderia cepacia complex, and Nocardia species and the fungi Aspergillus species, especially Aspergillus fumigatus and Aspergillus nidulans [2]. Other less common fungi, such as Paecilomyces species and Trichosporon inkin, are encountered more frequently in patients with CGD than among the general population, highlighting the fact that patients with CGD have a unique susceptibility pattern [1]. New pathogens are continually being identified as a result of improvements in microbiologic culture and identification techniques. The use of genomic sequencing and the increasing number of sequences available in databases have permitted identification of emerging pathogens (eg, Granulibacter bethesdensis) [3] in CGD patients, as well as the reassignment of misidentified pathogens (eg, Neosartorya udagawae) [4, 5] to their correct taxa. Here we describe, to our knowledge, the first report of invasive mycoses caused by another emerging pathogen, Geosmithia argillacea, in 7 CGD patients. This disease was aggressive, involving invasion across tissue planes or metastatic dissemination in 3 patients. As shown by genomic sequencing of stored fungal isolates, isolates from 4 of these patients had been misidentified originally as Paecilomyces variotii, which resembles G. argillacea morphologically. Correct identification has important therapeutic and prognostic implications.

Introduction: Cell enrichment and/or depletion by selection is critical for graft engineering in cellular and gene therapies. The semi-automated CliniMACS Plus Instrument (Miltenyi Biotec) is used in clinical cell processing to enrich or deplete a variety of hematopoietic cells including T, B, monocytes, NK cells, and/or CD34+ hematopoietic stem/progenitor cells (HSPCs). Antibody-conjugated paramagnetic beads attach to the desired cells which are separated from the rest of the apheresis or bone marrow product in a closed-system magnetic separation column. Either the selected cells retained in the separation column (positive selection) or those in the "flow-through" fraction (negative selection) may be used for downstream processing. There is sparse data comparing cell selection efficiency and purity across the various selection methods on the CliniMACS Plus Instrument. Further, the effect of donor demographics and other upstream processing events on final viable cell recovery (VCR)/ cell purity (CP) are unknown. This study systematically compared these parameters across various selection methods. Methods: From 2008 to 2018, data were obtained from 45 clinical processing protocols involving 991 consecutive adult and pediatric donor/patient apheresis products or bone marrow harvests, which underwent cell selections on the Miltenyi CliniMACS Plus device. Cell selections included 1.CD34+ (n=669), 2.CD4+ (n=179), 3.CD3-/56+ (n=79), 4.CD3+/19- (n=6), 5.CD4+/8+ (n=21), and 6.CD25- (n=37) cells. Based on protocol design or logistical needs, apheresis or bone marrow collections were processed/selected on the same day or held overnight for next day processing. Products were also either washed to remove platelets (PW) or loaded onto the selection device directly. Data on donor age, BMI, donor type (autologous vs. allogeneic), sex, race, pre-apheresis peripheral blood cell counts and concentrations, as well as apheresis bag blood counts were evaluated. Outcome variables included post-selection VCR and CP. Correlations and multivariate regression analyses were performed for the largest selection type, i.e CD34+ cell selections. Results: Summary data (means ± 1SD) on method-specific enrichments and depletions are shown in the Table. Median post-selection VCR and CP varied by selection method. VCR was the highest for the CD34+ cell selections (Fig. a). CP was the highest and most consistent for CD4+ selections (Fig. b). Platelet and red cell depletions also varied by selection method and averaged 3.9 ± 1.63 and 2.06 ± 0.74 logs, respectively. Selection procedures used for cell depletion resulted in near complete removal of the undesired cell fractions (Table). In multivariate regression analyses, products processed on the same day (compared to those held overnight) (Fig. c), those that underwent a PW (Fig. d), and had higher pre-apheresis peripheral blood CD34+ absolute cell counts and concentrations (Fig. e) were associated significantly with higher CD34+ CP (adjusted r2=0.2; p Conclusions: This is the largest study, to our knowledge, to systematically summarize and analyze Miltenyi CliniMACS Plus selections for clinical graft engineering. Cell selections on the Miltenyi CliniMACS Plus resulted in robust enrichment with near complete depletion of the undesired cell fractions. CD34+ CP was higher in patients with higher CD34+ cells counts following mobilization. Same day processing, as well as PW further improved CD34+ CP. PW also improved CD34+ VCR. This was likely due to a reduction in non-specific platelet binding to the paramagnetic beads. Higher pre-selection CD34+ cell concentrations in the product marginally worsened VCR. This was possibly due to bead saturation in each selection method. Improved CD34+ VCR over time was likely due to increased platelet washes in the latter years. Further studies are ongoing to assess impact of cell selection variations on downstream manufacturing steps (cell transduction, expansion) and clinical (cell engraftment, GVHD incidence) outcomes. Disclosures Larochelle: Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.

Destructive midline granulomatous disease characterized by necrotizing granulomas of the head and neck is most commonly caused by Wegener granulomatosis, natural killer/T-cell lymphomas, cocaine abuse, or infections. An adolescent patient with myasthenia gravis treated with thymectomy subsequently developed extensive granulomatous destruction of midface structures, palate, nasal septum, airways, and epiglottis. His lymphocyte numbers, total immunoglobulin G level, and T-cell receptor (TCR) repertoire appeared normal. Sequencing of Recombination activating gene-1 (Rag1) showed compound heterozygous Rag1 mutations; a novel deletion with no recombinase activity and a missense mutation resulting in 50% Rag activity. His thymus was dysplastic and, although not depleted of T cells, showed a notable absence of autoimmune regulator (AIRE) and Foxp3+ regulatory T cells. This distinct Rag-deficient phenotype characterized by immune dysregulation with granulomatous hyperinflammation and autoimmunity, with relatively normal T and B lymphocyte numbers and a diverse TCR repertoire expands the spectrum of presentation in Rag deficiency. This study was registered at www.clinicaltrials.gov as #NCT00128973.

Key Points Question What percentage of patients receiving long-term immediate-release (IR) opioid analgesic therapy in the United States adds or switches to an extended-release/long-acting (ER/LA) formulation? Findings In this cohort study, most patients (96.0%) receiving IR formulations for 90 days or longer continued IR opioid analgesic use without adding (3.3%) or switching (0.7%) to an ER/LA product. Furthermore, many patients received only 1 IR (40%) and/or 1 ER/LA (41%) prescription throughout the 12-year study period. Meaning Most patients receiving opioid analgesics use IR formulations, and patients receiving long-term IR opioid analgesic therapy are unlikely to add or switch to an ER/LA formulation., Importance Many stakeholders are working to improve the safe use of immediate-release (IR) and extended-release/long-acting (ER/LA) opioid analgesics. However, little information exists regarding the relative use of these 2 formulations in chronic pain management. Objectives To describe the distribution of IR and ER/LA opioid analgesic therapy duration and examine adding and switching patterns among patients receiving long-term IR opioid analgesic therapy, defined as at least 90 consecutive days of IR formulation use. Design, Setting, and Participants A retrospective cohort study of 169 million individuals receiving opioid analgesics from across 90% of outpatient retail pharmacies in the United States from January 1, 2003, to December 31, 2014, using the IQVIA Health Vector One: Data Extract Tool. Analyses were conducted from March 2015 to June 2017. Exposures Receipt of dispensed IR or ER/LA opioid analgesic prescription. Main Outcomes and Measures Distribution of therapy frequency and duration of IR and ER/LA opioid analgesic use, and annual proportions of patients receiving long-term IR opioid analgesic therapy who added an ER/LA formulation while continuing to use an IR formulation, switched to an ER/LA formulation, or continued receiving IR opioid analgesic therapy only. Results Among the 169 280 456 patients included in this analysis, 168 315 458 patients filled IR formulations and 10 216 570 patients filled ER/LA formulations. A similar percentage of women received ER/LA (55%) and IR (56%) formulations, although those receiving ER/LA formulations (72%) were more likely to be aged 45 years or older compared with those receiving IR formulations (46%). The longest opioid analgesic episode duration was 90 days or longer for 11 563 089 patients (7%) filling IR formulations and 3 103 777 patients (30%) filling ER/LA formulations. The median episode duration was 5 days (interquartile range, 3-10 days) for patients using IR formulations and 30 days (interquartile range, 21-74 days) for patients using ER/LA formulations. From January 1, 2003, to December 31, 2014, a small and decreasing proportion of patients with long-term IR opioid analgesic therapy added (3.8% in 2003 to 1.8% in 2014) or switched to (1.0% in 2003 to 0.5% in 2014) an ER/LA formulation. Conclusions and Relevance Most patients receiving opioid analgesics, whether for short or extended periods, use IR formulations. Once receiving long-term IR opioid analgesic therapy, patients are unlikely to add or switch to an ER/LA formulation., This cohort study characterizes use of immediate-release and extended-release/long-acting opioid analgesic therapy formulations and adding and switching patterns between the 2 formulations among patients in the United States between 2003 and 2014.

A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the ΔdblGATA mouse. We demonstrate here that bone marrow progenitors isolated from ΔdblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured ΔdblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured ΔdblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1EB), located ∼3700 bp downstream to the previously described first exon found in hemopoietic cells (1EA) and ∼42 bp upstream to another variant first exon, 1EC. These data suggest that cultured progenitors are able to circumvent the effects of the ΔdblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.

We describe haploidentical hematopoietic cell transplantation (HCT) with high-dose post-transplant cyclophosphamide (PTCy) in a boy with x-linked chronic granulomatous disease (CGD).A persistent and life-threatening fungal infection was the indication for HSCT. Non-myeloablative conditioning with PTCy (50 mg/kg days 3 and 4) was used in the absence of fully matched donors.Engraftment occurred on day 24. The patient experienced Grade 2 graft-versus-host disease of the skin and gastrointestinal tract and CMV infection, both of which were controlled. Chimerism was 100 % at days 30 and 6 months. Cessation of antifungal therapy was consistent with cure of the infection.Haploidentical HCT with high-dose PTCy for CGD is feasible and succeeded even in the context of active infection.

We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34(+) cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34(+) progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking.