Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

Enhancing implementation of the I-PASS Handoff Tool Using a Provider Handoff Task Force at a Comprehensive Cancer Center.

BACKGROUND: Communication failures are among the most common causes of harmful medical errors. At one Comprehensive Cancer Center, patient handoffs varied among services. The authors describe the implementation and results of an Organization-wide project to improve handoffs and implement an evidence-based handoff tool across all inpatient services. METHODS: The research team created a task force composed of members from 22 hospital services-advanced practice providers (APPs), trainees, some faculty members, electronic health record (EHR) staff, education and training specialists, and nocturnal providers. Over two years, the task force expanded to include consulting services and Anesthesiology. Factors contributing to ineffective handoffs were identified and organized into categories. The EHR I-PASS tool was used to standardize handoff documentation. Training was provided to staff on its use, and compliance was monitored using a customized dashboard. I-PASS champions in each service were responsible for the rollout of I-PASS in their respective services. The data were reported quarterly to the Quality Assessment and Performance Improvement (QAPI) governing committee. Provider handoff perception was assessed through the biennial Institution-wide safety culture survey. RESULTS: All fellows, residents, APPs, and physician assistants were trained in the use of I-PASS, either online or in person. Adherence to the I-PASS written tool improved from 41.6% in 2019 to 70.5% in 2022 (p < 0.05), with improvements seen in most services. The frequency of updating I-PASS elements and the action list in the handoff tool also increased over time. The handoff favorability score on the safety culture survey improved from 38% in 2018 to 59% in 2022. CONCLUSION: The implementation approach developed by the Provider Handoff Task Force led to increased use of the I-PASS EHR tool and improved safety culture survey handoff favorability.

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.

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.

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.

CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.

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.

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

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 nonhomologous 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.

Homozygous IL37 mutation associated with infantile inflammatory bowel disease.

Interleukin (IL)-37, an antiinflammatory IL-1 family cytokine, is a key suppressor of innate immunity. IL-37 signaling requires the heterodimeric IL-18R1 and IL-1R8 receptor, which is abundantly expressed in the gastrointestinal tract. Here we report a 4-mo-old male from a consanguineous family with a homozygous loss-of-function IL37 mutation. The patient presented with persistent diarrhea and was found to have infantile inflammatory bowel disease (I-IBD). Patient cells showed increased intracellular IL-37 expression and increased proinflammatory cytokine production. In cell lines, mutant IL-37 was not stably expressed or properly secreted and was thus unable to functionally suppress proinflammatory cytokine expression. Furthermore, induced pluripotent stem cell-derived macrophages from the patient revealed an activated macrophage phenotype, which is more prone to lipopolysaccharide and IL-1ß stimulation, resulting in hyperinflammatory tumor necrosis factor production. Insights from this patient will not only shed light on monogenic contributions of I-IBD but may also reveal the significance of the IL-18 and IL-37 axis in colonic homeostasis.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

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.

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

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.

Gene Editing in Chronic Granulomatous Disease.

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.

Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice.

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use the knockout of the X-linked Cybb gene as an example to describe our procedures for genetically modifying NSG mice using the CRISPR/Cas9 method. Briefly, two sgRNAs were designed and made to target exon 1 and exon 3 of the Cybb gene, and either sgRNA was then microinjected together with Cas9 mRNA into fertilized eggs collected from NSG mice. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. Offspring born to the foster mothers were genotyped by PCR and DNA sequencing. In this chapter, we describe our experiment procedures in detail and report our genotyping results for demonstrating that NSG mice can be genetically modified using the CRISPR/Cas9 technology in a highly efficient manner.

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit+ population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue.

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

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

Differentiation of human and murine induced pluripotent stem cells to microglia-like cells.

Microglia are resident inflammatory cells of the CNS and have important roles in development, homeostasis and a variety of neurologic and psychiatric diseases. Difficulties in procuring human microglia have limited their study and hampered the clinical translation of microglia-based treatments shown to be effective in animal disease models. Here we report the differentiation of human induced pluripotent stem cells (iPSC) into microglia-like cells by exposure to defined factors and co-culture with astrocytes. These iPSC-derived microglia have the phenotype, gene expression profile and functional properties of brain-isolated microglia. Murine iPSC-derived microglia generated using a similar protocol have equivalent efficacy to primary brain-isolated microglia in treatment of murine syngeneic intracranial malignant gliomas. The ability to generate human microglia facilitates the further study of this important CNS cell type and raises the possibility of their use in personalized medicine applications.

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

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

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.

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

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

Gene-edited pseudogene resurrection corrects p47phox-deficient 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.