Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells

Shin, Jiyung; Schröder, Markus; Caiado, Francisco; Wyman, Stacia K.; Bray, Nicolas; Bordi, Matteo; Dewitt, Mark; Vu, Jonathan T.; Kim, Won‐Tae; Hockemeyer, Dirk; Manz, Markus G.; Corn, Jacob E.

doi:10.1016/j.celrep.2020.108093

Cited by 66 publications

(70 citation statements)

References 96 publications

(145 reference statements)

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“…This resulted in the production of a relevant fraction of NHEJ-mediated InDels in the HBB gene, which can lead to β S -globin gene inactivation and the undesired generation of a β-thalassemic phenotype [ 52 ]. Interestingly, strategies to increase HDR efficiency in HSCs showed encouraging results and hold promise for a clinical application of gene correction strategies based on HDR [ 53 , 54 ].…”

Section: Nuclease-mediated Strategies For β-Globin Gene Repairmentioning

confidence: 99%

Genome Editing for β-Hemoglobinopathies: Advances and Challenges

Frati

Miccio

2021

JCM

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β-hemoglobinopathies are the most common genetic disorders worldwide and are caused by mutations affecting the production or the structure of adult hemoglobin. Patients affected by these diseases suffer from anemia, impaired oxygen delivery to tissues, and multi-organ damage. In the absence of a compatible donor for allogeneic bone marrow transplantation, the lifelong therapeutic options are symptomatic care, red blood cell transfusions and pharmacological treatments. The last decades of research established lentiviral-mediated gene therapy as an efficacious therapeutic strategy. However, this approach is highly expensive and associated with a variable outcome depending on the effectiveness of the viral vector and the quality of the cell product. In the last years, genome editing emerged as a valuable tool for the development of curative strategies for β-hemoglobinopathies. Moreover, due to the wide range of its applications, genome editing has been extensively used to study regulatory mechanisms underlying globin gene regulation allowing the identification of novel genetic and pharmacological targets. In this work, we review the current advances and challenges of genome editing approaches to β-hemoglobinopathies. Special focus has been directed towards strategies aimed at correcting the defective β-globin gene or at inducing fetal hemoglobin (HbF), which are in an advanced state of clinical development.

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Section: Nuclease-mediated Strategies For β-Globin Gene Repairmentioning

confidence: 99%

Genome Editing for β-Hemoglobinopathies: Advances and Challenges

Frati

Miccio

2021

JCM

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“…Cell cycle plays a critical role in DNA repair decisions after induction of DSB within the cellular genome. Quiescent HSPCs do not use HDR [ 95 ]. A two-day, low-density culture in medium supplemented with cytokines and small molecules that favor HSPC maintenance and self-renewal, such as prostaglandin E2 (PGE2) [ 96 ], StemRegenin1 (SR1) [ 97 ], and the pyrimidinone derivative UM171 [ 98 ] is thus routinely used to coax HSPCs into cycle and promote more efficient HDR [ 99 , 100 ].…”

Section: Strategies To Improve Hdr-mediated Gene Editing In Hspcsmentioning

confidence: 99%

“…However, HSPCs in the active phases of the cell cycle engraft poorly compared to quiescent cells [ 101 ]. A two-pronged strategy was recently proposed to address this quandary [ 95 ]. Cells were first allowed to cycle in culture medium containing cytokines to promote the accumulation of alleles corrected by HDR, and then reverted back to a G0 quiescent state to maintain stemness and long-term engraftment potential of edited cells.…”

Section: Strategies To Improve Hdr-mediated Gene Editing In Hspcsmentioning

confidence: 99%

“…Quiescence was re-induced after gene editing by a three-day culture in medium supplemented with regulators of Wnt (CHIR9901) and mammalian target of rapamycin (mTOR) (rapamycin) pathways [ 102 ]. This approach increased the number of HDR editing events by five-fold up to almost 30% of alleles in HSPCs capable of long-term engraftment in immune-deficient recipient mice, suggesting that HDR-edited stem cells had re-entered G0 and sustained long-term hematopoiesis in vivo [ 95 ]. Several strategies have also focused on small molecules that directly modulate regulators of the cell cycle.…”

Section: Strategies To Improve Hdr-mediated Gene Editing In Hspcsmentioning

confidence: 99%

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Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

Salisbury-Ruf

Larochelle

2021

JCM

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Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) is a promising strategy for the treatment of inherited blood disorders, obviating many of the limitations associated with viral vector-mediated gene therapies. The use of CRISPR/Cas9 or other programmable nucleases and improved methods of homology template delivery have enabled precise ex vivo gene editing. These transformative advances have also highlighted technical challenges to achieve high-efficiency gene editing in HSPCs for therapeutic applications. In this review, we discuss recent pre-clinical investigations utilizing homology-mediated gene editing in HSPCs and highlight various strategies to improve editing efficiency in these cells.

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“…Alternatively, Cas9 expression is synchronized in S/G 2 /M by controlled timing of CRISPR/Cas9 RNP complex delivery 71 or using a fusion of Cas9 and the N-terminal region of human Geminin 25 . HDR has also been successfully activated in quiescent stem cells and primary cells through controlled cycling and quiescence 72 . In this case, these quiescent cells are induced to briefly enter the cell cycle for HDR-based gene editing using the timed administration of a small-molecule cocktail (for example, SCF, TPO, and Flt3L).…”

Section: Homology-dependent Gene Knock-in and Gene Correction Strategmentioning

confidence: 99%

CRISPR-based strategies for targeted transgene knock-in and gene correction

Lau

Tin

Suh

2020

Fac Rev

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The last few years have seen tremendous advances in CRISPR-mediated genome editing. Great efforts have been made to improve the efficiency, specificity, editing window, and targeting scope of CRISPR/Cas9-mediated transgene knock-in and gene correction. In this article, we comprehensively review recent progress in CRISPR-based strategies for targeted transgene knock-in and gene correction in both homology-dependent and homology-independent approaches. We cover homology-directed repair (HDR), synthesis-dependent strand annealing (SDSA), microhomology-mediated end joining (MMEJ), and homology-mediated end joining (HMEJ) pathways for a homology-dependent strategy and alternative DNA repair pathways such as non-homologous end joining (NHEJ), base excision repair (BER), and mismatch repair (MMR) for a homology-independent strategy. We also discuss base editing and prime editing that enable direct conversion of nucleotides in genomic DNA without damaging the DNA or requiring donor DNA. Notably, we illustrate the key mechanisms and design principles for each strategy, providing design guidelines for multiplex, flexible, scarless gene insertion and replacement at high efficiency and specificity. In addition, we highlight next-generation base editors that provide higher editing efficiency, fewer undesired by-products, and broader targeting scope.

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Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells

Cited by 66 publications

References 96 publications

Genome Editing for β-Hemoglobinopathies: Advances and Challenges

Genome Editing for β-Hemoglobinopathies: Advances and Challenges

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

CRISPR-based strategies for targeted transgene knock-in and gene correction

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