The generation of T cells from pluripotent stem cells (PSCs) is attractive for investigating T cell development and validating genome editing strategies in vitro. X-linked severe combined ...immunodeficiency (X-SCID) is an immune disorder caused by mutations in the IL2RG gene and characterised by the absence of T and NK cells in patients. IL2RG encodes the common gamma chain, which is part of several interleukin receptors, including IL-2 and IL-7 receptors. To model X-SCID in vitro, we generated a mouse embryonic stem cell (ESC) line in which a disease-causing human IL2RG gene variant replaces the endogenous Il2rg locus. We developed a stage-specific T cell differentiation protocol to validate genetic correction of the common G691A mutation with transcription activator-like effector nucleases. While all ESC clones could be differentiated to hematopoietic precursor cells, stage-specific analysis of T cell maturation confirmed early arrest of T cell differentiation at the T cell progenitor stage in X-SCID cells. In contrast, genetically corrected ESCs differentiated to CD4 + or CD8 + single-positive T cells, confirming correction of the cellular X-SCID phenotype. This study emphasises the value of PSCs for disease modelling and underlines the significance of in vitro models as tools to validate genome editing strategies before clinical application.
Abstract The recent advances in cellular immunotherapies have revolutionized the treatment options for hematological malignancies. Among others, genetically engineered T cells to express chimeric ...antigen receptors targeting CD19 have shown clinical success in patients with B cell malignancies. However, depending on the study design only one to two thirds of the patients experience a complete response upon CD19 CAR T cells treatment. Antigen escape, immune suppressive microenvironment, CAR T cell dysfunction as well as lack of CAR T cell persistence have been discussed to be reasons for the lack of sustained therapy response. Well established models for the preclinical evaluation of cellular therapies are needed for the evaluation of next generation CAR T cells as well as novel combination therapies. Here, we describe a xenograft animal model of human acute lymphoblastic leukemia (NALM-6 cells) with a suboptimal treatment response to human CD19 CAR T cells from three different donors. First, the activation status and memory phenotype of the CAR T cells from the different donors was characterized by flow cytometry and the anti-tumoral efficacy of the CD19 CAR T cells was evaluated in an in vitro co-culture killing assay using luciferase expressing NALM-6 cells. Next, CD19 CAR T cells from three donors were injected in NALM-6_luc tumor bearing animals. The tumor growth was monitored by bioluminescence imaging and the phenotype of the transferred CAR T cells was checked by flow cytometry. The frequency of memory T cells differed between the donors whereas the T cells from all donors exhibited all low expression of activation markers. In vitro, all three donors showed high and comparable anti-tumoral killing efficacy independent of their memory status. In vivo, CD19 CAR T cells from all three donors delayed the tumor growth significantly although none of the animals had a complete remission. In line with the individual memory status of the CAR T cells prior to infusion, there were slight differences in the kinetics of the anti-tumoral response. The flow cytometric analysis revealed that the transferred CAR T cells were almost absent in the peripheral blood and spleen whereas for individual animals tumor cells and highly activated CAR T cells were detectable in the bone marrow of the animals. In summary, the NALM-6_luc xenograft model is a value tool for evaluating next generation cellular therapies and novel combination strategies to overcome the current limitations of cellular therapies. Citation Format: Philipp Metzger, Carla N. Castro, Jonas F. Hummel, Maria Silvia Roman Azcona, Dinu Antony, Claudio Mussolino, Daniel Zurr, Swetlana Adamsky, Holger Weber. Donor-dependent anti-tumoral efficacy of human CD19 CAR T cells in a leukemic xenograft mouse model abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4020.
Targeted genome editing in blood and immune cells enable new therapeutic applications, especially for infectious diseases. We present a GMP-compliant protocol to manufacture CCR5-edited CD34+ ...hematopoietic stem and precursor cells (HSPCs) with the goal to cure patients suffering from chronic infection with human immunodeficiency virus type 1 (HIV1). We hypothesize that genetic disruption of the CCR5 gene, which encodes the major HIV1 co-receptor, in HSPCs will give rise to an HIV-resistant immune system after transplantation. We have developed engineered nucleases based on transcription activator-like effector nucleases (TALENs) targeting CCR5. Electroporation of CD4+ T-cells and CD34+ HSPCs with mRNAs encoding TALENs revealed disruption of up to 80% of CCR5 alleles in CD4+ T-cells and over 90% of alleles in HSPCs. The high gene editing frequencies in T-cells and HSPCs were confirmed by deep sequencing, and no cleavage activity above background levels were detected at the top 20 predicted off-target sites. CCR5-edited CD4+ cells preserved their proliferation capacity and their biological function. Importantly, these cells showed significantly reduced CCR5 expression and became resistant to infection with the R5-tropic HIV-1JR-FL virus. The CCR5-edited HSPCs maintained their proliferation potential and their capacity to differentiate into the various blood lineages in vitro and in vivo, and clonal analysis revealed bi-allelic CCR5 disruption in more than 75% of cells. In summary, our developed protocol enables highly efficient and GMP-compliant knockout of the CCR5 locus in clinically relevant cells, so forming the foundation for a planned phase I/II clinical study.
Gautron:Cellectis SA: Employment. Busser:Cellectis: Employment, Patents & Royalties: Cellectis. Smith:Cellectis. Inc: Employment, Patents & Royalties. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis. Cathomen:TRACR Hematology: Consultancy; Cellectis: Research Funding; Miltenyi Biotec: Research Funding. Cornu:Cellectis: Research Funding; Miltenyi Biotec: Research Funding.
Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient ...bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.
Despite the recent success of gene‐based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain‐of‐function mutations poses great challenges. ...General therapeutic principles to correct these genetic defects mostly rely on post‐transcriptional gene regulation (RNA silencing). Engineered zinc‐finger (ZF) protein‐based repression of transcription may represent a novel approach for treating gain‐of‐function mutations, although proof‐of‐concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild‐type hRHO allele in a mutational‐independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc‐finger‐based mutation‐independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain‐of‐function mutations.
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