Most Common Erythroid Metabolic Inherited Diseases. BM transplantation and gene therapy approaches
Gene therapy is becoming a powerful tool to treat genetic diseases. Clinical trials performed during last two decades have demonstrated its usefulness in the treatment of several genetic diseases  but also the need to improve vector delivery, expression and safety . New vectors should reduce genotoxicity (genomic alteration due to vector integration), immunogenicity (immune response to gene delivery vectors and/or trangenes) and cytotoxicity (induced by ectopic expression and/or overexpression of the transgene).
In mature erythrocytes, most metabolic needs are covered by glycolysis, oxidative pentose phosphate pathway and glutathione cycle. Hereditary enzyme deficiencies of all these pathways have been identified, being most of them associated with chronic non-spherocytic hemolitic anemia (CNSHA). Hereditary hemolytic anemia exhibits a high molecular heterogeneity with a wide number of different mutations involved in the structural genes of nearly all affected enzymes. Deficiency in metabolic enzymes impairs energy balance in the erythrocytes, with or without changes in oxygen affinity of hemoglobin and delivery to the tissues. Despite of having a better understanding of their molecular basis, definitive curative therapy for Red Blood Cells (RBC) enzyme defects still remains undeveloped.
Conventional bone marrow transplantation allows the generation of donor-derived functional hematopoietic cells of all lineages in the host, and represents the standard of care or at least a valid therapeutic option for many inherited diseases . However, complications associated to allogeneic transplantation can be as severe as the enzymatic deficiency. The recessive inheriting trait of most of these metabolic diseases and the confined enzymatic defect to the hematopoietic/erythropoietic system, make them suitable diseases to be treated by gene therapy. Correction by gene therapy requires the stable transfer of a functional gene into the autologous self-renewing Hematopoietic stem cells (HSCs) and their mature progeny. Autologous BM transplantation of genetically corrected cells shows several advantages over the allogeneic procedure. First, it overcomes the limitation of human leukocyte antigen (HLA)-compatible donor availability, so it can be applied to every patient. Second, the reduction of morbidity and mortality associated with the transplant procedure, as there is no risk of graft versus host disease (GvHD) and consequently no need for post-transplant immunosuppression.
To date, gene therapy approaches for the treatment of inherited metabolic deficiencies are still limited, mainly because of the frequent lack of selective advantage of genetically corrected cells. This implies that high levels of transgene expression are required, as well as an efficient transduction of HSCs. This requirement have already been described in different RBC diseases as in the erytropoietic protoporphyria (EPP)  caused by the deficiency of the last enzyme of the heme biosintesis pathway or in the piruvate kinase deficiency (PKD) , where there is an impairment in the final yield of ATP in RBC. Additionally, some RBC pathologies require switching on expression of the transgene at only the proper stage of differentiation, which represents another challenge in the development of new gene therapy protocols.
2. Gene therapy attempts for inherited metabolic diseases of erythrocytes
Although more than 14 metabolic deficiencies have been identified causing CNSHA, approaches of gene therapy have been done only in a few of them (Table 1). Below, we are including a short description of the different diseases and the attempts addressed.
Among glycolytic defects causing CNSHA, Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic disease. More than 400 million people are affected world wide, showing a vast variability of clinical features. G6PD catalyzes the first reaction of the pentose phosphate pathway, in which Glucose 6-phosphate (G6P) is oxidized and Nicotinamide adenine dinucleotide phosphate is reduced (NADPH) resulting in decarboxylation of CO2 and pentose phosphate. G6PD plays a central role in the cellular physiology as it is the major source of NADPH, required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system and others. Indeed, G6PD is essential for cell survival.
|Glucose-6 Phosphate Dehydrogenase Deficiency (G6PD)||Xq28||X-linked||jaundice, spleno- and hepatomegaly, hemoglobinuria, leukocyte disfunction, and susceptibility to infections||D: C57BL/6 mice|
P: Transduction of 5-FU treated BM cells with MMLV-hG6PD or MPSV-hG6PD vectors and subsequent transplantation.
R: lethally irradiated C57BL/6 mice
|Pyruvate Kinase Deficiency (PKD)||1q21||A.R||Reticulocytosis, splenomegaly, ||D: normal CBA/N+/+ mice + 5FU|
R: CBA Pk-1slc/ PK-1slc mice
C: minimal (100 or 400 cGy)
|D: WT mice|
P: Transduction of 5-FU treated BM cells
with pMNSM-hLPK retroviral vector and subsequent transplantation
R: lethally irradiated mice
|D: normal CBA/N+/+ mice|
R: CBA Pk-1slc/ PK-1slc mice
C: no conditioning 
|D: CBA PK-1slc/PK-1slc mice|
P: Transgenic rescue using the μLCR-PKLR-hRPK construct
|D: normal Basenji dogs|
R: PKD Basenji dogs
C: sublethal dose (200 cGy) + mycophenolate memofetil + cyclosporine
|D: WT mice|
P: Transduction of Lin-Sca1+ BM cells with a MSFV-hRPK retroviral vector and subsequent transplantation
R: lethally irradiated WT mice 
|D: HLA-identical sister|
R: PKD severe patient
C: busulfan + cyclophosphamide 
|D: AcB55 mice|
P: Transduction of Lin-Sca1+ BM cells with a MSFV-hRPK retroviral vector and subsequent transplantation
R: lethally irradiated AcB55 mice 
|Glucose Phosphate Isomerase Deficiency (GPI)||19q13.1||A.R||neuromuscular disturbances|
|Triose Phosphate Isomerase Deficiency (TPI)||12p13||A.R||neuromuscular disorders, mental retardation, frecuent infections and death |
|Hexokinase Deficiency (HK)||10q22||A.R||defects in platelets|
|Phosphofructokinase Deficiency (PFK)||21q22.3||A.R||myopathy, storage disease type VII|
|Bisphosphoglycerate Mutase Deficiency (BPGM)||7q31-q34||A.R||erythrocytosis|
|Glutathion Synthetase Deficiency (GSD)||20q11.2||A.R||5-oxoprolinuria, metabolic acidosis, central nervous system impairment|
Patients with CNSHA suffer anemia and jaundice, but often tolerate their condition well. However, G6PD variants with low activity are related with alterations in the erythrocyte membrane facilitating its breakdown and causing intravasal hemolysis. These symptoms are often accompanied by spleno- and hepatomegaly and hemoglobinuria. Besides, leukocyte dysfunctions caused by lower concentration of NADPH appear when G6PD activity is below 5% of the normal activity, leading to an immune depression . Vives
Preclinical work from Rovira et al demonstrates that
Pyruvate kinase deficiency (PKD), the second most frequent abnormality of glycolysis causing CNSHA, has also been proposed as a potential disease to be treated by gene therapy. Pyruvate kinase (PK) catalyzes the second ATP generation reaction of the glycolysis pathway by converting phosphoenolpyruvate (PEP) into pyruvate, yielding nearly 50% of the total ATP production in red blood cells. PK plays a crucial role in erythrocyte metabolism, since mature RBC are absolutely dependent on the ATP generated by glycolysis, giving the loss of mitochondria, nucleus and endoplasmic reticulum in their mature state. RPK is therefore necessary for maintaining cell integrity and function. Reduced levels of erythrocyte Pyruvate kinase (RPK) lead to an accumulation of glycolytic intermediates that ultimately shortens the life span of mature RBC by metabolic block . Four tissue-specific isoenzymes of PK (M1, M2, R and L) encoded by two different genes (
The codifying region of
PK deficiency is transmitted as an autosomal recessive trait and although its global incidence is still unknown, it has been estimated in 1:20000 in the general caucasian population . Clinical symptoms appear in homozygous and compound heterozygous patients, which lead to a very variable clinical picture, ranging from mild or fully compensated forms to life-treating neonatal anemia necessitating exchange transfusions and subsequent continuous support . Pathological manifestations are usually observed when enzyme activity falls below 25% of normal PK activity , and severe disease has been associated with a high degree of reticulocytosis .
The feasibility of gene therapy in PKD was first reported by the group of Asano, who introduced the human LPK cDNA into C57BL/6 mouse bone marrow cells using a retroviral vector . They demonstrated the expression of the LPK transgene mRNA in both peripheral blood and hematopoietic organs after bone marrow transplantation. However, viral-derived expression in peripheral blood was detectable no longer than 30 days post-transplantation, indicating an insufficient transduction efficacy of the retroviral vector used or transduction of non-pluripotent BM cells. In a hemolytic anemia dog model, bone marrow transplantation of minimal conditioned receptors failed to correct the hematological symptoms . Other approaches to rescue RPK phenotype through a gene addition strategy have been also addressed using a PKD transgenic mouse model (
In addition, our work carried out in mouse models supported the therapeutic potential of viral vectors for the gene therapy of PK deficiency. Throughout the transduction of bone marrow cells using γ-retroviral vectors that carry the human RPK cDNA and subsequent transplantation, we reported a long-term expression of the human protein in RBC obtained from primary and secondary receptor mice, without detectable adverse effects . Recently, we have also reported a successful gene therapy approach using the same retroviral vectors in the congenital mouse strain AcB55, identified by Min-Oo in studies of alleles involved in malaria susceptibility . These mice carry a loss-of-function mutation (269T-> A) resulting in the amino acid substitution I90N in the
Glucose phosphate isomerase (GPI) deficiency is the third most common hereditary cause of CNSHA, due to mutations in
Other enzyme deficiencies causing CNSHA are Triose phosphate isomerase (TPI) deficiency, associated with neuromuscular disorders, mental retardation and frequent infections, Hexokinase deficiency (HK), affecting also platelet metabolism, phosphofructokinase (PFK) deficiency, 2,3-bisphosphoglycerate mutase (BPGM) deficiency and Glutathione synthetase (GS) deficiency (reviewed in [17,40,41]). Although the incidence of these diseases can be high (ie. TPI is considered as a frequent enzymopathy affecting 0.1% for caucasian populations and even 4.6% for black populations), they are considered rare or very rare diseases, because only few cases (~25 patients in the case of TPI) are diagnosed due to the severity of the clinical manifestations. No gene therapy approaches have been addressed up to now to treat these enzymopaties. However, due to their common characteristics, strategies developed in the other enzyme deficiencies could be applied directly to the treatment of all of them.
3. Optimization of vectors for the gene therapy of metabolic erythroid diseases: Erythroid specific expression vectors
The introduction of a cDNA, encoding for the correct version of the target mutated gene into patient cells using retroviral vectors has been successful for several inherited diseases. The initial integrative vectors for gene therapy design and used in clinical trials were based on Gamma(γ)-retroviral vectors in which the transgene expression was driven by the viral LTR promoter. γ-retroviruses preferably integrate in regions adjacent to the transcription initiation site . The expression of the transgene is promoted by the viral LTR, which drives a high expression that can affect gene regulation of the surrounding genes. Although a high efficiency of transduction and therapeutic effects have been described with these vectors in various monogenic disorders such as immunodeficiencies, adverse effects associated with insertional mutagenesis have also been observed. This has led to the development of the next generation of integrative vectors using self-inactivating-LTR lentiviral backbones. SIN-Lentiviral vectors tend to integrate in intergenic transcribing areas, which represent a safer integrative pattern than γ-retroviral vectors. Aditionally, the expression of the transgene is driven by internal promoters, offering a more physiological expression and a less genotoxic profile when using weak promoters . Current efforts to reduce the mutagenic potential of gene therapy vectors are focussed on not only the use of new viral backbones  but also on tissue-specific promoters to restrict the transgene expression to target cells  and insulators to confer position-independent expression . Additional regulatory DNA elements such as locus control regions (LCR), enhancers, or silencers have also been used to increase lineage specificity.
Gene therapy for RBC disorders requires, ideally, high erythroid-specific transgene expression in order to avoid side effects in progenitors or hematopoietic lineages other than the erythroid one. In inherited enzymophaties, the overexpression of metabolic enzymes in non-erythroid cells could provide these cells with a potential energetic advantage, with the consequent risk of disturbing the physiological generation of ATP in WBC. Also, the restriction of transcriptional activity to target cells with the use of either tissue-specific or physiologically regulated vectors decreasees the effect of the integrative vectors in the host genome. This goal is particularly important for erythrocyte metabolic deficiencies, as all the affected enzymes are highly regulated and connected with central metabolic pathways. Indeed, an expression limited to the erythroid progeny would reduce the genotoxic risk, as RBC become transcriptionally inactive during differentiation, and finally extrude their nucleus. To study tissue-specific gene therapy strategies for RBC diseases, hemoglobinophaties have been the most widely used.
Erythroid regulatory elements have been extensively used to manage targeted expression to RBC using reporter genes (Table 2). The Locus Control Regions (LCR), defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number-dependent manner at ectopic chromatin sites are commonly used. The components of the LCR normally colocalize to sites of DNase I hypersensitivity (HS) in the chromatin of expressing cells. Individual HS are composed of arrays of multiple ubiquitous and lineage-specific transcription factor-binding sites. In early experiments performed with retroviral backbones, the group of Ferrari developed an erythroid-specific vector by the replacement of the constitutive retroviral enhancer in the U3 region of the 3’ LTR with the HS2 autoregulatory enhancer of the erythroid GATA-1 transcription factor gene. The expression of this vector was restricted to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells [47,48]. Later, they showed that the addition of the HS1 enhancer to HS2, both from the GATA-1 gene, within the LTR of the retroviral vector significantly improved the expression of the reporter gene. Another enhancer element that has been used to achieve erythroid-specific expression is HS40, located upstream of the ζ-globin gene, since it is able to enhance the activity of heterologous promoters in a tissue-specific manner . It has been shown to be genetically stable in MMLV vectors and enhances expression comparable to that of a single -globin gene , although HS40 lacks some of the properties of the LCR, like position independence  or copy number dependence .
An additional improvement to provide safer vectors for RBC gene therapy was provided by the use of insulators elements, which have been shown to reduce position effects in transgenic animals . Insulators are genomic elements that can shelter genes from their surrounding chromosomal environment, by either blocking the action of a distal enhancer on a promoter [60,61], or by acting as barriers that protect the gene from the silencing effect of heterochromatin . The most well studied element is the chicken hypersensitive site 4 (cHS4), an insulator sequence of the chicken -like globin cluster. Studies performed by Chung et al with the γ-globin promoter and the neo reporter gene on selected cells lines, demonstrated the ability of cHS4 to insulate the expression cassette from the effects of a strong -globin LCR element  and therefore reducing its genotoxicity. Experiments from Arumugam et al showed a two-fold reduction in transforming activity with insulated LCR-containing lentiviral vectors comparing with vectors lacking the cHS4 element .
|HS2 GATA-1 enhancer within the LTR||ΔLNGFR and NeoR / EGFP||SFCM retroviral vector|||
|HS1 to HS2 GATA-1 enhancer within the LTR||EGFP and hΔLNGFR||SFCM retroviral vector|||
|Ankyrin-1 and α-spectrin promoters combined or not with HS40, GATA-1, ARE and intron 8 enhancers||EGFP||HIV-1 based vectors|||
|α-globin HS40 enhancer and Ankyrin-1 promoter||GFP / ||HIV-1 based vectors|||
|IHK, IHβp and HS3βp chimeric enhancers/promoters||hβ-globin cDNA||Sleeping beauty transposon|||
|HSFE and β-globin promoter||hβ-globin cDNA||MSCV retroviral vector|||
|LCR and β-globin promoter||hβ-globin cDNA or EGFP||HIV-1 based vectors||[56,57]|
|β-globin and θ-globin promoters combined or not with HS40, GATA-1, ARE and intron 8 enhancers||EGFP||HIV-1 based vectors|||
|LCR HS4, HS3, HS2, β-globin promoter and truncated β-globin intron 2||EGFP||HIV-1 based vectors|||
|LCR, cHS4 and β-globin promoter||hβ-globin cDNA||HIV-1 based vectors|||
|β-globin promoter, LCR HS2, HS3, HS4||hβ-globin cDNA||AAV2|||
Tissue-specific expression using alternative human promoters can be convenient or more efficient for some diseases, but driving the expression of the therapeutic genes using own promoters is still the most physiological approach to reduce the genotoxic risk of integrating gene vectors . The use of physiologically regulated vectors has been limited mainly because the promoter and the enhancer elements have to be obtained from the affected genes and they are often too large to be included in a lentiviral backbone, and also because the gene expression pattern depends partially on chromatin positioning . -globin LCR has been widely used when attempting to solve this limitation. The -globin LCR consists of 5 HS regions located upstream of the entire cluster of human -like globin genes, each containing a high density of erythroid-specific and ubiquitous transcription binding elements . Much of the transcriptional activity of the -globin LCR resides in HS2 and HS3 sites, but site 4 is important in adult globin expression . Previous studies
Other approaches to achieve consistent long-term expression of a transgene have been based on the use of HSFE element, an erythroid-specific chromatin remodelling element derived from the human β-globin LCR which contains binding sites for the erythroid-specific factors NF-E2, GATA-1, EKLF and the ubiquitous factor Sp-1, all of which are necessary to establish a hypersensitive chromatin domain. Work by Nemeth
Gene therapy for RBC metabolic diseases can also benefit from the new technologies based on the modification in mRNA stability or translation efficacy of the transgenes. The use of the post-transcriptional regulatory element (Wpre) from the woodchuck hepatitis virus (WHV) has significantly increased transgene expression in target cells [64,65], even in HSC  by stabilization of mRNA at post-transcriptional level. However, it may raise safety concerns, since it contains a truncated form of the WHV
In addition, the erythroid-specificity of short segments of the -globin LCR element has been documented in adeno-associated virus 2 (AAV2) system. Their efficacy to mediate an erythroid-restricted expression has been proved by Tan
In addition, the efficacy of some of these erythroid-specific elements and promoters has also been tested in non-viral vectors, such as transposons. Zhu et al, for instance, studied several hybrid promoters driving the expression of the human -globin gene using the sleeping beauty transposon (SB-Tn). They combined several erythroid elements to develop different chimeric promoters. Their results indicated that the ankyrin-1 minimum promoter was stronger than -globin’s, and the hALAS I8 enhancer (IH) was significantly more powerful that HS3 core element from -LCR and -globin promoter . SB-Tn system is a promising non-viral vector for efficient genomic insertion, even with erythroid-specificity. However, its efficiency for delivering transgenes into HSCs is still much lower than other engineering viral vectors.
4. Overcoming conventional gene therapy pitfalls: gene editing in induced plutipotent stem cells
4.1. Human induced pluripotent stem cells and reprogramming platforms
Since Yamanaka et al first reported the generation of mouse induced Pluripotent Stem Cells (iPSC) in 2006 by the ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and cMyc)  and one year later in human cells together with Thompson’s group [75,76], many laboratories around the world have been able to reprogram a large range of somatic cells into pluripotent stem cells, from neural stem cells  to terminally differentiated B-lymphocytes . The reproducibility and potentiallity (unlimited self-renewal and ability to differentiate into any cell type) of this technology has made the iPSC field to advance very rapidly. The human iPSC (hiPSC) technology brings together all the potential of hESC in terms of pluripotency without any ethical issue and the immunotolerance of the autologous cell treatment. Therefore, hiPSC technology is one of the most promising fields for future therapies for many human diseases. Safer reprogramming approaches have been designed and many patient specific hiPSC have been generated both to model human diseases and to correct by gene therapy approaches. Depending on the cell type to be reprogrammed, the number of factors used could be reduced and, what is more important, oncogenes or tumor related proteins included in the reprogramming cocktail, like
iPSC technology makes feasible the availability of patient specific cells to study the biology of the disease and develop advanced tools to cure the phenotype and could potentially be used as a therapeutic option (Figure 1). Focussing on metabolic diseases, the first reported metabolic disease patient specific hiPSC line was obtained one year after the first generation of hiPSCs. It was from a 42-year old female that suffered from Type I Diabetes mellitus  and it showed no differences compared to a wild type hiPSC line in terms of pluripotency. Next report in which liver metabolic disease patient samples were reprogrammed was carried out by the group of Ludovic Vallier , and showed the potential of this kind of approaches for disease modelling and new drug discovery. They reprogrammed fibroblast obtained from α-1 Antitrypsin deficiency (
The future therapeutic application of hiPSC will not only require non-integrative reprogramming system, but also a more precise gene correction. During last years, the cooperation between hiPSC technology and gene editing is being explored. Human iPSC technology has led to the opportunity to control the integration of viral vectors at a clonal level. As we have mentioned before, the analysis of lentiviral integration sites in β-thalassemia hiPSC allowed the identification of corrected hiPSC clones expressing β-globin transgene from a safe genomic site (also called Safe harbour), a site in which integration does not disturb the expression of any neighbouring genes during their erythroid differentiation . The therapeutic use of patient-specific hiPSC emerges then from the combination of gene and cell therapy. From this new research field,future gene therapy protocols will emerge.
4.2. Gene editing based on homologous recombination
Gene editing is a process in which a DNA sequence is introduced into a specific locus or a chromosomal sequence is replaced. This site-specific precise introduction requires an accurate recognition mechanism of the target site on the genome. Under normal conditions, the maintenance of the integrity of the genome requires that the cells repair DNA damage with high fidelity. One of the most harmful DNA damage is the generation of double-strand breaks (DSB). DSB are often resolved by non-homologous end joining (NHEJ), which joins the two ends of the DSB. However this DNA repair mechanism could introduce mutations. On the contrary, homologous recombination (HR) is a truly accurate DNA repair mechanism because it is basically a “copy and paste” mechanism. This process uses an undamaged homologous segment of DNA that can be exogenously provided as a template to copy the information across the DSB. The fidelity of HR gives us the specificity and accuracy that gene editing requires.
The natural HR process has been adapted by researchers to get the desirable addition of an exogenous cassette into the targeted locus. This techniques have been widely use for the generation of knock-out and knock-in transgenic animals . To correct or insert and express a transgene by HR we can consider three different strategies: i) Gene correction, a base or some bases can be substituted from the original strand using an homologous sequence where this base or bases are modified; it is the way to introduce/repair point or small mutations; ii) Safe harbour integration, a complete expression cassette (promoter, transgene and regulatory signals) is inserted in a safe place of the genome, without altering the expression of the surrounding genes and without being silenced by epigenetic mechanisms; this is the case for
Gene editing process can be separated in two different steps, generation of DSB and HR. The efficacy of gene editing in human cells depends on the generation of DSB at the specific target site and on the DNA repair mechanism that the cell uses to resolve the DSB. Unfortunately, NHEJ is the dominant pathway to solve these DNA lesions in human cells. Additionally, HR varies in different cell types and requires transit through S-G2 phase of the cell cycle . These limitations make gene editing in human cells difficult to achieve. However, different approaches are being used to improve gene editing by HR, like increasing the length of the DNA sequences homologous to target site (homology arms) , the use of adeno-associated vectors , the improvement of selection methods for edited cells or the stimulation of HR by inducing DSB using DNA nucleases.
Recently, engineered DNA nucleases have been developed to specifically induce DSB at a unique and defined sequence in the cell genome. These proteins are formed by a nuclease domain and a DNA binding domain whose sequence specificity can be engineered. The most widely used DNA nucleases are Zinc finger nucleases (ZFN), homing meganucleases (MN) and transcription activator-like effector nucleases (TALEN). They identify a potentially unique sequence in the genome and generate DSBs in the desired genomic site, aiming to promote the repair of the DSB by the cell machinery and, ideally by HR. The DNA binding domain of a ZFNs is derived from zinc-finger proteins and is linked to the nuclease domain of the restriction enzyme Fok-I. DNA-biding domain is a tandem repeat of Cys2His2 zinc fingers, each of which recognizes three nucleotides. ZFNs work as pairs of two monomers of ZFN, one in reverse orientation. This ZFN dimer can be designed to bind to genomic sequences of 18-36 nucleotides long. TALENs have a similar structure to ZFNs, but the DNA-binding domain comes from transcription activator-like effector proteins. The DNA-binding domain in TALENs is a tandem array of amino acid repeats. Each of these units is able to bind to one of the four possible nucleotides and this makes that the DNA binding domain can be designed to recognize any desired genomic sequence. TALENs also cleave as dimers. Contrary to these synthetic DNA-nucleases, MNs are a subset of homing endonucleases which recognize a DNA sequence from 14 to 40 nucleotides. Current MNs have been engineered from natural homing endonucleases to increase the number of target DNA sequences.
ZNFs have been widely used for gene editing in hESC and hiPSC. In 2007, Dr. Naldini’s laboratory showed the insertion of GFP into the CCR5 safe harbour in human stem cells (HSC and hESC) after inducing HR by ZFN expression. The CCR5-ZFN and donor DNAs were delivered into hESC by intergrative deficient lentiviruses. More interestingly, targeted hESC were able to differentiate into neurons keeping GFP expression . Soon, the proof of principle for the clinical application of ZFN-mediated gene editing was tested in hiPSC from patients affected by different genetic diseases. The first pre-clinical use of ZFN for gene therapy of a metabolic disease was performed by Yusa
One of the major limitations of ZFN is the generation of “off-target” DSB, due to unspecific sequence recognition. Different studies have highlighted this as a possible limitation in the clinical use of ZFN-mediated HR [100,101]. Recent works have explored the potential of other types of DNA-nucleases in order to prevent the “off-target” cleave limitations of the ZFN, being TALEN and MN the most promising ones. The feasibility of TALEN to mediate HR in hESC and hiPSC was assess by Jaenisch’s group when they designed TALEN targeting the
5. Complementary developments for the application of gene therapy to erythroid metabolic diseases
In vivotransduction using engineered envelopes
Another challenge for the clinical application of gene therapy relates to vector targeting. To achieve successful gene therapy, the appropriate gene must be delivered to target cells and specifically expressed in them, without harming non-targeted cells. The most common and easiest way to target specific cells is by
Many attempts have been made to develop targeted transduction systems using retroviral and lentiviral vectors by altering the envelope glycoprotein (Env), which is responsible for the binding of the virus to the cell surface receptors and for mediating viral entry into the cell. The plasticity of the surface domain of Env allows insertion of ligands, peptides or single-chain antibodies that can direct the vectors to specific cell types . However, this type of manipulation negatively affects the fusion domain of Env, resulting in low viral titers. To overcome this downside, a method to engineer lentiviral vectors has been developed. These vectors transduce specific cell types by breaking up the binding and fusion functions of the envelope protein into two distinct proteins . Instead of pseudotyping lentiviral vectors with a modified viral envelope protein, these lentiviral vectors co-display a targeting antibody and a fusogenic molecule on the same viral vector surface. Based on molecular recognition, the targeting antibody should direct lentiviral vectors to the specific cell type. The binding between the antibody and the corresponding cellular antigen should induce endocytosis resulting in the transport of lentiviral vectors into the endosomal compartment. Once inside the endosome, the fusogenic molecule should undergo a conformational change in response to the decrease in pH, thereby releasing the viral core into the cytosol . The use of fusion domain of the binding defective Sindbis virus glycoprotein together with an anti-CD20 antibody has been shown to mediate the targeted transduction of lentiviral vectors to CD20-expressing B cells .
However, two major challenges for
In vitroproduction of mature erythrocytes
Periodical blood transfusion is the previous to the last therapeutic option for severe cases of CNSHA patients. However, this clinical practice involves also adverse effects related to the immuneresponse against minor erythrocyte antigens which makes the patients refractory to additional blood transfusions in the long run. The availability of genetically corrected patient-specific iPSC would allow the possibility of generating disease free erythrocytes ready for transfusion, avoiding the adverse immune effects.
There have been numerous attempts to produce RBC
Erythroid metabolic diseases are well defined and well known diseases which main symptom is CNSHA. As they are monogenic diseases that can be cured by allogeneic bone marrow transplantation, they are very good candidates to be treated by gene therapy. However, the low number of patients with poor prognosis requiring BM transplantation and the absence of an apparent selective advantage of the corrected cells over the diseased ones have made their approach for gene therapy less attractive than other erythropaties. Up to now, no gene therapy clinical trial for erythroid metabolic diseseases has been accomplished. Gene therapy attempts in animal models have been applied to G6PD and PKD with successful results, emphasizing the usefulness of a gene therapy approach for these diseases. Although adverse effects due to ectopic expression of the metabolic enzyme have not been observed, an erythroid specific expression is preferred. Many developments have been made for the specific expression of globin genes that could be adapted to vectors developed for the discussed erythroid metabolic diseases. Similarly, any attempt directed to the improvement of HSC transduction, including the possibility of
A1ATD-1 antitripsin deficiency
AAV Adeno-associated virus
BM Bone marrow
BPGM 2,3-bisphosphoglycerate mutase
CNSHA Chronic non spherocytic hemaolotyc anemia
DSB Double strand breaks
Env Viral envelope
FH familiar hypercholesterolemia
G6PD Glucose-6-phopahate dehydrogenase
GPI Glucose phosphate isomerase
GS Glutathione synthetase
hESC human embryonic stem cell
hIF1 hypoxia-inducible factor-1
hiPSC Human induced pluripotent stem cell
HR Homologous recombination
HS DNase I hypersensitive sites
HSC Hematopoietic stem cell
iPSC Induced pluripotent stem cell
LCR Locus control region
LTR Long terminal repeats
MN homing meganuclease
NHEJ non-homologous end joining
SIN-LV Self-inactivated lentiviral vector
TALEN transcription activator-like effector nuclease
TPI Triose phosphate isomerase
ZFN zinc finger nuclease
The authors thank L. Cerrato, M.A. Martín and I. Orman for their technical assistance. We would also like to thank Dr. J. Bueren for careful reading and suggestion of the manuscript. M.G.G. was partially supported by a short-term fellowship from the European Molecular Biology Organization (EMBO ASTF 188.00-2010). Z.G. is a fellowship of the PhD program of the Departamento de Educación, Universidades e Investigación del Gobierno Vasco. This work was funded by grants from the Ministerio de Economía y Competitividad (SAF2011-30526-C02-01), Fondo de Investigaciones Sanitarias (RD06/0010/0015) and the PERSIST European project. The authors also thank the Fundación Botín for promoting translational research at the Hematopoiesis and Gene Therapy Division-CIEMAT/CIBERER.