scholarly journals A Small Key for a Heavy Door: Genetic Therapies for the Treatment of Hemoglobinopathies

2021 ◽  
Vol 2 ◽  
Author(s):  
Hidde A. Zittersteijn ◽  
Cornelis L. Harteveld ◽  
Stefanie Klaver-Flores ◽  
Arjan C. Lankester ◽  
Rob C. Hoeben ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Fetal Hemoglobin ◽  
Clinical Genetics ◽  
Genetic Modifiers ◽  
Promoter Regions ◽  
Fertile Ground ◽  
Adult Hemoglobin

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.

scholarly journals β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies

2020 ◽  
Vol 2 ◽  
Author(s):  
Gloria Barbarani ◽  
Agata Łabedz ◽  
Antonella Ellena Ronchi
Keyword(s):  
Genome Editing ◽  
Test Bench ◽  
Fetal Hemoglobin ◽  
Therapeutic Interventions ◽  
Globin Genes ◽  
Inherited Disorders ◽  
Hematopoietic Stem ◽  
Hereditary Persistence ◽  
Adult Hemoglobin ◽  
Α Subunits

Hemoglobin is a tetrameric protein composed of two α and two β chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and β-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two β subunits (α2β2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in β-globin production cause two of the most common monogenic-inherited disorders: β-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of β-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.

Crispr-Cas9 Saturating Mutagenesis Reveals an Achilles Heel in the BCL11A Erythroid Enhancer for Fetal Hemoglobin Induction (by Genome Editing)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 638-638 ◽  
Author(s):  
Daniel E. Bauer ◽  
Matthew C. Canver ◽  
Elenoe C. Smith ◽  
Falak Sher ◽  
Luca Pinello ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Genome Editing ◽  
Genetic Variants ◽  
Research Funding ◽  
Gene Editing ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Advisory Committees ◽  
Hbf Level

Abstract Common genetic variation associated with fetal hemoglobin (HbF) level and β-hemoglobin disorder clinical severity marks an erythroid enhancer within the BCL11A gene. The 12 kb intronic enhancer contains three ~1 kb erythroid DNase I hypersensitive sites (DHSs), termed +55, +58, and +62. Here we utilized a human adult-stage erythroid cell line to show by CRISPR-Cas9 mediated targeted deletion that the composite enhancer is required both for BCL11A expression and HbF repression. Because deletion of the entire enhancer is currently too inefficient to consider for a gene editing approach to hemoglobin disorders, we sought to define the critical features of the enhancer in its natural genomic context. We designed and synthesized a tiling pooled guide RNA (gRNA) library to conduct saturating mutagenesis of the enhancer sequences in situ using the CRISPR-Cas9 gene editing platform. The gRNAs direct Cas9 cleavage and non-homologous end-joining repair at discrete sites throughout the enhancer. By comparing the representation of lentiviral gRNA integrants in high and low HbF pools of the adult erythroid cells, we generated a functional map approaching nucleotide resolution of sequences within the enhancer influencing BCL11A regulation. We observed several discrete enhancer regions required for maximal expression. The largest effect was observed by producing mutations within a narrow functional core of the +58 DHS. These sequences include a GATA1 motif conserved among vertebrates located within a primate-specific context. This region constitutes an Achilles Heel for functional inactivation of the enhancer. We also identified rare genetic variants within the +58 DHS core in individuals with sickle cell disease that are associated with HbF level, independent of all known associations of common genetic variants. In parallel, we performed a similar saturating CRISPR mutagenesis screen of the corresponding murine Bcl11a enhancer. To our surprise, despite low-resolution evidence of conservation by primary sequence homology, syntenic genomic position, and shared chromatin signature, the mouse enhancer sequence determinants of BCL11A expression showed substantial functional divergence. The +58 orthologous sequences were dispensable whereas the +62 orthologous sequences were critically required in murine adult erythroid cells. These results were validated by producing targeted deletions in mouse and human adult erythroid cell lines. Furthermore we subjected cells to individual gRNAs to correlate individual nucleotide disruptions with loss of BCL11A expression. To substantiate the tissue-restricted effect of the enhancer mutations, we generated transgenic mice with deletion of the Bcl11a enhancer and found these sequences were dispensable for expression in developing neurons and B-lymphocytes (unlike conventional Bcl11a knockout) but essential for appropriate hemoglobin switching in vivo. We showed that in primary CD34+ hematopoietic stem and progenitor derived human erythroid precursors that delivery of an individual gRNA and Cas9 is sufficient to produce robust reinduction of HbF. These results validate the BCL11A erythroid enhancer as a promising therapeutic target. Our findings define the most favorable regions for generation of indel mutations in the BCL11A erythroid enhancer as a therapeutic genome editing strategy for HbF reinduction for the β-hemoglobin disorders. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zhang:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Horizon Discovery: Membership on an entity's Board of Directors or advisory committees. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

scholarly journals Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 281
Author(s):  
Catherine Baker ◽  
Matthew S. Hayden
Keyword(s):  
Human Genome ◽  
Genome Editing ◽  
Gene Editing ◽  
Human Diseases ◽  
Therapeutic Application ◽  
Cutaneous Disease ◽  
Crispr System ◽  
Novel Approaches

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research.  We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.

Role of H3K18ac-regulated nucleotide excision repair-related genes in arsenic-induced DNA damage and repair of HaCaT cells

2020 ◽  
Vol 39 (9) ◽  
pp. 1168-1177 ◽  
Author(s):  
AL Zhang ◽  
L Chen ◽  
L Ma ◽  
XJ Ding ◽  
SF Tang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nucleotide Excision Repair ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
The Body ◽  
Hacat Cells ◽  
Transcriptional Expression ◽  
Promoter Regions ◽  
Nucleotide Excision

Arsenic is an environmental poison and is a grade I human carcinogen that can cause many types of damage to the body. The skin is one of the main target organs of arsenic damage, but the molecular mechanisms underlying arsenic poisoning are not clear. Arsenic is an epigenetic agent. Histone acetylation is one of the earliest covalent modifications to be discovered and is closely related to the occurrence and development of tumors. To investigate the role of acetylated histone H3K18 (H3K18 ac) in arsenic-induced DNA damage, HaCaT cells were exposed to sodium arsenite (NaAsO2) for 24 h. It was found that arsenic induced the downregulation of xeroderma pigmentosum A, D, and F ( XPA, XPD, and XPF—nucleotide excision repair (NER)-related genes) expression, as well as histone H3K18 ac expression, and aggravated DNA damage. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) analysis showed that H3K18 acetylation in the promoter regions of XPA, XPD, and XPF was downregulated. In addition, the use of the histone deacetylase inhibitor trichostatin A (TSA) partially inhibited arsenic-induced DNA damage, inhibited deacetylation of H3K18 ac in the promoter regions of XPA, XPD, and XPF genes, increased acetylation of H3K18, and promoted the transcriptional expression of NER-related genes. Our study revealed that NaAsO2 induces DNA damage and inhibits the expression of NER-related genes, while TSA increases the H3K18 ac enrichment level and promotes the transcriptional expression of NER, thereby inhibiting DNA damage. These findings provide new ideas for understanding the molecular mechanisms underlying arsenic-induced skin damage.

scholarly journals ROLE OF CRISPR TO IMPROVE ABIOTIC STRESS TOLERANCE IN CROP PLANTS

2021 ◽  
Vol 2021 (1) ◽  
Author(s):  
MU Farooq ◽  
MF Bashir ◽  
MUS Khan ◽  
B Iqbal ◽  
Q Ali
Keyword(s):  
Abiotic Stress ◽  
Genome Editing ◽  
Gene Editing ◽  
Abiotic Stress Tolerance ◽  
Genetically Engineered ◽  
Transcription Activator ◽  
Guide Rnas ◽  
Field Crops ◽  
Genetically Engineered Crops

The study for genetic variation in plant genomes for a variety of crops, as well as developments of genome editing techniques, have made it possible to cultivate for about any desired trait. Zinc finger enzymes; have made strides in genome-editing. Molecular biologists can now more specifically target every gene using transcription activator-like effector nucleases and ZFNs. These methods, on the other hand, are expensive and time-consuming because they involve complex procedures. Referring to various genome editing techniques, CRISPR/Cas9 genetic modification is simple to construct and clone and the Cas9 could be used with different guide RNAs controlling different genes. Following solid evidence demonstrations using the main CRISPR-Cas9 unit in field crops, multiple updated Cas9 cassettes are often used in plant species to improve target precision and reduce off target cleavage. Nmcas9, Sacas9, as well as Stcas9 are a few examples. Furthermore, Cas9 enzymes are readily available from a variety of sources. Bacteria that had never been discovered before has found solutions available to improve specificity and efficacy of gene editing techniques. The choices are summarized in this analysis to plant's experiment to develop crops using CRISPR/Cas9 technology; the tolerance of biotic & abiotic stress may be improved. These strategies will lead to the growth of non-genetically engineered crops with the target phenotype, which will further improve yield capacity under biotic & abiotic stress environments.

scholarly journals Potential of Mitochondrial Genome Editing for Human Fertility Health

2021 ◽  
Vol 12 ◽  
Author(s):  
Lin Fu ◽  
Yu-Xin Luo ◽  
Ying Liu ◽  
Hui Liu ◽  
Hong-zhen Li ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Genome Editing ◽  
Gene Editing ◽  
Rapid Development ◽  
Normal Functioning ◽  
Complex Processes ◽  
Human Fertility ◽  
Fertility Disorders ◽  
Made In

Mitochondrial DNA (mtDNA) encodes vital proteins and RNAs for the normal functioning of the mitochondria. Mutations in mtDNA leading to mitochondrial dysfunction are relevant to a large spectrum of diseases, including fertility disorders. Since mtDNA undergoes rather complex processes during gametogenesis and fertilization, clarification of the changes and functions of mtDNA and its essential impact on gamete quality and fertility during this process is of great significance. Thanks to the emergence and rapid development of gene editing technology, breakthroughs have been made in mitochondrial genome editing (MGE), offering great potential for the treatment of mtDNA-related diseases. In this review, we summarize the features of mitochondria and their unique genome, emphasizing their inheritance patterns; illustrate the role of mtDNA in gametogenesis and fertilization; and discuss potential therapies based on MGE as well as the outlook in this field.

ILF3 Mediated BMP2 and STAT1 Transcription Is Responsible for Hyperlipidemia-Induced Arteriosclerotic Calcification

2020 ◽  
Author(s):  
Fei Xie ◽  
Zhao-yang Wang ◽  
Bin Liu ◽  
Wen Qiao ◽  
Na Li ◽  
...  
Keyword(s):  
Atherosclerotic Plaque ◽  
Molecular Mechanisms ◽  
Plaque Rupture ◽  
Bone Morphogenetic Protein 2 ◽  
Signal Transducer ◽  
Promoter Regions ◽  
Morphogenetic Protein ◽  
Binding Factor ◽  
Precise Role

Abstract Calcification is common in atherosclerotic plaque and can induce vulnerability, which further leads to myocardial infarction, plaque rupture and stroke. The mechanisms of atherosclerotic calcification are poorly characterized. Interleukin enhancer binding factor 3 (ILF3) has been identified as a novel factor affecting dyslipidemia and stroke subtypes. However, the precise role of ILF3 in atherosclerotic calcification remains unclear. Here we showed that ILF3 expression is increased in calcified human aortic vascular smooth muscle cells (HAVSMCs) and calcified atherosclerotic plaque in humans and mice. We then found that hyperlipidemia increases ILF3 expression and exacerbates calcification of VSMCs and macrophages by regulating bone morphogenetic protein 2 (BMP2) and signal transducer and activator of transcription 1 (STAT1) transcription. We further explored the molecular mechanisms of ILF3 in atherosclerotic calcification and revealed that ILF3 acts on the promoter regions of BMP2 and STAT1 and mediates BMP2 upregulation and STAT1 downregulation, which promotes atherosclerotic calcification. Our results demonstrate the effect of ILF3 in atherosclerotic calcification. Inhibition of ILF3 may be a useful therapy for preventing and even reversing atherosclerotic calcification.

scholarly journals Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuemeng Zhao ◽  
Fei Wang ◽  
Changhong Wang ◽  
Xiaobai Zhang ◽  
Cizhong Jiang ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genome Editing ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Related Gene ◽  
Malaria Parasites ◽  
Human Malaria Parasite ◽  
Genome Feature ◽  
First Time ◽  
Transgenic Strains

Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.

Chromosomal Loop Editing Reveals New Epigenomic Landscape in the β-Globin Locus with Implications for Hemoglobinopathy Therapies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 813-813
Author(s):  
Pamela Himadewi ◽  
Xiaotian Zhang ◽  
Haley Gore ◽  
Xue Qing David Wang
Keyword(s):  
Gene Expression ◽  
Chromatin Structure ◽  
Gene Editing ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Chromatin Loop ◽  
Erythroid Progenitors ◽  
Hemoglobin Gene ◽  
Ctcf Site

Human β-globin locus consists of at least six genes encoding components of the oxygen transport protein hemoglobin and an upstream locus control region (LCR) containing five DNase I hypersensitive sites. In addition, there are at least four conserved CTCF insulator elements surrounding the locus, which form dynamic chromatin interaction patterns across different cell types in the course of hematopoietic stem and progenitor cells (HSPCs) development. Chromatin conformation of the β-globin locus in normal adult CD34+ HSPCs reveals the formation of three topological associated domains (TADs), in which individual TAD boundaries are demarcated by CTCF sites (Figure 1). By comparing chromatin loop interactions between CD34+ HSPCs and its differentiated erythroid progenitors, we identify a chromatin loop that forms in erythroid progenitors and is not evident in the CD34+ HSPCs. Detailed examination of this loop shows that a DNase I hypersensitivity site, also called 3'HS1, overlaps with a CTCF site that forms a loop with another CTCF site adjacent to OR52A5 gene (Figure 2). To investigate the role of this specific chromatin loop in the regulation of hemoglobin gene expression, we knock out the 3'HS1 CTCF motif in adult CD34+ HSPCs under erythroid differentiation medium by CRISPR/Cas9-mediated gene editing. We find that deletion of 3'HS1 CTCF results in a 2-fold decrease of β-globin (HBB) and a 4-fold increase of the fetal hemoglobin gene encoded by γ-globin (HBG1/2) in erythroid colonies of edited CD34+ cells (Figure 3). Elevation of fetal hemoglobin upon 3'HS1 CTCF deletion was also confirmed in human umbilical cord blood-derived erythroid progenitor-2 cells (HUDEP-2), which results in a 12-fold increase of γ-globin expression (Figure 4). These results suggest that the 3'HS1 CTCF plays a crucial role in regulating the expression of fetal hemoglobin gene, however it remains unclear whether changes in chromatin structure is responsible for these changes. CTCF looping interactions have been described to form under convergent directionality. To validate the role of 3'HS1 CTCF in establishing chromatin interactions at the β-globin locus, we aim to invert this binding motif and evaluate how it disrupts chromatin organization and gene expression at this locus. Deciphering the underlying mechanisms will shed light on how three-dimensional chromatin structure is reorganized in differentiating erythroid cells as it undergoes nuclear condensation. Furthermore, elevation of fetal hemoglobin expression can potentially be a new therapeutic gene editing strategy to treat sickle cell disease and some cases of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 281
Author(s):  
Catherine Baker ◽  
Matthew S. Hayden
Keyword(s):  
Human Genome ◽  
Genome Editing ◽  
Gene Editing ◽  
Human Diseases ◽  
Therapeutic Application ◽  
Cutaneous Disease ◽  
Crispr System ◽  
Novel Approaches

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research.  We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.

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