|Year : 2020 | Volume
| Issue : 3 | Page : 146-154
Hemoglobinopathy correction with CRISPR or not; gene therapy is the solution
Bader Al Alwan, Arwa A Alsubait, Bahauddeen M Alrfaei
King Abdullah International Medical Research Center, Stem Cells Unit; Department of Cellular Therapy and Cancer Research, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
|Date of Submission||19-Nov-2019|
|Date of Decision||23-Feb-2020|
|Date of Acceptance||29-Feb-2020|
|Date of Web Publication||02-Jul-2020|
Bahauddeen M Alrfaei
Stem Cells and Regenerative Medicine Unit, Department of Cellular Therapy and Cancer Research, King Abdullah International Medical Research Center, Riyadh
Source of Support: None, Conflict of Interest: None
Hemoglobin (Hb) disorders or hemoglobinopathies are groups of blood conditions involving inherited genetic diseases – mostly as single-gene autosomal recessive – that lead to the formation of abnormal Hb structure or inadequate to no production of globin chains in Hbs. Disorders of Hb are a global concern since these diseases can cause severe morbidity and early mortality of the affected populations. Treatments vary between chemicals and molecular approaches. The most promising approach is Hb correction. However, the stability of the correction faces a big challenge along with safety concerns. It is worth noting that most of the inherited hemoglobinopathies share common clinical presentations and laboratory findings, although some have distinct features. Hemoglobinopathies with emphasis on recent advances in gene therapy targeting sickle cell disease and thalassemia are discussed in this review.
Keywords: CRISPR, hemoglobin disorders, thalassemia
|How to cite this article:|
Al Alwan B, Alsubait AA, Alrfaei BM. Hemoglobinopathy correction with CRISPR or not; gene therapy is the solution. J Nat Sci Med 2020;3:146-54
|How to cite this URL:|
Al Alwan B, Alsubait AA, Alrfaei BM. Hemoglobinopathy correction with CRISPR or not; gene therapy is the solution. J Nat Sci Med [serial online] 2020 [cited 2020 Aug 10];3:146-54. Available from: http://www.jnsmonline.org/text.asp?2020/3/3/146/287701
| Introduction|| |
Hemoglobin (Hb) disorders are composed of different inherited genotypes, classified into two broad categories. The first group has structural defect in one of the globin molecules of Hb such as sickle cell disease (SCD). The second group includes those with partial or complete defect in synthesis of globin chains such as β-thalassemia. Hb is a heterotetramer protein found within the cytoplasm of the mature red blood cells (RBCs). It is composed of four globin chains, and each globin chain is linked to a heme moiety that consists of protoporphyrin IX ring complexed with an iron atom in the form of ferrous (Fe 2+)., The classification of Hbs is according to the type of the globin chains present in each of them. The production of the globin chains is controlled by closely related two gene clusters that encode the human globins: α-globin-like gene cluster (HBZ, HBA1, and HBA2) which located on chromosome 16 and β-globin-like gene cluster (HBB, HBE1, HBG1, HBG2, and HBD) which located on chromosome 11., Each gene locus encodes different globin chains to produce different normal Hbs. For example, HBZ codes for ζ-globin (zeta-globin) chains that produce Hb Portland-1 during fetal life when combined with α-globins and HBB that encodes for β-globin chains. The Hb biosynthesis starts early during the embryonic life, which is not the focus of this review. To understand Hb disorders, we need to understand the composition of the normal Hbs. There are mainly three types of normal Hbs; HbA, HbA2, and HbF. HbA consists of α2β2, HbA2 consists of α2δ2, and HbF consists of α2γ2. During early days of life, all three types of Hb are found in the circulating RBCs, and the majority of the Hbs being HbF. As the age progresses, HbF synthesis starts to decline and replaced by HbA. This is known as “fetal switch” in which the synthesis of γ-globin chains is replaced by β-ones. Normally, HbF disappears from blood before adulthood, and only two types of the Hb remain in the circulation, HbA and HbA2. The predominant Hb is HbA which reaches more than 97% in normal cases. Hemoglobinopathies or Hb disorders occur when there is an inherited defect encoding the production of normal globin chains of Hb in terms of abnormal structure or inadequate quantity. As a result, abnormal Hbs' synthesis replaces the normal ones and produces clinical manifestations.
Hb disorders are caused by genetic mutation or deletion of α- and/or β-globin genes which fallout mainly into two types of inherited hemoglobinopathies, thalassemia and structural Hb variants. The defect in the synthesis of α- or β-globin chains causes thalassemia. The classification of thalassemia is depending on the chain involved. Structural Hb variants involve production of abnormal Hbs due to altered amino acid sequence in α- or β-globin chains that result in abnormal physical and chemical properties. Global estimation of Hb disorders diagnosed yearly in infants results in approximately 300000–400000 cases., Among these disorders, β-thalassemia (β-thal) and SCD are receiving more attention as they have a great impact on morbidity and mortality in the affected patients. Thalassemia and SCD affect around 5% of the world population. Some countries have adopted specific programs to prevent future incidences such premarital screening and the SCD prevention programs., Some countries have established newborn screening programs for SCD to implement early follow-up with the patients. In Saudi Arabia, the prevalence of β-thal and SCD are significantly increasing compared to other countries in the Middle East. Recent data published in 2017 about the prevalence of β-thal and SCD in Saudi Arabia shows a higher occurrence of SCD compared to β-thal major among tested populations: 0.38% and 0.07%, respectively.
| Structural Hemoglobinopathies|| |
Most frequent causes of abnormally structure Hbs are point mutations in the coding genes that result in exchange of single amino acid in the globin chains. For example in SCD, HbS is produced due to glutamic acid alteration to valine in β-globin chains of HbA1. One can find more than 1800 Hb variants in trusted databases such as database of human Hb variants and thalassemias, http://globin.bx.psu.edu/hbvar/menu.html. These hemoglobinopathies are further categorized into smaller groups: first: sickle cell disorders, which include sickle cell trait (SA), sickle cell anemia/disease (SS), HbSC disease (SC), S/β thal, and S with other Hb variants: D, O-Arab; second: unstable Hbs; third: Hbs with altered oxygen affinity; and fourth: acquired and congenital methemoglobinemia. Our interest in this work focused on SCD and thalassemia [Figure 1].
|Figure 1: Point mutation within hematopoietic stem cells may lead to structural hemoglobinopathies or thalassemia. The cellular phenotypes are more prominent when oxygen level is decreased|
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| Sickle Cell Disease|| |
SCD is an inherited hemolytic disease that affects millions of people worldwide, characterized by hemolytic anemia, vaso-occlusive crises, and premature death of RBCs., SCD is caused by a mutation in the β-globin genes of HbA1 that produces abnormal HbS due to exchange of the sixth glutamic acid in the β-globin chain for a valine residue. The SCD occurs with homozygous or compound heterozygous abnormal Hb. In the homozygous, the affected patients exclusively form HbS. However, in the compound heterozygous, the production of HbS is combined with production of other abnormal β-globin proteins such as HbC, HbD, or other Hb variants [Figure 1].
| Molecular Pathophysiology|| |
The amount of HbA1 converted into HbS in sickle cell trait is <40% in most cases with normal Hb levels and mild to rare pain occurrence. The produced HbS polymerizes when RBCs are deoxygenated and become insoluble  forming a gelatinous network of fibrous polymers in the red cell membrane that leads to the rigidity of RBCs. Then, RBC viscosity increases, which causes dehydration due to leaking of potassium and increasing calcium influx., These changes make the RBCs to have a sickle shape. In addition, the same changes including sickle shape generate adhesion tendency toward endothelial cells which cause accumulation in the small capillaries. The consequences are microvascular vaso-occlusion, ischemia, inflammation, and premature RBC destruction by spleen., Moreover, the sickled cells experience abnormal activation of intracellular signaling pathways, decreased ATP, declined antioxidant activity, dysregulated miRNAs, and altered gene expression during erythropoiesis. It is worth noting that the repeated cycles of deoxygenation and reoxygenation states lead to RBC surface changes that cause membrane damage and hemolysis. The acute and chronic pathology of SCD is developed through the interactions between sickle cells and leukocytes, endothelial adhesion molecules (E- and P-selectins), activated platelets, and nitric oxide depletion. The alteration in the membrane lipids of sickle cells leads to exposure of phosphatidylserine along with the formation of microparticles. This event increases aggregation property of the red cells inside blood vessels.
| Defective Biosynthesis of Globin Chains, Alpha-, Beta-, and Delta- (Thalassemias)|| |
Thalassemia is a group of inherited Hb disorder that results in a complete or partial deletion of the globin chains that are forming Hbs. For nomenclature, any absence or incomplete formation of the globin chains, the chain's name precedes the term thalassemia. The thalassemia hemoglobinopathies are subdivided into three categories; (1) β-thal group which includes β-thalassemia major, minor, and intermediate in addition to β-thal with other variants such as HbS/β-thalassemia and HbE/β-thalassemia; (2) α-thalassemia group which is further divided according to the number of α-globin chains affected: one allele: α+-thalassemia, two alleles in cis: α0-thalassemia, two alleles in trans: homozygous α+-thalassemia, three alleles: HbH disease, and four genes: hydrops fetalis with Hb Bart's; and (3) de novo and acquired α-thalassemia.
β-thalassemia is a major type of hemoglobinopathies affecting populations worldwide. More than 90% of thalassemias are β-thal. The disease is distributed widely in Mediterranean, Middle East, and Indian subcontinents, with estimated carrier for β-thal ranging from 1% to 20% of the population of these areas. It is an inherited Hb disorder that results in a complete or partial deletion of beta (β)-globin chains in HbA1. Those with a complete absence of β-globin chains are classified as β-thal major or β0 (beta null), while those with low β-globin chains are β+-thalassemia.
The main cause of the disease is point mutations in the β-genes that affect a single nucleotide with more than 200 different mutations described so far. The complete or partial absence of beta-genes leads to the deficiency or complete abolition in HbA1 synthesis. This results in accumulation of free alpha-globin subunits that are incapable to synthesize Hbs. This, in turn, leads to the production of low-level Hbs within the RBCs. Unlike α-thalassemia, most of the β-thal mutations are nondeletional. These nondeletional mutations include single base substitutions and small deletion or insertion of one or few bases. Some of these mutations result in inactivation of β-genes. Other mutations occur in the conserved DNA regions of the β-gene promoters, which result in minimal reduction in β-globin synthesis. Thalassemia is characterized by ineffective erythropoiesis, hypochromic microcytic anemia, hemolysis, extramedullary erythropoiesis, bone expansion, and iron deposition in different organs., The iron deposition in the tissues contributes to significant complications of the disease such as liver and cardiac diseases, endocrine malfunctions such diabetes, and adrenal disorders. A less severe form of β-thal is β-thalassemia intermediate, which has partially compensated levels of HbA1 in the circulation and does not require blood transfusion. The molecular bases of β-thal are heterogeneous, but the clinical symptoms and phenotype of the disease are similar.
| Detection of Hemoglobinopathies|| |
Hemoglobinopathies are diagnosed in the laboratory using different ways such as sickling or solubility test for HbS, complete blood count, Hb levels, red cell indices, and biochemical testing. All of which constitute the first-line detection methods., The most powerful techniques used to confirm the presence of hemoglobinopathies are Hb electrophoresis, isoelectric focusing, and high-performance liquid chromatography (HPLC).,,, Among these laboratory tests, HPLC is considered the most sensitive and powerful tool used to detect nearly all Hb disorders. Nonetheless, HPLC results should be confirmed with Hb electrophoresis or molecular techniques. Detection of carriers and parental status uses similar methods for confirmation.
Molecular techniques are advanced tools used to diagnose hemoglobinopathies. Sanger sequencing is typically for single-gene sequencing, but it is also useful for complex, multigene disorders, and gene locus heterogeneity. Gap-polymerase chain reaction, array comparative genome hybridization, and target locus amplification are also molecular methods designed to diagnose mutations., Next-generation sequencing is a technique used for whole genome, gene panels, and exome sequencing.,
| Therapeutic Regimens|| |
SCD is a severe syndrome that requires a continuous care. Current therapy is meant to treat and prevent the complications of SCD including hydroxyurea drug and blood transfusion. Hematopoietic stem cell transplantation (HSCT) is the only cure for the patients, and gene therapy is still under investigation. Targeting the cellular interactions constitutes one of the promising therapies to prevent the complications of SCD.,
Shortly after birth, new born babies usually stop producing the fetal Hb and switch over to the production of adult Hb. However, some people pursue to produce high levels of HbF throughout their lives. HbF can be presented in adults with benign condition, called hereditary persistence of fetal hemoglobin (HPFH). Usually, its prevalence in the RBCs ranges between 10% and 30% when it is evenly distributed among RBCs and it is described as pancellular HPFH. When HbF in adult has uneven distribution in the RBCs, it is called heterocellular HPFH and it is ranging between 1% and 10%.
Patients with relatively higher baseline of HbF have less disease severity and fewer complications. Hydroxyurea is an S-phase specific cytotoxic, antimetabolic, and neoplasm, and drug works as an inhibitor for the ribonucleoside diphosphate reductase that increases HbF synthesis , by 2–5 folds in patients with SCD. It promotes the reactivation of gamma-genes to produce HbF  through epigenetic modifications and combination of different signaling and transcriptional pathways that lead to increase synthesis of γ-globins. Elevated HbF in patients with SCD and β-thalassemia shows less severe disease. The Food and Drug Administration (FDA) has approved it for treating SCD in 1998 and by the European Medicines Agency in 2007. It decreases leukocytes, platelet adhesion, HbS polymerization, cellular changes, inflammation, and adhesion molecule expression. Hydroxyurea has been FDA approved to treat adult homozygous HbSS. However, there is a concern of increased tissue damage on long-term treated patients. There are several investigational drugs that are ongoing or completed trials used in combination to treat SCD. Some of them are inhibiting cell dehydration, increasing reduced nicotinamide adenine dinucleotide phosphate, elevating glutathione, increasing production of nitric oxide, decreasing platelets activation, and adhesion molecule expressions. Other drugs for HbF induction also exist. For example, butyrates, a drug, act as histone deacetylase inhibitor and thalidomides. Carbon monoxide, a drug, interacts with the Hb and prevents HbS polymerization while attached.,
The molecular changes of the RBCs in hemoglobinopathies contribute to hemolysis which eventually results in anemia. Blood transfusion represents the baseline to compensate for blood loss. Patients with β-thal major regularly require blood transfusion 3–4 times a week if their Hbs are lower than 7 g/dL. Frequent blood transfusion leads to serious complications such as alloimmunization that leads to hemolysis and formation of autoantibodies. Iron deposition in patients' vital tissues is also a complication affecting heart, liver, and lungs causing iron overload cardiomyopathy. Frequent hemolysis and ineffective erythropoiesis contribute to iron overload. The excess iron in the circulation enters the cells of these tissues through L-type calcium channels and interferes with normal antioxidative mechanism that causes injury to the cells. Iron-chelating therapy is crucial to treat and prevent transfusion-related iron overload. Typically, it should starts after 10–20 blood transfusion and when serum ferritin is above 1000 μg/L. Desferrioxamine is a highly effective parenteral iron-chelating drug that reduces the level of iron in the body. In 2006, an oral iron-chelating agent called deferasirox was approved which reduces the annoying subcutaneous puncture of parenteral administration of desferrioxamine., Deferasirox decreases the serum ferritin to baseline levels in blood-transfusing dependent patients as well as cardiac and liver iron. Deferiprone used as a second-line therapy with desferrioxamine when desferrioxamine alone is ineffective.
Targeting selectin-mediated adhesion molecules that lead to leukocyte activation and stimulate response to inflammation is a potential therapy reported to relieve the vaso-occlusion strike in murine model. There are different drugs under investigation that are targeting P-selectin adhesion molecules such as rivipansel (a low-molecular-weight heparin derivative).
Bone marrow transplantation (BMT) or HSCT is the curative regiment used to treat not only Hb disorders but also many types of blood disorders and malignancies. The term BMT was used previously in practice, but since the source of engraftment of hematopoietic stem cells (HSCs) has been expanded into umbilical cord blood (CB) and peripheral blood (PB), the term HSCT is the preferred one. Regardless of its preference, HSCT raises some concerns relevant to (1) patient selection (age, disease severity, and end-organ injury), (2) risk of short-term toxicity (infection, posterior reversible encephalopathy syndrome, and death), (3) graft failure, and (4) possibility of long-term adverse effects (graft-vs-host disease [GvHD] and infertility).
| Gene Therapy Targeting Sickle Cell Disease|| |
As mentioned above, RBC transfusion and hydroxyurea are the most common therapeutic treatments for SCD., Allogeneic HSCT is curative but limited. The major limitation of HSCT is the lack of compatible donors, since <14% of patients have a matched sibling donor. In addition, transplants with matched unrelated donors are limited by donor availability and immunologic barriers, such as graft rejection and GvHD. Therefore, the development of ultimate therapies targeting the causative gene would be a promising cure. Currently, research is trending toward genetic approaches that target SCD. Particularly, novel lentiviral and genome editing-based strategies are meant to reactivate endogenous fetal Hb (HbF) expression. In both systems, patient's HSC isolation is required. Then, the mutated globin gene is replaced with a healthy copy of the gene. Next, transplant those cells back into the patient. If the procedure was successful, the edited cells will be able to produce healthy RBCs rather than sickled ones.
| Development of Gene Therapy for Sickle Cell Disease|| |
The main advantage of gene therapy is the potential to cure individuals who have received the therapy. Treated patients are no longer at risk of getting health crisis from SCD. However, the disadvantages of gene therapy are the increase risk of toxicity, off-target effects, tumor formation, and incompatible immune response.
The first trial of human gene therapy for a hemoglobinopathy occurred in the early 1980s. In 1984, the pediatrician Janet Watson discovered the importance of HbF levels in SCD. She noted that the clinical complications in infants with SCD were uncommon before the age of 1 year old and that their deoxygenated RBCs took longer to sickle and did not misshape as massively as the sickle cell trait in their mother's cells. Thus, it was proposed that elevated level of HbF in infants' blood is associated with a milder SCD. Three decades later, this approach has changed dramatically when Ribeil et al. announced the world's first patient cured from SCD. This accomplishment was achieved after multiple attempts to overcome diverse major barriers including  the development of safe and effective viral vectors for therapeutic globin gene transfer to HSCs and  high and stable gene expression without interfering with the expression of the endogenous genes.
| Transcriptional Regulation of Fetal to Adult Hemoglobin Switch|| |
Transcriptional machinery is highly controlled for γ-to-β Hb switch. The switching process of HB (hemoglobin) depends on a 16-kb long sequence which is located 40–60 kb upstream of the β globin genes that known as the locus control region (LCR). This essential regulatory element contains a strong chromatin opening and DNA enhancer elements (DNase hypersensitive sites) which allow for high-level erythroid-specific globin expressionin vivo and regulate the switch from fetal to adult Hb.,, Understanding the molecular mechanisms required for the fetal to adult Hb switch have had a significant impact on discovering more efficient and specific approaches for HbF induction. A group of transcription factors acts as protein complexes contribute to the Hb switching process, including Kruppel-like factor, MYB, and the nuclear receptors TR2/TR4 and COUP-TFII. Genome-wide association studies have identified three major loci that account for genetic variation in HbF. The best described variant to date has been found within the zinc-finger transcription factor B-cell lymphoma/leukemia 11A (BCL11A).BCL11A is highly expressed in hematopoietic lineages, and it is critically required for HbF repression.,, It binds to the β-globin gene locus and suppresses γ-globin expression., In addition, BCL11A is essential for the function of several cell types. Its dysfunction can cause leukemias or lymphomas. Therefore, targeting BCL11A should be restricted to the erythroid lineage. Genetic linkage analysis has identified single-nucleotide polymorphisms (SNPs) in BCL11A enhancer that are associated with increased expression of γ-globin genes and ameliorate the SCD phenotypes. Several researchers have investigated BCL11A as a repressor of the fetal globin gene. When Wilber et al. used shRNA to knock down BCL11A expression in adult human erythroblasts, the RBC phenotype was not affected, but the production levels of HbF was increased. Furthermore, loss of BCL11a in mouse models of SCD was able of reversing the disease symptoms and increasing globin gene expression. These results support a key role for BCL11A in silencing the γ-globin genes during Hb switch. Furthermore, it has a potential role in reactivation of HbF in adult erythroblasts., Researchers at Dana-Farber/Boston Children's Cancer and Blood Disorders Center and the University of Massachusetts Medical School worked on optimizing the CRISPR/Cas9 system delivery. Their main goal was to efficiently edit mostly the entire blood stem cell population of an individual with SCD. When normal human HSCs were transplanted into a recipient mice with immunosuppressed system, ~80% of RBCs were corrected, and cell sickling was prevented since sufficient HbF was produced.
| β-Globin Gene Expressing Vectors|| |
In previous research studies, several viral vectors were tested to develop safe and efficient delivery systems, including retrovirus, adenovirus, adeno-associated virus, and lentivirus., So far, the most promising delivery system is the lentiviral vector, BB305, that has been used with some success in patients with SCD. In March 2017, a 13-year-old boy with SCD had been cured with transplantation of CD34+ HSPCs transduced with the BB305 vector expressing the mutant adult β-globin gene. βA-T87Q-globin gene disrupts lateral and axial interactions between Hb tetramers providing the antisickling property., Interestingly, the teenager remains free from sickle crises and other symptoms of the disease up to 15 months after receiving the treatment, which was conducted as part of a Phase ½ clinical trial (NCT02151526). This successful achievement prompted the start of two clinical trials in France and the United States targeting SCD patients. However, the multicenter U. S. trial failed to reproduce the success of the first patient. These outcomes proved that gene therapy through gene addition can be a powerful treatment for SCD patients even though it is challenging, and multiple factors are required for success.
| Gene Therapy Targeting Thalassemia|| |
The two main types of thalassemia are alpha and beta. In addition, rare categories of thalassemia also exist such as (δ, δ, δβ, and δδβ-thalassemias). As mentioned above, a person with thalassemia suffers from reduction of RBCs and shortage of Hb. In addition, patients present with smaller RBCs than normal. Those phenomena contribute to skeletal malformations in place of bone marrow production. Therefore, treatment of thalassemia involves the following: blood transfusions, iron chelation, bone marrow (stem cell) transplant, surgery, and gene therapy. Bone marrow transplant has been scoring the most success in curing the disease. However, this success is not enough due to multiple reasons relevant to patients' abnormalities such as central fibrosis in liver or transplant protocol such as finding a donor., Gene therapy is expected to be the potential saver. Although thalassemia mutations involve alpha- and beta-globin, most gene therapies are directed toward severe cases of thalassemia such as beta-thalassemia major. The repair of alteration in gene therapy favors retroviruses of both types: oncoretroviral and lentiviral. This method uses viruses to transfect and correct HSC because they are more stable while integrating into cells.
| Viral-Based Gene Therapy|| |
Oncoretroviral vectors have been triad on human previously on X-SCID, X-linked severe combined immunodeficiency, disease. Few children have been cured from X-SCID disease using this method. However, using this method to correct β-globin in thalassemia did not produce satisfactory success. The failure was sighted in animal studies as well due to many serious limitations such as cell replication requirement for oncoretroviral integration, DNA packaging limitation of viruses, and RNA instability of vector production that need to be processed and transferred to nucleus. Moreover, oncoretroviral possesses the risk of developing leukemia which was reported in three cases of human trials previously.
Lentiviral vectors have several advantages over oncoretroviral such as transducing nondividing cells which include transfer and process RNA transcripts efficiently. Animal models of β-globin correction using this method were successful. This method relies on simple replacement of altered sequence within HSCs. In addition, it includes facilitating products such as modulating proteins of the human β-globin LCR that regulate globin synthesis. This victory pushed more clinical trials to take place. The tendency of triggering leukemia was also overcome in this method by deleting functional LTR, long terminal repeat, which was proven to be involved in leukemia formation, [Figure 2].
|Figure 2: Viral and nonviral methods are competing to target abnormal hematopoietic stem cells causing an intended modification to be implanted into bone marrow|
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| Nonviral-Based Gene Therapy|| |
Nonviral approaches manifest advancements and advantages over viral based approaches, for example, ability to escape donor/host reaction and can be produced in bulk economically. However, they suffer greatly at delivery or transfection possibilities. The frequency and the odds of success are very low to generate a therapeutic dose. This resulted in less efficacy. Scientists tried to overcome those obstacles with new promising approaches including naked DNA insertion, magnetofection, sonoporation, the gene gun, electroporation, lipoplexes, oligonucleotides, nonorganic nanoparticles, and dendrimers. Most of these new approaches are relying on controlling certain subcellular trafficking.
CRISPR/Cas9 is another promising nonviral method for RNA and DNA editing. It can specifically target the β-globin gene (HBB) CD 41/42. Scientists proposed CRISPR to correct messenger RNA transcription, splicing, and/or translation to repair hemoglobinopathy. For example, Niu et al. demonstrated that the grouping of single-strand oligodeoxynucleotides and CRISPR/Cas9 had the ability to fix the HBB gene CD 41/42 mutation in thalassemia induced pluripotent stem cells. The application efficiency and safety were confirmed by sequencing of purified clones. Successful clones maintained pluripotency and ability to differentiate into erythroblast and restoration of HBB gene function. Although off-target effects exist with CRISPRs, they have minimal consequences which also can be predicted [Figure 2].,
More specific approaches to treat SCD patients is depending on the sequence-specific targeting of a nuclease to the genome and the repair of the double-strand break (DSB) by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The perfect therapy for a genetic disease would be to edit the mutationin vivo without leaving any trace left behind. However, the major problem with HDR-mediated strategy is that NHEJ is more often used to fix a DSB compared with HDR. For this reason, researchers examined the possibility of utilizing targeted nucleases in conjugation with NHEJ to inactivate proteins or DNA-regulatory elements that contributed to γ-globin gene expression.
| Clinical Trials for Sickle Cell Disease|| |
More than 600 clinical trials were conducted offering more than forty therapeutic options to develop the best cure for patients with SCD. Gene therapy clinical trials of SCD have been initiated since 2014. However, the enrollment and completion of clinical trials have been slow and costly, [Table 1].
|Table 1: Clinical trials of gene therapy for sickle cell disease since 2014 to August 2019|
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| Clinical Trials of Thalassemia Gene Therapy|| |
This first clinical trial relevant to thalassemia was registered in 2005. Since then, there were 336 clinical trials registered with different goals, but all of which were relevant to thalassemia. Out of them, 13 trials only were based on gene therapy [Table 2]. In those studies, nucleic acid inhibitors or stoppers were not considered as gene therapy in this study. In general, multiple successful safety and efficacy trials have been reported. Most gene therapy trials used marked globin to track successful implantation, for example, β-globin (βA-T87Q) and HbA T87Q.,
|Table 2: Thalassemia gene therapy clinical trials since 2005 - August 2019|
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| Conclusion|| |
Based on available knowledge, gene therapy will be the future of sickle cells and thalassemia treatment. Whether lentivirus or CRISPR applications will be used or not, both methods require improvements for the moment. Nonviral systems may compete better with capability to support multiple doses. However, we expect a satisfactory method to arise within the next 10 years. We may not need the perfect treatment, especially if the side effect and off-target effects can be controlled. The best example for this scenario is the generation of the FDA-approved spinal muscular atrophy 1 treatment – AVXS-101 by Novartis., One of the potential downsides for gene therapy is the cost. The estimated price tag for complete treatment might be between $500,000 to $1,000,000 per patient., Novartis treatment was far from perfect but prevented death of subjects and converted sever symptoms into mild, if any. It is a matter of time until a successful trial could mimic Novartis success. It's on the hands of insurance companies to decide whether the price tag is acceptable. Should the world wait for an extra 10 years on top of the predicted plan to have an affordable cure? The word “affordable” is subjective in the eye of insurance companies which limit globin disorders gene therapy at least partially.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]