**3. Novel therapeutic approaches for β-thalassemia**

Currently, clinical management of β-thalassemia and SCD patients is largely dependent on regular blood transfusions associated with chelating agents for the treatment of systemic iron overload. Indeed, except for allogeneic transplantation strategies, whose favorable outcome is anyway restricted to the availability of a wellmatched donor, or for the more recent haploidentical transplantation option that however carries substantial risks, no other curative option is currently available for hemoglobinopathies. Therefore, due to the relevant global health burden of these diseases, there is still a great need for effective and definitive large-scale treatments.

In the last decades, given the ameliorative effects of elevated HbF levels that can lead to decreased hemoglobin polymerization in SCD as well as to reduced α-chain imbalance in β-thalassemia, a great deal of effort has been devoted to the development of new therapies aimed at reactivating fetal globin gene expression. Therefore, according to the evidences that hypo-methylated DNA and hyper-acetylated histones are well-known epigenetic marks of transcriptionally active regions, inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) have been developed as novel pharmacologic approaches to support transfusion therapy. Among the first drugs to be identified, 5-azacytidine and decitabine were found to induce increased HbF levels through DNA hypo-methylation process. Subsequently, the use of hydroxyurea (HU) was also investigated as a promising HbF inducer agent in adults with severe SCD. Although the mechanism through which HU induces HbF reactivation is still not completely understood, HU treatment was found to increase HbF levels and reduce HbS polymerization, thus improving clinical symptoms and quality of life of these patients. Another epigenetic approach was also exploited through the use of HDAC inhibitors, including butyrate and its derivatives that have been demonstrated to stimulate HbF production in β-thalassemia patients [49].

More recently, many hopes were directed toward the development of gene therapy procedures designed to introduce a normal copy of the β-globin gene by viral vectors that offer the advantages of long-term benefits and, even more importantly, being based on autologous transplant, do not require a matched donor. However, in spite of great expectations for a rapid development of this approach, for many years it proved a difficult goal, and only recently it has been successfully introduced in clinical practice. Notably, during the last years, additional promising therapeutic options have been provided by progress in gene-editing technologies, including the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) (CRISPR-Cas9) tool, designed to introduce highly specific genome modifications for either gene disruption or correction [48, 50, 51]. At the same time, progress in understanding the molecular mechanisms underlying fetal globin gene silencing has contributed to identify several potential molecular targets, thus paving the way to novel therapeutic approaches for hemoglobinopathies that, besides correcting the defective β-globin gene, are now aimed at editing genomic segments that regulate fetal hemoglobin synthesis in hematopoietic stem cells (HSCs).

Interestingly, some of the most promising editing strategies for hemoglobinopathies that are being pursued include the reactivation of fetal globin genes that could be achieved either by silencing fetal globin gene repressors or, alternatively, by introducing known HPFH mutations in the HBB cluster. Since the beginning, targeting Myb or KLF1 appeared to be rather problematic given the pleiotropic roles played by these two factors in hematopoiesis and in erythropoiesis, respectively, whereas BCL11A emerged as one of the most appropriate candidate to be silenced, given its well-recognized role in HbF repression [48, 50, 51]. However, ubiquitous BCL11A knockdown rapidly turned out to be an unsuccessful strategy, given its role in other biological pathways including neuronal and B-lymphocyte development [28]. Consequently, alternative strategies are being underway focused on reducing its expression selectively in erythroid cells that could be achieved by disruption of its intronic erythroid-specific enhancer without affecting non-erythroid cells [51]. A similar approach could also be used to tune down Myb expression levels by editing SNPs at the *HBS1L-MYB* intergenic region that are known to disrupt the structure and the functionality of erythroid-specific enhancers causing reduced Myb expression levels. Anyway, other repressors such as CSDA or erythroidspecific cofactors could represent potential suitable candidates to be considered for novel genome-editing strategies. Furthermore, genome-editing procedures leading

**33**

*Transcriptional Repressors of Fetal Globin Genes as Novel Therapeutic Targets…*

to induce clinically significant increases in HbF levels [48, 50, 51].

to the introduction of KLF1 mutations that emerged as a natural model of impaired hemoglobin switching or HPFH mutations in the regulatory regions of fetal globin genes have been proposed either as alternative or supportive therapeutic strategies

The research in this field should be immediately focused on the development of safe, effective drug therapies that can be accomplished through fetal globin gene induction and at the same time on the prospect of cures through bone marrow transplantation using the promise of genome-editing strategies that will bring a vastly improved quality and quantity of life to patients who suffer from these

This work was supported by the Regione Campania SATIN grant, grant/award

, Maria Rosaria Storino1,2,

, Alessandra Lo Bianco1

,

, Rosa Catapano1

*DOI: http://dx.doi.org/10.5772/intechopen.90762*

**4. Conclusions**

devastating disorders.

**Acknowledgements**

number 2018–2020.

**Conflict of interest**

**Author details**

Marianna De Martino1

Mariarosaria Giuliano1

Paola Izzo1,2 and Michela Grosso1,2\*

Naples Federico II, Naples, Italy

2 CEINGE-Biotecnologie Avanzate, Naples, Italy

provided the original work is properly cited.

\*Address all correspondence to: michela.grosso@unina.it

The authors declare no conflict of interest.

, Raffaele Sessa1

, Silvia Trombetti1

1 Department of Molecular Medicine and Medical Biotechnology, University of

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Transcriptional Repressors of Fetal Globin Genes as Novel Therapeutic Targets… DOI: http://dx.doi.org/10.5772/intechopen.90762*

to the introduction of KLF1 mutations that emerged as a natural model of impaired hemoglobin switching or HPFH mutations in the regulatory regions of fetal globin genes have been proposed either as alternative or supportive therapeutic strategies to induce clinically significant increases in HbF levels [48, 50, 51].

## **4. Conclusions**

*Beta Thalassemia*

tion in β-thalassemia patients [49].

In the last decades, given the ameliorative effects of elevated HbF levels that can lead to decreased hemoglobin polymerization in SCD as well as to reduced α-chain imbalance in β-thalassemia, a great deal of effort has been devoted to the development of new therapies aimed at reactivating fetal globin gene expression. Therefore, according to the evidences that hypo-methylated DNA and hyper-acetylated histones are well-known epigenetic marks of transcriptionally active regions, inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) have been developed as novel pharmacologic approaches to support transfusion therapy. Among the first drugs to be identified, 5-azacytidine and decitabine were found to induce increased HbF levels through DNA hypo-methylation process. Subsequently, the use of hydroxyurea (HU) was also investigated as a promising HbF inducer agent in adults with severe SCD. Although the mechanism through which HU induces HbF reactivation is still not completely understood, HU treatment was found to increase HbF levels and reduce HbS polymerization, thus improving clinical symptoms and quality of life of these patients. Another epigenetic approach was also exploited through the use of HDAC inhibitors, including butyrate and its derivatives that have been demonstrated to stimulate HbF produc-

More recently, many hopes were directed toward the development of gene therapy procedures designed to introduce a normal copy of the β-globin gene by viral vectors that offer the advantages of long-term benefits and, even more importantly, being based on autologous transplant, do not require a matched donor. However, in spite of great expectations for a rapid development of this approach, for many years it proved a difficult goal, and only recently it has been successfully introduced in clinical practice. Notably, during the last years, additional promising therapeutic options have been provided by progress in gene-editing technologies, including the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) (CRISPR-Cas9) tool, designed to introduce highly specific genome modifications for either gene disruption or correction [48, 50, 51]. At the same time, progress in understanding the molecular mechanisms underlying fetal globin gene silencing has contributed to identify several potential molecular targets, thus paving the way to novel therapeutic approaches for hemoglobinopathies that, besides correcting the defective β-globin gene, are now aimed at editing genomic segments that regulate

Interestingly, some of the most promising editing strategies for hemoglobinopathies that are being pursued include the reactivation of fetal globin genes that could be achieved either by silencing fetal globin gene repressors or, alternatively, by introducing known HPFH mutations in the HBB cluster. Since the beginning, targeting Myb or KLF1 appeared to be rather problematic given the pleiotropic roles played by these two factors in hematopoiesis and in erythropoiesis, respectively, whereas BCL11A emerged as one of the most appropriate candidate to be silenced, given its well-recognized role in HbF repression [48, 50, 51]. However, ubiquitous BCL11A knockdown rapidly turned out to be an unsuccessful strategy, given its role in other biological pathways including neuronal and B-lymphocyte development [28]. Consequently, alternative strategies are being underway focused on reducing its expression selectively in erythroid cells that could be achieved by disruption of its intronic erythroid-specific enhancer without affecting non-erythroid cells [51]. A similar approach could also be used to tune down Myb expression levels by editing SNPs at the *HBS1L-MYB* intergenic region that are known to disrupt the structure and the functionality of erythroid-specific enhancers causing reduced Myb expression levels. Anyway, other repressors such as CSDA or erythroidspecific cofactors could represent potential suitable candidates to be considered for novel genome-editing strategies. Furthermore, genome-editing procedures leading

fetal hemoglobin synthesis in hematopoietic stem cells (HSCs).

**32**

The research in this field should be immediately focused on the development of safe, effective drug therapies that can be accomplished through fetal globin gene induction and at the same time on the prospect of cures through bone marrow transplantation using the promise of genome-editing strategies that will bring a vastly improved quality and quantity of life to patients who suffer from these devastating disorders.

#### **Acknowledgements**

This work was supported by the Regione Campania SATIN grant, grant/award number 2018–2020.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Marianna De Martino1 , Raffaele Sessa1 , Maria Rosaria Storino1,2, Mariarosaria Giuliano1 , Silvia Trombetti1 , Rosa Catapano1 , Alessandra Lo Bianco1 , Paola Izzo1,2 and Michela Grosso1,2\*

1 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy

2 CEINGE-Biotecnologie Avanzate, Naples, Italy

\*Address all correspondence to: michela.grosso@unina.it

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
