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 Table of Contents  
Year : 2012  |  Volume : 4  |  Issue : 3  |  Page : 194-201  

Recent advances in gene therapy for thalassemia

Department of Pharmacology, S. J. Thakkar Pharmacy College, Rajkot, Gujarat, India

Date of Submission08-Sep-2011
Date of Decision20-Oct-2011
Date of Acceptance10-Dec-2011
Date of Web Publication26-Jul-2012

Correspondence Address:
R H Gokani
Department of Pharmacology, S. J. Thakkar Pharmacy College, Rajkot, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.99020

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Thalassemias are genetically transmitted disorders. Depending upon whether the genetic defects or deletion lies in transmission of α or β globin chain gene, thalassemias are classified into α and β-thalassemias. Thus, thalassemias could be cured by introducing or correcting a gene into the hematopoietic compartment or a single stem cell. Initial attempts at gene transfer have proved unsuccessful due to limitations of available gene transfer vectors. The present review described the newer approaches to overcome these limitations, includes the introduction of lentiviral vectors. New approaches have also focused on targeting the specific mutation in the globin genes, correcting the DNA sequence or manipulating the development in DNA translocation and splicing to restore globin chain synthesis. This review mainly discusses the gene therapy strategies for the thalassemias, including the use of lentiviral vectors, generation of induced pluripotent stem (iPS) cells, gene targeting, splice-switching and stop codon readthrough.

Keywords: Gene targeting, lentiviral vectors, splice-switching, stop codon readthrough, thalassemia

How to cite this article:
Raja J V, Rachchh M A, Gokani R H. Recent advances in gene therapy for thalassemia. J Pharm Bioall Sci 2012;4:194-201

How to cite this URL:
Raja J V, Rachchh M A, Gokani R H. Recent advances in gene therapy for thalassemia. J Pharm Bioall Sci [serial online] 2012 [cited 2022 Dec 3];4:194-201. Available from:

The thalassemias are a diverse group of hereditary disorders in which there is a reduced rate of synthesis of one or more of the globin polypeptide chains. Thus, thalassemias, unlike hemoglobinopathies which are qualitative disorders of the hemoglobin, are quantitative abnormalities of polypeptides globin chain synthesis. [1]

The thalassemias are genetically transmitted disorders. Normally an individual inherits two β-globin genes located one each on two chromosomes 11, and two alpha globin genes one each on two chromosome 16, from each parent i.e. normal adult hemoglobin is α2β2 . Depending upon whether the genetic defects or deletion lies in transmission of α or β globin chain gene, thalassemias are classified into α and β-thalassemias. Thus, patient with α-thalassemias have structurally normal alpha globin chain but their production is impaired. Similarly, patient with beta thalassemias have structurally normal alpha globin chain but their production is decreased. Each of two main types of thalassemias may occur as heterozygous (minor) or homozygous state (major). The former is generally asymptomatic while the later is severe congenital hemolytic anemia.

   Signs and Symptoms Top

Signs and symptoms of thalassemia depend on the type of thalassemia occurred, i.e. depending on the type gene deletion or mutation. Although there are some common signs and symptoms of thalassemia which includes: severe tissue hypoxia, hemolytic anemia, marked hepatomegaly, marked erythroid hyperplasia, iron overload etc. [1]

   Molecular Pathogenesis Top


These are disorders in which there is defective synthesis of α-globin chains resulting in depressed production of hemoglobin that contains alpha chains i.e. HbA, HbA 2 and HbF. The alpha thalassemias are most commonly due to deletion of one or more of the alpha chain genes located on short arm of chromosome 16. Since there is a pair of alpha chain genes, the clinical manifestations of alpha thalassemia depend upon the number of genes deleted. [1]

Accordingly, alpha thalassemias are classified into four types:

  1. Hb bar's hydrops foetalis: There is deletion of all four alpha genes (homozygous state) that results in total suppression of alpha globin chain synthesis causing the most severe form of alpha thalassemia.
  2. HbH disease: Deletion of three alpha chain genes produces HbH which is a beta globin chain tetramer (β4 ) and markedly impaired α-chain synthesis. HbH is precipitated as Heinz bodies within the affected red cells.
  3. α-thalassemia trait: Two alpha gene deletion.
  4. α-thalassemia trait (carrier): One alpha gene deletion.

In β-thalassemia, there is decreased rate of β-chain synthesis resulting in reduced formation of HbA in the red cells. The molecular pathogenesis of the β-thalassemias is more complex than that of α-thalassemias. In contrast to α-thalassemia, gene deletions rarely ever cause β-thalassemia and is only seen in an entity called hereditary persistence of fetal hemoglobin (HPFH). Instead, most of β-thalassemias arise from different types of mutations of β-globin gene resulting from single base changes. The symbol β0 is used to indicate the complete absence of synthesis while β+ denotes partial synthesis of β-globin chains. More than 100 such mutations have been described affecting the preferred sites in the coding sequences e.g. (in promoter region, termination region, splice junctions, exons, introns).

Some of the important mutations having effect on β-globin chain synthesis are as under:

  1. Transcription defect: Mutation affecting transcriptional promoter sequence causing reduced synthesis of β-globin chain. Hence the result is partially preserved synthesis i.e. β+ thalassemia.
  2. Translation defect: Mutation in the coding sequence causing stop codon (chain termination) interrupting β-globin messenger RNA. This would result in no synthesis of β-globin chain and hence β0 thalassemia.
  3. mRNA splicing defect: Mutation leads to defective mRNA that is degraded in the nucleus. Depending upon whether part of splice-site remains intact or is totally degraded, it may result in β+ thalassemia or β0 thalassemia.
Depending on the extent of reduction in β-chain synthesis, there are three types of β-thalassemia:

  1. Homozygous form: β-thalassemia major:

    It is the most severe form of congenital hemolytic anemia. There are further two types:

    1. β0 thalassemia major characterized by complete absence of β chain synthesis.
    2. β+ thalassemia major having incomplete suppression of β chain synthesis.
  2. β-Thalassemia intermedia:

    It is β-thalassemia of intermediate degree of severity that does not require regular blood transfusions. These cases are genetically heterozygous (β0 /β or β0 /β).
  3. Heterozygous form: β-Thalassemia minor (trait):

    It is a mild asymptomatic condition in which there is moderate suppression of β-chain synthesis.

   Therapeutic Approaches Top

Current approaches

Hematopoietic stem cell transplantation

Current disease management of β-thalassemia consists of:

  1. Prenatal diagnosis
  2. Transfusion therapy
  3. Allogeneic bone marrow transplantation (BMT). [2],[3],[4]
Only the allogenic BMT is potentially curative. [5] Consequently, several centers have utilized this approach as definitive therapy. [6] The most extensive experience in treating β-thalassemia patients with BMT is that of Lucarelli and coworkers in Italy. [2] Established protocols can lead to a high success of thalassemia-free survival, although the chronic graft-versus-host disease is still a potential long-term complication of allogeneic hematopoietic stem cells (HSCs) transplantation. [6] However, development of new techniques to improve the management of graft-versus-host disease, to perform BMT from unrelated donors and cord blood stem cells may expand the pool of potential donors in the near future. Another complication is transplant-related mortality. Because of these two complications the use of allogenic bone marrow is limited.

In addition, availability of allogeneic bone marrow is limited by finding an identical human leucocyte antigen (HLA)-matched bone marrow donor. In addition, patients with severe β-thalassemia might benefit from new genetic and cellular approaches. From this prospective, β-thalassemia is the excellent candidate disease for genetically based therapies in autologous HSCs. Alternatively, somatic cells reprogrammed to induce pluripotent stem cells might also provide a possible new approach to treat β-thalassemia. [7],[8]

Genetic approaches

Gene transfer using onco-retroviral vectors

addition mediated by retroviral vectors is an attractive approach for monogenic disorder. However, when applied to hemoglobinopathies (Thalassemia), this strategy raises major challenges in terms of controlling transgene expression, which should be:

  1. Erythroid-specific;
  2. Elevated;
  3. Position-independent and
  4. Sustained over time.
In fact, many studies were performed before positive preclinical data were generated. The first attempts were done using Oncoviruses. These viruses belong to the large family of Retroviridae and are characterized by a genome that encodes the genes gag-pol and env. [9] Onco-retroviral vectors, such as those derived from Moloney murine leukemia virus, efficiently transfer therapeutic genes into murine HSCs without transferring any viral gene.

Recombinant onco-retroviruses were the first viral vectors used to transfer the human β-globin gene in mouse HSCs. These experiments resulted in tissue-specific but low and variable (position-dependent) human β-globin expression in bone marrow chimeras, usually varying between 0 and 2% of endogenous mouse β-globin mRNA levels. [10] Studies aimed at increasing expression levels of transferred β-globin genes have focused on including locus control region (LCR) elements of the human β-globin gene locus into onco-retroviral vectors. The LCR contains cis-acting DNase I hypersensitivity sites (HSs) that are critical for high-level, long-term, position-independent, and erythroid-specific expression. [11] These HS elements contain several deoxy ribo nucleic acid (DNA)-binding motifs for transcriptional and chromatin remodeling factors that facilitates chromatin opening. Also, these genomic regions allow for binding of other regulatory elements required for high-level expression of the β-globin gene. Incorporation of the core elements of HS2, HS3, and HS4 of the human β-globin LCR significantly increased expression levels in murine erythroleukemia (MEL) cells but failed to abolish positional variability of expression. [11]

Additional efforts aimed to include larger elements resulted in the inability of the vector to incorporate large quantities of genetic material, as shown by the rearrangements of the transferred sequences. [3] Since these rearrangements frequently occur because of activation of splicing sites of the LCR sequence contained in the retroviral ribo nucleic acid (RNA), additional attempts were done to eliminate these sites. However, even these new vectors failed to include HS elements sufficiently large to considerably increase expression of the β-globin gene. [11]

Additional erythroid-specific transcriptional elements were also investigated within onco-retroviral vectors, including the HS40 regulatory region from the human α-locus [12],[13] and alternative promoters. The promoter of ankyrin, a red cell membrane protein, has shown some promise in transgenic mice and in transduced MEL cells. [14] In mice, the ankyrin promoter has been used to drive expression of the human γ-globin gene, at double copy, resulting in an average expression of 8% of that of the endogenous α-globin genes. [15] To overcome transcriptional silencing of the γ-globin promoter in hematopoietic chimeras, mutant γ-globin promoters from patients with HPFH were also investigated. [16] The Greek mutation at position−117 thus appeared to substantially increase γ-globin expression in MEL cells. However, even these vectors failed to increase the level of γ-globin gene to therapeutic levels.

Although onco-retrovirus vectors integrate into the genome, many integrants undergo transcriptional silencing, posing an additional challenge to the success of gene therapy using these vectors.

Kalberer and co-workers attempted to avoid gene silencing by preselecting ex vivo retrovirally transduced hematopoietic stem cells on the basis of expression of the green fluorescent protein (GFP). In this vector, the GFP gene was driven by the phosphoglycerate kinase promoter, while the human β-globin gene by its own promoter and small elements from the LCR. [17]

Gene transfer using onco-retroviral vectors also have some limitations:

  1. Using this approach, in vivo hematopoietic stem cell gene silencing and age-dependent extinction of expression were avoided, although suboptimal expression levels and heterocellular position effects persisted.
  2. Another major limitation is that onco-retroviral vectors need to infect cells before and close to their division, otherwise the viral RNA cannot migrate into the nucleus due to the presence of a nuclear membrane. [17] Since most hematopoietic stem cells are in a quiescent state, they must be induced with cytokines to divide in order to achieve higher transduction efficiencies and overall expression levels. Stimulation of quiescent hematopoietic stem cells, however, impairs or halts their long-term repopulating capacities. [17]
Gene transfer using lentiviral vectors

With the extensive research on human immunodeficiency virus-1, it has been realized that lentivirus, engineered to be devoid of any pathogenic elements, can become efficient gene transfer vectors. Lentiviruses are characterized by a complex genome that encodes a number of accessory proteins besides the canonical retroviral genes gag-pol and env. They share all the common characteristic of retroviral replication including receptor-mediated entry, capsid uncoating, reverse transcription of the viral RNA and integration into the host cell genome. [9] In addition, they are able to transduce non-replicating cells, which confers to these viruses a special value for the development of clinically functional gene vectors. Compared to onco-retroviral vectors, the stabilization of the proviral mRNA genome by the interaction of the accessory protein Rev with its cognate motif Rev-responsive element (RRE) increases their range of application, since larger genomic elements can be introduced in their genome with limited or no sequence rearrangement. Therefore, lentiviral vectors are thus likely to be selected as vectors of choice for the stable delivery of regulated transgenes in stem cell-based gene therapy.

The use of lentiviral vectors has allowed the introduction of large genomic elements from the β-globin locus, different promoters, enhancers, and chromatin structure determinants that led to lineage-specific and elevated of β-, γ- and α-globin expression in vivo. This resulted in the amelioration or correction of anemia and secondary organ damage in several murine models of hemoglobinopathies, making the recombinant lentiviruses the most effective vector system to date for gene therapy of these disorders.

  • α-Thalassemia could potentially be a target for fetal gene therapy since fetuses with this disorder usually die between the third trimester of pregnancy and soon after birth. The potential use of lentiviral vectors to treat α-thalassemia was investigated as a vector containing the HS2, 3, and 4 of the LCR from the human β-globin locus, and the human α-globin gene promoter directing the human α-globin gene. Using this vector, Han and colleagues performed gene delivery in utero during mid-gestation targeting embryos affected by a lethal form of α-thalassemia. They showed that in newborn mice, the human α-globin gene expression was detected in the liver, spleen, and peripheral blood. [18] The human α-globin gene expression was at the peak at 3-4 months, when it reached 20% in some recipients. However, the expression declined at 7 months. Colony-forming assays in these mice showed low levels of transduction and lack of human α-globin transcript. Thus, lentiviral vectors can be an effective vehicle for delivering the human α-globin gene into erythroid cells in utero, but, in the mouse model, delivery at late mid-gestation could not transduce hematopoietic stem cells adequately to sustain gene expression.
  • Treatment of β-thalassemia, and other disorders through lentiviral-mediated gene transfer is studied in murine and primate models. [19],[20],[21],[22],[23] The original studies in mice showed that lentiviral-mediated human β-globin gene transfer can rescue mice affected by β-thalassemia intermedia and β-thalassemia major. [23],[24],[25] The mouse β-globin cluster has two adult β-globin genes, minor-β and major-β globin. Thalassemic mice were generated with deletion of both the minor-β and major-β globin on one allele, designated as th3/+. [26] These mice have a degree of disease severity (hepatosplenomegaly, anemia, aberrant erythrocyte morphology) comparable to that of patients affected by TI (Thalassemia intermedia).
May and colleagues tested two lentiviral vectors termed RNS1 (carrying minimal core LCR elements) and TNS9 (with large LCR fragments encompassing HS2, HS3 and HS4; approximately 3.2 kb in size) on th3/+ mice. Compared to RNS1, mice recipient of the larger TNS9 vector maintained higher human β-globin transcript levels over time showing amelioration of red cell pathology (anisocytosis and poikilocytosis) and significantly increased hemoglobin levels (from 8-9 g/dL to 11-13 g/dL). The massive splenomegaly found in chimeras engrafted with control th3/+ bone marrow was not observed in TNS9-treated animals. This correction was sustained in secondary mice. [27]

Mice completely lacking adult β-globin genes (th3/th3) die late in gestation, limiting their utilization as a model for Cooley's anemia. For this reason, adult animals affected by Cooley's anemia were generated by transplantation of hematopoietic fetal liver cells harvested from th3/th3 embryos by lethally irradiated syngeneic adult recipients. [23] Hematological analyses of engrafted mice performed 6 to 8 weeks post-transplant revealed severe anemia not due to pancytopenia but rather to low red blood cell and reticulocyte counts together with massive splenomegaly. [23],[24],[25]

These animals could be rescued using TNS9 or by blood transfusions, supporting the notion that their phenotype is due specifically to erythroid impairment. [23],[24],[25],[26] Miccio and colleagues also utilized an erythroid-specific lentiviral vector driving the expression of the human β-globin gene from a minimal promoter/enhancer element containing two hypersensitive sites from the β-globin LCR in mouse models of β-thalassemia. [28] They showed that genetically corrected erythroblasts underwent in vivo selection. The selected erythroblast that derived from progenitors harbored proviral integrations in genome sites and were more favorable to high levels of vector expression. These data suggested that a regimen of partially myeloablative transplantation might be sufficient to achieve a chimerism that would be therapeutic in a β-thalassemic patients. While correction of murine models of β-thalassemia has been achieved through lentiviral-mediated high levels of β-globin gene transfer into mouse HSCs, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells.

Zhao and coworkers therefore developed a double gene lentiviral vector encoding both;

(1) Human γ-globin under the transcriptional control of erythroid regulatory elements and (2) Methyl guanine methyltransferase (MGMT), driven by a constitutive cellular promoter. [29] MGMT is an alkyl transferase that normally functions to repair cellular DNA damage at the O6 position of guanine. The cytotoxic effects of alkylating agents, such as temozolomide and 1,3-bis-chloroethyl-1-nitrosourea (BCNU), can be prevented if there is adequate expression of MGMT, which removes the O6 adduct from the modified DNA.

Variant MGMT proteins with specific amino acid changes retain significant activity while possessing the useful property of resistance to inactivation by O6-benzylguanine (BG). BG can be used to inactivate endogenous MGMT to enhance the specificity of alkylator-mediated cell death to cells not expressing the variant form. Therefore, expression of these variant forms of MGMT provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced HSCs, whereas transduced cells are protected.

To test this hypothesis, mice transplanted with β-thalassemic HSCs transduced with a lentiviral γ-globin/MGMT vector were treated with BCNU. This led to significant increase in the number of γ-globin-expressing red cells, the amount of fetal hemoglobin and resolution of anemia. One important advantage of using the γ-globin gene, normally expressed exclusively during fetal life, is that high level γ-globin expression would be therapeutic not only for β-thalassemia, but also SCD. Interestingly, selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. These data suggest that co-expression of MGMT allowed autologous, γ-globin vector-transduced β-thalassemic HSCs to be enriched to therapeutic levels through either pre or post-transplantation selection. [30]

The frequency of proviral integration within genes observed in this study and the data from Miccio and co-workers that indicate that selected erythroblasts were derived from progenitors harboring proviral integrations more favorable to high levels of vector expression, indicate that regulated hematopoiesis might require additional safety modifications to prevent potential genotoxic effects. [28] This risk is inherent to the integration of foreign genetic material, and the risk of insertional oncogenesis has been established both in mice and humans. [31],[32],[33],[34],[35]

In light of these results, genetic elements with enhancer-blocking properties, such as insulators, could increase the safety of the clinical trails. These elements have been investigated to shelter the vector from the repressive influence of flanking chromatin by blocking interactions between regulatory elements within the vector and chromosomal elements at the site of integration. [34] This property of insulators can also be harnessed to diminish the risk that the vector will activate a neighboring oncogene. [35] The initial studies indicated that inclusion of the cHS4 insulator element into the 3′ LTR of recombinant murine leukemia virus increases the probability that randomly integrated proviruses will express the transgene. [15],[30]

Puthenveetil and coworkers tested a lentiviral vector carrying the human β-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human β-thalassemia major cells. [36] Using this vector, they demonstrated normal expression of human β-globin in erythroid cells produced in vitro. They also observed restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes β-thalassemia. The gene-corrected human β-thalassemia progenitor cells were also transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human β-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Based on all these preclinical studies on mouse models of β-thalassemia, clinical trials have been proposed or are underway [Figure 1]a. [37]
Figure 1: Schematic representation of the gene therapy approach mediated respectively by: (a) Gene transfer into hematopoietic stem cells (HSCs) using integration-competent lentiviral vector. (b) Gene transfer into hematopoietic stem cells (HSC) integrase defective lentiviral vectors. ZFN: Zinc figure protein. (c): Stem cell therapy reprogramming of adult cells to stem cells. iPS: Induced pluripotent stem cells

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Alternatively, the homologous recombination pathway can be harnessed to avoid random integration. Zinc-finger nucleases (ZFNs) can be used to enhance the frequency of gene correction. [32],[33],[34],[35] However, achieving the full potential of ZFNs for genome engineering in human cells requires their efficient delivery to the relevant cell types.

Lombardo and colleagues exploited the infectivity of integrase-defective lentiviral vectors (IDLVs) to express ZFNs and provide the template DNA for gene correction in different cell type. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common γ-chain gene (IL2RG) across different cell types as well as human embryonic stem cells (5%), allowing selection-free isolation of clonogenic cells with the desired genetic modification. Therefore, this technique opens new and exciting possibilities. By modifying the ZFN-binding specificity and selecting an appropriate donor sequence, one could target the IDLV-ZFN system to any individual site in the human genome avoiding random integration [Figure 1]b and potentially genome toxicity.

Gene transfer using lentiviral vectors also have some limitations:

  • Some of them includes: the need for improved efficiency of gene delivery, insertion of the gene into non-oncogenic sites and the potential negative or positive contributions of the b-thalassemic genotype and potential modifiers to the effectiveness of the gene transfer.
  • Original studies in animal models utilized mice with deletions of the β-globin genes. These mutations do not reflect the phenotypic variability observed in β-thalassemic patients. Thus, there is a gap in knowledge between our understanding of the primary mutation, the corresponding phenotype, and the approach to cure an individual patient based on his/her genotype (i.e. understanding of the disease and its treatment by genetic modalities). To date this variability has not been addressed and no studies have focused on the efficacy of gene therapy in relation to the different genotypes of the patients. Although gene therapy is an area of active clinical investigation, it also has some limitations in the management of thalassemia. Nonetheless, our review showed successful transfer of globin genes into hematopoietic cells of humans has been demonstrated and is encouraging.
Gene correction and induced pluripotent stem (ips) cells

Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. [37],[38]

Cotransfection of PNAs and recombinatory donor DNA fragments, Chin and co-workers demonstrated that these complexes can promote single base-pair modification at the start of the second intron of the beta-globin gene, which is the site of a common thalassemia-associated mutation.

This single base-pair change was detected by the restoration of proper splicing of transcripts produced from a GFP beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule.

They also showed that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. However, the enhanced recombination did not exhibit frequencies superior to 0.4%. [39],[40],[41],[42],[43] This technology could be a powerful tool in combination with the generation of stem cells. In particular, introduction of the genes Oct3/4, Sox2 with either Klf4 and c-Myc or Nanog and Lin28 genes can induce pluripotent stem cells.

Ye and co-workers showed that iPS cells can be generated from cells derived from skin fibroblasts, amniotic fluid or chorionic villus sampling of patients with β-thalassemia. Subsequently, the iPS cells were differentiated into hematopoietic cells that synthesized hemoglobin. Therefore, in the future the mutation in the β-globin gene of these iPS cells could be corrected by gene targeting and the cells differentiated into HSCs to be returned to the patient [Figure 1]c depicts this approach. In fact, mice affected by SCD (Sickle cell disease) were cured using this strategy. However, there are some obstacles that need to be overcome before iPS treatment of β-thalassemia will be utilized.


  • One of the most pressing problems is elimination of the transcription factors when they are no longer needed.
  • Second, it is necessary to reestablish the correct re-programming so that the iPS cells do not develop into tumors.
Splice-switching and stop codon readthrough

Defective β-globin gene expression and β-globin deficiency can be attributed to almost 200 thalassemic mutations.

However, only 10 mutations are responsible for the majority of cases worldwide and some of the most frequent cause aberrant splicing of intron 1 (IVS1-110, IVS1-6, IVS1-5) or intron 2 (IVS2-654, IVS2-745). [29]

These mutations lead to incorrectly spliced mRNAs, even though the correct splice sites remain undamaged and potentially functional. Use of small nuclear RNA (snRNA) and splice-switching oligo-nucleotides represents a promising approach since these molecules can restore the corrected splicing re-establishing the synthesis of the normal protein. Therefore blocking the aberrant splice sites with antisense oligonucleotides forces the splicing machinery to reselect the existing correct splice sites. Expression of antisense sequences targeted to the aberrant splice sites in thalassemic pre-mRNA has been successful, restoring the correct splicing pattern and ultimately restoring hemoglobin synthesis. [44],[45],[46],[47],[48],[49] This was demonstrated in HSCs and erythroid progenitor cells from a patient with IVS2-745/IVS2-1 thalassemia.

After transduction of the patient cells with a lentiviral vector that express an snRNA targeting the mutant RNA, the levels of correctly spliced β-globin mRNA and adult hemoglobin were approximately 25-fold over baseline. [29],[50] Similarly, the correct splicing pattern was restored in a mouse model of IVS2-654 thalassemia. This was achieved by delivery in vivo of a splice-switching oligonucleotide, a morpholino oligomer conjugated with an arginine-rich peptide. Repaired β-globin mRNA restored significant amounts of hemoglobin in the peripheral blood of the IVS2-654 mouse, improving the number and quality of erythroid cells. [29],[50],[51]

Another approach showing a great potential for the treatment of genetic disorders characterized by to premature termination codons (PTCs) is the use of drugs to induce stop codon readthrough. These modified RNA would protect against nonsense-mediated mRNA decay (NMD) and allow production of a protein.

Aminoglycoside antibiotics can decrease the accuracy in the codon-anticodon base pairing, inducing a ribosomal read-through of PTC. Aminoglycosides and analogous molecules were tested in their ability to restore β-globin protein synthesis on human erythroid cells (K562) carrying a lentiviral construct containing the 0-39 globin gene. Treatment of these cells with Geneticin (G418) and other aminoglycosides restored the production of β-globin. [51] Moreover, after FACS and high performance liquid chromatography (HPLC) analyses, G418 was also demonstrated to partially correct the biological function of the 0-39 globin mRNA in erythroid precursor cells from 0-39 homozygous thalassemia patients. This study strongly suggests that ribosomal read-through should be considered a novel approach for treatment of thalassemia caused by premature stop codon mutations and NMD. [52],[53],[54],[55]

   Conclusion Top

Thalassemias are diverse group of hereditary disorders in which there is a reduced rate of synthesis of one or more of the globin polypeptide chains. These are genetically transmitted disorders. Current approaches include hematopoietic stem cell transplantation. Disease management includes prenatal diagnosis, transfusion therapy, bone marrow transplantation (BMT); out of which only BMT is potentially curative. Transplant-related mortality and graft-vs-host disease are the limitation of the current approaches.

As thalassemia is genetically derived disorder, genetic and cellular targets are potential approaches in management of disease. Deliveries of transgenes in stem cell based gene therapy are effective in the therapeutic management. Gene transfer using onco-retroviral vectors and lentiviral vectors are beneficial. Lentiviral vectors have an advantage over onco-retroviral vector due to integration of larger element and minimal sequence rearrangement. Induced pluripotent stem cells, splice-switching and stop codon read-through are other genetic approaches which are showing advantages over the current therapy.

   References Top

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2.Giardini C, Lucarelli G. Bone marrow transplantation in the treatment of thalassemia. Curr Opin Hematol 1994;1:170-6.  Back to cited text no. 2
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6.Chin JY, Kuan JY, Lonkar PS, Krause DS, Seidman MM, Peterson KR, et al. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids. Proc Natl Acad Sci U S A 2008;105:13514-9.  Back to cited text no. 6
7.Dave UP, Akagi K, Tripathi R, Cleveland SM, Thompson MA, Yi M, et al. Murine leukemias with retroviral insertions at Lmo2 are predictive of the leukemias induced in SCID-X1 patients following retroviral gene therapy. PLoS Genet 2009;5:e1000491.  Back to cited text no. 7
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