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Cytotoxic immune cells mediate precise attacks against diseased cells to maintain organismal health. Their operational unit of killing and host defense is lytic granules (LGs), which are specialized lysosomal-related organelles. Precision in cytotoxicity is achieved by converging the many LGs to the microtubule-organizing center (MTOC) and polarizing these to the diseased cell for secretion. We identify unappreciated intimate relationships between the Golgi, MTOC, and LGs after cytotoxic cell activation, as well as the trans-Golgin protein GCC2 on the LG surface. GCC2 serves to tether LGs to the Golgi following convergence, and both GCC2 and the Golgi are required for the persistence of convergence. GCC2 allows LGs to utilize the Golgi as a docking station preventing LG dispersion and innocent bystander killing in complex three-dimensional environments. We also identify GCC2 variants causing human natural killer cell deficiency, further emphasizing the importance of LG convergence and Golgi linkage in precision targeting for human immunity., Competing Interests: Declaration of interests L.A.P., F.v.d.H., Y.L., and J.S.O. are inventors on a patent application (applied through the Trustees of Columbia University in the city of New York, application number PCT/US2023/01614, entitled “Compounds, targets, and methods for modulating lytic granule convergence in cytotoxic cells to promote bystander killing in cellular therapies”). J.R.L. has stock ownership in 23andMe and is a co-inventor on multiple patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders, and bacterial genomic fingerprinting., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human recombination-activating gene (RAG) deficiency can manifest with distinct clinical and immunological phenotypes. By applying a multiomics approach to a large group of RAG -mutated patients, we aimed at characterizing the immunopathology associated with each phenotype. Although defective T and B cell development is common to all phenotypes, patients with hypomorphic RAG variants can generate T and B cells with signatures of immune dysregulation and produce autoantibodies to a broad range of self-antigens, including type I interferons. T helper 2 (T H 2) cell skewing and a prominent inflammatory signature characterize Omenn syndrome, whereas more hypomorphic forms of RAG deficiency are associated with a type 1 immune profile both in blood and tissues. We used cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis to define the cell lineage-specific contribution to the immunopathology of the distinct RAG phenotypes. These insights may help improve the diagnosis and clinical management of the various forms of the disease.

Genetic manipulation of hematopoietic stem cells (HSCs) is being developed as a therapeutic strategy for several inherited disorders. This field is rapidly evolving with several novel tools and techniques being employed to achieve desired genetic changes. While commercial products are now available for sickle cell disease, transfusion-dependent β-thalassemia, metachromatic leukodystrophy and adrenoleukodystrophy, several challenges remain in patient selection, HSC mobilization and collection, genetic manipulation of stem cells, conditioning, hematologic recovery and post-transplant complications, financial issues, equity of access and institutional and global preparedness. In this report, we explore the current state of development of these therapies and provide a comprehensive assessment of the challenges these therapies face as well as potential solutions., Competing Interests: Declaration of competing interest Ashish Gupta is a site principal investigator at University of Minnesota for clinical trials in genome editing for sickle cell disease sponsored by Bluebird Bio (NCT04293185) and Beam Therapeutics (NCT05456880). He is also the site principal investigator at University of Minnesota for clinical trials in genome editing for Hurler syndrome (NCT06149403). He has received consultancy fees from Vertex Pharmaceuticals and is on speaker bureau for Emerging Therapies Solutions. He also has research funding from Jazz Pharmaceuticals for an investigator initiated trial. Akshay Sharma has received consultant fees from Spotlight Therapeutics, Medexus Inc., Vertex Pharmaceuticals, Sangamo Therapeutics and Editas Medicine. He is a medical monitor for an RCI BMT clinical trial for which he receives financial compensation. He has also received research funding from CRISPR Therapeutics and honoraria from Vindico Medical Education. Akshay Sharma is also the St. Jude Children's Research Hospital site principal investigator for clinical trials of genome editing for sickle cell disease sponsored by Vertex Pharmaceuticals/CRISPR Therapeutics (NCT03745287), Novartis Pharmaceuticals (NCT04443907) and Beam Therapeutics (NCT05456880). The industry sponsors provide funding for the clinical trials, which includes salary support paid to the investigator's institution. Akshay Sharma has no direct financial interest in these therapies. Jaap Jan Boelens has received consultant fees from Sanofi, Sobi, Merck, Immusoft and Smart Immune, is the MSKCC site PI for Novartis Pharmaceuticals (NCT04443907) and received research funding from Sanofi (for investigator-initiated research). Jaap Jan Boelens is also the chair of an independent data safety monitoring board (for bluebird bio gene therapy trials), for which he receives compensation (CRO; Advanced Clinical). Susan Prockop has received support for the conduct of clinical trials from AlloVir, Atara, and Jasper through Boston Children's Hospital. She has received an honorarium from Pierre Fabre and Regeneron and consults for CellEvolve, Smart Immune and Century. She has IP related to the implementation of third-party viral-specific T cells, with all rights assigned to Memorial Sloan Kettering Cancer Center. Chrystal U. Louis has equity in and is an employee of TScan Therapeutics. The other authors have nothing to disclose., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Abstract: Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment strategy for patients with inborn errors of immunities (IEIs). The objective of this study was to assess the optimal busulfan exposure before allogeneic HCT for patients with an IEI who received an IV busulfan-based conditioning regimen. Patients from 17 international centers were included. The main outcome of interest was event-free survival (EFS). Patients were categorized into 4 IEI subgroups: combined immunodeficiency (CID), severe combined immunodeficiency (SCID), neutrophil disorders, and hemophagocytic lymphohistiocytosis (HLH)-related disorders. Busulfan exposure was calculated by individual centers (area under the curve [AUC]CENTER) and re-estimated using a nonlinear mixed-effects model (NONMEM; exposure defined as AUCNONMEM). Overall, 562 patients were included: 173 (30.8%) with CID, 154 (27.4%) with SCID, 101 (18.0%) with HLH-related disorders, and 134 (23.8%) with neutrophil disorders. The median busulfan AUCNONMEM was 69.0 mg × h/L and correlated poorly with the AUCCENTER (r2 = 0.54). In patients with SCID, HLH-related, and neutrophil disorders with a busulfan AUCNONMEM of 70 to 90 mg × h/L, 2-year EFS was superior to <70 mg × h/L, and >90 mg ×h/L. Full donor chimerism increased with higher busulfan AUCNONMEM, plateauing at 90 mg × h/L. For patients with CID, the optimal AUCNONMEM for donor chimerism was found to be >70 mg × h/L. Improved EFS and higher donor chimerism may be achieved by targeting a cumulative busulfan AUCNONMEM of 80 mg × h/L (range, 70-90). Our study stresses the importance of uniformly using a validated population pharmacokinetic model to estimate AUCNONMEM., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)

There is lack of guidance for immune monitoring and infection prevention after administration of ex vivo genetically modified hematopoietic stem cell therapies (GMHSCT). We reviewed current infection prevention practices as reported by providers experienced with GMHSCTs across North America and Europe, and assessed potential immunologic compromise associated with the therapeutic process of GMHSCTs described to date. Based on these assessments, and with consensus from members of the International Society for Cell & Gene Therapy (ISCT) Stem Cell Engineering Committee, we propose risk-adapted recommendations for immune monitoring, infection surveillance and prophylaxis, and revaccination after receipt of GMHSCTs. Disease-specific and GMHSCT-specific considerations should guide decision making for each therapy., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)