Genome editing strategies for fetal hemoglobin induction in beta-hemoglobinopathies

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Abstract

Genome editing to correct a defective β-globin gene or induce fetal globin (HbF) for patients with beta-hemoglobinopathies has the potential to be a curative strategy available to all. HbF reactivation has long been an area of intense interest given the HbF inhibition of sickle hemoglobin (HbS) polymerization. Patients with HbS who also have high HbF tend to have less severe or even minimal clinical manifestations. Approaches to genetically engineer high HbF include de novo generation of naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations, editing of transcriptional HbF repressors or their binding sites and/or regulating epigenetic intermediates controlling HbF expression. Recent preclinical and early clinical trial data show encouraging results; however, long-term follow-up is lacking, and the safety and efficacy concerns of genome editing remain.

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Most cited references 70

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BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

Matthew Canver, Elenoe Smith, Falak Sher … (2015)

Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements.

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Identification of preexisting adaptive immunity to Cas9 proteins in humans

Carsten T. Charlesworth, Priyanka S Deshpande, Daniel Dever … (2019)

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

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Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor.

Wulan Deng, Jongjoo Lee, Hongxin Wang … (2012)

Chromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts, in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation. Copyright © 2012 Elsevier Inc. All rights reserved.

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Author and article information

Contributors

Selami Demirci: (View ORCID Profile)

Journal

Title: Human Molecular Genetics

Publisher: Oxford University Press (OUP)

ISSN (Print): 0964-6906

ISSN (Electronic): 1460-2083

Publication date Created: September 15 2020

Publication date Created: September 30 2020

Publication date Created: May 14 2020

Publication date Other: September 15 2020

Publication date (Print): September 30 2020

Publication date (Electronic): May 14 2020

Volume: 29

Issue: R1

Pages: R100-R106

Affiliations

[1 ]Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institutes, National Institutes of Health, Bethesda, MD, USA

Article

DOI: 10.1093/hmg/ddaa088

PubMed ID: 32406490

SO-VID: 9fa370dc-fa8c-4092-bd44-9ea5d935f0d9

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