Towards More Successful Gene Therapy Clinical Trials for -Thalassemia

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1 Send Orders for Reprints to Current Molecular Medicine 2013, 13, Towards More Successful Gene Therapy Clinical Trials for -Thalassemia E. Drakopoulou 1,2, E. Papanikolaou 1,2, M. Georgomanoli 1,2 and N.P. Anagnou *,1,2 1 Laboratory of Cell and Gene Therapy, Centre for Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece 2 Laboratory of Biology, University of Athens School of Medicine, Athens, Greece Abstract: -thalassemias constitute hereditary blood disorders characterized by reduced or absence of -globin synthesis resulting in mild to severe anemia, depending on the genotype. More than 200 mutations in the -globin gene are responsible for their specific features leading to a very heterogeneous phenotype. Current therapies for -thalassemia include blood transfusions, usually along with iron chelation and in selected cases with bone marrow transplantation (BMT) of HLA-matched hematopoietic stem cells (HSCs). However, these approaches are limited by factors, such as iron overload and donor availability, respectively. Since 2000, when globin lentiviral vectors (LVs) were first successfully tested for transfer efficiency of the therapeutic transgene, which led to disease amelioration in murine models, attention was drawn towards the improvement of such vectors for -thalassemia gene therapy. Constantly improving vector design and efficient HSC manipulation led recently to the first successful clinical trial in France, which further proved that this genetic approach can be curative. Furthermore, improved new efficient vectors and methods to safely monitor integration sites and therapeutic transgene position effects, promise a new era for -thalassemia gene therapy, with more and safer clinical trials yet to come. Keywords: -thalassemia, clinical trial, CD34 + cells, gene therapy, -globin, hematopoietic stem cells (HSCs), lentiviral vectors (LVs). 1. INTRODUCTION -Thalassemia syndromes represent a group of hereditary, monogenic, blood disorders inherited in an autosomal recessive manner. There are more than 200 different mutations affecting the human -globin gene, which are responsible for the significant heterogeneity of the thalassemic phenotype [1], leading either to reduction or absence of -chain synthesis. As a result, -globin chain molecules are produced in excess and precipitate in red blood precursors, leading to impaired erythrocyte maturation, mechanical damage and ultimately to apoptosis [2]. Current therapies for -thalassemia include mainly blood transfusions, usually together with life-long iron chelation and in specific cases combined with hydroxyurea treatment, which induces fetal hemoglobin (HbF) production leading to decrease of the unpaired -globin chains. The only available curative therapy so far, is the allogeneic hematopoietic stem cell (HSC) transplantation of human leukocyte antigen (HLA)- matched sibling donors [3]; however, this is restricted due to limited matched-related donors and the need for long-term immunosuppression to prevent or delay Address correspondence to this author at the Laboratory of Cell and Gene Therapy, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens , Greece; Tel: ; , ; Fax: ; anagnou@med.uoa.gr graft-versus-host disease (GVHD). Therefore, a molecular approach, such as gene therapy, seems quite promising, as it overcomes a significant number of the afore-mentioned limitations and exploits the beneficial effects that originate from the re-infusion of the corrected cells. For a successful -thalassemia gene therapy, the need for improved vector design exhibiting safety, lacking genotoxicity and leading to higher numbers of corrected HSC yields, is imperative. This paper summarizes the current state of the field, including all the in vitro studies using globin lentiviral vectors (LVs) and human thalassemic CD34 + cells, the first successful clinical trial in France by the team of Philippe Leboulch, as well the strategies for improved gene transfer and effective HSC manipulation, mainly ex vivo, which will eventually lead to more successful clinical trials for -thalassemia gene therapy. Although, a lot of pioneering experiments, which significantly contributed to our current knowledge and success in gene therapy for -thalassemia, were carried out in mice, they are not described in full here. Lastly, this review highlights the newly developed methods for vector safety and integration site analysis, which point towards a safe and controlled gene therapy outcome /13 $ Bentham Science Publishers

2 2 Current Molecular Medicine, 2013, Vol. 13, No. 8 Drakopoulou et al. 2. LENTIVIRAL VECTOR DESIGN FOR GLOBIN GENE TRANSFER 2.1. Leaving Oncoretroviruses Behind A critical step for a successful -thalassemia gene therapy is the identification and development of the most effective vector system for globin gene transfer. More than two decades ago, investigators utilized -retroviral vectors to transfer -globin genes into murine HSCs [4-6]; however the results of these studies were not satisfactory since the gene transfer efficiency was poor and expression of the transgene reached only up to 2% of the endogenous RNA levels. Even when Novak et al. [7] incorporated the newly identified DNA-enhancer elements from the -globin locus control region (LCR), shown to drive high-level expression of globin genes [8, 9], they failed to improve expression, as again poor vector production and genetic instability of the viral vector genome were observed. Pioneering experiments on retroviral vectors from Leboulch et al. [10] and Sadelain et al. [11] indicated that vector instability might be caused by splicing of the retroviral RNA genome, a consequence of the presence of cryptic splice sites within the incorporated human genomic sequences. Apparently, lentiviral vectors were less prone to splicing due to the expression of the Rev gene that allows the production of full-length unspliced viral RNA genomes [12]. Therefore, the use of a lentiviral backbone in globin vectors and Rev expression in a lentiviral packaging cell line, could minimize splicing of globin vectors containing large genomic fragments, thus leading to vector stability. The significant breakthrough in the field of globin gene therapy took place when May et al. [13] and Pawliuk et al. [14] constructed a human immunodeficiency virus (HIV)-based vector combining the -globin gene along with its LCR elements. This vector was able to transmit the significantly larger LCR and -globin configuration without rearrangement, and be produced in high titers, sufficient to ameliorate the disease in -thalassemic mice. Following this approach, several groups working also on hemoglobinopathies employed -globin LVs in their studies, leading to correction of murine -thalassemia [15]. Furthermore, the observation that compound thalassemic patients with the syndrome of hereditary persistence of fetal hemoglobin (HPFH) typically have less anemia, milder clinical symptoms and are often transfusion-independent, drew the attention towards constructing -globin vectors [16-18]. Derek Persons and colleagues were the first to design and test such vectors; the latter contained the extended -globin LCR and succeeded in demonstrating significant correction of the thalassemic phenotype [18]. However, the inconsistent globin expression by these vectors due to chromosomal position effects, eventually led to the use of chromatin insulators [19], which represent DNA elements capable to shield the therapeutic gene from the negative and/or positive effects of the surrounding DNA Self-Inactivating (SIN) Vectors, Insulators and Insertional Mutagenesis Integration of viral DNA constitutes the most basic step in the retroviral life cycle, since the viral genomes are permanently fixed as pro-viruses into the DNA of the host. During this process, the retroviral DNA is present in a large complex with a subset of retroviral and cellular proteins referred to as the pre integration complex (PIC). For -retroviruses, such as Murine Leukemia Virus (MLV), uncoating, DNA synthesis, and formation of PIC occur at the same rate, both in dividing and non-dividing cells, but integration fails to occur. During mitosis, however, due to disassembly of the nuclear membrane, the chromosomes are rendered accessible to the virus and integration occurs, suggesting that infection by oncoretroviruses, such as MLV, requires cell division [20]. This does not seem to be the case for lentiviruses, as it has been extensively documented that they are able to infect both dividing and non-dividing cells, due to their capacity to cross the nuclear membrane of interphasic cells. This represents a crucial asset for genetically modifying tissues, especially those considered as the main potential targets of gene therapy, such as the brain, muscle, liver and the hematopoietic system [21]. As with any new therapeutic approach, gene transfer using retroviral vectors may also induce novel kinds of side effects due to integration of the viral DNA within or near a proto-oncogene, a phenomenon called insertional mutagenesis. The report of lymphoproliferation due to insertional activation of the LMO2 gene following gene therapy for X1-linked severe combined immunodeficiency (SCID-X1) [22], as well as the detection of unexpected increase in the proportion of gene-corrected cells carrying retroviral insertion into the MDS1-EVI1 region in both nonhuman primates [23] and in patients in the German X-linked chronic granulomatous disease (X-CGD) clinical trial [24], has led to a re-evaluation of the mechanisms of insertional mutagenesis. While insertional mutagenesis using replicationdefective vectors is possible [25], such risks were originally underestimated [26], primarily based on the assumption that proviral integration into the genome is random. However, with the new and readily accessible human genome sequence data, mapping studies of retroviral integration sites in cell lines have uncovered semi-random integration patterns, using wild-type HIV, HIV-derived, or MLV-derived vectors [27-30]. Moreover, the integration patterns have only been recently investigated in HSCs [31-33] and have shown that while MLV integrants were located predominantly around transcription start sites, HIV integrants strongly favoured transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration, as well as distinct safety implications for oncoretroviral versus lentiviral vectors and imply a correlation between the stage of chromatin condensation and transcription [34]. The basis for these preferences is unknown, but they may reflect interaction of the PIC with specific proteins or

3 Thalassemia Gene Therapy Clinical Trials Current Molecular Medicine, 2013, Vol. 13, No. 8 3 with specific DNA sequences or structures that are associated with transcription. While copy number can be largely controlled with retroviral technology, the choice of the insertion site within the genome cannot be easily achieved with the current technology. This establishes a considerable risk for insertional up-regulation, silencing or disruption of cellular genes, a subset of which (>200) may act as proto-oncogenes or belong to the considerably smaller group of tumour suppressor genes. However, oncogene de-regulation requires a specific epigenetic and systematic context to foster tumour development. Therefore, mutagenesis is not necessarily predicting oncogenesis. However, the onset of leukemia in the SCID-X1 trial has been considered as the most severe side-effect of gene transfer, urging the gene therapy society and the monitoring organisms to create new methods in order to overcome the respective obstacle. The first strategy adopted was the construction of selfinactivating (SIN) vectors. This strategy essentially includes deletion of the enhancer sequences located within the U3 segment of the long terminal repeats (LTRs) of retroviruses. By employing the SIN configuration combined with the use of tissue-specific regulatory elements to drive expression of the transgene, activation of adjacent cellular genes becomes less likely. The second strategy involved the use of insulators. Transcriptional insulators are DNA elements that set boundaries on the actions of enhancer and silencer elements and thereby organize the eukaryotic genome into regulatory domains [35]. Thus, when an insulator is incorporated in the U3 region of the LTR, thereby flanking the transgene at both sides, the expression of the transgene is greatly protected, regardless of the integration site (position effect). At the same time, it provides extra safety, concerning the activation of adjacent proto-oncogenes. Arumugam et al. [19] improved globin gene expression by incorporating into the globin vector the 1.2 kb DNA element from the chicken hypersensitive site 4 (chs4) -globin locus; however, the vector s practical use was hampered, as the incorporation of the above element, significantly reduced the titer. The latter was rescued when Hanawa et al. [36] included only the 0.25 kb core element of chs4 and managed to obtain significantly increased transgene expression from an internal promoter, due to improved transcriptional termination. This element, in an orientation-dependent manner, demonstrated also some barrier activity, reducing variability of expression due to position effects, indicating that some insulation can be achieved with this core element. Furthermore, Lisowski and Sadelain [37] showed that addition of the HS1 element enhances the therapeutic efficacy of the globin gene transfer in murine -thalassemia, compared to the HS2-HS3-HS4 cassette, leading to even higher globin expression, along with lower vector copy numbers Inhibiting HbF Repressors The implication of the expression of -globin in thalassemia has long been recognized [1]. Firstly, the observation that thalassemic patients with HPFH syndromes show milder clinical symptoms of the disease and secondly, the great variability in -globin persistence in adults, led to efforts towards mapping modifier loci in these individuals, focusing on patients that do not have a mutation within the -globin locus on chromosome 11 [38]. These studies employed both murine and human cells and uncovered the role of BCL11A located on chromosome 2, on hemoglobin switching and inevitably on -globin expression [39]. More specifically, the pioneering work, of Sankaran et al. showed that low HbF expression in adult erythroid cells is associated with increased BCL11A expression. Moreover, knocking down BCL11A, by employing small interfering RNA (sirna) technology, led to the elevation of HbF levels, postulating that directed downregulation of BCL11A in thalassemic patients could ameliorate the severity of the disease through the re-activation of HbF in adult erythroid cells. Nearly a year later, two independent studies shed more light to HbF repression by BCL11A. Particularly, Borg et al. [40] with the aim to define the HPFH cause in a large Maltese family used a genome-wide singlenucleotide polymorphisms (SNPs) scan and found that KLF1 binds to human BCL11A in vivo and that its levels in affected individuals are directly proportional to BCL11A and inversely proportional to HbF levels. The above data indicated that BCL11A might regulate HbF expression via KLF1. A similar paper by Zhou et al. [41] published at the same time, verified the above, as it demonstrated that knockdown of KLF1 leads to reduced BCL11A expression in both murine and human adult erythroid cells, which in turn results in increased -globin expression with a concomitant decrease in -globin levels. Therefore, these data imply that KLF1, primarily in the adult life, is directly activating -globin and indirectly repressing -globin expression via the activation of BCL11A. The latter was shown to involve long-range interactions and cooperation with SOX6 [42]. The above results suggest that there is great therapeutic potential for interfering with KLF1 and/or BCL11A expression in the context of -thalassemia treatment. Construction of -globin LVs that combine the therapeutic gene, along with small hairpin RNA (shrna) that downregulate these HbF transcriptional repressors would, in theory, result in robust HbF expression. Our group is currently constructing such vectors. Recently, the Orkin group inactivated BCL11A in SCD transgenic mice and demonstrated correction of the hematologic and pathologic defects associated with SCD through a highlevel pancellular induction of HbF [43]. The above experiments verified that BCL11A can be a therapeutic target for -thalassemia gene therapy. Apart from the above -globin repressors, van Dijk et al. [44] showed that the chromatin factor Friend of

4 4 Current Molecular Medicine, 2013, Vol. 13, No. 8 Drakopoulou et al. Prmt1 (FOP) is also a critical modulator of HbF expression, since knockdown of the specific factor in adult erythroid precursors led to significantly elevated HbF levels. The above HbF induction was shown to occur independently of BCL11A, as BCL11A levels remained unaffected following FOP knockdown, but it involved modulation of SOX6, whose expression was reduced upon the specific experimental conditions [44]. Thus, utilization of shrnas against FOP in therapeutic LVs, alone or in combination with shrna against BCL11A and/or KLF1 could eventually lead to higher HbF expression levels. 3. IN VITRO STUDIES USING HUMAN THALAS- SEMIC CD34 + CELLS Both -globin and -globin vectors have been used in correcting thalassemic erythropoiesis in erythroid cultures. These studies are summarised in Fig. (1) and Table 1. The first study testing a -globin LV flanked by a chromatin insulator in human thalassemic CD34 + cells was carried out by Puthenveetil and co-workers [45] using transfusion-dependent human thalassemia major cells, where they demonstrated that the human -globin transgene was expressed at normal levels in erythroid cells produced in vitro. An in vivo significant outcome was also demonstrated, as these corrected progenitors were able to establish normal erythropoiesis when transplanted in immunodeficient mice. In addition, Roselli et al. [46] using the GLOBE vector, reported successful correction of thalassemia major in human cells, by achieving high transduction efficiency, restoration of hemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The same group, quite recently demonstrated that incorporation of the 200 bp erythroid-specific enhancer GATA1-HS2 into GLOBE, allows persistent and position-independent expression of -globin by reducing expression variegation and epigenetic silencing [47]. The improved globin vector, designated as G-GLOBE, managed to correct murine thalassemia with a lower vector copy number (VCV) than that of GLOBE (VCN = 1 vs VCN = 1.7). Initial experiments using human CD34 + cells from cord blood, suggest that the GATA1-HS2 element maintains high transgene expression in erythroid progeny of human HSCs, both in vitro and in vivo. Also, recently Breda et al. [48] developed a novel -globin lentiviral vector that carries the 200 bp erythroid-specific ankyrin 5 hypersensitive barrier insulator element and used it to transduce erythroid progenitor and CD34 + cells, originating from peripheral blood (PB) of thalassemic patients. AnkT9W was shown to contribute in increasing HbA synthesis from 0% to 62% in CD34 + derived cells (VCN = 0.92) or to 73% in erythroid progenitors-derived cells (VCN = 0.97). The group concluded that AnkT9W increased the synthesis of globin in most thalassemic specimens to levels comparable to carriers and control samples. However, 0 - thalassemic erythroid cells required higher VCN to reach therapeutic levels. Furthermore, the Persons group by employing the -globin vector V5m3-400 in an in vitro model of human erythropoiesis, showed recently that both LV-mediated -globin gene addition and genetic reactivation of endogenous -globin genes, either via a synthetic zinc finger transcription factor designed to interact with the -globin promoter, or via BCL11A inhibition, have the potential to provide therapeutic HbF levels to patients with -globin deficiency [49, 50]. Finally, our group, generated a novel insulated SIN LV, designated as GGHI (Gamma-globin HPFH insulated), containing the A -globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer [51]. This vector managed to produce efficient amounts of HbF, resulting in phenotypic correction in erythroid cultures of CD34 + HSCs isolated from PB or bone marrow (BM) of thalassemic patients [51]. It is noteworthy that in this specific vector design, the incorporation of the full length 1.2 kb chs4 in the U3 region and the SIN configuration had no apparent effect on viral titer, as it reached 2x10 8 TU/ml. GGHI is the first LCR-free LV that has demonstrated efficient correction of thalassemia in vitro. 4. THE FIRST SUCCESSFUL CLINICAL TRIAL The first human clinical trial for -thalassemia commenced in France on June 7, 2007 and employed LentiGlobin, a -globin LV with a variant of the human -globin gene that carries a single base change at amino acid position 87. The specific variant, which was initially developed as an anti-sickling agent [14] was used in this setting to distinguish vector-encoded from endogenous -globin. Cavazzana-Calvo and colleagues selected two -thalassemia patients for BM transplantation (BMT) of LV-transduced HSCs [52, 53]. The initial patient, a 28-year old male, experienced a period of prolonged aplasia due to the low number of HSCs engrafted, and therefore the need for administration of his untransduced cells, which were kept as a back-up, was imperative. As a consequence, neither the LV-transduced cells reached a significant number in PB, nor did the therapeutic hemoglobin levels, thus leading to an inconclusive outcome for the specific patient, who was designated as patient 0. The next patient in the study (designated as patient 1) was a 18-year old male, a compound heterozygote for -thalassemia and HbE (HbE/ o ), requiring 160 ml of packed erythrocytes/kg/year, since the age of three. In this trial, he received 4x10 6 CD34 + cells/kg [53]. Within the first few months, the levels of the genetically modified cells increased to 2%, while they reached 11% at 33 months post-transplant, a rise that was also observed in the levels of the therapeutic -globin variant, which was expressed in 10 20% of genetically modified HSCs. As a result, improved production and quality of red blood cells was observed. The above patient was last transfused on June 6, 2008 and for more than four years post BMT he remains transfusion-

5 Thalassemia Gene Therapy Clinical Trials Current Molecular Medicine, 2013, Vol. 13, No. 8 5 Fig. (1). Schematic representation of the proviral elements of the / -globin lentiviral vectors currently used or planned for phase I/II clinical trials for -thalassemia gene therapy. All vectors have been constructed using a lentiviral backbone driven by a CMV or HIV promoter located at the 5' LTR. Following reverse transcription, the 398 bp enhancer U3 deletion (light grey triangle) of the 3' LTR is transferred to the 5' LTR, rendering the vector self-inactivating. All / -globin expression cassettes are in a reverse orientation compared to the orientation of the lentiviral vector reverse transcription. The proviral elements of each lentiviral vector used in preclinical trials, are depicted in detail. BG-I vector is insulated with the full length 1.2 kb chs4 insulator, carries the human -globin gene linked to a -globin 3' enhancer element, the 254 bp core element of the -globin promoter, and extended fragments of HS2, HS3 and HS4 hypersensitive sites of the LCR element [19]. LentiGlobin vector encodes a mutated -globin gene at codon 87, carries a double copy of the core 250 bp element of the chs4 insulator, a 266 bp -globin 3' enhancer element and different regions of HS2, HS3 and HS4 hypersensitive sites of the LCR [52, 53]. Both BG-I and LentiGlobin vectors have a 372 bp deletion in the intervening sequence (IVS) 2 of the -globin gene. GLOBE vector is the smallest in length lentiviral vector pre-clinically assessed, carries a minimal -globin gene with a 562 bp deletion in the IVS 2 (represented as a dark grey triangle) regulated by a 265 bp -globin promoter and contains only the HS2 and HS3 elements of the LCR, whereas it is not insulated [15]. G-GLOBE vector, which was recently constructed, has the same elements with GLOBE, but carries also the GATA1-HS2 element in both LTRs (shown between dotted lines), so as to allow persistent and position-independent expression of the -globin transgene [47]. TNS9 vector has similar control elements as the LentiGlobin, but it is not insulated and has control elements of different sizes, as depicted [13]. Its therapeutic efficiency has been tested only in mouse thalassemia models, and has not been clinically assessed yet. Notwithstanding, the novel version of TNS9, designated as TNS (not shown) is scheduled to be clinically assessed. AnkT9W vector represents a novel globin vector, which carries the 200 bp erythroid-specific ankyrin insulator element and was recently shown to increase HbA synthesis to therapeutic levels in human erythroid progenitors cells from thalassemic patients [48]. V5m3-400 and GGHI are -globin vectors, carrying the human A -globin coding sequence, with a 713 bp deletion in the IVS 2 region (black triangle), as well as the -globin or the -globin 3' UTR, respectively. V5m3-400 LV vector is flanked by the 400 bp core element, expresses the A -globin gene under the control of a minimal 130 bp -globin promoter and the HS2, HS3, HS4 fragments of the LCR [49, 50]. GGHI vector is a LCR-free, fulllength insulated -globin vector, which expresses A -globin coding sequences under the control of an A -globin promoter, carrying the -117 HPFH activating point mutation, and flanked by the HPFH-2 and HS-40 enhancer elements [51]. Triangles of different shades indicate deletions of different sizes as shown in figure; asterisks indicate point mutation; RRE, Rev Response Element; HS2, HS3, HS4, DNAase Hypersensitive sites 2, 3, 4 of the -globin LCR, the length of which is also indicated in each case; cppt, central Polypurine Tract; cppt/flap, DNA flap at the center of the viral cdna in between the central polypurine tract (cppt) and the Central Termination Sequence (CTS); LTR, Long Terminal Repeat; 3' Enh, -globin 3' enhancer element;, packaging signal; p, -globin gene promoter; A p, A -globin gene promoter; chs4, insulator from the chicken -like globin gene cluster; Ank, Ankyrin insulator; SD, Splice Donor site; SA, Splice Acceptor site; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element; UTR, Untranslated Region; HPFH-2, Human Persistence of Fetal Hemoglobin-2 enhancer; HS-40, DNAase Hypersensitive Site-40 enhancer element of the human -like globin gene cluster; gag, 2 stop codons in gag; The length of the lentiviral vectors in kb is indicated adjacent to each vector.

6 6 Current Molecular Medicine, 2013, Vol. 13, No. 8 Drakopoulou et al. Table 1. Summary of the Key Findings of Preclinical and Clinical Assessment of Different and -Globin Vectors Used for -Thalassemia Gene Therapy LV Preclinical Assessment Using CD34 + Cells Clinical Trial MFI % Hb + Cells HPLC % Apoptosis CD34 + Hospital/Preliminary Source * % MTE VCN/Cell Results or Current Control Thal /LV Control Thal /LV Control Thal/LV Control Thal /LV Status BG-I LentiGlobin GLOBE AnkT9W V5m3-400 GGHI BM (n=4) CB (ND) BM (n=30) PB n=19 PB and BM (ND or Thal) (n=3) BM (n=20) ± ± ± 6 60 ± 5 7.3% 75% 49 ± ± 429 (ND) 2076 ± 647 (ND) 3170 ± ± ± 7.2 (ND) 44.6 ± (ND) (ND) (GFPtransduced) (V5m3-400 transduced) (ND) 0.79 (ND) 0% (ND) 39.76% (ND) 1.4 ± 0.4% (mock) 27.5% 25.9% % 74.63% 15.4 ± 4.9% 35.4% 34.9% 33.5 ± 5.4 GpA+/ AnnV 18.1 ± 3.1 PI 2 ± ± ± (mock) CHC Cincinatti Pending Necker Paris 21 months post BMT Hb: 9-10 g dl -1 HSR-TIGET Milano Pending Saint Jude Memphis Pending * CD34 + cell source for each lentiviral vector. For BG-I assessment, bone marrrow (BM) samples of o / o, + / + and o / + thalassemia patients were used [19]; for LentiGlobin preclinical assessment, cord blood (CB) samples from normal donors (ND) were used, while thalassemic BM were used in its clinical trial [52, 53]; For GLOBE vector, BM samples from o / o, o / + and + / + thalassemia patients were used [15, 47]. AnkT9W was tested on erythroid progenitor (data not shown) and CD34 + cells from peripheral blood (PB) of o / o, o / + and + / + thalassemic patients [48]. V5m3-400 was tested on mobilized PB or BM from ND or thalassemia major donors [49, 50]. Lastly, GGHI vector was tested on BM samples from o / o, + / o or + / + patients [51]. For GLOBE, AnkT9W and GGHI assessment, thalassemic patients with o / o genotypes are designated as o, while patients with + / o and/or + / + genotypes are included in one group, designated as +. The number of patients involved in each trial is provided. Mean transduction efficiency (MTE) is presented as percentage per vector-positive cells, calculated at clonal level by PCR and primers against vector-specific sequences. Average Vector Copy Number (VCN) per cell determined by quantitative PCR or Southern blot analysis, using genomic DNA isolated from LV-transduced CD34 + cells. For BG-I vector a corrected value for gene transfer efficiency is provided. Mean Fluorescence Intensity (MFI) is measured by FACS and intracellular staining of Thal cells from erythroid cultures transduced with either -globin or -globin LVs and compared to control samples. The latter are either untransduced Thal cells or cells from ND, as indicated in brackets. Percentage of HbA or HbF positive fraction of transduced cells from erythroid cultures determined by FACS analysis, with the exception of V5m3-400 vector, for which calculation of globin LV-transduced and GFP-transduced cells was determined by densitometry. For all other LVs, control is either untransduced Thal cells or cells from ND, as indicated in bracket. High Performance Liquid Chromatography (HPLC) for -globin or -globin chains, as estimated from transduced cell lysates from erythroid liquid cultures and compared to control (Thal, ND or mock), as indicated in brackets. For BG-I, the percentage of chains is estimated by reverse phase HPLC; for GLOBE vector the ratio of / chains is provided, as estimated with reverse phase HPLC analysis for radiolabelled neo-synthesized globin chains; for V5m3-400 the percentage of chains is estimated by HPLC and normalized per VCN and lastly for GGHI the percentage of -chains is estimated by HPLC. Percentage of apoptotic cells in erythroid liquid cultures assessed by FACS for apoptotic markers in Thal or mock-transduced CD34 + versus transduced Thal CD34 + cells, as indicated; for BG-1 and GGHI vectors, it was estimated by Annexin-V single staining; for GLOBE vector, it was estimated by both GpA+/Annexin-V double staining and propidium iodine (PI) staining, as indicated; for V5m3-400, the percentage of apoptotic cells was determined by TdT-mediated dutp nicked endlabelling (TUNEL) assay, which did not show a significant reduction, but just a trend. independent, while he is exhibiting sustained amelioration of the thalassemic phenotype for 64 months. Despite the above success, the group [53] reported clonal dominance of a single hematopoietic clone, harbouring a vector insertion into the HMGA2 gene. The specific clone, 20 months post transplant, accounted for 50% of the genetically modified HSCs, though with only 3% contribution to the circulating red blood cells. The potential clinical relevance of the aberrant HMGA2 expression alteration by the vector integration is highlighted by the fact that this gene may function as a potential oncogene in various types of cancer. However, as the above observation may simply reflect the consequences of engraftment from a small number of transduced HSCs, it remains unclear whether the insertion into the specific gene is responsible for the contribution of this clone to hematopoiesis [53].

7 Thalassemia Gene Therapy Clinical Trials Current Molecular Medicine, 2013, Vol. 13, No HEMATOPOIETIC STEM CELL (HSC) SOURCES FOR CLINICAL TRIALS IN -THALASSEMIA GENE THERAPY Before the French gene therapy thalassemia trial, allogeneic HSC transplantation was the only radical cure for -thalassemia major. Since gene therapy for thalassemia requires optimal numbers of vector transduced HSCs to be infused to the patient, the issue of supplying sufficient numbers of HSCs remains critical, while the originating source is a matter of debate. Until recently, there were two sources of procurement of HSCs, i.e. BM and mobilized PB. However, several research groups are now investigating the possibility of using alternative sources such as induced pluripotent stem (ips) cells Bone Marrow (BM) Bone marrow is undoubtedly the most well known source for CD34 + HSCs, as the majority of these cells under steady-state and normal conditions, reside in plethora within BM, organised in distinct niches [54]. Therefore, HSC transplantation has become synonymous to BMT. Until recently, BM was not considered a preferred source of HSCs in thalassemia gene therapy, primarily due to the invasive nature of the technique, requiring general anesthesia while providing insufficient yield of CD34 + cells. A recent study by Frittolli et al. [55] however, demonstrated for the first time that BM can indeed serve as a source of HSCs for gene therapy. Specifically, 20 BM harvests were carried out from 20 transfusion-dependent thalassemic patients undergoing allogeneic HSC transplantation, 29 BM harvests from thalassemia heterozygote donors and 20 from healthy donors. The group managed to collect sufficient amounts of CD34 + cells from the thalassemic patients, with concentrations being similar to that of healthy subjects, i.e. median 0.26 x 10 6 /ml in thalassemic patients, 0.36 x 10 6 /ml in thalassemia heterozygotes donors and 0.24 x 10 6 /ml in normal donors. All harvests were analyzed further for routine BM morphology. The group found an expected expansion in the erythroid compartment in thalassemic patients (70%) compared to heterozygotes (40%) and normal donors (35%). However, this expansion influenced neither the yield of CD45 + or CD34 + cells nor the proportion of myeloid and erythroid progenitors. Moreover, the HSC yield in the thalassemic patients was also not affected by disease-specific characteristics or iron overload. Lastly, none of the subjects suffered from any serious adverse effects following aspiration, suggesting that BM harvesting is safe and free from severe side effects. Based on the above clinical data, the Italian group has applied to the authorities for a phase I/II gene therapy clinical trial, using BM as a source of HSCs Mobilized Peripheral Blood Stem Cells (PBSCs) It represents the most widely used source of HSCs for autologous or allogeneic transplantation, primarily due to the higher yield of CD34 + cells, and to the noninvasive nature of the procedure compared to conventional BM harvest. The term PBSC mobilization refers to the forced migration of HSCs from the BM to the periphery. Granulocyte colony-stimulating factor (G- CSF) represents the major inducer of PBSC mobilization, and can be administered with or without chemotherapy [56, 57]. More specifically, Li et al. [58] administered G-CSF to mobilize stem and progenitor cells in thalassemia major pediatric patients and compared the kinetics of CD34 + cells and lymphocyte subsets with those of healthy PBSC donors. Results showed that G-CSF in concentrations of g/day/kg of body weight, succeeded in effectively mobilizing CD34 + cells of both thalassemic patients and healthy donors. Additionally, no significant difference was observed in the levels of daily stem cell counts between the two groups, indicating that efficient CD34 + cell levels in PB and collection of PBSC in -thalassemia patients are feasible [58]. In a different study, Yannaki et al. [59] used G-CSF to mobilize murine HSCs and concluded that thalassemic mice mobilized less efficiently than their control counterparts, possibly due to increased splenic trapping of HSCs and progenitor cells. The reduced mobilization efficiency was restored in splenectomized subjects. Recently, the same group published the results of a clinical trial using either G-CSF or plerixafor in splenectomized and nonsplenectomized individuals [60]. They concluded that for -thalassemia gene therapy, both G-CSF and plerixafor could be used as mobilization agents in nonsplenectomized subjects, whereas plerixafor is preferred in the case of splenectomized patients, both in terms of efficacy and safety. The French ongoing clinical trial, following authorization from the regulatory agency, could use either BM-derived or PB G-CSF mobilized CD34 + cells [52]. Also, Michel Sadelain in his strategy for phase I clinical trial using vector TNS9.3.55, is planning to mobilize PBSCs [61] Induced Pluripotent Stem (ips) Cells Induced pluripotent stem (ips) cells result from reprogramming of a differentiated somatic cell into a pluripotent embryonic-like stem cell (ESC) [62]. They exhibit many features of human ESCs and can theoretically generate any cell type in the human body. They can also serve as suitable cell types for the correction of mutant cells or tissues, mainly by homologous recombination. For ips cell generation, somatic cells from a patient are isolated and reprogrammed to a stem cell-like status, using the Oct3/4, Sox2 genes, with either Klf4 and c-myc or Nanog and Lin28. Following genetic manipulation and differentiation to the desired cell lineage, they can be administered back to the patient [62]. Several approaches have been described in detail for the generation of ips cells [62]. These mainly include direct reprogramming using viral vectors, primarily lentiviruses and adenoviruses and small molecules, such as plasmids and transposon elements. For -thalassemia gene therapy, ips cells can provide an alternative and patient-friendly strategy for

8 8 Current Molecular Medicine, 2013, Vol. 13, No. 8 Drakopoulou et al. obtaining higher number of HSCs for genetic manipulation and transplantation. Hanna et al. [63] were the first group to report gene correction in a murine SCD model, using ips cells. Cells from the skin of the SCD mouse were harvested and reprogrammed to ips cells, using a retrovirus to deliver Oct4, Sox2, Klf4 and c-myc genes. After removing the potential oncogenic c-myc gene, ips cells were cultured to produce hematopoietic stem cell precursors that carried the normal gene and were then transplanted back to SCD mice. The data documented disease amelioration in treated mice. ips cells were also used as an HSC source in human studies, with Ye et al. [64] being the first to show that skin fibroblasts from a thalassemic patient with o -thalassemia could be reprogrammed into ips cells. Specifically, fibroblasts from skin biopsies derived from a thalassemic patient homozygous for a CTTT deletion-mutation causing a frameshift that results in no -globin synthesis, were used for retroviral infections both in the presence and absence of c-myc gene, along with the Oct4, Sox2 and Klf4 transcription factors. The obtained colonies, expressed pluripotency markers, and were shown to carry the same 4-bp mutation found in the thalassemic patient, confirming that they derived from the patient s skin fibroblasts. Furthermore, these ips cells demonstrated the ability to differentiate towards hematopoietic lineage, upon cultivation in cytokine-containing differentiation medium and produced cells that stained positive for fetal hemoglobin. Taken together, the above findings suggest that reprogramming of skin fibroblasts from a thalassemic patient into ips cells is feasible [64]. Following this proof of principle, Zou et al. [65] recently reported a homologous recombination-based approach to correct SCD mutation in ips cells obtained from an adult patient carrying two mutated -globin alleles ( S ). Precise conversion of one mutated S gene to wild type A allele in the patient s ips cells was achieved, using a plasmid containing a drug-resistant gene cassette, flanked by loxp sites and S -specific zinc finger endonucleases. Up to 40% transcript expression upon differentiation to erythrocytes was documented, demonstrating that single nucleotide substitution using human ips cells is feasible in humans, representing an efficient strategy for gene correction [65]. Furthermore, in a recent study, besides the correction of the -thalassemia mutation, a successful transplantation of the corrected human ips cell-derived HSCs into an immune deficient mouse model was achieved [66]. More specifically, Wang et al. [66] reprogrammed skin fibroblasts into ips, derived from a 2-year old thalassemic patient homozygote for the CTTT deletion-mutation in codons 41-42, by introducing Oct3/4, Sox2 and Klf4 using retroviral vectors. Following successful correction of the mutation by specific gene targeting and homologous recombination, the ability of the specific cell lines to differentiate was assessed both in vitro and in vivo. It was shown that the gene-corrected cells showed improved hematopoietic differentiation capacity in vitro and could induce hematopoiesis in sub-lethally irradiated SCID mice. Furthermore, these cells could also produce adult human -globin after hematopoietic differentiation in vivo at about 9% of the endogenous mouse -globin. Furthermore, ips cells were also used as means of gene addition, leading to correction of -thalassemia in vitro. In a pioneering work by Papapetrou and colleagues [67], skin fibroblasts or BM mesenchymal cells from four patients with -thalassemia major were used to obtain -globin-expressing ips cell clones, in which the transgene was inserted in safe harbors and expressed in therapeutic levels. More specifically, somatic cells from thalassemia major patients were reprogrammed to patient-specific ips cells and following removal of the reprogramming vectors and transduction with the TNS9-derived -globin vector at safe harbors, they were differentiated towards erythroid lineage. Twelve out of the 13 ips cell clones used expressed detectable vector-encoded -globin, the average expression of which was 53% of the normal endogenous -globin allele. The above data proved that correction through addition of a therapeutic gene expressed in therapeutic levels into safe harbors in patient-specific ips cells is feasible and potentially could be used for the clinical treatment of -thalassemia. However, a major limitation to their use remains their inability for long-term engraftment, following generation and genetic modification. In 2009, Raya et al. [68] reported that disease-corrected ips cells originating from a Fanconi anemia patient, despite their successful differentiation into HSCs, they failed to engraft and reconstitute severe immunocompromised mice. It was only recently, that promising data arose, with Wang et al. [66] managing to produce patientspecific ips that could differentiate in HSCs and induce hematopoiesis in sub-lethally irradiated SCID mice, as seen above. In general however, ips cell use in human applications remains elusive. Despite extensive studies in mice, long-term engraftment of ES-originating HSCs in vivo has been reported only by ectopic expression of the transcription factor HoxB4 [69], however as the latter factor is implicated in various leukemias in both mice and primates [70, 71], this approach cannot be considered in human clinical applications. All the above strategies, which involved reprogramming using integrating viral vectors, require various genetic modifications of the target cells to stably express a transgene, and therefore the need for semi random and controlled integration of the transgene of interest is imperative to ensure safety. In their recent work, Papapetrou and Sadelain [67, 72, 73] demonstrated how the above issues could be addressed. Specifically, by employing bioinformatics and functional analysis, they screened patient-specific ips cell clones and retrieved safe integration sites, lowering thus the risk for semi random vector intergration, which could interfere with the expression of neighbouring genes, including oncogenes. The above safe harbors met the following five criteria: (i) distance of at least 50 kb from the 5 end of any gene, (ii) distance of at least 300 kb from any cancer-related

9 Thalassemia Gene Therapy Clinical Trials Current Molecular Medicine, 2013, Vol. 13, No. 8 9 gene, (iii) distance of at least 300 kb from any microrna (mirna) coding gene, (iv) location outside a transcription unit, and (v) location outside ultraconserved regions (UCRs) of the human genome [67]. Furthermore, they devised a 7-week protocol, which enables efficient derivation of genetically modified human ips cells, carrying the transgene at known genomic sites [72]. The group also proposed a 12 to 14-week protocol for generating transgene-free ips cells, employing the polycistronic LV pml-fsv2a, in which Oct4, Sox2, Klf4 and c-myc genes are flanked by loxp sites. Upon establishment of ips cell clones, in which vector integrations are again mapped to the human genome, expression of Cre recombinase, driven by an integrase-deficient vector, mediates excision of the above genes, leading thus to transgenefree ips cells [73]. Reprogramming of ips cells can be also achieved using methods that are devoid of integration, such as episomal plasmids, non-integrating vectors -such as adenoviruses- and transposons [74]. Use of such methods is promising for generation of ips cells, as they are free from issues, such as insertional mutagenesis and incomplete silencing of reprogramming factors. However, great concerns are also raised in certain occasions, as various genetic alterations, some affecting oncogenes during reprogramming phase under these conditions, have been reported. Specifically, in their recent report, Howden and colleagues [75], using an episomal reprogramming method, reported that comparison of ips cell lines before and after gene targeting revealed an accumulation of a number of mutations originating during reprogramming procedures. It is therefore worthwhile to extend our knowledge towards safe ips cell generation and genetic modification methods, before their use can be implemented in human applications. 6. ENHANCING GENE TRANSFER BY HSC MANIPULATION Human HSCs are more resistant to lentiviral gene transfer compared to murine ones, and so far the maximum number of genetically modified PB cells obtained is 15%, as demonstrated by various laboratories. Specifically, Kim et al. [76] working on rhesus macaques and using a GFP LV showed that only 7% of PB cells were transduced, an observation that was also made by Trobridge et al. [77] and Hanawa et al. [78], who worked on pigtail macaques. Lastly, in the French clinical trial [53], genetically modified HSCs did not exceed 15% on average, leading to the conclusion that higher percentages of genetically modified HSCs in the case of human and non-human primates might not be easily obtainable. Below, a description of the main methods for ex-vivo manipulation currently used for increasing HSC gene transfer suitable for clinical trials is presented Traditional vs Newly Developed Envelope Glycoproteins Significant efforts have been made towards improving the ability of globin LVs pseudotyped with different envelope glycoproteins to transduce human HSCs. Kim et al. [79] performed comparative transduction studies of human HSCs with GFP lentiviral vectors pseudotyped with either the vesicular stomatitis virus glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein (RD114TR) or the feline leukemia virus type C envelope protein (FLVCTR). They concluded that VSV-G is more effective in transducing engrafting cells compared to other -retroviral glycoproteins, as it led from 2 to 10-fold higher transduction rates, with no indication of toxicity, despite the relatively high multiplicities of infection used, thus supporting its use in clinical-grade vectors [79]. Pioneering experiments by Verhoeyen et al. [80] demonstrated that HSC gene transfer is significantly increased when lentiviral particles are engineered to display early-acting cytokines on their surface, such as SCF and TPO, alone or combined. In this setting, the modified LVs would particularly stimulate the HSCs within the CD34 + pool, leading to increased gene transfer rates, with relatively low MOI and short incubation times. This procedure minimizes the risk for insertional mutagenesis resulting from high vector doses and also helps to preserve HSC stemness, which is usually lost, after cytokine cocktail stimulation, that is usually employed to sustain survival in vitro. Furthermore, Frecha et al. [81] from the same group, performed comparative analysis studies using novel LVs co-displaying SCF and TPO glycoproteins, fused to the N-terminus of the hemagglutinin protein of influenza virus (HA), and the mutant endogenous feline retroviral glycoprotein RDTR. The data disclosed that particularly RDTR/SCFHA can selectively target and transduce cord blood CD34 + cells, even in the absence of retronectin and with MOI as low as 1. Also, as RDTR and RDTR/SCFHA LVs, unlike VSV-G LVs, resist inactivation by complement factors present in human BM aspitates, they were further tested in unfractionated BM of healthy and Fanconi anemia donors, leading to selective transduction, again in the absence of retronectin and in low MOIs. Moreover, the RDTR/SCFHA lentiviral vector, when used to drive expression of a FANCA gene, resulted in 40% selective transduction of human CD34 + in the patients BM, suggesting that it can confer efficient gene targeting, allowing correction of CD34 + cells. Furthermore, Neff et al. [82], Ghani et al. [83] and Bell et al. [84], also proposed that the specific glycoprotein could potentially substitute the more cytotoxic VSV-G envelope protein and enhance viral transduction, as it demonstrates stability, its receptor is widely expressed on HSCs, and most importantly, it demonstrates no toxicity. More specifically, in a recent study by Bell and colleagues [84], the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G was compared. It was concluded that RD114-pseudotyped viruses, despite having one log