CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

Geoffroy, Marine; Pili, Louna; Buffa, Valentina; Caroff, Maëlle; Bigot, Anne; Gicquel, Evelyne; Rouby, Grégory; Richard, Isabelle; Fragnoud, Romain

doi:10.3390/cells12202444

Cited by 2 publications

(1 citation statement)

References 53 publications

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“…Recently, Tremblay's lab produced DMD lines with specific point mutations in exons 9, 20, 35, 43, 55, and 61 using prime editing [121]. These cell lines and associated subsequent assays [120,122,123] represent powerful tools to evaluate and optimize therapeutic strategies, screen for effective drugs, and gain insights in the disease pathophysiology.…”

Section: Crispr/cas9 and Generation Of Dmd Study Modelsmentioning

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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Section: Crispr/cas9 and Generation Of Dmd Study Modelsmentioning

confidence: 99%

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

Laurent,

Geoffroy,

Pavani

et al. 2024

Cells

Self Cite

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Establishment of FUT8 gene knockout CHO cell line with stable expression of monoclonal antibody

Gao,

Zhang,

et al. 2023

Preprint

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Background: Afucosylated antibodies have been become increasingly popular in clinical trials for therapeutic purposes owing to the improved efficacy compared to the fucosylated counterparts. The elimination of fucose on the heavy chain of an antibody can enhance the activity of antibody-dependent cellular cytotoxicity (ADCC). However, antibodies produced by Chinese hamster ovary (CHO) cells are heavily fucosylated, and the modification reduces the activity of ADCC. FUT8,GMD and FX encoding major fucose modification enzymes, which were knockout respectively in CHO cells using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). These gene knockout cell lines were evaluated for cell growth, antibody yield, N-glycan profiles and ADCC activities. Results: The results showed that the gene knockout cell lines exhibited morphology and growth kinetics similar to those of the wild CHO cells, and produced completely afucosylated recombinant antibodies. Among them, the ADCC activity of cell pool and monoclonal cell line with FUT8 gene knocked out (FUT8KO) were increased 2-2.5-fold comparing to conventional fucosylated antibodies. Conclusions: Our results indicated that FUT8KO monoclonal clone cell lines (#08E9, 05E9, 20D8, 12C4, 01E9, and 01F4) were ideal hosts for stable production of completely afucosylated high-ADCC activity antibodies, with considerable therapeutic quality and efficacy.

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CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

Cited by 2 publications

References 53 publications

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

Establishment of FUT8 gene knockout CHO cell line with stable expression of monoclonal antibody

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