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BACKGROUND: Prospective genetic evaluation of patients at our referral research hospital presents clinical research challenges. OBJECTIVE: This study sought not only a single-gene explanation for participants’ immune-related presentations, but viewed each participant holistically, with the potential to have multiple genetic contributions to their immune-phenotype and other heritable comorbidities relevant to their presentation and health. METHODS: We developed a program integrating exome sequencing, chromosomal microarray, phenotyping, results return with genetic counseling, and reanalysis in 1505 individuals from 1000 families with suspected or known inborn errors of immunity. RESULTS: Probands were 50.8% female, 71.5% ≥18 years, and had diverse immune presentations. Overall, 327/1000 probands (32.7%) received 361 molecular diagnoses. These included 17 probands with diagnostic copy number variants, 32 probands with secondary findings, and 31 probands with multiple molecular diagnoses. Reanalysis added 22 molecular diagnoses, predominantly due to new disease-gene associations (9/22, 40.9%). One-quarter of the molecular diagnoses (92/361) did not involve immune-associated genes. Molecular diagnosis was correlated with younger age, male sex, and a higher number of organ systems involved. This program also facilitated the discovery of new gene-disease associations such as SASH3-related immunodeficiency. A review of treatment options and ClinGen actionability curations suggest that at least 251/361 (69.5%) of these molecular diagnoses could translate into ≥1 management option. CONCLUSION: This program contributes to our understanding of the diagnostic and clinical utility whole exome analysis on a large scale.

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 Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

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.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversionmutation. 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 20years. 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.

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.

XMEN disease, defined as “X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect,” is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

A 4–8 year follow-up of vector integration site (VIS) analysis in blood lineages from patients with X-linked severe combined immune deficiency receiving lentivector gene therapy found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of HMGA2. Some patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but with no disturbance of hematopoiesis. Molecular analysis demonstrated a cryptic splice site within the cHS4 insulator generating truncated mRNA transcripts from any transcriptionally active gene containing forward-oriented intronic insert, but with detectable effect on VIS frequency only for intron 3 HGMA2 inserts. A two base-pair change at the splice site eliminated splicing activity while retaining vector functional capability. Functional analysis of lentivectors for cryptic splicing should become routine for pre-clinical safety assessment.

Background Development of a diverse T-cell receptor β (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution. Objectives We investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID. Methods We used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements. Results TRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention. Conclusions TRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

Primary immunodeficiency diseases (PIDs) encompass a range of diseases due to mutations in genes that are critical for immunity. Haploinsufficiency and gain-of-function mutations are more complex than simple loss-of-function mutations; in addition to increased susceptibility to infections, immune dysregulations like autoimmunity and hyperinflammation are common presentations. Hematopoietic stem cell (HSC) gene therapy, using integrating vectors, provides potential cure of disease, but genome-wide transgene insertions and the lack of physiological endogenous gene regulation may yet present problems, and not applicable in PIDs where immune regulation is paramount. Targeted genome editing addresses these concerns; we discuss some approaches of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system applicable for gene therapy in PIDs. Preclinical repair of gene mutations and insertion of complementary DNA restore endogenous gene regulation and they have shown very promising data for clinical application. However, ongoing studies to characterize off-target genotoxicity, careful donor designs to ensure physiological expression, and maneuvers to optimize engraftment potential are critical to ensure successful application of this next-gen targeted HSC gene therapy.

Mutations in NCF1 (p47phox) cause autosomal recessive chronic granulomatous disease (CGD) with abnormal dihydrorhodamine (DHR) assay and absent p47phox protein. Genetic identification of NCF1 mutations is complicated by adjacent highly conserved (>98%) pseudogenes (NCF1B and NCF1C). NCF1 has GTGT at the start of exon 2, whereas the pseudogenes each delete 1 GT (ΔGT). In p47phox CGD, the most common mutation is ΔGT in NCF1 (c.75_76delGT; p.Tyr26fsX26). Sequence homology between NCF1 and its pseudogenes precludes reliable use of standard Sanger sequencing for NCF1 mutations and for confirming carrier status. We first established by flow cytometry that neutrophils from p47phox CGD patients had negligible p47phox expression, whereas those from p47phox CGD carriers had ∼60% of normal p47phox expression, independent of the specific mutation in NCF1. 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, TERT. We showed that 84% of p47phox CGD patients were homozygous for ΔGT NCF1. 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 (NCF1/NCF1B/NCF1C) copies, designated 2/6; 14.7% had 3/6, and 1.6% had 4/6 GTGT copies. In summary, flow cytometry for p47phox expression quickly identifies patients and carriers of p47phox CGD, and genomic ddPCR identifies patients and carriers of ΔGT NCF1, the most common mutation in p47phox CGD.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)–ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with

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 gp91phox 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 gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2–13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox 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 non-homologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox 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.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by mutations encoding the NADPH oxidase complex.1 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).

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.

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.

BACKGROUND Approximately, one-third to one-half of children with chronic granulomatous disease (CGD) develop gastrointestinal inflammation characteristic of idiopathic inflammatory bowel disease (IBD), usually Crohn's disease. We hypothesized that the overall IBD genetic risk, determined by IBD genetic risk score (GRS), might in part determine IBD development in CGD. METHODS We reviewed medical records to establish IBD diagnoses in CGD subjects seen at NIAID. IBD risk single nucleotide polymorphism genotypes were determined using the Immunochip, and GRS were estimated by Mangrove. RESULTS Among 157 white patients with CGD, 55 were confirmed, 78 excluded, and 24 were uncertain for IBD. Two hundred one established, independent European IBD risk single nucleotide polymorphisms passed quality control. After sample quality control and removing non-IBD CGD patients with perianal disease, mean GRS for 40 unrelated patients with CGD-IBD was higher than 53 CGD non-IBD patients (in log2-scale 0.08 ± 1.62 versus -0.67 ± 1.64, P = 0.026) but lower than 239 IBD Genetics Consortium (IBDGC) young-onset Crohn's disease cases (0.76 ± 1.60, P = 0.025). GRS for non-IBD CGD was similar to 609 IBDGC controls (-0.69 ± 1.60, P = 0.95). Seven established IBD single nucleotide polymorphisms were nominally significant among CGD-IBD versus CGD non-IBD, including those near LACC1 (P = 0.005), CXCL14 (P = 0.007), and TNFSF15 (P = 0.016). CONCLUSIONS The weight of the common IBD risk alleles are significant determinants of IBD in CGD. However, IBD risk gene burden among CGD children with IBD is significantly lower than that in nonsyndromic pediatric Crohn's disease, congruent with the concept that defective superoxide production in CGD is also a major IBD risk factor. Individual IBD genes might interact with the CGD defect to cause IBD in CGD.

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.

BACKGROUND: Although autoimmunity and hyperinflammation secondary to recombination activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series. OBJECTIVE: Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency. METHODS: In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology. RESULTS: Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. Most patients (55.6%) presented with more than 1 autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median, 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in most cases (64.7%, 73.7%, and 71.4% for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/ hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients. CONCLUSIONS: Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multilineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management. (C) 2019 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.

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.

Made available in DSpace on 2019-09-12T16:53:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2019 American Lebanese Syrian Associated Charities California Institute of Regenerative Medicine [CLIN2-09504] National Heart, Lung, and Blood Institute [P01 HL053749] National Cancer Institute [CA21765] National Institute of Allergy and Infectious Diseases (NIAID) [Z01-AI-00988] NIAID [U54-AI082973] Assisi Foundation of Memphis 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 gamma-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 IL2RG 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.) [Mamcarz, Ewelina; Triplett, Brandon; Janssen, William; Gottschalk, Stephen] St Jude Childrens Res Hosp, Dept Bone Marrow Transplantat & Cellular Therapy, 262 Danny Thomas Pl,Mail Stop 1130, Memphis, TN 38105 USA [Zhou, Sheng; Condori, Jose; Dowdy, Jola; Tang, Xing; Ryu, Byoung Y.; Weiss, Mitchell J.; Sorrentino, Brian P.] St Jude Childrens Res Hosp, Dept Hematol, 332 N Lauderdale St, Memphis, TN 38105 USA [Lockey, Timothy; Meagher, Michael M.] St Jude Childrens Res Hosp, Dept Therapeut Prod & Qual, 332 N Lauderdale St, Memphis, TN 38105 USA [Abdelsamed, Hossam; Youngblood, Benjamin] St Jude Childrens Res Hosp, Dept Immunol, 332 N Lauderdale St, Memphis, TN 38105 USA [Cross, Shane J.] St Jude Childrens Res Hosp, Dept Pharmaceut Sci, 332 N Lauderdale St, Memphis, TN 38105 USA [Kang, Guolian; Li, Chen] St Jude Childrens Res Hosp, Dept Biostat, 332 N Lauderdale St, Memphis, TN 38105 USA [Maron, Gabriela] St Jude Childrens Res Hosp, Dept Infect Dis, 332 N Lauderdale St, Memphis, TN 38105 USA [Aldave Becerra, Juan C.] Hosp Nacl Edgardo Rebagliati Martins, Allergy & Clin Immunol Div, Lima, Peru [Church, Joseph A.] Childrens Hosp Los Angeles, Dept Pediat, Div Allergy Immunol, Los Angeles, CA 90027 USA [Long-Boyle, Janel R.; Puck, Jennifer M.; Cowan, Morton J.] Univ Calif San Francisco, Dept Pediat, Benioff Childrens Hosp, Div Pediat Allergy Immunol Bone Marrow Transplant, San Francisco, CA USA [Dokmeci, Elif] Univ New Mexico, Dept Pediat Pediat Allergy & Immunol, Albuquerque, NM 87131 USA [Love, James T.] Univ Oklahoma, Hlth Sci Ctr, Tulsa, OK USA [da Matta Ain, Ana C.] Universidade de Taubaté (Unitau), Dept Pediat, Conselho Nacl Med, Sao Paulo, Brazil [van der Watt, Hedi] Copperfield Childcare, Claremont, South Africa [De Ravin, Suk See; Malech, Harry L.] NIAID, Genet Immunotherapy Sect, Lab Clin Immunol & Microbiol, NIH, 9000 Rockville Pike, Bethesda, MD 20892 USA

Introduction Chronic granulomatous disease (CGD) is a rare immunodeficiency caused by mutations in the NADPH oxidase complex. Dysregulated immune function may cause inflammatory bowel disease (IBD). Patients with CGD-associated IBD may not respond to or may develop serious infections as a result of traditional IBD therapies such as vedolizumab and infliximab. Ustekinumab is approved for use in Crohn’s disease and ulcerative colitis however there is scarce data on its efficacy and safety in CGD. Aims To evaluate the efficacy and safety of ustekinumab for CGD-associated IBD. Methods A retrospective chart review was conducted on CGD patients followed at a single center who had consented to participate in a natural history study. Clinical, laboratory, and endoscopic data were extracted in those that had received ustekinumab for IBD. Results Eight patients were found. Four were male and four were female. Five were white, one was Asian, one was black, and one was mixed race. Median age at diagnosis of CGD was 3 years (IQR 8) and of IBD was 15.5 years (IQR 20). Median age at initiation of ustekinumab was 27.5 years (IQR 14) and median duration on ustekinumab was 10 months (IQR 7). Six had colonic disease, two had ileocolonic disease, and six had perianal disease. Six failed other biologics (n=5 for vedolizumab, n=1 for infliximab, n=1 for adalimumab). Six patients symptomatically improved whereas two had no improvement. Changes in hemoglobin and C-reactive protein were equivocal. Three patients had improved endoscopic findings, two had unimproved findings, and three patients lacked this data. Overall, four patients achieved clinical remission. However, none of the five patients with endoscopic reevaluation achieved endoscopic remission. Three patients discontinued therapy due to lack of response: two required surgery and one underwent stem cell transplant. Fungal pneumonia (n=2), otitis media (n=1), oral herpes simplex virus 1 (n=1), and viral gastroenteritis (n=1) were reported. One infusion reaction occurred. Discussion In our cohort of eight patients with CGD-associated IBD receiving ustekinumab, results were mixed with four patients experiencing some degree of clinical or endoscopic improvement including four who achieved clinical remission. Multiple CGD-related variables may account for the mixed laboratory findings. Four of the five patients with endoscopic reevaluation had pre-existing strictures that would be unlikely to reverse with medical therapy alone. Of these, two had otherwise resolved endoscopic inflammation. Only two patients had no endoscopic improvement. Two serious infections occurred however CGD confers increased infectious susceptibility and no infections lead to discontinuation of therapy. Given these promising results, further formalized study of ustekinumab in CGD-associated IBD is needed.

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Early clinical studies of gene therapy for patients with X-linked Severe Combined Immunodeficiency (XSCID) only restored T cell immunity and carried a significant risk of iatrogenic leukemia. We developed a new gene therapy approach that utilizes a safety-modified lentiviral (LV) vector together with reduced exposure busulfan conditioning for newly diagnosed infants with XSCID (NCT01512888). Of the first enrolled 8 patients, 7 demonstrated robust reconstitution of T-, NK-, and B-cells with a median follow up of 16.4 months (range: 6.7 to 24.9 months; Mamcarz et al, N Engl J Med, 2019). Here we provide an update on our clinical study, which now includes 3 more patients (n=11 total), 8 months additional median follow-up (23.6 months; range: 1.5 to 33.9 months), more extensive analysis of T and B cell functional recovery, and detailed vector integration site studies. Overall, we successfully generated transduced autologous bone marrow (BM) CD34+ cells for all patients with a median vector copy number (VCN) of 0.45 VCN/cell (range: 0.16-1.13). Prior to the infusion of transduced CD34+ cells (median cell dose: 8.7 x106/kg; range: 4.5-19.0), patients received two daily doses of busulfan to target a cumulative area-under-the-curve (cAUC) of 22 mg*hr/L (achieved median: 22.3 mg*hr/L; range: 20.0-23.0). No severe adverse events, other than hematologic related to busulfan, were observed. All 11 patients had robust hematopoietic recovery within 3-4 weeks post cell infusion without blood product support. Nine patients, with a follow up of >3 months, achieved normal for age T-cell and NK-cell numbers within 3-4 months post gene therapy. T-cells matured appropriately as assessed by normal receptor excision circles (TREC) levels and TCRvb repertoire analysis. In addition, phytohemagglutinin (PHA) stimulation assays demonstrated normal T-cell function. So far, 5 patients are off IVIG of whom 3 responded to vaccines. As previously reported, patient #1 demonstrated poor immune reconstitution. He received a 2nd infusion of transduced CD34+ cells without conditioning one year after his initial infusion, which resulted in functional T-cell immune reconstitution. Clinically, all patients with a follow up >3 months recovered from pre-existing infections, are off protective isolation and prophylactic antimicrobials, and have normal growth in respect to height and weight. The median VCN at 12 months post gene therapy in seven patients, who have been followed for >12 months, was 2.25 VCN/cell (range: 1.24-3.03) in T cells, 0.34 VCN/cell (range: 0.23-1.25) in B cells, 1.55 VCN/cell (range 1.27-3.39) in NK cells, and 0.08 VCN/cell (range: 0.03-0.76) in myeloid cells in peripheral blood, and 0.10 (range: 0.05-0.66) in CD34+ bone marrow cells, respectively. Detailed integration sites analysis for the first 7 patients, who received a single infusion of transduced CD34+ cells, revealed that the majority of sites were located in introns and intergenic regions throughout the human genome. The integration site pattern was highly consistent across patients with integration site clusters that had been previously described by us and others after LV transduction. In conclusion, LV gene therapy for XSCID using low dose busulfan conditioning and a novel LV vector is well tolerated and results in the development of a functional normal immune system without evidence of malignant transformation with a median follow up of almost 2 years. Thus, our approach may present a promising alternative to current therapies, which rely in part on high dose chemotherapy followed by allogeneic hematopoietic cell transplantation. Disclosures Mamcarz: American Lebanese Syrian Associated Charities: Research Funding; UpToDate: Honoraria; NHLBI: Research Funding; 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. Zhou: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. Lockey: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. Boi: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. Koon-Kiu: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. Cross: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; NIH: Research Funding. Kang: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. Ma: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. Condori: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. Dowdy: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. Metais: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. Langfitt: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. Triplett: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. Li: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. Zhao: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. Maron:Chimerix: 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; Astellas: Research Funding. Janssen: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. Weiss:GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy. Youngblood: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. 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. Puck:Pfeizer: Other: spouse serves on Rare Disease Advisory Board; NIAID: Research Funding; Invitae: Other: spouse employment. Cowan:NIH NIAD: Research Funding; Leadiant: Consultancy; Rocket Pharma: Consultancy; bluebird bio: Consultancy; California Institute Of Regenerative Medicine: Research Funding; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria. Gottschalk:Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; TESSA Therapeutics: Other: Research Collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; EMD Serono: Honoraria; California Institute for Regenerative Medicine: Research Funding; Sanofi: Honoraria; NHLBI: Research Funding; Inmatics: Membership on an entity's Board of Directors or advisory committees; 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; America Lebanese Syrian Associated Charities: Research Funding; ViraCyte: Consultancy; ASSISI fundation of Memphis: Research Funding.

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene. State-of-the-art genome editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. We have demonstrated efficient gene correction in SCD CD34+ cells with SCD mutation-specific guide RNA, Cas9 mRNA/protein, and single strand donor DNA, resulting in ~30% gene correction and ~50% indels at the DNA level, and ~60% normal β-globin production at the protein level in in vitro erythroid differentiation (ASH 2018). Gene correction by homology directed repair is thought to be enhanced by cell proliferation; however, cell proliferation might reduce stemness of HSCs. To investigate this hypothesis, we sought to evaluate engraftment of gene-edited CD34+ HSCs in a non-human primate model. To model SCD gene correction, a β-to-βs globin conversion was designed in rhesus macaques. Mobilized rhesus CD34+ cells (n=2) were electroporated using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver rhesus β-globin-targeting guide RNA (the same target site as the SCD mutation-specific guide RNA), SpCas9 protein, and single strand donor DNA including a SCD mutation (20A>T). We also added an adjuvant to improve gene conversion efficiencies. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. We observed high-efficiency genome editing without the adjuvant (20-30% gene conversion and 61-64% indels), and further enhanced genome editing with the adjuvant (51-59% gene conversion and 36-39% indels). After erythroid differentiation, we observed production of βs-globin protein (~100%) but not normal β-globin in gene-edited cells. We then evaluated engraftment of gene-edited rhesus CD34+ cells with β-to-βs globin conversion (n=2, 13U005 and 12U011). Mobilized rhesus CD34+ cells (3.4-3.8e7) were pre-stimulated for 2 days, and edited cells were cryopreserved after electroporation with editing tools. Small aliquots of edited cells (before and after cryopreservation) were differentiated into erythroid cells in vitro, resulting in 17-26% of gene conversion and 57-71% of indels at the DNA level and 50-100% of β-globin production at the protein level, with no difference observed between aliquots taken before and after cryopreservation. Following 9.5 Gy total body irradiation, the frozen edited CD34+ cells (1.6-2.2e7) were injected into autologous macaques. We observed robust recovery of blood counts in 13U005, while peripheral blood recovery was delayed in 12U011, who was supported by serial whole blood transfusion. We observed 7-11% of gene conversion and 44-54% of indels in both granulates and lymphocytes in 13U005 1 month post-transplant. Around 15% sickle Hb production in red blood cells was detected by Hb electrophoresis in 13U005 three months post-transplant and ~7% in 12U011 two months post-transplant. Interestingly, ~10% of fetal Hb production was observed in 12U001, likely due to stress hematopoiesis. In summary, we developed a rhesus β-to-βs globin conversion model with HSC-targeted genome editing strategies. The gene-edited rhesus CD34+ cells are engraftable for at least 3 months post-transplant. Although further follow-up is necessary for transplanted animals, these findings are helpful in designing HSC-targeted gene correction trials. Figure Disclosures Li: MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Peshwa:MaxCyte, Inc: Employment.

Introduction 'X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia' (XMEN) disease is a primary immunodeficiency disease caused by loss-of-function mutations in the MAGT1 gene encoding for the magnesium transporter 1. This leads to the absence of expression of the "Natural-Killer Group 2, member D" (NKG2D) receptor in natural killer (NK) and CD8+ T cells, which is essential for their antiviral and antitumoral cytotoxic activity. In consequence, XMEN patients develop chronic EBV infections and EBV-related lymphoproliferative disorders. Allogeneic bone marrow transplant has been associated with significant mortality, and there are no other effective treatments. In that context, we aimed at developing two complementary approaches to treat XMEN patients: 1) Adoptive transfer of XMEN T/NK cells corrected by transient mRNA therapy or longer-lasting gene editing therapy in order to control infections, and 2) Transplantation of gene-edited CD34+ cells in order to permanently restore production of functional immune cells. Material and methods CD34+ cells and PBMCs were collected from XMEN patients and healthy donors (HD) (NIH Protocol 94-I-073). For mRNA therapy, we expanded T cells with anti-CD3/anti-CD28 beads in RPMI + 10% serum supplemented with 200 IU/mL IL2 for 5-7 days and NK cells with 100 IU/mL IL2 and 10 ng/mL IL15 in culture with K562-mb15-41BBL for 10-15 days. Both XMEN T and NK cells were electroporated (EP) with MAGT1 mRNA and cultured for up to 28 days. For gene editing, XMEN CD34+ or stimulated T cells were electroporated with Cas9 mRNA and sgRNA; a rAAV6 donor encoding for the codon-optimized MAGT1 cDNA was added after EP. Two days post-EP, CD34+ cells were differentiated into NK cells for 35 days in vitro. Results MAGT1 mRNA-based therapy. We first showed a restored MAGT1 expression by western blot at 6h and 24h post-EP of the MAGT1 mRNA. In consequence, NKG2D expression analyzed by flow cytometry was restored in expanded CD8+ T and NK cells starting within the 6h post-EP (20-40%), with a peak at 48h (>85%) and a progressive decrease of the expression over time (still 40% and 75%, respectively, of CD8+ T and NK cells of cells at day 14 post-EP respectively). The cytotoxic activity of mRNA-corrected XMEN NK cells was analyzed by culture with K562 target cells at several effector:target (E:T) ratios and shown to be restored at a level similar to HD NK cells (mRNA-treated: 66.7% ±5.8%; HD: 67.8% ±5.9% at E:T 2:1 ratio) compared to untreated cells (49.0% ±7.2%) (Fig 1a). Anticipating the potential use of these cells for repeated infusions as a treatment modality to control infections, we demonstrated that MAGT1 mRNA-corrected CD8+ T and NK cells that have been cryopreserved and thawed exhibit the same NKG2D expression kinetics following thaw and culture. Gene editing therapy. XMEN CD34+ cells electroporated with Cas9 mRNA and a sgRNA targeting exon 1 of MAGT1 gene showed an in vitro average integration rate of the MAGT1 cDNA AAV donor of 35.6% (range: 33.8-41.9%). The NKG2D expression in AAV-treated CD34+-derived NK cells was approximatively of 23% (range: 14.2-27.9%). Interestingly, their cytotoxic activity was similar to the level of NKG2D expression (23.1% ±4.3%), significantly higher than in untreated cells (9.7% ±2.8%) (Fig 1b). Similar rates of targeted integration and NKG2D expression were also obtained in AAV-treated CD8+ T cells. Conclusion For the first time, we demonstrate the efficiency of two approaches for development of potential cell therapy treatments of XMEN patients. MAGT1 mRNA electroporation can restore efficient transient expression of NKG2D in CD8+ T and NK cells, thus fully restoring the cytotoxic activity of NK cells. In addition, cells electroporated with MAGT1 mRNA can be cryopreserved, thus allowing repeated infusions. In parallel, we showed that efficient targeted insertion can be achieved in CD8+ T cells and CD34+ cells by using an AAV donor although the level of NKG2D expression is lower. Optimizations are currently ongoing in order to reach higher levels of correction. Both approaches could be combined in order to propose a new therapeutic strategy for the treatment of XMEN patients: repetitive adoptive transfer of mRNA-corrected autologous T/NK cells for the prevention or control of intractable infections, and transplantation of gene-edited CD34+ cells for the definitive treatment of these patients. Disclosures Meis: CELLSCRIPT, LLC: Employment. Li:MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Clark:CELLSCRIPT, LLC: Employment. Dahl:CELLSCRIPT, LLC: Other: Owner and officer.

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.

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

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.

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

Key teaching points •We report a case of a 6-year-old African American boy with X-linked CGD who developed DLE–like skin lesions after starting voriconazole. •Voriconazole is commonly used to treat fungal infections in patients with CGD. •Lupus erythematosus–like skin lesions have been reported in carriers of X-linked CGD and, less commonly, in patients with CGD. •Voriconazole is a significant cause of drug-induced photosensitivity, and may play a role in unmasking an underlying predisposition to lupuslike skin lesions. •Photoprotective measures and routine examinations to monitor for skin toxicity, including skin cancer, are prudent during voriconazole treatment.

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene, and can be cured by therapeutic β-globin gene addition into hematopoietic stem cells (HSCs) with lentiviral transduction. However, this method relies upon random integration, leaving the SCD mutation intact and potentially inducing insertional mutagenesis. Genome editing technologies have the potential to correct the SCD mutation without integration, producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. In this study, we investigated CRISPR/Cas9-based gene correction for SCD CD34+ cells. Plerixafor-mobilized SCD CD34+ cells were transfected by electroporation using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver SCD mutation-specific guide RNA at 200mg/ml, SpCas9 mRNA at 200mg/ml or protein at 120mg/ml, and single strand donor DNA with a normal β-globin sequence at 80, 120, or 200mg/ml. We chose Cas9 mRNA and single strand donor DNA due to the ease of clinical grade large-scale production and to avoid the need for viral vector manufacturing. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. Cell viability was reduced to 76-87% after electroporation, compared to 90% in the control. We observed high-efficiency genome editing (29-34% gene correction and 49-58% indels) with Cas9 mRNA, showing donor DNA concentration dependence, and editing levels were comparable to Cas9 protein (39% correction and 43% indels). 15-23% Biallelic and 17-26% monoallelic gene correction were detected at the clonal level by colony assay. After erythroid differentiation, up to 54% normal β-globin production was observed with Cas9 mRNA (Figure), comparable to Cas9 protein (67%), while βs-globin amounts were markedly reduced under both conditions (6-10%). Similar correction efficiencies were obtained from two additional SCD patients' CD34+ cells at DNA levels (28-35%) and protein levels (33-56%). These data demonstrate that Cas9 mRNA and single strand donor DNA allow for efficient gene correction in SCD CD34+ cells, exceeding the therapeutic threshold of 20% in SCD. We then evaluated off-target effects on the δ-globin gene, which was reported as a major off-target site in β-globin gene editing due to high homology; however, almost no off-target effects (0.6-1.3% indels) were detected. Interestingly, gene conversion in the 9T>C polymorphism (11bp upstream of SCD mutation) on the β-globin gene was observed, and this conversion always occurred with SCD gene correction (26-33% of SCD gene correction), suggesting that gene conversion is strongly affected by distance from the target site. In addition, we evaluated genome editing among subpopulations of CD34+ cells from 3 healthy donors under the same conditions (normal β-globin to SCD mutation). We observed similar editing efficiencies (conversion and indels) among more immature (CD34+CD133+CD90+) and relatively differentiated populations (CD34+CD133+CD90-, CD34+CD133-, and CD34-) as well as among cells at different phases of the cell cycle (G0/G1, S, and G2/M), suggesting that similar gene correction efficiencies are obtained in all CD34+ cell populations, including the HSC population. We have begun efforts to evaluate gene-corrected SCD CD34+ cell engraftment in the mouse xenograft model, as similarly corrected X-CGD CD34+ cells were engrafted in immunodeficient mice. To examine the effects of indels in the β-globin gene, we next evaluated Hb production from genome-edited SCD CD34+ cells (2 patients) without donor DNA. Editing without donor DNA resulted in 63-70% indels (compared to 26-29% correction and 46-53% indels with donor DNA) and increased non-adult Hb production (small amounts of fetal Hb and significant amounts of a Hb variant), which will require further investigation to characterize. In summary, we observed efficient gene correction in SCD CD34+ cells with a simple Cas9 mRNA, single strand donor DNA, and guide RNA method, resulting in ~30% gene correction and ~50% indels. After erythroid differentiation, the majority of Hb detected was adult Hb; we detected up to 54% normal β-globin production with a marked reduction of βs-globin to ~10%. Evaluation of engraftment potential is required for gene-corrected CD34+ cells, but these methods would be clinically applicable for gene correction in SCD. Figure. Figure. Disclosures Li: MaxCyte, Inc.: Employment. Allen:MaxCyte, Inc.: Employment. Peshwa:MaxCyte, Inc.: Employment.

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91 phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91 phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1–13 minigene into CYBB exon 1 resulted in no detectable gp91 phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2–13 minigene into exon 2 restored both gp91 phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2–13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.

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.

Targeted gene therapy has been hampered by the inability to correct mutations in stem cells that can reconstitute the immune system after transplant into patients. De Ravin et al . now report that CRISPR, a DNA editing technology, corrected blood stem cells from patients with an immunodeficiency disorder (chronic granulomatous disease) caused by mutations in NOX2. CRISPR-repaired human stem cells engrafted in mice after transplant and differentiated into leukocytes with a functional NOX2 protein for up to 5 months. The authors did not detect off-target treatment effects, suggesting that this gene repair strategy may benefit patients with chronic granulomatous disease or other blood disorders.

X-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.