Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Allogeneic hematopoietic cell transplantation is effective for p47phox chronic granulomatous disease: A Primary Immune Deficiency Treatment Consortium study.

BACKGROUND: P47phox (neutrophil cytosolic factor-1) deficiency is the most common cause of autosomal recessive chronic granulomatous disease (CGD) and is considered to be associated with a milder clinical phenotype. Allogeneic hematopoietic cell transplantation (HCT) for p47phox CGD is not well-described. OBJECTIVES: We sought to study HCT for p47phox CGD in North America. METHODS: Thirty patients with p47phox CGD who received allogeneic HCT at Primary Immune Deficiency Treatment Consortium centers since 1995 were included. RESULTS: Residual oxidative activity was present in 66.7% of patients. In the year before HCT, there were 0.38 CGD-related infections per person-years. Inflammatory diseases, predominantly of the lungs and bowel, occurred in 36.7% of the patients. The median age at HCT was 9.1 years (range 1.5-23.6 years). Most HCTs (90%) were performed after using reduced intensity/toxicity conditioning. HCT sources were HLA-matched (40%) and -mismatched (10%) related donors or HLA-matched (36.7%) and -mismatched (13.3%) unrelated donors. CGD-related infections after HCT decreased significantly to 0.06 per person-years (P = .038). The frequency of inflammatory bowel disease and the use of steroids also decreased. The cumulative incidence of graft failure and second HCT was 17.9%. The 2-year overall and event-free survival were 92.3% and 82.1%, respectively, while at 5 years they were 85.7% and 77.0%, respectively. In the surviving patients evaluated, ≥95% donor myeloid chimerism at 1 and 2 years after HCT was 93.8% and 87.5%, respectively. CONCLUSIONS: Patients with p47phox CGD suffer from a significant disease burden that can be effectively alleviated by HCT. Similar to other forms of CGD, HCT should be considered for patients with p47phox CGD.

Posttransplantation late complications increase over time for patients with SCID: A Primary Immune Deficiency Treatment Consortium (PIDTC) landmark study.

BACKGROUND: The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children in the United States and Canada onto a retrospective multicenter natural history study of hematopoietic cell transplantation (HCT). OBJECTIVE: We investigated outcomes of HCT for severe combined immunodeficiency (SCID). METHODS: We evaluated the chronic and late effects (CLE) after HCT for SCID in 399 patients transplanted from 1982 to 2012 at 32 PIDTC centers. Eligibility criteria included survival to at least 2 years after HCT without need for subsequent cellular therapy. CLE were defined as either conditions present at any time before 2 years from HCT that remained unresolved (chronic), or new conditions that developed beyond 2 years after HCT (late). RESULTS: The cumulative incidence of CLE was 25% in those alive at 2 years, increasing to 41% at 15 years after HCT. CLE were most prevalent in the neurologic (9%), neurodevelopmental (8%), and dental (8%) categories. Chemotherapy-based conditioning was associated with decreased-height z score at 2 to 5 years after HCT (P < .001), and with endocrine (P < .001) and dental (P = .05) CLE. CD4 count of ≤500 cells/µL and/or continued need for immunoglobulin replacement therapy >2 years after transplantation were associated with lower-height z scores. Continued survival from 2 to 15 years after HCT was 90%. The presence of any CLE was associated with increased risk of late death (hazard ratio, 7.21; 95% confidence interval, 2.71-19.18; P < .001). CONCLUSION: Late morbidity after HCT for SCID was substantial, with an adverse impact on overall survival. This study provides evidence for development of survivorship guidelines based on disease characteristics and treatment exposure for patients after HCT for SCID.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency.

ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αß and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Small bowel disease prevalence on video capsule endoscopy in chronic granulomatous disease-associated inflammatory bowel disease.

BACKGROUND AND AIM: Patients with chronic granulomatous disease (CGD) may develop inflammatory bowel disease (IBD). Characterization of small bowel disease in this cohort is scarce. Here, we sought to determine the prevalence and characteristics of small bowel disease and evaluate the clinical utility of video capsule endoscopy (VCE) for its diagnosis. METHODS: A retrospective study was performed on patients with CGD who were evaluated for gastrointestinal disease with VCE as a part of ongoing natural history studies at a single academic center. VCEs were reviewed for inflammatory findings and severity of disease utilizing the Capsule Endoscopy Crohn's Disease Activity Index. Radiographic studies and endoscopies performed within 30 days of VCE were compared with small bowel inflammatory findings. RESULTS: Twenty-six VCEs corresponding to 25 patients were found. The majority of patients were male and White; mean age was 28 years old. The majority (85%) demonstrated presence of small bowel inflammatory findings on VCE including strictures, ulcers, erosions, and erythema. Duodenal and ileal inflammatory disease on endoscopy did not correlate with disease on VCE. Moderate-severe colonic disease correlated with moderate-severe disease on VCE. Radiography did not correlate with disease on VCE. Prolonged small bowel transit time correlated with moderate-severe small bowel disease. CONCLUSIONS: Small bowel IBD was highly prevalent in this cohort of patients with CGD. Limitations included small sample size. Given that radiology and duodenal/ileal disease did not correlate with VCE findings, VCE-driven investigation of small bowel disease should be considered in patients with CGD-associated IBD, particularly those with colonic disease.

A phase III randomized crossover trial of plerixafor versus G-CSF for treatment of WHIM syndrome.

BACKGROUNDWarts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is a kind of neutropenia caused by neutrophil retention in bone marrow and in WHIM syndrome is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, its safety and efficacy in WHIM syndrome are undefined.METHODSIn this investigator-initiated, single-center, quadruple-masked phase III crossover trial, we compared the total infection severity score (TISS) as the primary endpoint in an intent-to-treat manner in 19 patients with WHIM who each received 12 months treatment with plerixafor and 12 months treatment with granulocyte CSF (G-CSF, the standard of care for severe congenital neutropenia). The treatment order was randomized for each patient.RESULTSPlerixafor was nonsuperior to G-CSF for TISS (P = 0.54). In exploratory endpoints, plerixafor was noninferior to G-CSF for maintaining neutrophil counts of more than 500 cells/µL (P = 0.023) and was superior to G-CSF for maintaining lymphocyte counts above 1,000 cells/µL (P < 0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life or the incidence of drug failure or serious adverse events.CONCLUSIONPlerixafor was not superior to G-CSF in patients with WHIM for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.TRIAL REGISTRATIONClinicaltrials.gov NCT02231879.FUNDINGThis study was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells.

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Gene therapy for inborn errors of immunity: Base editing comes into play.

CRISPR-Cas9-based base editing allows precise base editing to achieve conversion of adenosine to guanine or cytosine to thymidine. In this issue of Cell, McAuley et al. use adenine base editing to correct a single base-pair mutation causing human CD3δ deficiency, demonstrating superior efficiency of genetic correction with reduced undesired genetic alterations compared with standard CRISPR-Cas9 editing.

CRISPR/Cas9-mediated Cxcr4 disease allele inactivation for gene therapy in a mouse model of WHIM syndrome.

WHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4+/+) and WHIM (Cxcr4+/w) HSCs and that a patient with WHIM was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4+/w and Cxcr4+/+ alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wild-type allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge, this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.

Gastrointestinal and Hepatic Manifestations of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare inborn error of immunity, resulting from a defect in nicotinamide adenine dinucleotide phosphate oxidation and decreased production of phagocyte reactive oxygen species. The main clinical manifestations are recurrent infections and chronic inflammatory disorders. Current approaches to management include antimicrobial prophylaxis and control of inflammatory complications. Hematopoietic stem cell transplantation or gene therapy can provide definitive treatment. Gastrointestinal and hepatic manifestations are common in CGD and include structural changes, dysmotility, CGD-associated inflammatory bowel disease, liver abscesses, and noncirrhotic portal hypertension. The findings can be heterogeneous, and the management is complex in light of the underlying immune dysfunction. This review describes the various clinical findings and the latest studies in management of gastrointestinal and hepatic manifestations in CGD, as well as the management experience at the National Institutes of Health.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

Lentivector cryptic splicing mediates increase in CD34+ clones expressing truncated HMGA2 in human X-linked severe combined immunodeficiency.

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken ß-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

Evolution of Gene Therapy, Historical Perspective.

The earliest conceptual history of gene therapy began with the recognition of DNA as the transforming substance capable of changing the phenotypic character of a bacterium and then as the carrier of the genomic code. Early studies of oncogenic viruses that could insert into the mammalian genome led to the concept that these same viruses might be engineered to carry new genetic material into mammalian cells, including human hematopoietic stem cells (HSC). In addition to properly engineered vectors capable of efficient safe transduction of HSC, successful gene therapy required the development of efficient materials, methods, and equipment to procure, purify, and culture HSC. Increased understanding of the preparative conditioning of patients was needed to optimize the engraftment of genetically modified HSC. Testing concepts in pivotal clinical trials to assess the efficacy and determine the cause of adverse events has advanced the efficiency and safety of gene therapy. This article is a historical overview of the separate threads of discovery that joined together to comprise our current state of gene therapy targeting HSC.

Granulocyte Transfusions in Patients with Chronic Granulomatous Disease Undergoing Hematopoietic Cell Transplantation or Gene Therapy.

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

Febrile neutropenia management and outcomes in hematopoietic cell transplantation for chronic granulomatous disease.

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

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

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).

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

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.

CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells.

Introduction: Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. Methods: We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus. Results and discussion: In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.

Hematologically important mutations: The autosomal forms of chronic granulomatous disease (third update).

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe, recurrent bacterial and fungal infections. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is subsequently metabolized to hydrogen peroxide and other reactive oxygen species (ROS). These products are essential for intracellular killing of pathogens by phagocytic leukocytes (neutrophils, eosinophils, monocytes and macrophages). The leukocyte NADPH oxidase is composed of five subunits, four of which are encoded by autosomal genes. These are CYBA, encoding p22phox, NCF1, encoding p47phox, NCF2, encoding p67phox and NCF4, encoding p40phox. This article lists all mutations identified in these genes in CGD patients. In addition, cytochrome b558 chaperone-1 (CYBC1), recently recognized as an essential chaperone protein for the expression of the X-linked NADPH oxidase component gp91phox (also called Nox2), is encoded by the autosomal gene CYBC1. Mutations in this gene also lead to CGD. Finally, RAC2, a small GTPase of the Rho family, is needed for activation of the NADPH oxidase, and mutations in the RAC2 gene therefore also induce CGD-like symptoms. Mutations in these last two genes are also listed in this article.