Recently, scientists have conducted many studies to identify the natural remedies that could possibly be applied for the treatment of β-thalassemia (Table 2) [176]. Several studies have found that the extracts from medicinal plants for biomedical purposes [177–186] including therapeutic strategies can be used for the treatment of a number of diseases.

In case of hemoglobinopathies, only a few examples are available. For instance, the extract of Aegle Marmelos containing bergatene was found to activate erythroid differentiation and HbF induction in human leukemic K562 cells [187–188]. Citropten and bergatene are the two active ingredients in bergamot juice. They are potent inducers of Erythroid cell differentiation, Gγglobin gene expression and fetal hemoglobin synthesis in human erythroid cells. Thus, it is known as a possible therapeutic approach for both β-thalassemia and Sickle cell anemia [187].

Another example of HbF inducer from the natural world is Angelicin, which can be found in the fruit of *Angelica arcangelica*. There is evidence demonstrating that angelicin is a strong inducer of Erythroid cell differentiation, improvement of the HbF synthesis, and γ-globin mRNA accumulation of human leukemia K562 cells [84, 179]. Red wine, particularly the skin of black grapes, contains resveratrol which mimics the HbF-inducing activity of HU. Its role in increasing the γ-globin mRNA in human erythroid precursors has been confirmed [84]. Since β-thalassemia cells present a high level of oxidative stress resulting in short time survival of erythroid cells in β-thalassemia patients, resveratrol which exhibits both antioxidant activity and HbF inducing property can be a very good HbF inducer from the natural world [88].

Rapamycin, which is isolated from *Streptomyces hygroscopicus*, is a bacterial species found in soil of Easter Island has the capability of increasing HbF production in cultures of erythroid precursors from β-thalassemia patients without any cytotoxicity or growth-inhibitory effect. Apart from Rapamycin, Mithramycin is a DNA-binding drug that is easily isolated from Streptomyces has the potential to induce γ-globin mRNA accumulation and HbF production in erythroid cells from healthy subjects as well as β-thalassemia patients [189].

**Table 2.** Inducers of HbF from natural sources

#### **6. Discussion and conclusion**

The Human Genome Project has improved efforts of developing gene-based therapy for βhemoglobinopathies alongside chemical inducers. GWAS have found certain major genetic modifiers of γ-globin, including -158 XmnI HBG2, HBS1-MYB and BCL11A accounting for ~50% of inherited HbF variance [190–191]. Orkin and colleagues advanced the field signifi‐ cantly by defining mechanisms through which BCL11A repressed γ-globin expression and holds the promise for the development of gene-based therapy in the future.

The occurrence of inherited mutations in KLF1 that produce HPFH suggested that this factor is a viable target for gene therapy and might be accomplished by RNAi technology to create a haploinsufficiency state. Promising molecular targets such as KLF1 and BCL11A for thera‐ peutic efforts aimed at HbF induction have been identified: however, additional pre-clinical data are needed before manipulation of transcription factors can be translated into therapeutic options at the bedside.

Agents which improve HbF synthesis represent a rational approach for the treatment of βthalassemia. For more than three decades, many chemical agents have been tested in tissue culture as HbF inducers, but few have crossed the path from clinical trials and have reached to the bedside. One major roadblock is the lack of suitable pre-clinical models to test agents in vivo. Baboon studies led to the clinical development of HU, butyrate, decitabine, and HQK-1001 in SCD and β-thalassemia [93, 120–121, 124–125, 192–197]. With the establishment of SCD mouse models, newer agents such as pomalidomide have been progressed to clinical trials, however, the limited availability of the knockout SCD mouse model has affected progress in this area [155]. Townes and colleagues had designed a second knockin SCD mouse model with γ-globin and β-globin constructs [157]. However, HbF induction has not been effectively achieved with HU therapy (Pace, unpublished data). Therefore, the knock-in mouse may not be ideal for future pre-clinical drug screens. An alternative model, the β-YAC mouse was used to confirm in vivo HbF induction by 5-azacytidine, scriptaid, and transcyclomide showing the utility of this mouse for pre-clinical drug screening [161, 198–199]. Currently, very few agents are in clinical trials targeting HbF induction and drugs aimed at other complications of SCD, such as nitric oxide deficiency, endothelium antagonist, anti-platelet agents, anti-sickle agents, and so forth are still under development (clinicaltrials.gov). These can be combined with HU or other HbF inducers to produce an additive or synergistic clinic benefit [155].

**Inducer Source Biological effects References** Bergaptene *A. marmelos* Erythroid differentiation of K562 cells Lampronti et al. (2003) Angelicin *A. arcangelica* HbF production Lampronti et al. (2003) Rapamycin *S. hygroscopicus* HbF production Mischiati et al. (2004)

Mithramycn *Streptomyces* species HbF production Bianchi et al. (1999)

*Terminalia catappa leaves* Erythropoiesis, cell proliferation, and

The Human Genome Project has improved efforts of developing gene-based therapy for βhemoglobinopathies alongside chemical inducers. GWAS have found certain major genetic modifiers of γ-globin, including -158 XmnI HBG2, HBS1-MYB and BCL11A accounting for ~50% of inherited HbF variance [190–191]. Orkin and colleagues advanced the field signifi‐ cantly by defining mechanisms through which BCL11A repressed γ-globin expression and

The occurrence of inherited mutations in KLF1 that produce HPFH suggested that this factor is a viable target for gene therapy and might be accomplished by RNAi technology to create a haploinsufficiency state. Promising molecular targets such as KLF1 and BCL11A for thera‐ peutic efforts aimed at HbF induction have been identified: however, additional pre-clinical data are needed before manipulation of transcription factors can be translated into therapeutic

Agents which improve HbF synthesis represent a rational approach for the treatment of βthalassemia. For more than three decades, many chemical agents have been tested in tissue culture as HbF inducers, but few have crossed the path from clinical trials and have reached to the bedside. One major roadblock is the lack of suitable pre-clinical models to test agents in vivo. Baboon studies led to the clinical development of HU, butyrate, decitabine, and

holds the promise for the development of gene-based therapy in the future.

darakchasava HbF production Rodrigue et al. (2001)

Erythroid survival, proliferation, and terminaldifferentiation of K562 cells and HbF production

HbF production and Erythroid differentiation of K562cells

Zhang and Wu (2008)

Liu et al. (2010); Li et al. (2011)

transcription Aimola et al. (2014)

Resveratrol Redwine, grape skin and

Ethanol extract of Fructus trichosanthis, which is the fruit of Trichosanthes kirilowii MAXIM

YiSui ShengXue Granule

118 Inherited Hemoglobin Disorders

Cucurbitacn D

Terminalia catappa distilled water active fraction (TCDWF)

options at the bedside.

**Table 2.** Inducers of HbF from natural sources

**6. Discussion and conclusion**

Despite the success of HU in clinical trials conducted on adults and children and its proven safety and efficacy, it remains underutilized in SCD [163–164, 166, 168–170, 173, 200–201]. Possible causes include: (1) limited access to comprehensive sickle cell medical care; 2) lack of coordination between subspecialists and community based clinicians; 3) concern over poten‐ tial genotoxicity; and 4) lack of patient adherence with the medication regimen. Future studies are needed to address early initiation of HU, the role of HU therapy in prevention of chronic complications, and improved methods for HU therapy delivery to all patients with SCD. There remains a question about the optimal age to initiate HU therapy. The Baby HUG trial estab‐ lished safety in young children, but additional clinical safety data are needed for children <6 months at the highest risk of infection and spleen and kidney dysfunction. Whether the early HU therapy administration will prevent the γ- to β-globin switch has not been demonstrated.

Curative therapy for SCD and β-thalassemia includes hematopoietic stem cell transplantation, however, this option is limited by the availability of suitable donors in <20% of children with SCD [202–203]. Ongoing clinical trials exploring alternative approaches such as matched unrelated donors and the development of new regimens using haplo-identical donors in the future will increase the transplant option for the majority of SCD patients [204]. Another approach to cure β-hemoglobinopathies is efforts to develop gene therapy, e.g., recently there was a successful treatment of two β-thalassemia patients with a modified β-globin lentivirus based vector. This progress holds promise for SCD patients [205–206].

Landmark studies have developed an excitement that fully differentiated somatic cells can be reprogrammed to make induced pluripotent stem cells [207]. Subsequent studies demonstrat‐ ed rectification of a mouse model of SCD using this innovative approach, therefore opened the way to the use of these cells to cure β-hemoglobinopathies beside few limitations [208]. One limitation was the inability to restore all hematopoietic lineages with induced pluripotent stem cells which precludes using human therapy [209]. Therefore, until transplantation and gene therapy is more widely available, chemical inducers of HbF remain the most effective way to treat β-thalassemia and SCD.

#### **Author details**

Anjana Munshi1\*, Sneha Dadeech2 , M. Sai Babu2 and Preeti Khetarpal1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Centre for Human Genetics, Central University of Punjab, Bathinda, India

2 Department of Molecular Biology, Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, India

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**Author details**

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### **Novel Therapy Approaches in β-Thalassemia Syndromes — A Role of Genetic Modifiers**

Sonja Pavlovic, Milena Ugrin and Maja Stojiljkovic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61023

#### **Abstract**

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136 Inherited Hemoglobin Disorders

The β-thalassemia syndromes are heterogeneous autosomal recessive hereditary disorders, caused by alterations in the *HBB* gene and characterized by absent or reduced β-globin chain synthesis. The β-thalassemia phenotypes are variable, ranging from severe, transfusion-dependent thalassemia major to mild, asymptomatic thalassemia trait. This interpatient clinical variability has swayed researchers toward identifying genetic modifiers for these disorders. Primary modifiers refer to type of alterations affecting β-globin gene. Secondary modifiers include variations in genes affecting α/β-globin chain equilibrium, such as genes involved in the γ-globin gene expression and genes affecting the amount and stability of α-globin chains. Tertiary modifiers are gene variations affecting the phenotype with regard to the complications caused by β-thalassemia syndromes. A role of secondary genetic modifiers in ameliorating the clinical phenotype has been observed. Secondary genetic modifiers are the most common targets for modern therapy and could be located within α- and γ-globin genes or outside globin gene cluster. The most potent secondary modifier genes are γ-globin genes. Production of fetal hemoglobin (HbF) trough adulthood ameliorates the severity of β-thalassemia phenotype. Large family and genome-wide association studies have shown that regions outside of the β-globin gene cluster are also implicated in γ-globin gene expression regulation. *HBS1-MYB* intragenic region and *BCL11A* gene have been particularly studied. Variants within these loci, along with γ-globin gene variants, account for approximately 50% of the HbF level variation, suggesting that additional factors are involved (transcription regulators (KLF1), regulators of α-globin chain stability (AHSP), epigenetic regulators (FoP)). Until recently a definitive cure for β-thalassemia could be achieved with bone marrow transplantation. However, it is available for less than 30% of the patients and bears a significant risk of morbidity and mortality. Alternative strategies, such as gene

© 2015 The Author(s). Licensee InTech. 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.

therapy and development of induced pluripotent stem cells (iPSCs) have been explored. The targets for gene therapy are hematopoietic stem cells, which are harvested from patient bone marrow or peripheral blood, purified by immunoselec‐ tion, transduced by "therapeutic gene" aimed at correcting the effect of defective βglobin gene, and returned to the patient. Various types of vectors have been considered for gene transfer, including non viral (tRNK and ribozymes) and viral (retroviral and lentiviral vectors). In the past few years, iPSCs emerged as an interesting candidate for gene transfer. The feature that makes these cells appealing in the field of gene therapy is their susceptibility to gene correction by homologous recombination. Therapy protocols based on molecular basis of β-thalassemia are the best example of novel approaches in disease treatment.

**Keywords:** β-thalassemia, modifier genes, gene therapy, iPSC

### **1. Introduction**

#### **1.1. Hemoglobinopathies**

Hemoglobinopathies are very heterogenic group of hereditary anemias and are classified according to the qualitative nature of the resulting hemoglobin and the quantitative amount of hemoglobin produced.

"Qualitative" hemoglobinopathies, or structural hemoglobin variants, are characterized by amino acid sequence variation of the hemoglobin (Hb) molecule which influences biochemical features of Hb. "Quantitative" hemoglobin disorders or thalassemias, are classified according to the deficient globin chain and, hence, include α-thalassemia and β-thalassemia.

Moreover, there is a distinct group of disorders classified as β -thalassemia syndromes which comprises β-thalassemia and certain group of hemoglobin variants whose synthesis is reduced due to amino acid sequence variation in the regions responsible for regulation of transcription or peptide stability.

Molecular mechanisms leading to formation of "quantitative" Hb variants include a δ-β hybrid gene (Hb Lepore) [1] and mutations causing hyperunstable beta globin chain synthesis (Hb Sabine) [2]. These highly unstable Hb variants precipitate before assembling with the α-globin chains to produce the Hb tetramer, resulting in excess of the α-globin chains and, thus, ineffective erythropoiesis [3, 4].

#### **1.2. β-thalassemia syndromes**

The β-thalassemia syndromes are one of the most common autosomal recessive hereditary disorders worldwide, with high prevalence in the populations of the Mediterranean, Middle-East, Central Asia, Indian subcontinent and Far East [3]. The β-thalassemias are caused by alterations in the β-globin gene and characterized by absent (β<sup>0</sup> -thalassemia) or reduced (β<sup>+</sup> - thalassemia) synthesis of β-globin chain of adult hemoglobin (HbA; α2β2). The result of this reduced globin chain synthesis is reduced production of functional hemoglobin tetramers which leads to hypochromia (decrease in the hemoglobin content of the erythrocytes) and microcytosis (reduces mean corpuscular volume of erythrocytes). Also, the excess of unbound α-globin chains precipitate in erythroid precursors in the bone marrow and red blood cells in circulation, leading to their premature death and, hence, to ineffective erythropoiesis and hemolytic anemia [5].

The clinical manifestations of β-thalassemias are extremely diverse, ranging form the severe, transfusion-dependent thalassemia major to the mild, asymptomatic thalassemia trait. Diverse phenotypes between the two extremes of thalassemia major and thalassemia trait constitute the clinical syndrome of thalassemia intermedia [6].

#### **1.3. β-globin gene locus**

therapy and development of induced pluripotent stem cells (iPSCs) have been explored. The targets for gene therapy are hematopoietic stem cells, which are harvested from patient bone marrow or peripheral blood, purified by immunoselec‐ tion, transduced by "therapeutic gene" aimed at correcting the effect of defective βglobin gene, and returned to the patient. Various types of vectors have been considered for gene transfer, including non viral (tRNK and ribozymes) and viral (retroviral and lentiviral vectors). In the past few years, iPSCs emerged as an interesting candidate for gene transfer. The feature that makes these cells appealing in the field of gene therapy is their susceptibility to gene correction by homologous recombination. Therapy protocols based on molecular basis of β-thalassemia are the

Hemoglobinopathies are very heterogenic group of hereditary anemias and are classified according to the qualitative nature of the resulting hemoglobin and the quantitative amount

"Qualitative" hemoglobinopathies, or structural hemoglobin variants, are characterized by amino acid sequence variation of the hemoglobin (Hb) molecule which influences biochemical features of Hb. "Quantitative" hemoglobin disorders or thalassemias, are classified according

Moreover, there is a distinct group of disorders classified as β -thalassemia syndromes which comprises β-thalassemia and certain group of hemoglobin variants whose synthesis is reduced due to amino acid sequence variation in the regions responsible for regulation of transcription

Molecular mechanisms leading to formation of "quantitative" Hb variants include a δ-β hybrid gene (Hb Lepore) [1] and mutations causing hyperunstable beta globin chain synthesis (Hb Sabine) [2]. These highly unstable Hb variants precipitate before assembling with the α-globin chains to produce the Hb tetramer, resulting in excess of the α-globin chains and, thus,

The β-thalassemia syndromes are one of the most common autosomal recessive hereditary disorders worldwide, with high prevalence in the populations of the Mediterranean, Middle-East, Central Asia, Indian subcontinent and Far East [3]. The β-thalassemias are caused by

alterations in the β-globin gene and characterized by absent (β<sup>0</sup>

to the deficient globin chain and, hence, include α-thalassemia and β-thalassemia.

best example of novel approaches in disease treatment.

**1. Introduction**

138 Inherited Hemoglobin Disorders

**1.1. Hemoglobinopathies**

of hemoglobin produced.

or peptide stability.

ineffective erythropoiesis [3, 4].

**1.2. β-thalassemia syndromes**

**Keywords:** β-thalassemia, modifier genes, gene therapy, iPSC

The β-globin gene (*HBB*) maps in the short arm of chromosome 11, in the 70 kb region that also contains four other functional globin genes: embryonic, ε-globin gene (*HBE*), the fetal Aγand Gγ-globin genes (*HBG1* and *HBG2*) and adult δ-globin gene (*HBD*) as well as ψβ pseudo‐ gene. The five functional globin genes are arranged in the order of their developmental expression [7]. Upstream of the β globin complex is the locus control region (LCR), important regulatory region which consists of five erythroid-specific DNase1 hypersensitive (HS) sites (HS 1–5) distributed between 6 and 20 kb 5' of the ε-globin gene (Fig.1). These HS sites interact with each other and two additional 5' and one 3' HSs to form an active chromatin hub (ACH) through looping, which interacts with the specific globin gene in any given moment of development. The role of the individual HS is still unclear, although HS2 and HS3 appear to be the most important sites for the efficiency of transcription, each leading to an ∼30% loss of transcription when deleted [6, 8].

**Figure 1.** Structure of β-globin gene cluster. Human β-globin gene locus showing embryonic (ε), fetal (<sup>A</sup>γ;<sup>G</sup>γ) and adult (δ;β) globin genes, controlled by locus control region (LCR) and additional hypersensitive sites (3'HS; 5'HS). Region between fetal and adult globin gene contains ψβ pseudogene. The five functional globin genes are arranged in the or‐ der of their developmental expression.

During fetal development and the first six months of neonatal life, a complex pattern of globin gene expression occurs called the hemoglobin switch. Namely, in the early part of the first trimester, there is high expression of an embryonic globin gene within the primitive lineage of erythrocytes in the yolk sac. During fetal development, however, predominantly expressed globin genes are γ-globin genes, coding for the γ-globin polypeptides, produced within fetal liver. These γ-globin chains combine with adult α-globin chains into a stable tetramer forming fetal hemoglobin (HbF; α2γ2). Shortly after the time of birth this fetal hemoglobin is progres‐ sively replaced by the adult hemoglobin (HbA), which is mediated by a transcriptional switch in definitive erythroid progenitors from γ- to β-globin [9, 10].

#### **2. Genetic modifiers of β-thalassemia**

The β-thalassemia is caused by more than 200 point mutations and, rarely, by deletions [11]. However, genotypic variability at known loci is often insufficient to explain the phenotypic variability between individuals with the same genotype [12]. This interpatient clinical variability in the β-thalassemia syndromes has swayed researchers toward identifying genetic modifiers of severity for these disorders. Such genetic modifiers could potentially lead to the development of more specific and effective therapies [13]. Genetic modifiers exert their potential on three levels: primary, secondary and tertiary.

Primary modifiers usually refer to a type of alterations affecting β-globin gene. Location of the mutations within different gene regions determines the phenotypic severity, therefore the point mutations affecting the β-globin expression belong to three different categories: muta‐ tions leading to defective β-globin gene transcription (promoter and 5' UTR mutations); mutations affecting mRNA processing (splice-junction and consensus sequence mutations, polyadenylation, and other 3' UTR mutations); and mutations resulting in abnormal mRNA translation (nonsense, frameshift, and initiation codon mutations) [3]. Mutations affecting transcription usually result in a mild deficit of β-globin production that reflects the relatively mild phenotype of these β<sup>+</sup> -thalassemias. The example of transcription affecting mutation is the C>T mutation at position -101 to the β-globin gene which appears to cause an extremely mild deficit of β-globin, such that it is asymptomatic in heterozygotes who have normal HbA<sup>2</sup> (α2δ2) levels [6]. Mutations affecting β-globin mRNA processing are located within 5' and 3'-splice junction (donor and acceptor site), as well as within splice junctions' consensus sequences. Mutations altering the donor and acceptor splice site lead to deficiency of functional mRNA production resulting in complete absence of β-globin polypeptide chains and, hence, to β0 -thalassemia. On the other hand, mutations affecting consensus sequences surrounding splice-junction, decrease the efficiency of the normal splicing to varying degrees, hence producing β-thalassemia phenotype that ranges from mild to severe [7]. Also, these mutations could affect cryptic splice site, sequence that mimics a consensus sequence, leading to low efficiency splicing and therefore milder form of β-thalassemia. Cap-site mutations, as well as mutations affecting polyadenylation also lead to mild, β<sup>+</sup> -thalassemia phenotype [14]. Muta‐ tions disrupting the mRNA translation ether in initiation or elongation phase, result in β0 thalassemia phenotype. Most of these defects result from the introduction of premature termination codons due to frameshift or nonsense mutations and nearly all terminate within first and second exon [15].

Secondary modifiers include variations in genes affecting α/β globin chain equilibrium such as α- and γ-globin genes, as well as genes involved in the γ-globin gene expression (*HBS1-* *MYB*, *BCL11A*, *KLF1, C1orf77*) and genes affecting the amount and stability of α-globin chains (*AHSP*). These genetic modifiers could be located within (α- and γ-globin genes) or outside globin gene cluster. In the recent years, there has been significant advancement in the fields of secondary genetic modifiers ameliorating the clinical phenotype of β-thalassemia syndromes. Specifically, production of fetal hemoglobin (HbF) trough adulthood could ameliorate the severity of β-thalassemia phenotype since γ-globin polypeptide chains compensate for the lack of the functional β-globin polypeptide chains. This is why γ-globin genes, along with other secondary modifiers, represent the most common targets for modern therapeutic.

globin genes are γ-globin genes, coding for the γ-globin polypeptides, produced within fetal liver. These γ-globin chains combine with adult α-globin chains into a stable tetramer forming fetal hemoglobin (HbF; α2γ2). Shortly after the time of birth this fetal hemoglobin is progres‐ sively replaced by the adult hemoglobin (HbA), which is mediated by a transcriptional switch

The β-thalassemia is caused by more than 200 point mutations and, rarely, by deletions [11]. However, genotypic variability at known loci is often insufficient to explain the phenotypic variability between individuals with the same genotype [12]. This interpatient clinical variability in the β-thalassemia syndromes has swayed researchers toward identifying genetic modifiers of severity for these disorders. Such genetic modifiers could potentially lead to the development of more specific and effective therapies [13]. Genetic modifiers exert their

Primary modifiers usually refer to a type of alterations affecting β-globin gene. Location of the mutations within different gene regions determines the phenotypic severity, therefore the point mutations affecting the β-globin expression belong to three different categories: muta‐ tions leading to defective β-globin gene transcription (promoter and 5' UTR mutations); mutations affecting mRNA processing (splice-junction and consensus sequence mutations, polyadenylation, and other 3' UTR mutations); and mutations resulting in abnormal mRNA translation (nonsense, frameshift, and initiation codon mutations) [3]. Mutations affecting transcription usually result in a mild deficit of β-globin production that reflects the relatively

the C>T mutation at position -101 to the β-globin gene which appears to cause an extremely mild deficit of β-globin, such that it is asymptomatic in heterozygotes who have normal HbA<sup>2</sup> (α2δ2) levels [6]. Mutations affecting β-globin mRNA processing are located within 5' and 3'-splice junction (donor and acceptor site), as well as within splice junctions' consensus sequences. Mutations altering the donor and acceptor splice site lead to deficiency of functional mRNA production resulting in complete absence of β-globin polypeptide chains and, hence,

tions disrupting the mRNA translation ether in initiation or elongation phase, result in β0

thalassemia phenotype. Most of these defects result from the introduction of premature termination codons due to frameshift or nonsense mutations and nearly all terminate within

Secondary modifiers include variations in genes affecting α/β globin chain equilibrium such as α- and γ-globin genes, as well as genes involved in the γ-globin gene expression (*HBS1-*

in definitive erythroid progenitors from γ- to β-globin [9, 10].

potential on three levels: primary, secondary and tertiary.

mutations affecting polyadenylation also lead to mild, β<sup>+</sup>

**2. Genetic modifiers of β-thalassemia**

140 Inherited Hemoglobin Disorders

mild phenotype of these β<sup>+</sup>

first and second exon [15].

to β0

Tertiary modifiers are gene variations affecting the phenotype with regard to some of the complications caused by β-thalassemia syndromes such as hyperbilirubinemia, propensity to gallstone formation, bone diseases, thrombophilia and cardiopathies [6].

Hyperbilirubinemia and gallstone formation (cholelithiasis) is a common complication of βthalassemia and is attributed to the rapid turnover of the red blood cells, bilirubin being a breakdown product of hemoglobin. It occurs with variable incidence in homozygous βthalassemia with the reported variation being partly related to the age of the patients and to its clinical severity, as it is more common in thalassemia intermedia than thalassemia major. Studies have shown that the levels of bilirubin and the predisposition to gallstones in βthalassemia is related to a polymorphic variant in the promoter of the uridine diphosphateglucoronyltransferase A1 (*UGT1A1*) gene, also referred to as Gilbert's syndrome. Individuals who are homozygous for the this (TA)7 variant instead of the usual (TA)6, tend to have higher levels of bilirubin and increased predisposition to gallstone [16, 17].

Progressive osteoporosis and osteopenia is another increasingly common complication observed in young adults with β-thalassemia and it is determined by a combination of genetic and environmental factors. Anemia and bone marrow expansion which are prevalent in βthalassemia are major contributors in inadequately treated patients. Bone mass, the main indicator of the osteoporosis and osteopenia, is another quantitative trait known to be under strong genetic control involving multiple loci including estrogen receptor gene, vitamin D receptor (*VDR*), collagen type a1 and type a2 genes (*COL1A1*, *COL1A2*), and transforming growth factor β1 (*TGFβ1*) [15].

The presence of a higher than normal incidence of thromboembolic events, mainly in βthalassemia intermedia patients, and the existence of prothrombotic hemostatic anomalies in the majority of the patients, have led to the recognition of the existence of a chronic hyper‐ coagulable state in thalassemic patients [18]. Genetic risk factors for thrombosis, such as mutations in the gene for Factor II and Factor V, as well as variations in the *MTHFR* gene, could significantly influence the phenotype of the β-thalassemia syndromes [19].

Cardiac diseases are the main cause of death in β-thalassemia patients and are attributed to iron overload because of regular transfusions, increased iron intestinal absorption, and ineffective erythropoiesis during the life span of the patients. One of the studies depicted apolipoprotein E4 and it decreased antioxidant activity, as a risk factor for left ventricular failure (LVF) in thalassemia patients. However, the presence of this E4 allele does not guarantee the development of LVF in β-thalassemia but, when present, does affect the severity of the disease [20, 21].

#### **3. Secondary genetic modifiers of β-thalassemia**

#### **3.1. Genetic modifiers of β-thalassemia within globin gene loci**

#### *3.1.1. α-globin genes*

The α-globin genes encoding the α-globin chains are duplicated (α1- and α2-globin genes) and localized in the telomeric region of chromosome 16 (16p13.3), in a cluster containing also an embryonic, ζ-gene, encoding the embryonic globin chains, three pseudogenes (pseudo ζ-, pseudo α1- and pseudo α2-gene) and θ-gene of unknown function [22]. The level of transcrip‐ tion of the two α-globin genes differs, as the α2 gene encodes two to three times more α-globin than α1 gene. The different expression of the two α-globin genes has implications for the amount of hemoglobin variant present in carriers of α1- or α2-globin mutations and for the pathophysiology of the deletional and nondeletional forms of α-thalassemia [23]. As with βthalassemia, α-thalassemias are characterized by absent (α<sup>0</sup> -thalassemia) or reduced (α<sup>+</sup> thalassemia) production of α-globin chains, thus resulting in globin chain imbalance. The majority of the α-thalassemia defects result from deletions involving one or both α-globin genes on the same chromosome whereas point mutations affecting the functional expression of one of the two α-globin genes are not as common [24].

In many populations in which β-thalassemia is prevalent, α-thalassemia occurs at high frequency as well, resulting in coinheritance of both conditions. In these cases, homozygotes or compound heterozygotes for β-thalassemia will exert less severe phenotype since they would have less redundant α-globin chains. Therefore, the degree of amelioration of βthalassemia phenotype depends on the severity of the β-thalassemia alleles and the number of functional α-globin genes. At one extreme, homozygous β-thalassemia patients who have only one functional α-globin gene, have thalassemia intermedia. On the other hand, the presence of increased α-globin product, as a result of triplicated or quadruplicated α-globin genes in β-thalassemia heterozygotes, increase the globin chain imbalance, converting a typically clinically asymptomatic state to that of thalassemia intermedia [25]. However, the phenotype of a single extra α gene (ααα/αα) with heterozygous β-thalassemia is more variable and depends on the severity of the β-thalassemia allele [26, 27].

#### *3.1.2. γ -globin genes*

As already discussed, after a brief period of embryonic globin gene expression, the γ-globin chain of fetal hemoglobin (HbF) is predominantly expressed for much of gestation. Shortly after the time of birth, γ-globin is progressively replaced by the β-globin chain of adult hemoglobin (HbA).This complex process of globin gene expression called hemoglobin switch, is clinically important because the persistence or reactivation of γ-globin gene expression could mitigate the severity of the symptoms caused by β-thalassemia syndromes [9, 10].

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by continued γ-globin gene expression and, therefore, synthesis of high levels of HbF (1.6-30%) during adult life without other hematological abnormalities in affected heterozygotes. Two major types of HPFH have been described. Very high levels of fetal hemoglobin synthesis and uniform distribution of HbF among all RBCs characterize pancellular HPFH. On the other hand, heterocellular HPFH results from inherited increase in the number of erythrocytes with persisting production of fetal hemoglobin, termed F cells [28, 29].

the development of LVF in β-thalassemia but, when present, does affect the severity of the

The α-globin genes encoding the α-globin chains are duplicated (α1- and α2-globin genes) and localized in the telomeric region of chromosome 16 (16p13.3), in a cluster containing also an embryonic, ζ-gene, encoding the embryonic globin chains, three pseudogenes (pseudo ζ-, pseudo α1- and pseudo α2-gene) and θ-gene of unknown function [22]. The level of transcrip‐ tion of the two α-globin genes differs, as the α2 gene encodes two to three times more α-globin than α1 gene. The different expression of the two α-globin genes has implications for the amount of hemoglobin variant present in carriers of α1- or α2-globin mutations and for the pathophysiology of the deletional and nondeletional forms of α-thalassemia [23]. As with β-

thalassemia) production of α-globin chains, thus resulting in globin chain imbalance. The majority of the α-thalassemia defects result from deletions involving one or both α-globin genes on the same chromosome whereas point mutations affecting the functional expression

In many populations in which β-thalassemia is prevalent, α-thalassemia occurs at high frequency as well, resulting in coinheritance of both conditions. In these cases, homozygotes or compound heterozygotes for β-thalassemia will exert less severe phenotype since they would have less redundant α-globin chains. Therefore, the degree of amelioration of βthalassemia phenotype depends on the severity of the β-thalassemia alleles and the number of functional α-globin genes. At one extreme, homozygous β-thalassemia patients who have only one functional α-globin gene, have thalassemia intermedia. On the other hand, the presence of increased α-globin product, as a result of triplicated or quadruplicated α-globin genes in β-thalassemia heterozygotes, increase the globin chain imbalance, converting a typically clinically asymptomatic state to that of thalassemia intermedia [25]. However, the phenotype of a single extra α gene (ααα/αα) with heterozygous β-thalassemia is more variable

As already discussed, after a brief period of embryonic globin gene expression, the γ-globin chain of fetal hemoglobin (HbF) is predominantly expressed for much of gestation. Shortly after the time of birth, γ-globin is progressively replaced by the β-globin chain of adult hemoglobin (HbA).This complex process of globin gene expression called hemoglobin switch, is clinically important because the persistence or reactivation of γ-globin gene expression could

mitigate the severity of the symptoms caused by β-thalassemia syndromes [9, 10].

**3. Secondary genetic modifiers of β-thalassemia**

**3.1. Genetic modifiers of β-thalassemia within globin gene loci**

thalassemia, α-thalassemias are characterized by absent (α<sup>0</sup>

and depends on the severity of the β-thalassemia allele [26, 27].

of one of the two α-globin genes are not as common [24].

disease [20, 21].

142 Inherited Hemoglobin Disorders

*3.1.1. α-globin genes*

*3.1.2. γ -globin genes*

Pancellular HPFH could further be divided into two classes. The deletional HPFH is caused by large deletions 3' to the γ-globin genes, removing δ- and β-globin genes and part of the γδ intergenic DNA. These deletions appear to bring enhancer sequences into the proximity of the remaining γ-globin genes or remove specific regulatory sequences that play role as silencing elements for γ-globin genes, which in both cases promote high expression of fetal globin genes [4, 30]. Nondeletional form of HPFH are usually the result of point mutations (including small deletions) 5' from the *cap* site of γ-globin gene promoters [28]. These point mutations occur in transcription factor binding motifs in the γ-globin gene promoters, clustered in three regions, around positions -114 to -117, at -175, and from -195 to -202 nucleotide [31]. The first region contains distal CCAAT box, responsible for binding basal CP1 and CDP transcriptional factor and erythroid-specific GATA1 and NF-E3 transcriptional factors [32]. Region which contains mutation g.-175 T>C contains binding site for GATA1 transcriptional factor while mutations affecting -195 to -202 region alter the affinity for binding of the Sp1 transcriptional factor [33-35]. Recent studies on γ-globin gene promoter, showed the presence of mutations associated with HPFH, outside of the known, -202 to -110, region. Namely, mutation g.-567 C>G upstream of the γ G-globin gene (*HBG2*) alters a GATA1 binding motif which acts as a silencer of *HBG2* and is, thus, associated with increased γ-globin gene expression in affected adults [36].

Heterocellular HPFH is usually a result of mutations outside the β-globin gene cluster, such as locus located in the X chromosome. Namely, it was shown that a locus controlling F-cell production (FCP locus) is localized on Xp22.2 [37]. Despite these original findings, Xp22.2 loci affecting HbF levels were not supported by later genome-wide association studies (GWAS) [38, 39]. In some cases, elevated levels of HbF are observed in otherwise normal individuals, while in others, high HbF levels become apparent only when erythroid stress-producing factors are present [4, 30].

One of the common genetic variants that has little effect in normal individuals but favors a higher HbF response in the conditions of erythroid stress is XmnI-γG (g.-158 C>T) variant. This may explain why the same mutations on different chromosomal backgrounds are associated with different clinical severity. However, the HbF response associated with the XmnI-γG site is usually moderate and may not be sufficient to explain the wide difference in phenotype observed in some cases [26, 40]. Also, numerous studies do not support this positive effect of XmnI-γG, as a sole determinant on γ-globin gene expression [41, 42], which makes this issue very controversial.

Recently, large family and genome-wide association studies have shown that regions outside of the β-globin gene cluster are also implicated in γ-globin gene expression and HbF regulation. Two of them have been particularly studied: *HBS1*-*MYB* intragenic region and *BCL11A* gene.

#### **3.2. Genetic modifiers of β-thalassemia outside globin gene loci**

#### *3.2.1. HBS1L-MYB intragenic region*

Variants within *HBS1L*-*MYB* intragenic region, located on chromosome 6q23, account for more than 20% of the HbF level variance in northern Europeans. Most of these variants are distributed within 79 kb long region, which consist of three linkage disequilibrium blocks, referred to as *HBS1L-MYB* intergenic polymorphism (HMIP) blocks 1, 2, and 3. A small number of the variants shown to display an especially strong association with the in‐ creased levels of HbF, are concentrated in 24 kb of HMIP block 2, located 33 kb up‐ stream of *HBS1L* and 65 kb upstream of *MYB* and include polymorphisms rs28384513, rs9399137 and rs4895441 [43, 44]. Despite strong genetic evidence and extensive studies, a clear mechanism trough which these variants are causing variation in HbF levels has remained elusive, although the two flanking genes, *HBS1L* and *MYB* are candidate target genes. Namely, recent data suggest that this intragenic area contains GATA1 binding motifs and is thought to have properties of regulatory element [44].

*HBS1L* gene is thought to be housekeeping gene because it is ubiquitously expressed. The function of *HBS1L*, a possible member of the "GTPases" superfamily, in the red blood cell development, and therefore in the regulation of HbF levels, is not immediately apparent and could be manifested indirectly, through its effect on the expression of various cyto‐ kines and transcription factors that impact erythroid cell growth [38, 45]. On the other hand, *MYB* gene, encoding the c-MYB transcriptional factor, is a well known regulator of hematopoiesis and erythropoiesis. This transcriptional factor plays an essential role in controlling the erythroid cellular proliferation/differentiation and acts as a potent nega‐ tive regulator of HbF expression, trough which other genes, such as miR-15a and miR-16-1, play a role in HbF level variations [46, 47].

Although 6q22–23 locus, containing *HBS1L*-*MYB* intragenic region, have been extensively studied for over a decade, not enough attention had been paid in regard to other positional candidate genes in this quantitative trait loci (QTL). These include phosphodiesterase 7 (*PDE7B*)*,* mitogen-activated protein kinase kinase kinase 5 (*MAP3K5*), and peroxisomal biogeneses factor 7 (*PEX7*) genes with their SNPs showing strong association with different HbF levels [48]. It was shown that a short tandem repeat in the *MAP3K5* promoter, as well as intronic variations within both *MAP3K5* and *PDE7B* genes could be associated with lower HbF levels and thus, more severe β-thalassemia phenotype [49]. MAP3K5, encoded by *MAP3K5* gene, is a member of the MAPK family and, as such, a part of the MAPK pathway. This signaling cascade is one of the most important mechanisms for the cytoplasmic transduction of extracellular signals. As such, MAP3K5, has not been clearly associated with mechanisms governing erythropoiesis. On the other hand, *PDE7B* could be considered a strong modifier gene candidate given its high affinity and specificity for cAMP, which has an inhibitory effect on γ-globin expression and, thus, plays a role in fetal-to-adult globin gene switching [50].

#### *3.2.2. BCL11A gene*

Recently, large family and genome-wide association studies have shown that regions outside of the β-globin gene cluster are also implicated in γ-globin gene expression and HbF regulation. Two of them have been particularly studied: *HBS1*-*MYB* intragenic region and *BCL11A* gene.

Variants within *HBS1L*-*MYB* intragenic region, located on chromosome 6q23, account for more than 20% of the HbF level variance in northern Europeans. Most of these variants are distributed within 79 kb long region, which consist of three linkage disequilibrium blocks, referred to as *HBS1L-MYB* intergenic polymorphism (HMIP) blocks 1, 2, and 3. A small number of the variants shown to display an especially strong association with the in‐ creased levels of HbF, are concentrated in 24 kb of HMIP block 2, located 33 kb up‐ stream of *HBS1L* and 65 kb upstream of *MYB* and include polymorphisms rs28384513, rs9399137 and rs4895441 [43, 44]. Despite strong genetic evidence and extensive studies, a clear mechanism trough which these variants are causing variation in HbF levels has remained elusive, although the two flanking genes, *HBS1L* and *MYB* are candidate target genes. Namely, recent data suggest that this intragenic area contains GATA1 binding motifs

*HBS1L* gene is thought to be housekeeping gene because it is ubiquitously expressed. The function of *HBS1L*, a possible member of the "GTPases" superfamily, in the red blood cell development, and therefore in the regulation of HbF levels, is not immediately apparent and could be manifested indirectly, through its effect on the expression of various cyto‐ kines and transcription factors that impact erythroid cell growth [38, 45]. On the other hand, *MYB* gene, encoding the c-MYB transcriptional factor, is a well known regulator of hematopoiesis and erythropoiesis. This transcriptional factor plays an essential role in controlling the erythroid cellular proliferation/differentiation and acts as a potent nega‐ tive regulator of HbF expression, trough which other genes, such as miR-15a and miR-16-1,

Although 6q22–23 locus, containing *HBS1L*-*MYB* intragenic region, have been extensively studied for over a decade, not enough attention had been paid in regard to other positional candidate genes in this quantitative trait loci (QTL). These include phosphodiesterase 7 (*PDE7B*)*,* mitogen-activated protein kinase kinase kinase 5 (*MAP3K5*), and peroxisomal biogeneses factor 7 (*PEX7*) genes with their SNPs showing strong association with different HbF levels [48]. It was shown that a short tandem repeat in the *MAP3K5* promoter, as well as intronic variations within both *MAP3K5* and *PDE7B* genes could be associated with lower HbF levels and thus, more severe β-thalassemia phenotype [49]. MAP3K5, encoded by *MAP3K5* gene, is a member of the MAPK family and, as such, a part of the MAPK pathway. This signaling cascade is one of the most important mechanisms for the cytoplasmic transduction of extracellular signals. As such, MAP3K5, has not been clearly associated with mechanisms governing erythropoiesis. On the other hand, *PDE7B* could be considered a strong modifier

**3.2. Genetic modifiers of β-thalassemia outside globin gene loci**

and is thought to have properties of regulatory element [44].

play a role in HbF level variations [46, 47].

*3.2.1. HBS1L-MYB intragenic region*

144 Inherited Hemoglobin Disorders

BCL11A (B-cell CLL/lymphoma 11A) is transcriptional repressor expressed in most hemato‐ poietic cells and critically important in the T and B cell development. BCL11A is a zinc-finger protein with usually a C2N2 zinc finger at the N-terminal, and six other Krüppel-like C2H2 zinc fingers near the C-terminal and it has at least 4 predicted isoforms (XS, S, L, XL) due to the alternative splicing. It is encoded by *BCL11A* gene, which spans over 102 kb on chromo‐ some 2p16 [51]. Genome-wide association studies (GWAS) have demonstrated that a mean‐ ingful fraction of the variations in HbF levels is accounted for by variants within *BCL11A* gene [39]. More precisely, close to 15% of the phenotypic variation in the HbF levels could be explained by variations in intron 2 of the *BCL11A* gene, such as rs4671393 and rs11886868 [42, 43]. Further, it was shown that BCL11A (L and XL isoforms) acts as a potent silencer of γ-globin gene expression [52] by binding not to γ-globin gene promoter, but to LCR and γ<sup>A</sup>-δ intragenic region with known role in repression of fetal globin genes [53]. Its role as a repressor, BCL11A most probably exerts trough association with various partners within erythroid multiprotein complexes, including the repressive nucleosome remodeling and deacetylase complex (NuRD), GATA1, the erythroid master regulator, and SOX6, a transcription factor previously shown to repress embryonic globin genes in mice [54]. Evidence that BCL11A acts as a γ-globin gene repressor also lays in a fact that *BCL11A* expression levels are much higher in adult compered to fetal developmental stage [54, 55]. As a direct repressor of fetal globin genes, BCL11A is the first genetically and biochemically validated regulator of the fetal to adult globin switch in humans [42] and, as such, represents potential target protein for HbF induction. However, since BCL11A acts as a transcriptional factor in non erythroid cells as well, recent studies have proposed GWAS-identified erythroid *BCL11A* enhancers as a particularly promising target for gene therapy in the β-thalassemia syndromes. This way, disruption of the *BCL11A* enhancers would only affect *BCL11A* expression in erythroid cells, while this gene expression would be intact in the non erythroid cells [56].

Genome-wide association studies indicate that variants in the *HBB*, *HSB1L- MYB* and *BCL11A* loci account for approximately 50% of the HbF level variation, suggesting that additional factors are involved [38, 39, 43, 57]. These factors may involve regulators of β-globin genes' expression (KLF1), proteins responsible for α-globin chain stability (AHSP) or factors involved in epigenetic regulation of fetal globin gene expression (FoP).

#### *3.2.3. KLF1 gene*

Erythroid Krüppel-like factor, (KLF1), previously known as EKLF, is one of the key erythroidspecific transcriptional factor that interacts with the CACCC box, important regulatory element of many erythroid genes, including adult β-globin gene and *BCL11A* [58, 59]. This interaction is carried out via three zinc finger domains, necessary for binding and activation of KLF1 target genes. KLF1 also contains proline-rich transactivation domain tough which this transcriptional factor preferentially activates the *HBB* gene at the expense of *HBG1*/*HBG2* gene expression by interacting directly with regulatory elements in *HBB* promoter [60-62]. Prolinerich region contains functionally distinct activation (AA 20-124) and inhibitory domains (AA 195-291). The minimal activation domain (AA 20-124) can be further divided into a subregion (AA 20-60) that itself does not activate, but interacts trough intermolecular interactions with another cellular protein, providing optimal transactivation potential to its adjacent (AA 60- 124) sequence. At the same time, the inhibitory domain (AA 195-291) operates intramolecu‐ larly, preventing efficient binding of the DNA-binding zinc finger region within the same molecule [63]. Mutations affecting any of these functions of the KLF1 transcriptional factor, could potentially, directly or indirectly, alter expression of β-globin genes.

The direct association of mutations in human *KLF1* with hemoglobin regulation was first described by the Borg *et al.* [59], who reported a single point mutation in *KLF1* gene (p.K288X) responsible for completely abolishing the DNA binding domain resulting in KLF1 haploin‐ sufficiency. Results from this study showed that this reduced KLF1 activity results in decreased *BCL11A* expression and hence, diminished synthesis of BCL11, γ-globin gene repressor. This, in turn, leads to increased HbF levels, observed in the carriers of p.K288X mutation [59, 64]. Similarly, novel *KLF1* mutation (c.914-4\_914-1 del CTAG), hypothesized to affect splicing, causes haploinsufficiency of *KLF1*, leading to reduced expression levels of KLF1 target genes [65]. Results form our study on the first *KLF1* promoter mutation also go in favor of a KLF1 being a potent regulator of HbF levels [66]. Namely, results form this study suggest that this KLF1:g.-148G>A mutation leads to reduced *KLF1* gene transcription, which could explain, at least in part, the observed HPFH phenotype, further underlining the significant role of KLF1 on human fetal globin genes switching [66, 67].

Number of mutations affecting *KLF1* gene, as well as our understanding of different pheno‐ types associated with these mutations, are very obscure [68]. However, studies on *KLF1* gene mutations, including the ones without an obvious phenotype, contribute to the better under‐ standing of the human erythropoiesis in general [59, 69-71].

#### *3.2.4. AHSP gene*

The αHb-stabilizing protein (AHSP), also known as erythroid-associated factor (ERAF), is erythroid-specific protein with an important role in erythropoiesis. Namely, it is involved in folding of the α-globin chains for β-globin association, heme binding, transfer for β-globin association and stabilization of α-globin chains. AHSP specifically binds multiple forms of αglobin including the apo form (no heme present) and α-hemoglobin (αHb) (α-globin with heme). Its role as a specific molecular chaperone that binds α-globin chain of hemoglobin preventing its precipitation, imply that alterations in *AHSP* gene expression or protein function could influence β-thalassemia phenotypes [72-74]. While some studies reported that reduced expression of *AHSP* was associated with a more severe phenotype among individuals with identical β-thalassemia and α-globin [75, 76], others indicated that AHSP is not a disease modifier of β-thalassemia [77, 78]. Research on *AHSP* gene mutation showed that structural mutations are uncommon, and therefore, not likely to be major modifiers of β-thalassemia. However, it remains a possibility that rare *AHSP* null or missense mutations, genetic or epigenetic factors unlinked to the gene, could modulate coexisting β-thalassemia [73, 77].

#### *3.2.5. FoP protein*

expression by interacting directly with regulatory elements in *HBB* promoter [60-62]. Prolinerich region contains functionally distinct activation (AA 20-124) and inhibitory domains (AA 195-291). The minimal activation domain (AA 20-124) can be further divided into a subregion (AA 20-60) that itself does not activate, but interacts trough intermolecular interactions with another cellular protein, providing optimal transactivation potential to its adjacent (AA 60- 124) sequence. At the same time, the inhibitory domain (AA 195-291) operates intramolecu‐ larly, preventing efficient binding of the DNA-binding zinc finger region within the same molecule [63]. Mutations affecting any of these functions of the KLF1 transcriptional factor,

The direct association of mutations in human *KLF1* with hemoglobin regulation was first described by the Borg *et al.* [59], who reported a single point mutation in *KLF1* gene (p.K288X) responsible for completely abolishing the DNA binding domain resulting in KLF1 haploin‐ sufficiency. Results from this study showed that this reduced KLF1 activity results in decreased *BCL11A* expression and hence, diminished synthesis of BCL11, γ-globin gene repressor. This, in turn, leads to increased HbF levels, observed in the carriers of p.K288X mutation [59, 64]. Similarly, novel *KLF1* mutation (c.914-4\_914-1 del CTAG), hypothesized to affect splicing, causes haploinsufficiency of *KLF1*, leading to reduced expression levels of KLF1 target genes [65]. Results form our study on the first *KLF1* promoter mutation also go in favor of a KLF1 being a potent regulator of HbF levels [66]. Namely, results form this study suggest that this KLF1:g.-148G>A mutation leads to reduced *KLF1* gene transcription, which could explain, at least in part, the observed HPFH phenotype, further underlining the significant role of KLF1

Number of mutations affecting *KLF1* gene, as well as our understanding of different pheno‐ types associated with these mutations, are very obscure [68]. However, studies on *KLF1* gene mutations, including the ones without an obvious phenotype, contribute to the better under‐

The αHb-stabilizing protein (AHSP), also known as erythroid-associated factor (ERAF), is erythroid-specific protein with an important role in erythropoiesis. Namely, it is involved in folding of the α-globin chains for β-globin association, heme binding, transfer for β-globin association and stabilization of α-globin chains. AHSP specifically binds multiple forms of αglobin including the apo form (no heme present) and α-hemoglobin (αHb) (α-globin with heme). Its role as a specific molecular chaperone that binds α-globin chain of hemoglobin preventing its precipitation, imply that alterations in *AHSP* gene expression or protein function could influence β-thalassemia phenotypes [72-74]. While some studies reported that reduced expression of *AHSP* was associated with a more severe phenotype among individuals with identical β-thalassemia and α-globin [75, 76], others indicated that AHSP is not a disease modifier of β-thalassemia [77, 78]. Research on *AHSP* gene mutation showed that structural mutations are uncommon, and therefore, not likely to be major modifiers of β-thalassemia. However, it remains a possibility that rare *AHSP* null or missense mutations, genetic or epigenetic factors unlinked to the gene, could modulate coexisting β-thalassemia [73, 77].

could potentially, directly or indirectly, alter expression of β-globin genes.

on human fetal globin genes switching [66, 67].

*3.2.4. AHSP gene*

146 Inherited Hemoglobin Disorders

standing of the human erythropoiesis in general [59, 69-71].

Friend of Prmt1 (FoP) is a small arginine/glycine rich protein encoded by the *C1orf77* gene. This protein, through its association with protein arginine methyltransferase 1 (PRMT1), is involved in transcriptional regulation of globin genes via histone methylation [79]. Studies have shown that this protein is a critical modulator of HbF levels, since knockdown of this factor leads to elevated γ-globin gene expression. Although it is unclear how FoP regulates γ-globin gene expression, it is presumed that this induction of fetal globin genes occurs trough modulation of SOX6 which acts as BCL11A cofactor, and not trough BCL11A itself. These results identify FoP as a novel potential therapeutic target in β-thalassemia syndromes, as well as in other hemoglobin-related disorders [80].

**Figure 2.** γ-globin gene regulators as potential targets for therapeutic induction of HbF. Human β-globin gene locus showing embryonic, fetal and adult globin genes, controlled by LCR. Transcription factors involved in regulation of γglobin genes are shown. A line with an arrow at the end denotes positive regulation (activation). A line with a black box at the end denotes negative regulation (repression). Dashed line denotes ambiguous interaction. A line with ar‐ rows at two end points denotes mutual interaction. For details see the text.

#### **4. Gene therapy**

Despite immense achievement in the traditional care and treatment of β-thalassemia, including transfusion and drug therapy, until recently, a definitive cure for these disorders could only be achieved by bone marrow transplantation (BMT) from related or unrelated donors. However, BMT is available for only a small fraction of β-thalassemia patients and is charac‐ terized by relatively high mortality and morbidity, especially in the case of unrelated donors [27]. As an answer to these limitations of BMT and the need for more permanent solution, arose transfer of a therapeutic gene using autologous hematopoietic stem cells (HSC) as potential definitive cure for β-thalassemia syndromes.

The goal of this gene therapy is substitution of a defective or missing protein by introducing an intact copy of the faulty gene in question or by introduction of a gene which modifies the effect defected gene has on a cell such as β-thalassemia gene modifiers do.

For the successful gene therapy for β-hemoglobinopathies, several requirements need to be met. Those include high-efficiency gene transfer and high HSC engraftment; consistent levels of β-globin gene expression, independent of the site of integration; high expression levels of β-globin or γ-globin genes; regulated expression in the erythroid lineage; safe expression with little or no risk of insercional mutagenesis/oncogenesis [81].

Hematopoietic stem cells, as the targets for gene transfer, are harvested from patient bone marrow or peripheral blood following cytokine mobilization, purified by immunoselection, transduced and returned to the patient. Various types of vectors have been considered for gene transfer, including viral and non viral vectors [82].

#### **4.1. Non viral vectors**

Indication that gene therapy, as a therapy of the future for thalassemia syndromes, could be possible, came from the study on suppressor tRNA [83]. In this study a human tRNALys gene was converted to an amber suppressor by site-specific mutagenesis of the anticodon. As a result, a tRNA that suppressed the UAG amber nonsense mutation in β<sup>0</sup> -thalassemia mRNA was produced giving rise to functional β-globin polypeptide. Although promising, the use of such genes in gene therapy would be limited to β<sup>0</sup> -thalassemias due to nonsense mutations. Also, amber suppressor tRNA could suppress termination of the proteins with UAG as their normal termination codon making these genes not an ideal candidate for use in gene therapy.

As a potential approach to gene therapy for hemoglobin disorders, the catalytic properties of ribozymes to alter the defective mRNA produced by the mutated β-globin gene was also explored [84]. Namely, *trans*-acting group 1 ribozyme was created in a such a way that it was able to convert mutated β-globin transcript into RNA encoding γ-globin. This way, not only would the mutated gene be removed but it would be replaced with gene producing fetal globin chain. Although these results were very promising, several problems emerged, one of which is long-term effect of the therapy since it is directed at transcription product and not at the mutated gene itself [85].

Contrary to these non viral vectors, viral vectors showed higher gene transfer efficiency, since they do not require harsh physical means, such as electroporation, to enter the cell [86].

#### **4.2. Viral vectors**

The first viral vectors used to transfer the human β-globin gene in the mouse HSCs were oncoretroviruses or gamma-retroviruses (γRV), which efficiently transfer therapeutic gene into HSCs without transferring any viral genes. Gamma-retroviruses belong to the family of Retroviridae, which, among others, also include lentivirus (LV) and foamy virus (FV), both used in clinical trials or preclinical testing. These viral vectors are constructed in such a way that the genetic elements needed for pathogenicity and replication are removed and are replaced with the cellular transgene of interest [86, 87].

#### *4.2.1. Retroviral vectors*

The goal of this gene therapy is substitution of a defective or missing protein by introducing an intact copy of the faulty gene in question or by introduction of a gene which modifies the

For the successful gene therapy for β-hemoglobinopathies, several requirements need to be met. Those include high-efficiency gene transfer and high HSC engraftment; consistent levels of β-globin gene expression, independent of the site of integration; high expression levels of β-globin or γ-globin genes; regulated expression in the erythroid lineage; safe expression with

Hematopoietic stem cells, as the targets for gene transfer, are harvested from patient bone marrow or peripheral blood following cytokine mobilization, purified by immunoselection, transduced and returned to the patient. Various types of vectors have been considered for gene

Indication that gene therapy, as a therapy of the future for thalassemia syndromes, could be possible, came from the study on suppressor tRNA [83]. In this study a human tRNALys gene was converted to an amber suppressor by site-specific mutagenesis of the anticodon. As a

was produced giving rise to functional β-globin polypeptide. Although promising, the use of

Also, amber suppressor tRNA could suppress termination of the proteins with UAG as their normal termination codon making these genes not an ideal candidate for use in gene therapy. As a potential approach to gene therapy for hemoglobin disorders, the catalytic properties of ribozymes to alter the defective mRNA produced by the mutated β-globin gene was also explored [84]. Namely, *trans*-acting group 1 ribozyme was created in a such a way that it was able to convert mutated β-globin transcript into RNA encoding γ-globin. This way, not only would the mutated gene be removed but it would be replaced with gene producing fetal globin chain. Although these results were very promising, several problems emerged, one of which is long-term effect of the therapy since it is directed at transcription product and not at the

Contrary to these non viral vectors, viral vectors showed higher gene transfer efficiency, since they do not require harsh physical means, such as electroporation, to enter the cell [86].

The first viral vectors used to transfer the human β-globin gene in the mouse HSCs were oncoretroviruses or gamma-retroviruses (γRV), which efficiently transfer therapeutic gene into HSCs without transferring any viral genes. Gamma-retroviruses belong to the family of Retroviridae, which, among others, also include lentivirus (LV) and foamy virus (FV), both used in clinical trials or preclinical testing. These viral vectors are constructed in such a way that the genetic elements needed for pathogenicity and replication are removed and are

effect defected gene has on a cell such as β-thalassemia gene modifiers do.

result, a tRNA that suppressed the UAG amber nonsense mutation in β<sup>0</sup>

little or no risk of insercional mutagenesis/oncogenesis [81].

transfer, including viral and non viral vectors [82].

such genes in gene therapy would be limited to β<sup>0</sup>

replaced with the cellular transgene of interest [86, 87].

**4.1. Non viral vectors**

148 Inherited Hemoglobin Disorders

mutated gene itself [85].

**4.2. Viral vectors**

The γRV vectors contain intact viral long terminal repeats (LTRs) which enclose U3, R and U5 regions. The U3 region has strong promoter and enhancer activity and is usually used by the virus to reverse transcribe and incorporate the genetic material into the host genome (Figure 3.) [86]. Early attempts to transfer human β-globin gene in the mouse HSCs using these vectors, resulted in tissue-specific, but low and variable human β-globin expression in bone marrow chimeras, usually varying between 0% and 2% of endogenous mouse β-globin mRNA levels [88]. In order to increase expression levels of transferred β-globin genes, efforts were made to include LCR elements of the β-globin gene locus into these γRV vectors. Incorporation of the LCR's DNase I hypersensitivity sites, HS2, HS3 and HS4, significantly increased expression levels in the murine erythroleukemia (MEL) cells, but failed to abolish positional variability expression and resulted in vectors with low titers [81, 87]. Gamma-retroviral vectors were also limited by their size, need for cell division before integration, as well as their stable transmis‐ sion since they were so unstable that the transduced globin gene was very rarely integrated intact into the genome, thus losing any therapeutic efficacy [27].

#### **4.2.2. Lentiviral vectors**

Bioengineering of HIV-1 devoid of any pathogenic elements resulted in the development of lentiviral (LV) vectors as suitable vectors for high-efficiency gene transfer. In order to improve its safety, self-inactivating (SIN) LV vector was constructed by deleting the viral promoter/ enhancer in the U3 region of the 3' LTR, without significant loss in titers or infectivity. During reverse transcription of the viral RNA, this deletion that gets copied to the 5' LTR, minimizing transactivation of neighboring cellular promoters, thus improving the safety of the vector itself [89]. Instead of U3 region in the 5' LTR, SIN LV vector contains a cytomegalovirus (CMV) promoter which is only used in packaging the vector and is not transmitted to the host cell (Figure 3.). This new generation of LV vectors display variety of advantages compared to γRV vectors including the ability to infect quiescent, nondividing long-term HSC (HSC capable of self renewal). Unlike γ RV vectors, SIN LV can stably carry larger and more complex transgene cassettes containing introns and regulatory elements, necessary for high globin gene expres‐ sion. LV vectors usually insert these cassettes within gene introns, avoiding promoters and 5' regulator regions which RV vectors have high affinity for [86, 90].

With the discovery of LV vectors as a potent transporter of gene of interest, research turned to the globin cassette itself. The first study to demonstrate stable transmission and high-level β-globin gene expression in a mouse model of β-thalassemia intermedia, tested two types of LV vectors. Larger, TNS9 vector, contained large LCR fragments encompassing HS2, HS3 and HS4 and was approximately 3.2 kb in size, while smaller, RNS1 vector carried only a minimal core LCR elements. It was shown that cells transduced with TNS9 vector sustained higher human β-globin transcript levels. Also, this vector achieved significant improvement in the hematocrits, red blood cell and reticulocyte count, as well as hemoglobin levels in β-thalasse‐ mia mice [91]. Today, vectors' globin cassette usually contains β-globin gene with deleted destabilizing Rsal fragment located within intron 2 and several sequences surrounding HS of the LCR. However, the number and the length of the HS sites vary in all globin cassettes, since

**Figure 3.** A. Genome organization of MLV γRV and γRV vector; B. Genome organization of HIV LV and SIN LV vec‐ tor.

it's proven very difficult to define really important regulatory sequence within each HS, while omitting potentially destabilizing elements. Also, some of the vectors in use are flanked by insulators, genomic element that can shelter genes from their surrounding chromosomal environment, resulting in position-independent expression [27].

#### **5. Gene therapy ofβ -thalassemia: Success or fail?**

The first successful human gene therapy for β-thalassemia was achieved in 2007., when HbE/ β0 -thalassemia major patient was transduced with vector containing antisickling β-globin (βA(T87Q)), a 260 bp long globin promoter, HS2, HS3, HS4 and two copies of insulator flanking the globin cassette. Although the patient became totally transfusion independent, it was discovered a dominant cell clone with integration site into the *HMGA2* gene, a potential oncogene. While it was shown that overexpression of *HMGA2* is mainly associated with benign tumors, this observation points out some of the limitation of lentiviral vector gene transfer, which include the need for improved efficiency of gene delivery and insertion of the gene into non-oncogenic sites [92].

As the secondary modifier genes begin to take a center stage in the fight against β-thalassemia syndromes, researchers turned to these genes as potential target genes in gene therapy for βthalassemia. Namely, transduction of CD34 cells with lentiviral vector carrying a short hairpin RNA (shRNA) targeting the γ-globin gene repressor BCL11A, led to significant increase of HbF in differentiated erythroblasts, around 10% in cells derived from normal donors and from 33% to 45% in β-thalassemic cells [93].

In the past few years, induced pluripotent stem cells (iPSCs) emerged as an interesting candidate for gene transfer. iPSCs are generated from mature somatic cells derived from skin fibroblasts, amniotic fluid or chorionic villus of β-thalassemia patients, by transduction with number of specific transcriptional factors. More specifically, it was shown that fibroblasts derived from tail-tip of a mouse, homozygote for human sickle cell allele (βS /βS ), and infected with retroviruses encoding for Oct4, Sox2, and Klf4 factors, as well as with a lentivirus encoding a 2-lox c-Myc cDNA, were transformed into iPSC. These iPS cells, after being infected with an adenovirus encoding Cre-recombinase to delete the lentivirus-transduced c-Myc copies, in order to reduce the potential risk of tumor formation due to c-Myc transgene expression, stained positive for pluripotency markers. They also had a normal karyotype, and generated teratomas and chimeras. iPSC obtained in such a way were later successfully used in specific correction of the sickle cell allele, by being electroporated with a construct containing the human wild type β-globin gene. [94].

iPSCs are easily obtainable and represent an endless source of stem cells for gene manipulation and correction strategies. Therefore, the major advantage of these cells, when it comes to gene therapy, is the possibility to screen and choose the ideal clone with safe integration and high transgene expression profile. Unfortunately, several obstacles stand in the way of iPSCs being successfully used in gene therapy, one of which is elimination of the transcriptional factors used for induction, once they are no longer needed. More importantly, it is necessary to establish the correct re-programming so that the iPSCs do not develop tumors [27, 86, 87].

#### **6. Conclusion**

it's proven very difficult to define really important regulatory sequence within each HS, while omitting potentially destabilizing elements. Also, some of the vectors in use are flanked by insulators, genomic element that can shelter genes from their surrounding chromosomal

**Figure 3.** A. Genome organization of MLV γRV and γRV vector; B. Genome organization of HIV LV and SIN LV vec‐

The first successful human gene therapy for β-thalassemia was achieved in 2007., when HbE/

environment, resulting in position-independent expression [27].

**5. Gene therapy ofβ -thalassemia: Success or fail?**

β0

tor.

For details see the text.

150 Inherited Hemoglobin Disorders

non-oncogenic sites [92].

The β-thalassemias are the best understood disorders at the molecular level. Accordingly, therapy protocols based on molecular basis of β-thalassemia have been designed as an example of novel approaches in disease treatment. However, there is no well established gene therapy protocol for β-thalassemia to date. Why is so difficult to design an appropriate "therapeutic gene", even for this monogenic disorder, and to deliver it to hematopoietic stem cells to achieve therapeutic effect in β-thalassemia patients? Despite extensive research, modern science does not understand a complex gene expression regulation of globin genes yet. First of all, there is very specific regulation of expression of globin genes during ontogenesis. Also, tissue (erythroid)-specific regulation is present. Moreover, globin genes are regulated in a cellspecific manner since they are expressed only in the certain stages during differentiation of erythroid cell lineage. Finally, there is coordination between expression of α- and β-globin genes.

Besides all that, our knowledge is accumulating, and there is no doubt that gene manipulation will begin to cure in near future. Certainly, only somatic gene therapy is considered, since germ-line gene therapy raises many unique ethical concerns. Hopefully, thanks to gene therapy, a large number of people suffering from β-thalassemia will have a long and better life, despite the predispositions.

#### **Acknowledgements**

This work was supported by Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. III41004).

#### **Author details**

Sonja Pavlovic\* , Milena Ugrin and Maja Stojiljkovic

\*Address all correspondence to<EMAIL_ADDRESS>
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia

#### **References**

Besides all that, our knowledge is accumulating, and there is no doubt that gene manipulation will begin to cure in near future. Certainly, only somatic gene therapy is considered, since germ-line gene therapy raises many unique ethical concerns. Hopefully, thanks to gene therapy, a large number of people suffering from β-thalassemia will have a long and better life, despite the predispositions. That way, an old proverb will finally become true: "Fato

This work was supported by Ministry of Education, Science and Technological Development,

Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade,

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### **Sickle Cell Disease – Current Treatment and New Therapeutical Approaches**

Thais Regina Ferreira de Melo, Lucas dos Reis Ercolin, Rafael Consolin Chelucci, Aylime Castanho Bolognesi Melchior, Carolina Lanaro, Chung Man Chin and Jean Leandro dos Santos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60515

#### **Abstract**

Sickle cell disease (SCD) is one of the most common genetic disorders worldwide. It is caused by a point mutation that changes glutamic acid (Glu6) to valine (Val6) in the β chain of hemoglobin. Vaso-occlusion is the most well-known problem associated with SCD. Despite recent advances in understanding the disease at the molecular level, few therapeutic strategies are available. Hydroxyurea is the only drug currently approved by the U.S. Food and Drug Administration for the disease, and it has serious adverse effects and lack of efficacy in some patients. However, new therapeutic approaches are under investigation in the hope of discovering new drugs to treat SCD. These include agents that: a) increase nitric oxide bioavailability; b) modify the rheological properties of the blood; c) bind covalently to hemoglobin; d) prevent hemoglobin dehydration; e) reduce iron overload; and f) induce the expression of gamma globin and fetal hemoglobin. In this chapter, we discuss the current treatment of SCD and the advances made in medicinal chemistry to find new drugs to treat this neglected hematological disease.

**Keywords:** Sickle cell disease, hemoglobinopathy, gamma globin, fetal hemoglobin, drug discovery

© 2015 The Author(s). Licensee InTech. 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.

#### **1. Introduction**

#### **1.1. General background**

Sickle cell disease (SCD) is one of the most prevalent hemoglobinopathies worldwide. It has been hypothesized that this disease originated millions of years ago, in the sub-Saharan countries in mid-western Africa, eastern Asia, and some regions of India [1]. The distribution of the disease correlates with the malaria-endemic regions because it confers a protective effect against infection by the plasmodium [1-3].

Many biochemical and immunological mechanisms have been investigated to explain the protection conferred by hemoglobin S (HbS) against malaria. Infected sickle erythrocytes are known to be phagocytized faster than normal erythrocytes, thereby reducing parasitemia [4]; however, despite the many studies of the complex mechanisms involved, their relevance *in vivo* remains unclear [5].

According to the World Health Organization, about 5% of the world population carried a gene mutation for a hemoglobinopathy in 2011, particularly those mutations causing SCD and thalassemia. Today, SCD is not restricted to Africa and parts of India, but is found in the America and Europe, mainly as a result of migration and racial intermingling. In the United States, the disease afflicts approximately 1:500 Afro-American and 1:4000 Hispanic-American neonates [6].

The Brazilian National Program of Neonatal Screening estimates that around 2 million individuals carry the HbS trait in that country and 25,000–50,000 individuals are homozygous for HbS. About 3,500 children are thought to be born with SCD every year and 200,000 are heterozygous for the HbS gene [7-8].

#### **1.2. Pathophysiology**

Sickle cell disease is characterized by a point mutation in the sixth codon of the β-globin gene. The replacement of a thymine residue with an adenine (GTG to GAG) results in the substitution of glutamic acid for valine in the β-chain of hemoglobin, thus producing an anomalous hemoglobin (β<sup>s</sup> -globin). After several cycles of deoxygenation and oxygenation, the HbS molecule polymerizes. This process is facilitated during the deoxygenation state of hemoglobin S (HbS) by hydrophobic interactions between the β subunits of the hemoglobin tetramer. The polymers thus formed can damage the erythrocyte structure, leading to sickle-shaped erythrocytes [9].

The polymerization of HbS represents the primary event in the molecular pathogenesis of the disease, and this process is dependent on several factors, including the concentrations of HbS and oxygen, the presence of high levels of normal hemoglobin, pH, temperature, and ionic strength [10]. HbS polymerization is responsible for: a) altering the structure and flexibility of the erythrocytes; b) promoting erythrocyte dehydration; and c) physical and oxidative stress [11, 12]. All of these events contribute to the hemolysis of the erythrocytes. The heme group present in the hemoglobin is then released into the circulation and can capture the nitric oxide (NO) molecules present in the vascular endothelium, generating a "vasoconstriction effect" in the patient [13]. Low levels of NO contribute to the vasculopathy and hypercoagulability characteristic of the disease, and have been related to its clinical manifestations, including pulmonary hypertension, leg ulcers, priapism, and cerebrovascular disease [14, 15].

**1. Introduction**

162 Inherited Hemoglobin Disorders

**1.1. General background**

*vivo* remains unclear [5].

heterozygous for the HbS gene [7-8].

neonates [6].

**1.2. Pathophysiology**

hemoglobin (β<sup>s</sup>

erythrocytes [9].

against infection by the plasmodium [1-3].

Sickle cell disease (SCD) is one of the most prevalent hemoglobinopathies worldwide. It has been hypothesized that this disease originated millions of years ago, in the sub-Saharan countries in mid-western Africa, eastern Asia, and some regions of India [1]. The distribution of the disease correlates with the malaria-endemic regions because it confers a protective effect

Many biochemical and immunological mechanisms have been investigated to explain the protection conferred by hemoglobin S (HbS) against malaria. Infected sickle erythrocytes are known to be phagocytized faster than normal erythrocytes, thereby reducing parasitemia [4]; however, despite the many studies of the complex mechanisms involved, their relevance *in*

According to the World Health Organization, about 5% of the world population carried a gene mutation for a hemoglobinopathy in 2011, particularly those mutations causing SCD and thalassemia. Today, SCD is not restricted to Africa and parts of India, but is found in the America and Europe, mainly as a result of migration and racial intermingling. In the United States, the disease afflicts approximately 1:500 Afro-American and 1:4000 Hispanic-American

The Brazilian National Program of Neonatal Screening estimates that around 2 million individuals carry the HbS trait in that country and 25,000–50,000 individuals are homozygous for HbS. About 3,500 children are thought to be born with SCD every year and 200,000 are

Sickle cell disease is characterized by a point mutation in the sixth codon of the β-globin gene. The replacement of a thymine residue with an adenine (GTG to GAG) results in the substitution of glutamic acid for valine in the β-chain of hemoglobin, thus producing an anomalous

molecule polymerizes. This process is facilitated during the deoxygenation state of hemoglobin S (HbS) by hydrophobic interactions between the β subunits of the hemoglobin tetramer. The polymers thus formed can damage the erythrocyte structure, leading to sickle-shaped

The polymerization of HbS represents the primary event in the molecular pathogenesis of the disease, and this process is dependent on several factors, including the concentrations of HbS and oxygen, the presence of high levels of normal hemoglobin, pH, temperature, and ionic strength [10]. HbS polymerization is responsible for: a) altering the structure and flexibility of the erythrocytes; b) promoting erythrocyte dehydration; and c) physical and oxidative stress [11, 12]. All of these events contribute to the hemolysis of the erythrocytes. The heme group present in the hemoglobin is then released into the circulation and can capture the nitric oxide

NO is an important mediator of cell functions, with various effects, including vasodilatation, the inhibition of platelet aggregation, and the reduced expression of adhesive molecules (Figure 1). This mediator also stimulates the expression of the gamma globin gene and consequently increases the production of fetal hemoglobin (HbF). This mechanism seems to involve soluble guanylyl cyclase (sGC), which increases the expression of γ-globin in eryth‐ roleukemic cells and primary erythroblasts [16, 17].

**Figure 1.** Effects of nitric oxide on the vascular endothelium, inflammatory cells, and platelets.

Another manifestation of SCD is vaso-occlusion (Figure 2), which is the major cause of morbidity in patients with SCD, causing tissue infarct, painful crises, acute thoracic syndrome, and nephropathy. The interactions of the sickle erythrocytes, leucocytes, neutrophils, and platelets with the vascular endothelium increase, leading to the formation of heterocellular aggregates, which are responsible for vaso-occlusion [18]. Mechanistically, the interaction between the erythrocytes and the endothelium involves α4β1 integrin, expressed on the erythrocyte surface, and fibronectin, vascular cell adhesion protein 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), and E-selectin, expressed on the endothelial cell surface [19]. Other ligands, including thrombospondin, von Willebrand factor, immunoglobulins, and fibrinogen, also seem to contribute to this adherence [20].

This vaso-occlusion is aggravated by ischemic cycles, which cause oxidative and inflammatory stress and increase the production of proinflammatory cytokines [21, 22].

**Figure 2.** Pathophysiology of sickle cell disease.

Inflammation is another central feature of the vasculopathy of SCD (Figure 2). The adhesion and activation of leucocytes increase the production of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-8, which contribute to chronic inflammation and vaso-occlusive crises [23, 24]. SCD patients show increased levels of proinflammatory cytokines, including TNF-α [25], and high levels of TNF-α can increase the chemotactic proprieties of cells and amplify inflammation. This cytokine is also responsible for: a) increasing neutrophil adherence to the vascular endothelium; b) stimulating the production of free radicals; c) stimulating the synthesis of other inflammatory mediators, such as IL-1β and prostaglandin E2 (PGE2); and d) modulating coagulation and anticoagulation functions [26, 27]. Therefore, the increased plasma levels of TNF-α in SCD patients contribute to their vaso-occlusive crises and inflammatory episodes [25, 28-29].

#### **1.3. Treatment**

Despite current advances in medical technology, there is still no specific treatment for SCD. The drugs available can only reduce the symptoms and increase the patient's quality of life. The complexity of SCD is an obstacle to the scientific development of new selective and effective therapies. This is coupled with the lack of interest within the pharmaceutical industry in searching for new drugs for this disease, another major impediment to the discovery of new treatments [30, 31]. Here, we discuss the main strategies and current advances in the search for new drugs with which to treat SCD.

Several strategies and therapies can be explored for the treatment of SCD. Among these, we include: a) the induction of HbF synthesis; b) the inhibition of erythrocyte dehydration; c) the inhibition of cellular adhesion; d) vasodilators; e) adenosine agonists; f) hemoglobin modifiers; g) Rho-kinase inhibition; and h) chelating agents (Figure 3). All of these strategies are discussed below.

**Figure 3.** Therapeutic strategies for the treatment of SCD.

#### *Induction of HbF synthesis*

**Figure 2.** Pathophysiology of sickle cell disease.

164 Inherited Hemoglobin Disorders

**1.3. Treatment**

Inflammation is another central feature of the vasculopathy of SCD (Figure 2). The adhesion and activation of leucocytes increase the production of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-8, which contribute to chronic inflammation and vaso-occlusive crises [23, 24]. SCD patients show increased levels of proinflammatory cytokines, including TNF-α [25], and high levels of TNF-α can increase the chemotactic proprieties of cells and amplify inflammation. This cytokine is also responsible for: a) increasing neutrophil adherence to the vascular endothelium; b) stimulating the production of free radicals; c) stimulating the synthesis of other inflammatory mediators, such as IL-1β and prostaglandin E2 (PGE2); and d) modulating coagulation and anticoagulation functions [26, 27]. Therefore, the increased plasma levels of TNF-α in SCD patients contribute

Despite current advances in medical technology, there is still no specific treatment for SCD. The drugs available can only reduce the symptoms and increase the patient's quality of life. The complexity of SCD is an obstacle to the scientific development of new selective and effective therapies. This is coupled with the lack of interest within the pharmaceutical industry

to their vaso-occlusive crises and inflammatory episodes [25, 28-29].

The induction of HbF synthesis is a promising strategy for the treatment of SCD [32, 33]. The elevated levels of HbS and low levels of HbF in patients with SCD are related to the clinical severity of the disease and the early mortality of the patients. This effect is related to high levels of HbS polymerization and its increased adherence to the vascular endothelium, which aggravate the vaso-occlusive process [34, 35]. Agents that increase HbF levels include hydrox‐ yurea (HU), decitabine, azacitidine, NO donors, butyric acid and its derivatives, erythropoie‐ tin, and thalidomide and its derivatives.

#### **•** Hydroxyurea

Hydroxyurea (Figure 4) is a chemotherapeutic agent and selective inhibitor of ribonucleoside diphosphate reductase, an enzyme that converts ribonucleoside diphosphates to deoxyribo‐ nucleoside diphosphates. Therefore, HU inhibits the G1/S phase transition of the cell cycle [36]. Currently, it is the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of SCD.

**Figure 4.** Chemical structure of hydroxyurea.

HU reduces the number of vaso-occlusive crises, episodes of acute thoracic syndrome, and urgent requirement for blood transfusion [37]. One nine-year clinical study demonstrated a reduction of 40% in the number of fatalities among patients treated with this drug [38].

The beneficial effects of HU in SCD are related to the increase of HbF levels. Some data suggest that the mechanism whereby HU increases the levels of HbF involves its biotransformation of NO, which activates the soluble guanylate cyclase (sGC) on erythroid cells [17, 39]. The activation of sGC increases the expression of γ-globin in erythroleukemic cells and primary human erythroblasts [16]. Other effects of HU include the reduction of leukocytes, reticulo‐ cytes, and platelets, and a reduction in the adhesiveness of erythrocytes and leukocytes to the vascular endothelium [40]. However, HU has several adverse effects, such as myelotoxicity, cutaneous hyperpigmentation, and ulcerative lesions on lower limbs [41].

Despite these adverse effects, the benefits of HU use are supported by evidence of its efficacy in reducing morbidity and mortality. However, importantly, about one third of patients do not respond to HU treatment [42]. In this context it is important to introduce new drugs that recapitulate the beneficial effects of HU without its potential toxicity.

**•** NO donors