Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo

Asperti, Claudia; Canarutto, Daniele; Porcellini, Simona; Sanvito, Francesca; Cecere, Francesca; Vavassori, Valentina; Ferrari, Samuele; Rovelli, Elisabetta; Albano, Luisa; Jacob, Aurelien; Sergi Sergi, Lucia; Montaldo, Elisa; Ferrua, Francesca; González-Granado, Luis Ignacio; Lougaris, Vassilios; Badolato, Raffaele; Finocchi, Andrea; Villa, Anna; Radrizzani, Marina; Naldini, Luigi

doi:10.1016/j.omtm.2023.08.020

Cited by 4 publications

(4 citation statements)

References 22 publications

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“…Given the lower geno‐toxicity concerns observed for IDLV over AAV6 as template delivery vehicle in the context of HSPC gene editing (Ferrari et al , 2022), we favored the former for the development of scalable GMP compliant T‐cells manufacturing process (Asperti et al , 2023), whose editing reagents could be portable to HSPCs. We chose to include the low affinity nerve growth factor receptor (LNGFR) selector (Fig 1A) in the CD40LG corrective cassette, functionally coupled to gene correction by an Internal Ribosome Entry Sites (IRES) sequence to allow for in vitro enrichment of edited cells and better reconstitution of CD40L expression (Vavassori et al , 2021) and function, based on the following data.…”

Section: Resultsmentioning

confidence: 99%

“…On day 2 cells were transduced at 1 × 10 6 cells/ml with CD40LG‐IRES_LNGFR‐IDLV at MOI 40, and after 8 ± 1 h electroporated with SpyFi™ high fidelity (HiFi) Cas9 complexed with a single guide RNA (gRNA) targeting the first intron of CD40LG . On day 6 cells were optionally enriched by immunomagnetic CD271 beads (Mitenyi Biotec) (Asperti et al , 2023).…”

Section: Methodsmentioning

confidence: 99%

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Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4⁺ T‐cells for the treatment of Hyper IgM1

Canarutto,

Asperti,

Vavassori

et al. 2023

The EMBO Journal

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Hyper IgM1 is an X‐linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T‐cell gene editing with Cas9 and a “one‐size‐fits‐most” corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long‐range gene editing, and no consensus on the relevant assays. We developed drop‐off digital PCR assays for unbiased detection of large on‐target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on‐target deletions were counter‐selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on‐target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4⁺ T‐cells for the treatment of Hyper IgM1

Canarutto,

Asperti,

Vavassori

et al. 2023

The EMBO Journal

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“…Importantly, CD40L activity was restored in gene-edited cells in vitro and in vivo, without signs of aberrant differentiation. Recently, large-scale good manufacturing practice (GMP)-compliant production processes have been developed, bringing this therapy closer to clinical application [81].…”

Section: Primary Immunodeficienciesmentioning

confidence: 99%

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

Laurent,

Geoffroy,

Pavani

et al. 2024

Cells

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In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome.

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“…Asperti et al. 14 develop a GMP-compliant process using an integrase-defective lentiviral vector for gene editing of CD4 + T cells, enhancing safety and effectiveness in treating hyper-IgM1, a severe immunodeficiency. In a similar vein, Lydeard et al.…”

Section: Main Textmentioning

confidence: 99%

Biomanufacturing in gene and cell therapy

Stone,

Wang,

Abou-el-Enein

2024

Molecular Therapy - Methods & Clinical Development

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Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo

Cited by 4 publications

References 22 publications

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4⁺ T‐cells for the treatment of Hyper IgM1

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4⁺ T‐cells for the treatment of Hyper IgM1

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

Biomanufacturing in gene and cell therapy

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Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo

Cited by 4 publications

References 22 publications

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T‐cells for the treatment of Hyper IgM1

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T‐cells for the treatment of Hyper IgM1

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

Biomanufacturing in gene and cell therapy

Contact Info

Product

Resources

About

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4⁺ T‐cells for the treatment of Hyper IgM1

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4⁺ T‐cells for the treatment of Hyper IgM1