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Gene Editing for Therapy and Reverse Genetics of Blood Diseases

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A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Human Genomics and Genetic Diseases".

Deadline for manuscript submissions: closed (20 March 2023) | Viewed by 11651

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Special Issue Editor

Dr. Carsten W. Lederer

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Guest Editor

Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus
Interests: advanced therapies; genetic modifiers; regulation of globin expression; rare anemias
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Gene editing is increasingly taking center stage for both basic research and translational studies. The key driver of this development is the rapidly growing adoption of editing technology and, in particular, fast-evolving RNA-guided CRISPR/Cas tools, which, with their versatility and ease of use, have facilitated the development of double-strand-break-independent editors and the exploration of new fields of application. In particular blood biology and disorders are a favorite focus of gene editing, motivated by a relatively high prevalence of monogenic, infectious and complex diseases affecting blood cells, and helped by the accessibility of hematopoietic stem and progenitor cells for manipulation.

This Special Issue aims to showcase the application of gene editing technology in therapy development and research for blood diseases. In this context, we welcome Articles, Communications and Reviews providing new insights into (i) developmental and disease mechanisms; (ii) the establishment or study of disease models; (iii) the creation of new editing platforms and molecules for diagnosis, functional study or therapy; and (vi) the refinement of corresponding delivery, targeting and culture procedures.

The topics of interest in the context of gene editing and blood biology include but are not limited to:

Editing primary disease-causing mutations;
Editing disease modifiers, developmental regulators and pathologically relevant pathways;
The treatment or development of cellular and animal models by editing;
Improvements in post-editing cell survival, stemness and engraftment behavior;
Improvements in the in vivo or in vitro efficiency and specificity of delivery for editing tools;
The improvement of or insights into in vivo and ex vivo selection mechanisms for editing events;
Comparisons of editing platforms, of gene therapy strategies and of vectors for the delivery of editors and/or templates;
New or improved editing technology for RNA, the nuclear or mitochondrial genome and the epigenome;
The application of editing technology for the diagnosis and for the detection and verification of disease biomarkers;
The modification of the balance between homology-directed repair, non-homologous end joining and other editing events;
Safety assessment and optimization for on- and off-target activity.

Dr. Carsten W. Lederer
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas))
TALEN (Tal effector nuclease)
ZFN (zinc finger nuclease)
Triplex-forming nucleic acid analog
Meganuclease/homing endonuclease
Nanoparticles
Viral vector
Gene therapy
Hematopoiesis
Erythropoiesis

Published Papers (4 papers)

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Research

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15 pages, 1537 KiB

Open AccessArticle

Allele-Specific Disruption of a Common STAT3 Autosomal Dominant Allele Is Not Sufficient to Restore Downstream Signaling in Patient-Derived T Cells

by Saskia König, Manfred Fliegauf, Manuel Rhiel, Bodo Grimbacher, Tatjana I. Cornu, Toni Cathomen and Claudio Mussolino

Genes 2022, 13(10), 1912; https://doi.org/10.3390/genes13101912 - 20 Oct 2022

Cited by 1 | Viewed by 1558

Abstract

Dominant negative mutations in the STAT3 gene account for autosomal dominant hyper-IgE syndrome (AD-HIES). Patients typically present high IgE serum levels, recurrent infections, and soft tissue abnormalities. While current therapies focus on alleviating the symptoms, hematopoietic stem cell transplantation (HSCT) has recently been proposed as a strategy to treat the immunological defect and stabilize the disease, especially in cases with severe lung infections. However, because of the potentially severe side effects associated with allogeneic HSCT, this has been considered only for a few patients. Autologous HSCT represents a safer alternative but it requires the removal of the dominant negative mutation in the patients’ cells prior to transplantation. Here, we developed allele-specific CRISPR-Cas9 nucleases to selectively disrupt five of the most common STAT3 dominant negative alleles. When tested ex vivo in patient-derived hematopoietic cells, allele-specific disruption frequencies varied in an allele-dependent fashion and reached up to 62% of alleles harboring the V637M mutation without detectable alterations in the healthy STAT3 allele. However, assessment of the gene expression profiles of the STAT3 downstream target genes revealed that, upon activation of those edited patient cells, mono-allelic STAT3 expression (functional haploinsufficiency) is not able to sufficiently restore STAT3-dependent signaling in edited T cells cultured in vitro. Moreover, the stochastic mutagenesis induced by the repair of the nuclease-induced DNA break could further contribute to dominant negative effects. In summary, our results advocate for precise genome editing strategies rather than allele-specific gene disruption to correct the underlying mutations in AD-HIES. Full article

(This article belongs to the Special Issue Gene Editing for Therapy and Reverse Genetics of Blood Diseases)

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18 pages, 1849 KiB

Open AccessArticle

Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β⁰39-Globin Gene Editing and Induction of Fetal Hemoglobin

by Lucia Carmela Cosenza, Cristina Zuccato, Matteo Zurlo, Roberto Gambari and Alessia Finotti

Genes 2022, 13(10), 1727; https://doi.org/10.3390/genes13101727 - 26 Sep 2022

Cited by 4 | Viewed by 2267

Abstract

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β⁰39-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β⁰39-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β⁰39-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF. Full article

(This article belongs to the Special Issue Gene Editing for Therapy and Reverse Genetics of Blood Diseases)

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Review

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24 pages, 2372 KiB

Open AccessReview

Epigenetic Regulation of β-Globin Genes and the Potential to Treat Hemoglobinopathies through Epigenome Editing

by Letizia Fontana, Zoe Alahouzou, Annarita Miccio and Panagiotis Antoniou

Genes 2023, 14(3), 577; https://doi.org/10.3390/genes14030577 - 25 Feb 2023

Cited by 2 | Viewed by 3749

Abstract

Beta-like globin gene expression is developmentally regulated during life by transcription factors, chromatin looping and epigenome modifications of the β-globin locus. Epigenome modifications, such as histone methylation/demethylation and acetylation/deacetylation and DNA methylation, are associated with up- or down-regulation of gene expression. The understanding of these mechanisms and their outcome in gene expression has paved the way to the development of new therapeutic strategies for treating various diseases, such as β-hemoglobinopathies. Histone deacetylase and DNA methyl-transferase inhibitors are currently being tested in clinical trials for hemoglobinopathies patients. However, these approaches are often uncertain, non-specific and their global effect poses serious safety concerns. Epigenome editing is a recently developed and promising tool that consists of a DNA recognition domain (zinc finger, transcription activator-like effector or dead clustered regularly interspaced short palindromic repeats Cas9) fused to the catalytic domain of a chromatin-modifying enzyme. It offers a more specific targeting of disease-related genes (e.g., the ability to reactivate the fetal γ-globin genes and improve the hemoglobinopathy phenotype) and it facilitates the development of scarless gene therapy approaches. Here, we summarize the mechanisms of epigenome regulation of the β-globin locus, and we discuss the application of epigenome editing for the treatment of hemoglobinopathies. Full article

(This article belongs to the Special Issue Gene Editing for Therapy and Reverse Genetics of Blood Diseases)

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23 pages, 443 KiB

Open AccessReview

In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations

by Nikoletta Psatha, Kiriaki Paschoudi, Anastasia Papadopoulou and Evangelia Yannaki

Genes 2022, 13(12), 2222; https://doi.org/10.3390/genes13122222 - 27 Nov 2022

Cited by 6 | Viewed by 2827

Abstract

The tremendous evolution of genome-editing tools in the last two decades has provided innovative and effective approaches for gene therapy of congenital and acquired diseases. Zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR-Cas9 have been already applied by ex vivo hematopoietic stem cell (HSC) gene therapy in genetic diseases (i.e., Hemoglobinopathies, Fanconi anemia and hereditary Immunodeficiencies) as well as infectious diseases (i.e., HIV), and the recent development of CRISPR-Cas9-based systems using base and prime editors as well as epigenome editors has provided safer tools for gene therapy. The ex vivo approach for gene addition or editing of HSCs, however, is complex, invasive, technically challenging, costly and not free of toxicity. In vivo gene addition or editing promise to transform gene therapy from a highly sophisticated strategy to a “user-friendly’ approach to eventually become a broadly available, highly accessible and potentially affordable treatment modality. In the present review article, based on the lessons gained by more than 3 decades of ex vivo HSC gene therapy, we discuss the concept, the tools, the progress made and the challenges to clinical translation of in vivo HSC gene editing. Full article

(This article belongs to the Special Issue Gene Editing for Therapy and Reverse Genetics of Blood Diseases)

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