Gene Therapy And Its Potency In Treatment Biology Essay


Thalassaemias are a group of disorders occur as a result of genetic mutations that incorporate a decrease of gene synthesis or a complete reduction and exclusion of one or more of the hemoglobin globin chains (Rodak, 2002). Thalassaemia name is derived from the Greek word ''Sea'' which states Thalassa (Whipple and Bradford, 1936) and it is a hereditary disease which occurs due to a globin gene mutation which leads to a disturbance of synthesis of haemoglobins' globin chains and, consequently, disturbs the level of globin genes leading to an erythroopoietic and pathologic tribulations. In 1925, the first case of thalassaemia was discovered by a Detroit physician, Thomas B. Cooley, who defined infants who are diagnosed with severe anaemia with spleen enlargement/splenomegaly (Hoffbrand et al., 2005).

Moreover, thalassaemia has been divided, as shown in Table 1.1, into several types according to the phenotype or genotype (Bain, 2006). The application of gene therapy in treatment for β-thalassaemia is based on the point of mutations and the expression level of β-globin gene (Liu et al., 1990; Forget, 1998). Furthermore, treatment of β-thalassaemia by using blood transfusion and bone marrow transplantation is still inadequate, which makes gene therapy the most preferred option (Romano et al., 1999). However, a number of studies have shown some severe outcomes due to gene therapy that have limited its use for thalassaemia (Liu et al., 1990) and (Forget, 1998) which will be described later in this review.

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This review will describe thalassaemia disorder, focusing on β-thalassaemia taking into consideration its pathogenesis, etiology including the genetic defects, and the current treatments and new therapeutic trials on thalassaemia in view of some aspects and potency of genetic therapy on β-thalassaemias in comparison to other current treatments.

1.1. Haemoglobin:

The red blood cells contain particular protein haemoglobin; which assist red cells' role in gaseous exchange by carrying O2 to body tissues and carrying carbon dioxide (CO2) back from tissues to the lungs. Moreover, one red cell contains around 460 million Hb molecules; and each molecule of normal adult Hb A composed of four polypeptide chains α2 β2, each contain its haem group (Figure 1.1). In addition, Hb A is a dominant Hb present in blood after 3-6 months of age with a molecular weight approximately of 68000. Moreover, Adult Hb conatins small number of Hb F and Hb A2; these two haemoglobins contain α chain but with γ and δ chains, respectively, instead of the β globin chain. The switch from fetal to adult Hb begins at 3-6 months after birth (Hoffbrand et al., 2000).

Synthesis of haem is mainly occurring in the mitochondria through biochemical reactions initiating with the condensation of glycine and succinyl coenzyme-A which is controlled by the δ-aminolaevulinic acid (ALA) synthase. Moreover, Vitamin B6 (pyridoxal phosphate) a coenzyme for this reaction will be activated by erythropoietin. Finally, the haem will be produced as a result of binding of protoporphyrin with ferrous (Fe2+); and each molecule of haem link to a globin chain produced on the polyribosomes. A produced tetramer of the four globin chains packed each with its own haem group will form a haemoglobin molecule (Hoffbrand et al., 2000).

Hb structure.gif

Figure 1.1.Haemoglobin structure (Mader, 1997).

1.2. General Epidemiology and pathophysiology of thalassaemia:

Thalassaemias are a group of disorders which occur as a result of genetic mutations that incorporate a decrease of gene synthesis or a complete reduction and exclusion of one or more of haemoglobin globin chains (Rodak, 2002). According to which of the globin chain had the mutation or has been deleted, alpha or beta, as a result thalassaemia has been recognised into two forms: α- and β-thalassaemia. The α- and β-thalassaemia happen due to an absent or reduced alpha or beta globin chain, correspondingly. Patients who carry the thalassaemia globin chains or the ''silent carriers'' and those who are with alpha or beta thalassaemia trait are often asymptomatic and therefore do not require a therapy (Muncie et al., 2009).

The β-thalassaemia has been found mostly common in the Mediterranean region, Africa, the Middle East, the Indian subcontinent Burma, South East Asia as well as Southern China, the Malay Peninsula and Indonesia (Romano et al., 1999). However, thalassaemia has been considered as the world's widespread disease and not anymore specific to the tropical countries as a result of migration (Weatherall and Clegg, 1996; Rund and Rachmilewitz, 2000). In addition, incidence of thalassaemia in males and females appeared to be equal and found to arise in around 4.4 of 10,000 live births. Also, it has been identified that α-thalassaemia occurs more often in Africans and south Asians, while β-thalassaemia shows a broad incidence in Mediterraneans, Africans, and South Asians. Moreover, about 5% of the world's population has been shown with globin variant, while just 1.7% with α- or β- thalassaemia trait (Rund and Rachmilewitz, 2005). Alpha-thalassaemia: The α-thalassaemia syndromes are caused by gene deletions. In normal status there are four copies of α-globin gene and for that severity of the disease could be categorised in relation to the quantity of the missing or resting genes. Moreover, as the α-globin chain is important for fetal and adult haemoglobin, a comprehensive loss of all four genes often cause suppression of α-chain synthesis and later death in the utero which described as hydrops fetalis condition (Hoffbrand et al., 2001). Furthermore, deletion of three α-globin genes usually causes moderately severe microcytic hypochromic anaemia with low Hb (7-11 g/dl) associated with splenomegaly. This form called as Haemoglobin H disease; because Hb H (β4) could be identified in erythrocytes via electrophoresis or within reticulocyte preparations. Hb H disease can be investigated in peripheral blood smear stained with brilliant cresyl blue allowing seeing glof ball red blood cells appearance characterised with multiple fine and deeply stained components which are participated β-globin chain aggregates (Figure 1.2). In addition, Hb Barts (γ4) exist during fetal life (Hoffbrand et al., 2001).

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Figure 1.2. Hb H disease α-thalassaemia seen in a peripheral blood film stained with brilliant cresyl blue (x100) (NériI et al., 2005).

HB H.jpg

The normal α/β chain synthesis ration is usually 1:1 which could be reduced in the α-thalassaemia and elevated in β-thalassaemias. Furthermore, loss of one or two genes causes α-thalassaemia traits which are frequently not combined with anaemia; however the erythrocytes number are over 5.5 x 1012/l, the mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) both are reduced. Moreover, Hb electrophoresis is normal and studies of α/β synthesis or DNA analyses are required for a definite diagnosis. Alpha thalassaemia intermedia or termed also as Haemoglobin H disease usually leads into haemolytic anaemia; while alpha thalassaemia major with haemoglobin Bart's usually causes fatal hydrops fetalis condition (Hoffbrand et al., 2004).hydrops fetalis.gif

Figure 1.3. Hydrpos Fetalis.


Either no β-chain (β0) or small quantities (β+) can be synthesized; and as a result, excess α-chain precipitate in erythroblasts and in mature erythrocytes leading to severe haemolysis and ineffective erythropoiesis; the greater excess of α-chain the more severe the anaemia occur. In addition, majority of genetic lesions of β-thalassaemia are characterized with the point mutations rather than gene deletions as in α-thalassaemia. These mutations occur in the gene itself or within the promoter or enhancer sites. Furthermore, there are specific mutations that often occurring in various societies; this could assist antenatal screening intended for detection of the mutations in fetal DNA (Hoffbrand et al., 2001). In addition, β-thalassaemia usually characterized with severe anaemia which becomes obvious at 3-6 months after birth. Hepatomegaly and splenomegaly could arise due to extreme erythrocytes destruction, extramedullary haemopoiesis and later on because excess iron. Moreover, bones expansion occur as a result of intensive bone hyperplasia causing a thalassaemic facies, with thinning of the cortex and bossing of the skull as well with a characteristic 'hair-on-end' appearance on X-ray (Fig 1.5).

There are three types of β-thalassaemia and each one has been subdivided into three types depending on its phase of state which vary from minor to thalassaemia intermedia and to thalassaemia major. Moreover, thalassaemia-minor is the mild form which often does not require a therapy; while thalassaemia intermedia is more severe and needs blood transfusion which might progresses patient's life. The most severe form is thalassaemia-major which requires a long term therapy and it has been found that children with this form of thalassaemia are on demand of blood transfusion and chelation therapy (Catlin, 2003). Furthermore, the first case of thalassaemia has been discovered as a severe type of anaemia in which children were investigated with a huge spleen or splenomegaly and modified bones (Rodak, 2002).

β-thalassaemia major

The β-thalassaemia major found to be appearing in one in four infants if both parents are β-thalassaemia trait carriers (Hoffbrand et al., 2001). The β-thalassaemia major occurs due to inheritance of two different mutations each deteriorating β-globin synthesis In addition, deletion of the β gene, δ and β genes or δ, β and γ genes might arise (Figure 1.4). In other cases, unequal crossing-over could generate δβ fusion genes which termed as Lepore syndrome (called lepore according to the name of the first family identified with this form of disorder) (Hoffbrand et al., 2001).

β-thalassaeima intermedia:

This type of β-thalassaemia considered being not as much severe as beta thalassaemia major and patients tend to have a sustained Hb of around 7 g/dl (Weatherall, 1981; Orkin and Nathan, 1998); therefore they might need a periodic blood transfusion (Muncie et al., 2009). Moreover, β-thalassaemia intermedia patients could be a heterozygous for mutations that lead to a mild reduction in β-globin expression, or might be doubly heterozygous for these mutations and a mutation that result in a more decrease in β-globin expression (Wainscoat et al., 1987). The severe form of beta thalassaemia, beta thalassaemia major, usually results in a weak growth, haemolytic anaemia, and bone deformities in children (Muncie et al., 2009). Furthermore, peripheral blood film of β-thalassaemia intermedia has similar red blood cells morphology features to β-thalassaemia major. Electrophoresis can illustrate 2-100% HbF, approximately 7% HbA2, and 0-80% HbA according to the phenotype of the individual (Orkin and Nathan, 1998). It has been found that patients with β-thalassaemia intermedia might have iron overload which can occur due to accelerated ineffective erythropoiesis which leads to amplify plasma iron turnover, stimulating an elevation in gastrointestinal iron absorption. In consequence, these patients tend to be diagnosed with cardiac and endocrine problems occur within 10-20 years later rather than those patients who receive regular blood transfusion (Orkin and Nathan, 1998).

β-thalassaemia minor:

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It occurs as a result of heterozygous mutations in β-globin synthesis. It frequently shows mild, asymptomatic, haemolytic anaemia (Weatherall, 1981; Orkin and Nathan, 1998). One of β-genes is mutated causes a reduction or elimination of its function, while the other genes remain normal. Also, peripheral full blood count can show Hb level within 10-13 g/dl range, and red cells count is usually normal or slightly increased. Β-thalassaemia minor appears with microcytic hypochrmoic anaemia, with some poikilocytosis including target cells and elliptocytosis. Generally, β-thalassaemia diseases were described with an elevated HbA2 which might range from 3.5% to 8.0%. HbF levels often vary from 1% to 5%. Less common Hb variants of β-thalassaemia traits subsist; one has an increased HbA2 level but with HbF within 5-20% range (Gilman et al., 1984). In addition, bone marrow examination might show a mild to moderate erythroid hyperplasia with slight ineffective erythropoiesis. Splenomegaly and hepatomegaly could be found in small group of patients.


Figure 1.4. Peripheral blood film from a patient with

β- thalassaemia major (Moinuddin, 2008).

Patients with β-thalassaemia usually need sustained blood transfusion; but iron overload might occur following repeated transfusions which can be avoided via using chelation therapy alongside long-term blood transfusions. In addition, food iron absorption can lead to elevated iron in β-thalassaemia which might occur as a result of ineffective erythropoiesis. For each 500 ml of transfused blood approximately 250 mg iron is present. Thus, iron damages the liver, the endocrine organs and the myocardium. Death can occur in the second or third year in absence of iron chelation therapy; as a consequence to heart failure or cardiac arrhythmias.

Furthermore, due to excessive melanin and haemosiderin skin pigmentation occur and causes a gray appearance at an early phase of iron overload. Also, during infancy, with lacking of sufficient transfusion, the anaemic infants are likely to get bacterial infections; for example, pneumococcal, haemophilus and meningococcal infections are potential to occur of splenectomy has been performed and the prophylactic penicillin is not provided. Moreover, iron overlaoded patients who are taking deferoxamine treatment are likely to bcome infected with Yersinia enterocolitica; which can lead to severe gastroenteritis. Viral infections is possibly can be transmitted throughout blood transfusion such as liver disease frequently present in thalassemic patients due to hepatitis C. Furthermore, Human immunodeficiency virus (HIV) can also be transmitted via blood transfusion (Hoffbrand et al., 2001).

Figure 1.5. β-thalassaemia major with bone face changes (left) and ''hair-on-end'' appearance on X-ray (right). thalassemia_head.jpg

Patients who receive regular and permanent blood transfusion might come out with iron overload condition in which the chelation therapy is applied in order to get rid of the overload iron. Classically, by the age of 30, beta thalassaemia major patients might die from heart problems as a result of the excess iron. Moreover, persons with alpha thalassaemia trait have a normal life expectancy. Besides, chorionic villus sampling might be performed for a purpose to analyse infants with Hb Bart's which raise the incident of toxemia and postpartum bleeding (Muncie et al., 2009).

An α-haemoglobin-stabilizing protein has been recognized, lately, binds to and stabilises free α-chains and consequently block the production of reactive oxygen species and reduce oxidative damage to red blood cells. This protein has not been identified able to modify the clinical picture of β-thalassaemia in human studies (Rund and Rachmilewitz, 2005). Thalssemia patients' bone marrow have five to six times the number of erythroid precursors as does the B.M of healthy controls (Centis et al., 2000) with 15 times the number of apoptotic cells in the polychromatophilic and orthochromic stages (Centis et al., 2000; Mathias et al., 2000). In unproductive erythropoiesis, hastened apoptosis occur as a result of the over α-chain deposition in erythroid precursors (Pootrakul et al., 2000).

1.5. Clinical manifestations and diagnosis:

1.5.1. Laboratory diagnosis:

Peripheral blood films appear with features of haemolysis; however red blood cells might be normal. Mild hypochromia and microcytosis are usually present. Heinz bodies also might be present in peripheral blood film (Figure 1.3) following splenectomy. Moreover, for the unstable haemoglobins the most typical feature is that they are heat stable, that is unstable haemoglobin precipitate as a dense cloud at 50o C. Some of these variants could be seen by haemoglobin electrophoresis (Hoffbrand et al., 2005).

1.5.2. Current therapies:

Patients' with β-thalassaemia major need an episodic long-term blood transfusion therapy to keep their Hb stable more than 9.5 g/dl (Rund and Rachmilewitz, 2005; Cazzola et al., 1997). It has been assumed that blood transfusion is necessary at the early six months. Moreover, for persons with α-thalassaemia intermedia or named as Hb H disease, mild to moderate haemolysis is highly to occur consequently; therefore, blood transfusion might be crucial, sporadically, relaying on the severity of the patients' clinical grade. For β-thalassaemia intermedia patients, the transfusion could be a biased medical evaluation. Furthermore, transfusion obligations are periodic and necessary once the patient's Hb is very low and/or the anaemia defects grow (Roberts et al., 2007). Usual blood transfusion therapy to keep hemoglobin levels of at least 9-10 g/dl could improve growth and development. In addition, it can also decreases hepatosplenomegaly occurrence because of abnormal extramedullary haematopoiesis and bone malformation (Cunningham et al., 2004; Old et al., 2001).

Type of thalassaemia

Synthesized chain or chains at

a reduced rate

Synthesized haemoglobin or haemoglobins at a reduced rate

Alpha: α0 or α+


A, A2 and F

Beta: β0 or β



Gamma: γ



Delta: δ0 or δ



Delta beta: δβ0 or δβ+

δ and β

A and A2

A Gamma delta beta:

Aγ δ β0

Aγ δ and β

A and A2

Epsilon gamma delta beta: εGγAγδβ0

ε, Gγ, Aγ, δ and β

A, A2 and F

Haemoglobin Lepore

δ and β

A and A2+

Table 1.1. Classification of thalassaemias (Bain, 2006).

Figure 1.3. Heinz bodies on a peripheral blood smear, crystal violet stain (1000x) (Hess, 2008).Heinz-bodies-100x2-website-arrow.jpg

It has been found that hypogonadism, which is an endocrinopathy, and abnormal growth to be common in thalassaemia patients due to chronic anaemia and excess iron. These disorders are found wide-spread in the eldest patients whose chelation treatment is inadequate (Cunningham et al., 2004; De Sacntis, 2002; Raiola et al., 2003). Furthermore, De Sacntis (2002) and Raiola et al (2003) state that the hormonal replacement which has been signified for residual endocrine insufficiency was effective in male patients with hypogonadotropic hypogonadism and appropriate to cure their fertility damage which considered being a consequence of this disorder. Fatality in old patients has been found due to bone disorder which results from osteopenia and osteoporosis (Voskaridou and Terpos, 2004) (Figure 1.4).

Figure 1.4. Anteroposterior radiograph of the lumbar spine. Osteopenia is present.336139-396792-197.jpg

Iron-chelation therapy found to be able to improve life of thalassaemia major patients (Hoffbrand et al., 2003; Borgna-Pignatti et al., 2004). A common iron-chelation agent still in use is Deferoxamine; however, it has different restrictions such as requiring parental administration, side effects and high-cost especially in underdeveloped countries (Olivieri, 1999). Furthermore, attempts to improve and produce new orally active chelators have been devoted. Although Deferoprone, an orally chelator, was considered to be an insufficient chelator and might exacerbate liver fibrosis, the wide-reaching practice on this drug confirmed its potency and safe usage (Franchini and Veneri, 2004).

Moreover, deferiprone has several advantages over than deferoxamine, as it can migrate through the cell membrane and then chelate toxic intracellular iron species (Shalev et al., 2008). Some new oral chelation agents undergo improvement (Cohen et al., 2004). Some new data verified that deferoprone can be more potent than deferoxamine in exclusion of myocardial iron (Anderson et al, 2002; Piga et al., 2003; Hershko et al., 2005). Moreover, a combined administration of deferiprone and deferoxamine could be a new promising innovation in chelation therapy. So, deferoprone can be considered as an acceptable alternative for patients who are incapable to receive deferoxamine (Brittenham et al., 1999). The mixture of deferoxamine and deferiprone found encouraging but needs more confirmation. Also, developed noninvasive technologies such as imaging and blood tests intended to measure excess iron might give dependable information for the evaluation of the potency of present and future treatments (Deborah and Rachmilwitze, 2005). In general, it has been expected for thalassaemia's patients to have an improved life expectancy (Rund and Rachmilewitz, 1995).

Multiple blood transfusions might lead to iron overload condition in thalassaemia patients as a result of that they do not have a physiologic process to remove excess iron. For that reason, they usually need chelation therapy between age starts from five to eight years old (Roberts et al., 2007). Hence, Deferoxamine, intravenously or subcutaneously, has been selected as the treatment of choice and it is moderately nontoxic. However, it is an expensive treatment. Moreover, the United States and Drug Administration lately permitted oral deferasirox as a substitute treatment. Moreover, defereasirox undesirable side effects were found being transient and gastrointestinal, and there were no cases of granulocytosis have been reported. Bone marrow transplantation is another important therapy for thalassaemia patients. It has been estimated to be the only healing therapy for β-thalassaemia major in childhood. Haematopoietic stem cells transplantation usually leads to a good outcome in low-risk persons who have no hepatomegaly, no portal fibrosis on liver biopsy, and recurrent chelation therapy, or generally, with two of these defects (Rund and Rachmilewitz, 2005).

Thalassaemia patients might require management for certain conditions such as for hypersplenism that enhance transfusion requirements, splenectomy might be recommended. Also, growth hormone therapy for β-thalassaemia major and intermedia has shown successful achievements and it is commonly not recommended. Preconception genetic counseling is highly recommended for all persons with thalassaemia (ACOG Committee on Obstetrics, 2007). As each parent with β-halassaemia trait have a one in four chance to give a child with β-thalassaemia major and a three in four chance the child might have thalassaemia trait or be normal. Therefore, chorionic villus sampling using polymerase chain reaction in order to identify point of mutations or deletions would recognise infants with β-thalassaemia. In addition, because the Hb Bart's in parents with α-thalassaemia trait can enhance toxemia and postpartum bleeding. Preimplantation genetic diagnosis will be available in conjunction with in vitro fertilization (Braude et al., 2002)

Also, serum ferritin can be used as a marker of iron storage to detect cardiac problems. (De Sanctis, 2002). Moreover, for patients with high risk of thromboembolic conditions, there remain no studies about anticoagulation, antiplatelet therapy, or both have been illustrated. So, there is no therapy to be advised. However, persons with a history of thrombosis they can be treated with low-molecular-weight heparin. It has been displayed that anticoagulation therapy is necessary for persons at risk conditions before surgery and during pregnancy (Rund and Rachmilewitz, 2005).

1.6. Gene therapy for β-thalassaemia:

1.6.1. Molecular pathology in β-thalassaemia:

Rund and Rachmilewitz (1995) illustrate that around 150 mutations have been discovered. In addition, advanced technique such as polymerase chain reaction (PCR, with direct sequencing and other variations such as SSPE) (Erlieh, 1989) in molecular genetics provided for rapid investigation of point mutations at the molecular level of β-thalassaemia. Moreover, small size of the β-globin gene (less than 2 kilobases) involving regulatory and dominant sequences makes it compliant to inclusive sequencing. The straightforwardly recognizable phenotype of homozygous and heterozygous patients in whom hypochromia and microcytosis were identified, assisted the detection of applicants for powerful analysis (Rund et al., 1992). So, detection of heterozygous could be possible in ethnic populations not often diagnosed with β-thalassaemia; which described new mutations (Oppcnheim et al., 1993). Thus, this has considered the β-globin gene amongst the best studied of human genes (Kazazian, 1990).

There are several types of mutations can occur due to various forms of nucleotides aberrations in certain sequences have been defined which describe that aspects of β-globin RNA transcription and processing has been found to be occurred due to point mutations leading to β-thalassaemia. The several classes of β-thalassaemia mutations might occur at any step in globin gene production and for that they can be classified into: transcriptional mutations, RNA modification mutations, RNA processing mutations, mutations result in non-functional RNA and mutations lead to unstable globins (Rund and Rachmilewitz, 1995). Each type is describes as following: Transcriptional mutations:

These mutations include deletions and point mutations linked to the globin gene promoter regions (Hoffbrand et al., 2005). They illuminated certain sequences essential for accurate transcription initiaton; and they are mainly concentrated in the TATA box located 30 nucleotides upstream of the cap site also in the two CACACCC sequences at -90 and -105 to the cap site,. All of these mutations found cause β+ -thalassaemia as they damage transcription to a mild and moderate level (Rund and Rachmilewitz, 1995). RNA modification mutations:

Point mutations have been recognized in the cap site at which the 5' end modification of the RNA takes place, and as well as in the polyadenylation signal, AATAAA, a hexanucleotide which is essential for transcription termination, 3' end cleavage and poly (A) tail addition. These mutations described to be leading to β+ phenotype (Rund and Rachmilewitz, 1995). RNA processing mutations:

These mutations might result in either β+- or β0-thalassaemia, the latter was identified if normal splicing is fully stopped. Such mutations is based on that several mutations can affect splice junctions, consensus sequences, or internal IVS sequences leading the correct splicing be reduced or eliminated (Rund and Rachmilewitz, 1995). Mutations lead to non-functional RNA:

These can cause either an in frame abolished codon or a frameshift because of nucleotide deletion or insertion. These RNAs are unable to be translated and for that, these mutations can lead to β0-thalassaemia. Furthermore, these β0 mutations have been studied broadly as they are interesting at the molecular level because they might cause decreased accumulation of mRNA with short half-life. Also, it has been found that the instability of these untranslatable mRNA molecules remain indistinct (Rund and Rachmilewitz, 1995). Mutations result in unstable globins:

On the contrary to the recessive form of β-thalassaemia types, it has been identified that most of these mutations can affect exon 3 and cause dominant inheritance of inclusion-body haemolytic anaemia which is quietly opposite to the form of inheritance of β-thalassaemia (Rund and Rachmilewitz, 1995).

In addition, a wide range of mutations can affect the primary mRNA transcript. Mutations occur in introns or exons, or on their intersections, might interfere with the splicing mechanism of the exons following introns removal. Moreover, single-base replacements take place at the invariant GT or AG sequences at intron-exon intersections stop splicing leading to β0-thalassaemia. The sequences near to the GT in the introns are quite conserved and so called consensus sequences. Numerous β-thalassaemia mutations might exist in these sequences or other sites of introns and are linked to several levels of impaired β-globin construction; substitute splicing regions are produced leading to normal and abnormal mRNA species are synthesised. Mutations related to translation of β-globin mRNA can be described in two types. First group include nonsense mutations indicates single-base alterations that generate stop codons in the middle of the coding site of mRNA; thus, might lead to premature termination of globin chain synthesis. Moreover, other exon mutations cause frameshifts, which is at least one or more bases, are lost or inserted and the reading frame for the genetic code is thrown out of phase (Hoffbrand et al., 2005). Lastly, mutations that involve post-translational stability might result in some types of thalassaemia as a result of instability of the β-globin gene product. Unstable β-globin gene products can form inclusion bodies in erythrocyte precursor; and this is the origin for dominant β-thalassaemia. Moreover, highly unstable β-chains could be produced, even though they make a viable haemoglobin molecule; are quickly broken down in blood circulation, causing a chronic haemolytic anaemia (Hoffbrand et al., 2005).

According to various types of genetic lesions causing β-thalassaemia, phenotypes of patients and carriers could be varied (Huisman, 1990). There are a number of mechanisms by which specific point mutations causes reduced β-globin mRNA have been described via expression analysis of mutant genes. These analyses can be used via different approaches such as, assessment of RNA derived from peripheral blood red cell precursors (normoblasts) or bone marrow of patients. Analysis of the derived RNA could help in studying the function of mutant genes (14). Moreover, different methods of RNA analysis could be applied, for example Northern blotting which might illustrate the reduced amounts of RNA or abnormal sized transcripts (15), RNAse and S1 mapping which were considered for in depth diagnosis of RNA minor sections. In addition, it is important to take into account having homozygous patients to assess the effect of certain alleles; but for rare mutations it can be difficult to find such persons.

1.6.2. Gene therapy: Brief review:

Gene therapy is a type of molecular medicine which could improve human health and provide novel therapies for a wide range of diseases (Friedman, 1999). Principally, the theory of gene therapy is based on transferring a piece of genetic material into the target cells aiming to provide a treatment for the disease or reduce the progression of the disease. Therefore, gene therapy needs particular methods and techniques that are capable for gene transfer into different types of cells, tissues and organs (Verma and Weitzman, 2005). Furthermore, the procedure of gene transfer and expression was named as 'transduction'. There are many difficulties that are can be frequently associated with all vector systems and, therefore, must be conquered in order to obtain a successful transduction (Somia and Verma, 2000). The production of vectors considered one of the main obstacles. So, a superlative vector was described to be the vector which can be generated in high concentrations via using a proper and reproducible production method. Moreover, the vector should be able to targeting the most appropriate cell type for the disease, whichever it is dividing or non-dividing cells (Verma and Weitzman, 2005). In addition, appreciating of the transduction in studies of vector uptake, intracellular trafficking and gene regulation has assisted the production of potent vehicles for gene transfer. Hence, through many cases it could be required to attain a constant, prolonged gene expression, which needs incorporation of the vector DNA into the DNA of the host or maintenance as an episome (Verma and Weitzman, 2005).

There were some vectors that have been generated with a purpose to conquer most of these problems were classified into two major classes: viral and non-viral vectors (Friedman, 1999). Furthermore, viral vectors were described as those derived from viruses with either DNA or RNA genomes and are identified as both integrating and non-integrating vectors. On the other hand, the non-viral vectors are composed of naked DNA delivered via injection, liposomes (which are cationic lipids combined with nucleic acids), nano-particles, and others. Also, non-viral vectors could be produced in large concentrations and are tend to provide low fatal or immunological troubles; however, they undergo with inefficient gene delivery (Verma and Weitzman, 2005). Viral vectors:

Basically, viral vectors are intended to control the innate immunity of viruses to transfer genetic material into the target cell; and the main concern of using viruses is to replicate and produce abundant quantities of progeny. Moreover, most viruses tend to kill the host; however several viral infections can cause harmful effects on the host along with damage of infected host cells. Deleterious effects might be due to introduction of genes whose own products are toxic or via gaining host genomic substance which might result in pathogenesis. Furthermore, the conversion of these pathogenic substances into transferring methods mainly depends on the elements required for replication from those causing diseases. Non-viral vectors:

Because β-thalassaemia occurs due to decreased or absent role of a specific gene, it was one of the initial applicants that triggered demand for treatment of thalassaemia with gene therapy (Rund and Rachmilewitz, 2000). Hence, this was carried out by a successful transfer of the β-globin gene into mice in order to correct thalassaemia in a transgenic mouse model (Constantini et al., 1986) (Figure 1.4).


Figure 1.4: Gene therapy involves insertion of genes into an individual's cells/tissues

for treatment of a disease.

In fact, gene therapy is considered as one of the most preferred techniques in treatment of β-thalassaemia inside the utero, transforming stem cells into normal noncarrier cells (Catlin, 2003). However, there were some aspects verified that the aim of using β-globin gene in gene therapy is hard to achieve (Sadelain, 1997). Those aspects were related to strict necessities for β-globin gene expression; such as the high levels were needed at a certain time during growth of haematopoietic stem cells, which are the target for the transfer of β-globin gene (Rund and Rachmilewitz, 2000). However, Rund and Rachmilewitz, 2000 state in their paper, which discusses the treatment of β-thalassaemia, that there are three strategies are identified as strategies to perform genetic therapy for β-thalassaemia. The first strategy is gene transfer; this includes addition of an exogenous gene to human haematopoietic cells. Certainly, viral mediated gene transfer has been considered as the most widely used method for a purpose to attain the aim of this strategy. Retroviruses and adeno-associated viruses are the mainly used ones (Sadelain, 1997).

The second strategy of genetic therapy for thalassaemia is application of different molecular biological actions to either correct the DNA, thus correcting the mutation, or via correcting the mutatnt RNA transcript. Such new strategy might develop the cell's own repair mechanism, via bringing in the oligonucleotides that are inserted to certain parts in the DNA double helix which include the mutations (Wang et al., 1996). The final strategy includes using of certain procedures that aim to inhibit the production of α-globin gene, decreasing globin-chain inequity. This strategy might be useful for the treatment of thalassaemia intermedia in which there is adequate products of the β-globin gene (Rund and Rachmilewitz, 2000). FOR WHAT?? HOW?

The application of gene therapy in treatment for β-thalassaemia is based on the point of mutations and the expression level of β-globin gene (Liu et al., 1990; Forget, 1998). A study done by (Li et al., 2001) discussed application of targeted correction of the point mutation of β-thalassaemia by DNA/DNA oligonucleotides which were suspected to be potential for β-thalassaemia gene therapy; and it was observed that RNA/DNA chimeric designed oligonucleotides found to be very potent in targeted correction of single bases in genome of mammalian cells both in vivo and in vitro 21-24. Thus, this study might support using of the gene therapy for human genetic disorders as a result of point mutations or single base deletions. Li et al., 2001 states that as the targeted regions are changed in situ in the cells, this procedure could be the suitable one for some human genetic diseases rather than viral-based gene therapy; especially for diseases such as β-thalassaemia in which genetic therapy is controlled with an extremely complex form. Although chimerplasty strategy seems promising for human gene therapy, there are some queries regarding this strategy (Capecchi, 1989), Li et al. (2001) revealed that effectiveness differ with several observational studies.

However, the main problem which might affect the success of genetic therapy applications was identified to be the improvement of potent and secure vectors through which to introduce genetic materials into the target cells