Off-target effects in CRISPR/Cas9 gene editing

Frontiers in Bioengineering and Biotechnology, Journal Year: 2023, Volume and Issue: 11

Published: March 9, 2023

Gene editing stands for the methods to precisely make changes a specific nucleic acid sequence. With recent development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, gene has become efficient, convenient and programmable, leading promising translational studies clinical trials both genetic non-genetic diseases. A major concern in applications CRISPR/Cas9 system is about its off-target effects, namely deposition unexpected, unwanted, or even adverse alterations genome. To date, many have been developed nominate detect sites CRISPR/Cas9, which laid basis successful upgrades derivatives with enhanced precision. In this review, we summarize these technological advancements discuss current challenges management effects future therapy.

Language: Английский

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Genotoxic effects of base and prime editing in human hematopoietic stem cells

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 42(6), P. 877 - 891

Published: Sept. 7, 2023

Base and prime editors (BEs PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses genotoxicity. Here, we compared state-of-the-art BEs PEs Cas9 in human hematopoietic stem progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes on-target genome-wide induced detrimental transcriptional that reduced efficiency repopulation xenotransplants also generated breaks genotoxic byproducts, including deletions translocations, at a lower frequency Cas9. These effects were strongest for cytidine due suboptimal inhibition of base excision repair mitigated by tailoring delivery timing editor expression through optimized mRNA design. altered mutational landscape across genome increasing load relative proportions nucleotide variants. findings raise concerns genotoxicity warrant further investigation view clinical application.

Language: Английский

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Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice

Nature Biomedical Engineering, Journal Year: 2023, Volume and Issue: 7(5), P. 616 - 628

Published: April 17, 2023

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β-globin gene (HBB). Here we show that prime editing can correct SCD allele (HBB

Language: Английский

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Epitope editing enables targeted immunotherapy of acute myeloid leukaemia

Nature, Journal Year: 2023, Volume and Issue: 621(7978), P. 404 - 414

Published: Aug. 30, 2023

Abstract Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia 1,2 , broader applicability of adoptive immunotherapies is hampered by absence tumour-restricted antigens 3–5 . Acute myeloid target genes expressed haematopoietic stem/progenitor cells (HSPCs) or differentiated cells, resulting intolerable on-target/off-tumour toxicity. Here we show that epitope engineering donor HSPCs used for bone marrow transplantation endows lineages with selective resistance to chimeric antigen receptor (CAR) T monoclonal antibodies, without affecting protein function regulation. This strategy enables are essential survival regardless shared expression on HSPCs, reducing risk tumour immune escape. By performing mapping and library screenings, identified amino acid changes abrogate binding therapeutic antibodies FLT3, CD123 KIT, optimized base-editing approach introduce them into CD34 + which retain long-term engraftment multilineage differentiation ability. After CAR treatment, confirmed epitope-edited haematopoiesis concomitant eradication patient-derived xenografts. Furthermore, multiplex feasible more effective against multiple targets incurring overlapping off-tumour toxicities. We envision this will provide opportunities treat relapsed/refractory enable safer non-genotoxic conditioning.

Language: Английский

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Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(13), P. 7333 - 7333

Published: July 4, 2024

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it enabled precise genome editing with unprecedented accuracy and efficiency, paving way for clinical applications to treat otherwise incurable genetic disorders. Typically, requires delivery multiple components target cells that, depending on platform used, may include messenger RNA (mRNA), complexes, DNA fragments. For purposes, these have be efficiently delivered into transplantable cells, such primary T lymphocytes or hematopoietic stem progenitor that are typically sensitive exogenous substances. This challenge limited broad applicability those strategies which efficient methods available. Electroporation-based methodologies been generally applied applications, but procedure-associated toxicity represented a major burden. With advent novel less disruptive deliver cargo is now possible safely editing, thus expanding strategies. In this review, we describe different systems available components, including viral non-viral systems, highlighting their advantages, limitations, recent applications. Recent improvements achieve cell specificity represent critical development enable in vivo targeting future will certainly play pivotal role field.

Language: Английский

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Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: April 8, 2024

Language: Английский

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Advanced Delivery Systems for Gene Editing: A Comprehensive Review from the GenE-HumDi COST Action Working group

Molecular Therapy — Nucleic Acids, Journal Year: 2025, Volume and Issue: 36(1), P. 102457 - 102457

Published: Jan. 18, 2025

Language: Английский

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Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells

Blood, Journal Year: 2023, Volume and Issue: 142(9), P. 812 - 826

Published: June 9, 2023

Ex vivo gene editing in T cells and hematopoietic stem/progenitor (HSPCs) holds promise for treating diseases. Gene encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex via electroporation, when aiming homology-driven correction DNA template, provided by viral vectors together with nuclease editor. Although HSPCs activate robust p53-dependent damage response upon nuclease-based editing, responses triggered remain poorly characterized. Here, we performed comprehensive multiomics analyses found that electroporation is main culprit cytotoxicity cells, causing death cell cycle delay, perturbing metabolism, inducing an inflammatory response. Nuclease using lipid nanoparticles (LNPs) nearly abolished ameliorated growth, improving tolerance to procedure yielding higher number edited compared electroporation. Transient transcriptomic changes LNP treatment were mostly caused cellular loading exogenous cholesterol, whose potentially detrimental impact could be overcome limiting exposure. Notably, LNP-based HSPC dampened p53 pathway induction supported clonogenic activity similar reconstitution long-term repopulating reaching comparable efficiencies. Overall, LNPs may allow efficient harmless human

Language: Английский

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Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy

Cell stem cell, Journal Year: 2023, Volume and Issue: 30(5), P. 549 - 570

Published: May 1, 2023

Language: Английский

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Hemophilia Gene Therapy: The End of the Beginning?

Human Gene Therapy, Journal Year: 2023, Volume and Issue: 34(17-18), P. 782 - 792

Published: Sept. 1, 2023

Extensive preclinical research over the past 30 years has culminated in recent regulatory approval of several gene therapy products for hemophilia. Based on efficacy and safety data a recently conducted phase III clinical trial, Roctavian® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing B domain deleted factor VIII (FVIII) complementary DNA, was approved by European Commission Food Drug Administration (FDA) treatment patients with severe hemophilia A. In addition, Hemgenix® (etranacogene dezaparvovec) also Medicines Agency FDA B. This product is based AAV5 hyper-active IX (FIX) transgene (FIX-Padua) transgene. All AAV-based trials to date show significant increase FVIII or FIX levels majority treated patients, consistent substantial decrease bleeding episodes concomitant reduction usage obviating need prophylaxis most patients. However, interpatient variability remains that not fully understood. Moreover, encountered short-term asymptomatic liver inflammation immune suppression corticosteroids other suppressants. all date, expression appeared relatively more stable than FVIII, though individual had prolonged expression. Whether lifelong clotting factors can be realized after requires longer follow-up studies. Further development next-generation editing technologies offers new prospects sustained cure hemophilia, only adults, but ultimately children too.

Language: Английский

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