Publications by authors named "Harry L Malech"

171 Publications

Preclinical evaluation for engraftment of CD34 cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.

Cell Rep Med 2021 Apr 20;2(4):100247. Epub 2021 Apr 20.

Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD, USA.

Sickle cell disease (SCD) is caused by a 20A > T mutation in the β-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34 cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34 cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus β-to-βs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34 cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34 HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.
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http://dx.doi.org/10.1016/j.xcrm.2021.100247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080237PMC
April 2021

Gastrointestinal Computed Tomography Findings in Chronic Granulomatous Disease with Subgroup Clinicopathologic Analysis.

Dig Dis Sci 2021 May 1. Epub 2021 May 1.

Abdominal Imaging, Division of Diagnostic Radiology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency which can lead to gastrointestinal (GI) complications including inflammatory bowel disease. Radiographic findings in this cohort have not been well described.

Aims: To describe the frequency and spectrum of gastrointestinal abnormalities seen on computed tomography (CT) in patients with CGD and determine whether radiography was predictive of endoscopic or histopathologic inflammatory findings.

Methods: A retrospective review was conducted on 141 consecutive CGD patients seen at the National Institutes of Health between 1988 and 2011. All corresponding CTs were reviewed for gastrointestinal abnormalities including wall thickening. Endoscopic and histopathologic findings were reviewed in subjects with documented endoscopy within 30 days of an imaging study. Findings were compared between patients with and without wall thickening on CT to determine whether bowel wall thickening was predictive of endoscopic or histologic inflammatory findings.

Results: Two hundred and ninety-two CTs were reviewed. GI wall thickening was present on CT in 61% of patients (n = 86). Among a subgroup of 20 patients who underwent endoscopy at the time of their imaging, there was a statistically significant correlation between radiographic gastrointestinal wall thickening and endoscopic inflammation in the same intestinal segment (p = 0.035). Additionally, there was a significant correlation between radiographic gastrointestinal wall thickening and inflammatory features on histopathology (p = 0.02).

Conclusions: GI abnormalities are commonly observed on CT in CGD patients. Bowel wall thickening correlates with endoscopic and histopathologic evidence of inflammation. These findings may be used to better facilitate directed endoscopic assessment and histopathologic sampling in patients with CGD.
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http://dx.doi.org/10.1007/s10620-021-06978-4DOI Listing
May 2021

Homozygous variant p. Arg90His in NCF1 is associated with early-onset Interferonopathy: a case report.

Pediatr Rheumatol Online J 2021 Apr 23;19(1):54. Epub 2021 Apr 23.

The Hospital for Sick Children, University of Toronto, Toronto, Canada.

Background: Biallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome in adult patients. This study demonstrates the association of the homozygous p.Arg90His variant with interferonopathy with features of autoinflammation and autoimmunity in a pediatric patient.

Case Presentation: A 5-year old female of Indian ancestry with early-onset recurrent fever and headache, and persistently elevated antinuclear, anti-Ro, and anti-La antibodies was found to carry the homozygous p.Arg90His variant in NCF1 through exome sequencing. Her unaffected parents and three other siblings were carriers for the mutant allele. Because the presence of two NCF1 pseudogenes, this variant was confirmed by independent genotyping methods. Her intracellular neutrophil oxidative burst and NCF1 expression levels were normal, and no clinical features of CGD were apparent. Gene expression analysis in peripheral blood detected an interferon gene expression signature, which was further supported by cytokine analyses of supernatants of cultured patient's cells. These findings suggested that her inflammatory disease is at least in part mediated by type I interferons. While her fever episodes responded well to systemic steroids, treatment with the JAK inhibitor tofacitinib resulted in decreased serum ferritin levels and reduced frequency of fevers.

Conclusion: Homozygosity for p.Arg90His in NCF1 should be considered contributory in young patients with an atypical systemic inflammatory antecedent phenotype that may evolve into autoimmunity later in life. The complex genomic organization of NCF1 poses a difficulty for high-throughput genotyping techniques and variants in this gene should be carefully evaluated when using the next generation and Sanger sequencing technologies. The p.Arg90His variant is found at a variable allele frequency in different populations, and is higher in people of South East Asian ancestry. In complex genetic diseases such as SLE, other rare and common susceptibility alleles might be necessary for the full disease expressivity.
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http://dx.doi.org/10.1186/s12969-021-00536-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063424PMC
April 2021

Safety and Efficacy of Ustekinumab in the Inflammatory Bowel Disease of Chronic Granulomatous Disease.

Clin Gastroenterol Hepatol 2021 Apr 1. Epub 2021 Apr 1.

Translational Hepatology Section, Liver Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland. Electronic address:

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by mutations encoding the NADPH oxidase complex. Those affected are at increased risk of bacterial and fungal infections and require antimicrobial prophylaxis. Dysregulated inflammation may cause inflammatory bowel disease (IBD), termed CGD-associated IBD or CGD colitis, a distinct entity from Crohn's disease (CD) or ulcerative colitis (UC).
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http://dx.doi.org/10.1016/j.cgh.2021.03.039DOI Listing
April 2021

, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid-Lipid A Glycolipid (Ko-Lipid A).

Int J Mol Sci 2021 Mar 24;22(7). Epub 2021 Mar 24.

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact () is hypostimulatory compared to , i.e., cytokine production in human blood requires 10-100 times more CFU/mL than . To better understand the pathogenicity of , we isolated its lipopolysaccharide (LPS) and characterized its lipid A. Unlike with typical , the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Man-(1→4)-β-GlcN3N-(1→6)-α-GlcN-(1⇿1)-α-GlcA tetra-saccharide substituted with five acyl chains: the amide-linked N-3' 14:0(3-OH), N-2' 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of LPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of Ko-lipidA compared to lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the Ko-lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.
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http://dx.doi.org/10.3390/ijms22073303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036547PMC
March 2021

Preclinical Optimization and Safety Studies of a New Lentiviral Gene Therapy for p47-Deficient Chronic Granulomatous Disease.

Hum Gene Ther 2021 May 6. Epub 2021 May 6.

Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom.

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 p47-deficient CGD (p47CGD), 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 p47CGD 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 p47CGD.
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http://dx.doi.org/10.1089/hum.2020.276DOI Listing
May 2021

Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair.

Gene Ther 2021 Mar 12. Epub 2021 Mar 12.

Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91 protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1-13 or 2-13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1-13 cDNA did not restore physiologic gp91 levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2-13 cDNA fully restored gp91 and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91 expression in exon 2-13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91 and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.
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http://dx.doi.org/10.1038/s41434-021-00251-zDOI Listing
March 2021

The response to vedolizumab in chronic granulomatous disease-related inflammatory bowel disease.

Gastroenterol Rep (Oxf) 2020 Oct 10;8(5):404-406. Epub 2020 Mar 10.

Liver Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.

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http://dx.doi.org/10.1093/gastro/goaa005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603867PMC
October 2020

Homozygous mutation associated with infantile inflammatory bowel disease.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892;

Interleukin (IL)-37, an antiinflammatory IL-1 family cytokine, is a key suppressor of innate immunity. IL-37 signaling requires the heterodimeric IL-18R1 and IL-1R8 receptor, which is abundantly expressed in the gastrointestinal tract. Here we report a 4-mo-old male from a consanguineous family with a homozygous loss-of-function mutation. The patient presented with persistent diarrhea and was found to have infantile inflammatory bowel disease (I-IBD). Patient cells showed increased intracellular IL-37 expression and increased proinflammatory cytokine production. In cell lines, mutant IL-37 was not stably expressed or properly secreted and was thus unable to functionally suppress proinflammatory cytokine expression. Furthermore, induced pluripotent stem cell-derived macrophages from the patient revealed an activated macrophage phenotype, which is more prone to lipopolysaccharide and IL-1β stimulation, resulting in hyperinflammatory tumor necrosis factor production. Insights from this patient will not only shed light on monogenic contributions of I-IBD but may also reveal the significance of the IL-18 and IL-37 axis in colonic homeostasis.
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http://dx.doi.org/10.1073/pnas.2009217118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958356PMC
March 2021

Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells.

Blood 2021 Feb 23. Epub 2021 Feb 23.

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.
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http://dx.doi.org/10.1182/blood.2020008503DOI Listing
February 2021

Treatment by CRISPR-Cas9 Gene Editing - A Proof of Principle.

Authors:
Harry L Malech

N Engl J Med 2021 01;384(3):286-287

From the Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

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http://dx.doi.org/10.1056/NEJMe2034624DOI Listing
January 2021

NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease.

Blood Adv 2020 12;4(23):5976-5987

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

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.
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http://dx.doi.org/10.1182/bloodadvances.2020003224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724899PMC
December 2020

JAGN1 mutations in severe congenital neutropenia.

Br J Haematol 2021 01 18;192(1):9-10. Epub 2020 Nov 18.

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

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http://dx.doi.org/10.1111/bjh.17135DOI Listing
January 2021

MAGT1 messenger RNA-corrected autologous T and natural killer cells for potential cell therapy in X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasia disease.

Cytotherapy 2021 Mar 10;23(3):203-210. Epub 2020 Oct 10.

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Electronic address:

Background Aim: X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect' (XMEN) disease is caused by mutations in the magnesium transporter 1 (MAGT1) gene. Loss of MAGT1 function results in a glycosylation defect that abrogates expression of key immune proteins such as the NKG2D receptor on CD8 T and NK cells, which is critical for the recognition and killing of virus-infected and transformed cells, a biomarker for MAGT1 function. Patients with XMEN disease frequently have increased susceptibility to EBV infections and EBV-associated B cell malignancies, for which no specific treatment options are currently available. Experimental transfer of donor EBV-specific cytotoxic T cells may be beneficial but carries the risks of eliciting alloimmune responses. An approach for cell therapy to address viral infections and associated complications that avoids the risks of alloimmunity is needed.

Methods: Here the authors assess the feasibility and efficiency of correcting autologous lymphocytes from XMEN patients by MAGT1 mRNA electroporation (EP) that avoids genomic integration and can be scaled for clinical application.

Results And Conclusions: Restoration of NKG2D expression was demonstrated in XMEN patient lymphocytes after MAGT1 mRNA electroporation that reach healthy donor levels in CD8 T and NK cells at 1-2 days after EP. NKG2D expression persisted at ∼50% for 2 weeks after EP. Functionally, mRNA-correction of XMEN NK cells rescued cytotoxic activity also to healthy donor NK cell level. The restored NKG2D receptor expression and function were unaffected by cryopreservation, which will make feasible repeat infusions of MAGT1 mRNA-corrected autologous XMEN CD8 T and NK cells for potential short term therapy for XMEN patients without the risks of alloimmunization.
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http://dx.doi.org/10.1016/j.jcyt.2020.08.013DOI Listing
March 2021

Successful SCID gene therapy in infant with disseminated BCG.

J Allergy Clin Immunol Pract 2021 Feb 16;9(2):993-995.e1. Epub 2020 Sep 16.

Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, Tenn. Electronic address:

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http://dx.doi.org/10.1016/j.jaip.2020.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870513PMC
February 2021

Low Plasma Gelsolin Concentrations in Chronic Granulomatous Disease.

Inflammation 2021 Feb 4;44(1):270-277. Epub 2020 Sep 4.

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

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.
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http://dx.doi.org/10.1007/s10753-020-01330-wDOI Listing
February 2021

Correction: Chronic Granulomatous Disease-Associated IBD Resolves and Does Not Adversely Impact Survival Following Allogeneic HCT.

J Clin Immunol 2020 Nov;40(8):1211-1213

Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford School of Medicine, Stanford, CA, USA.

The original version of this article unfortunately contained the missing author, Caridad Martinez. The authors would like to correct the list. We apologize for any inconvenience that this may have caused. The correct author list is shown above.
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http://dx.doi.org/10.1007/s10875-020-00852-0DOI Listing
November 2020

Progressive B Cell Loss in Revertant X-SCID.

J Clin Immunol 2020 10 17;40(7):1001-1009. Epub 2020 Jul 17.

Department of Pediatrics, Division of Immunology, Allergy, and Rheumatology, University of California Los Angeles, Los Angeles, CA, 90095, USA.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20 years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.
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http://dx.doi.org/10.1007/s10875-020-00825-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508923PMC
October 2020

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

Blood Adv 2020 06;4(12):2611-2616

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD.

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.
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http://dx.doi.org/10.1182/bloodadvances.2020001730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322962PMC
June 2020

Failure to Prevent Severe Graft-Versus-Host Disease in Haploidentical Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide in Chronic Granulomatous Disease.

J Clin Immunol 2020 05 20;40(4):619-624. Epub 2020 Apr 20.

National Institute of Allergy and Infectious Diseases, National Institutes of Health, 10 Center Dr., Rm. 6-3754, MSC 1763, Bethesda, MD, 20892-1456, USA.

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.
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http://dx.doi.org/10.1007/s10875-020-00772-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507116PMC
May 2020

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Nat Med 2020 02 27;26(2):200-206. Epub 2020 Jan 27.

Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells. 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.
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http://dx.doi.org/10.1038/s41591-019-0735-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115833PMC
February 2020

The Role of Neutrophils in the Immune System: An Overview.

Methods Mol Biol 2020 ;2087:3-10

Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA.

Neutrophils, also known as polymorphonuclear neutrophils (PMNs), have long been considered as the short-lived, nonspecific white cells that form pus-and also happen to kill invading microbes. Indeed, neutrophils were often neglected (and largely not considered) as immune cells. This historic view of neutrophils has changed considerably over the past several decades, and we now know that in addition to playing the predominant role in the clearance of bacteria and fungi, they have a major role in shaping the host response to infection and immune system homeostasis. The change in our view of the role of neutrophils in the immune system has been due in large part to the study of these cells in vitro. Such work has been made possible by new and/or improved methods and approaches used to investigate neutrophils. These methods are the focus of this volume.
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http://dx.doi.org/10.1007/978-1-0716-0154-9_1DOI Listing
January 2021

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

J Clin Invest 2020 01;130(1):507-522

National Human Genome Research Institute, and.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαβ+ T cells (αβDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.
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http://dx.doi.org/10.1172/JCI131116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934229PMC
January 2020

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases.

Biol Blood Marrow Transplant 2020 01 4;26(1):94-106. Epub 2019 Sep 4.

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.
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http://dx.doi.org/10.1016/j.bbmt.2019.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942248PMC
January 2020

Chronic Granulomatous Disease-Associated IBD Resolves and Does Not Adversely Impact Survival Following Allogeneic HCT.

J Clin Immunol 2019 10 2;39(7):653-667. Epub 2019 Aug 2.

Division of Pediatric Allergy and Immunology, Mayo Clinic, Rochester, MN, USA.

Introduction: Inflammatory 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.

Methods: We 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.

Results: Forty-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 2 years following allogeneic HCT.

Conclusions: In 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.
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http://dx.doi.org/10.1007/s10875-019-00659-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754755PMC
October 2019

Gene Editing in Chronic Granulomatous Disease.

Methods Mol Biol 2019 ;1982:623-665

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Chronic granulomatous disease (CGD) is an immune deficiency characterized by defects in the production of microbicidal reactive oxygen species (ROS) by the phagocytic oxidase (phox) enzyme complex in neutrophils. We have previously described targeted gene editing strategies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases for gene targeting with homology-directed repair in CGD patient stem cells to achieve functional restoration of expression of phox genes and NADPH oxidase activity in differentiated neutrophils. In this chapter, we describe detailed protocols for targeted gene editing in human-induced pluripotent stem cells and hematopoietic stem cells and for subsequent differentiation of these stem cells into mature neutrophils, as well as assays to characterize neutrophil identity and function including flow cytometry analysis of neutrophil surface markers, intracellular staining for phox proteins, and analysis of ROS generation.
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http://dx.doi.org/10.1007/978-1-4939-9424-3_36DOI Listing
January 2020

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

Mol Ther 2019 06 9;27(6):1074-1086. Epub 2019 Apr 9.

Translational Stem Cell Biology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.
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http://dx.doi.org/10.1016/j.ymthe.2019.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554657PMC
June 2019

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

N Engl J Med 2019 04;380(16):1525-1534

From the Departments of Bone Marrow Transplantation and Cellular Therapy (E.M., B.T., W.J., S.G.), Hematology (S.Z., Z.M., J.C., J.D., X.T., B.Y.R., M.J.W., B.P.S.), Therapeutics Production and Quality (T.L., M.M.M.), Immunology (H.A., B.Y.), Pharmaceutical Sciences (S.J.C.), Biostatistics (G.K., C.L.), and Infectious Diseases (G.M.), St. Jude Children's Research Hospital, Memphis, TN; the Allergy and Clinical Immunology Division, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru (J.C.A.B.); the Department of Pediatrics, Allergy-Immunology Division, Children's Hospital Los Angeles, Los Angeles (J.A.C.), and the Department of Pediatrics, Division of Pediatric Allergy-Immunology-Bone Marrow Transplantation, University of California, San Francisco (UCSF) Benioff Children's Hospital, San Francisco (J.R.L.-B., J.M.P., M.J.C.) - both in California; the Department of Pediatrics, Pediatric Allergy and Immunology, University of New Mexico, Albuquerque (E.D.); University of Oklahoma Health Sciences Center, Tulsa (J.T.L.); Departamento de Pediatria da Universidade de Taubaté, Conselho Nacional de Medicina, São Paulo (A.C.M.A.); Copperfield Childcare, Claremont, South Africa (H.W.); and the Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (S.S.D.R., H.L.M.).

Background: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia.

Methods: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1.

Results: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven.

Conclusions: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).
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http://dx.doi.org/10.1056/NEJMoa1815408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636624PMC
April 2019

(p47)-deficient chronic granulomatous disease: comprehensive genetic and flow cytometric analysis.

Blood Adv 2019 01;3(2):136-147

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

Mutations in (p47) cause autosomal recessive chronic granulomatous disease (CGD) with abnormal dihydrorhodamine (DHR) assay and absent p47 protein. Genetic identification of mutations is complicated by adjacent highly conserved (>98%) pseudogenes ( and ). has GTGT at the start of exon 2, whereas the pseudogenes each delete 1 GT (ΔGT). In p47 CGD, the most common mutation is ΔGT in (c.75_76delGT; p.Tyr26fsX26). Sequence homology between and its pseudogenes precludes reliable use of standard Sanger sequencing for mutations and for confirming carrier status. We first established by flow cytometry that neutrophils from p47 CGD patients had negligible p47 expression, whereas those from p47 CGD carriers had ∼60% of normal p47 expression, independent of the specific mutation in We developed a droplet digital polymerase chain reaction (ddPCR) with 2 distinct probes, recognizing either the wild-type GTGT sequence or the ΔGT sequence. A second ddPCR established copy number by comparison with the single-copy telomerase reverse transcriptase gene, We showed that 84% of p47 CGD patients were homozygous for ΔGT The ddPCR assay also enabled determination of carrier status of relatives. Furthermore, only 79.2% of normal volunteers had 2 copies of GTGT per 6 total () copies, designated 2/6; 14.7% had 3/6, and 1.6% had 4/6 GTGT copies. In summary, flow cytometry for p47 expression quickly identifies patients and carriers of p47 CGD, and genomic ddPCR identifies patients and carriers of ΔGT , the most common mutation in p47 CGD.
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http://dx.doi.org/10.1182/bloodadvances.2018023184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341190PMC
January 2019

Addressing the Value of Gene Therapy and Enhancing Patient Access to Transformative Treatments.

Mol Ther 2018 12 30;26(12):2717-2726. Epub 2018 Oct 30.

American Society of Gene and Cell Therapy, Milwaukee, WI, USA. Electronic address:

Although high upfront costs for the high value of gene therapy have resulted in concerns about sufficient reimbursement to allow patient access to these therapies, the significant benefits of gene therapies will not be realized unless patients have access to them. Stakeholders are discussing these issues, and the payment models being developed for the newly approved gene therapies provide an early indication of the flexibility that will be needed from treatment manufacturers, payers, and policy makers to optimize patient access. Maximizing patient access to effective gene therapies is one integral part of the overall mission of the American Society of Gene and Cell Therapy, along with maximizing the quality of therapies and minimizing their costs.
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http://dx.doi.org/10.1016/j.ymthe.2018.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277509PMC
December 2018