Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

Ravi, Nithin Sam; Wienert, Beeke; Wyman, Stacia K.; Bell, Henry W.; George, Anila; Mahalingam, Gokulnath; Vu, Jonathan T.; Prasad, Kishore Chandra; Bandlamudi, Bhanu Prasad; Devaraju, Nivedhitha; Rajendiran, Vignesh; Syedbasha, Nazar; Pai, Aswin Anand; Nakamura, Yukio; Kurita, Ryo; Narayanasamy, Muthuraman; Balasubramanian, Poonkuzhali; Thangavel, Saravanabhavan; Marepally, Srujan; Velayudhan, Shaji R; Srivastava, Alok; Dewitt, Mark; Crossley, Merlin; Corn, Jacob E.; Mohankumar, Kumarasamypet M.

doi:10.7554/elife.65421

Cited by 32 publications

(33 citation statements)

References 75 publications

(126 reference statements)

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“…Previous studies have demonstrated the use of base editors to create or correct specific mutations in different genomic loci in various genetic disorders (28,(36)(37)(38). Most studies in β hemoglobinopathies have demonstrated the therapeutic up-regulation of fetal globin expression by introducing HPFH-like mutations or by disruption of fetal globin repressors using base editors to compensate for the deficient β-globin (23). Despite this, only a few attempts have been performed to address the correction of β-thalassemia point mutations using a base editing strategy.…”

Section: Discussionmentioning

confidence: 99%

“…HUDEP-2 cell line were cultured in Stemspan SFEM-II (StemCell Technologies) supplemented with 1 μM dexamethasone (Alfa Aesar), 1 μg/ml doxycycline (Sigma-Aldrich), 1X L-glutamine 200mM (Gibco™), 1X pen-Strep (Gibco™), SCF 50ng/ml (ImmunoTools) and 3 U/ml EPO (Zyrop 4000 IU injection) ( Kurita et al, 2013). HUDEP-2 cells were further differentiated into erythroid lineage using eight days differentiation protocol as previously described (23).…”

Section: Methodsmentioning

confidence: 99%

“…For preparing ABE, CBE, and ABE8e stables in HBB deleted HUDEP-2 cell harbouring thalassemia point mutations, lentivirus produced with pLenti-FNLS-P2A-Puro (Addgene#110841-CBE), pLenti-ABERA-P2A-Puro (Addgene#112675-ABE ) gift from Lukas Dow (42) and pLenti-ABE8e-Puro were used. The pLenti-ABE8e-Puro plasmid was constructed from ABE8e (TadA-8e V106W) (Addgene#138495-ABE8e), a gift from David Liu (43), as previously reported (23).…”

Section: Plasmid Constructmentioning

confidence: 99%

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Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

Prasad

Devaraju

George

et al. 2022

Preprint

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β-thalassemia and HbE result from mutations in the β-globin locus that impedes the production of functional β-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of β-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Plasmid Constructmentioning

confidence: 99%

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Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

Prasad

Devaraju

George

et al. 2022

Preprint

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“…However, for diseases such as SCD (20A<T) or the β-thalassemia A>G variant that is caused by well-documented point mutations, a new technology known as ‘base editing’ provides a suitable non-DSB-reliant alternative. Additionally, the upstream region of γ-globin promoter which includes transcriptional factor binding sites, such as BCL11A , LRF , KLF1 , and GATA1 are also possible base-editing targets for HbF induction to treat SCD and β-thalassemia [ 112 , 113 , 114 ].…”

Section: Hsc-targeted Gene-editing Therapy In Scdmentioning

confidence: 99%

Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease

Germino-Watnick

Hinds

et al. 2022

Cells

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Autologous hematopoietic stem cell (HSC)-targeted gene therapy provides a one-time cure for various genetic diseases including sickle cell disease (SCD) and β-thalassemia. SCD is caused by a point mutation (20A > T) in the β-globin gene. Since SCD is the most common single-gene disorder, curing SCD is a primary goal in HSC gene therapy. β-thalassemia results from either the absence or the reduction of β-globin expression, and it can be cured using similar strategies. In HSC gene-addition therapy, patient CD34+ HSCs are genetically modified by adding a therapeutic β-globin gene with lentiviral transduction, followed by autologous transplantation. Alternatively, novel gene-editing therapies allow for the correction of the mutated β-globin gene, instead of addition. Furthermore, these diseases can be cured by γ-globin induction based on gene addition/editing in HSCs. In this review, we discuss HSC-targeted gene therapy in SCD with gene addition as well as gene editing.

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“…The fact is that β-thalassemia and SCD patients suffer from a less severe disorder when HbF is increased; therefore, HbF re-expression could be considered as a beneficial therapeutic approach for these diseases. Some animal and human studies have reported the usage of GETs for HbF elevation [ 42 , 43 , 44 ]. Strategies for HbF induction in adult RBCs deploying GETs can be summarized as inducing natural mutations regarding enhanced HbF level, knocking out repressors of HbF and modifying intermediates of epigenetic to control HbF synthesis [ 45 ].…”

Section: Hbf or Gamma Globin Induction Using Gene Editing Toolsmentioning

confidence: 99%

Gene Editing-Based Technologies for Beta-hemoglobinopathies Treatment

Rahimmanesh

Boshtam

Kouhpayeh

et al. 2022

Biology

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Beta (β)-thalassemia is a group of human inherited abnormalities caused by various molecular defects, which involves a decrease or cessation in the balanced synthesis of the β-globin chains in hemoglobin structure. Traditional treatment for β-thalassemia major is allogeneic bone marrow transplantation (BMT) from a completely matched donor. The limited number of human leukocyte antigen (HLA)-matched donors, long-term use of immunosuppressive regimen and higher risk of immunological complications have limited the application of this therapeutic approach. Furthermore, despite improvements in transfusion practices and chelation treatment, many lingering challenges have encouraged researchers to develop newer therapeutic strategies such as nanomedicine and gene editing. One of the most powerful arms of genetic manipulation is gene editing tools, including transcription activator-like effector nucleases, zinc-finger nucleases, and clustered regularly interspaced short palindromic repeat–Cas-associated nucleases. These tools have concentrated on γ- or β-globin addition, regulating the transcription factors involved in expression of endogenous γ-globin such as KLF1, silencing of γ-globin inhibitors including BCL11A, SOX6, and LRF/ZBTB7A, and gene repair strategies. In this review article, we present a systematic overview of the appliances of gene editing tools for β-thalassemia treatment and paving the way for patients’ therapy.

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Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

Cited by 32 publications

References 75 publications

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease

Gene Editing-Based Technologies for Beta-hemoglobinopathies Treatment

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