Globin gene transfer for treatment of the β-thalassemias and sickle cell disease

Best Practice & Research Clinical Haematology Vol. 17, No. 3, pp. 517–534, 2004 doi:10.1016/j.beha.2004.08.002 available online at http://www.sciencedirect.com

12 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease Michel Sadelain* Stefano Rivella

MD, PhD

PhD

Leszek Lisowski

BS (PhD Student)

Selda Samakoglu

PhD

Isabelle Rivie`re PhD Laboratory of Gene Transfer and Gene Expression, Gene Transfer and Somatic Cell Engineering Facility, Box 182 Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

The b-thalassemias and sickle cell disease are severe congenital anemias that are caused by mutations that alter the production of the b chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a functional globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling the expression of the globin transgene, which ideally should be erythroid specific, differentiation-stage restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, we have demonstrated that an optimised combination of proximal and distal transcriptional control elements permits lineage-specific, elevated expression of the b-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in b-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors represent a general paradigm for the regulation of transgene function and the improvement of vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy. Key words: gene therapy; gene regulation; centiviral vector; stem cell; hemoglobinopathy; insertional oncogenesis.

* Corresponding author. Tel.: C1-212-639-6190. E-mail address: [email protected] (M. Sadelain). 1521-6926/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

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The b-Thalassemia major and sickle cell disease (SCD) are severe congenital anemias that result from the deficient or altered synthesis of the b chain of hemoglobin. In the b-thalassemias, the b-chain deficit leads to the intracellular precipitation of excess a-globin chains, causing ineffective erythropoiesis.1–4 In the most severe forms found in homozygotes or compound heterozygotes, the anemia is lethal within the first years of life in the absence of any treatment.5 Transfusion therapy is life saving and aims to correct the anemia, suppress the massive erythropoiesis and inhibit increased gastrointestinal absorption of iron.1–4 However, transfusion therapy leads to iron overload, which is lethal if untreated. The prevention and treatment of iron overload are the major goals of current patient management.6 The only means to cure the disease is through allogeneic bone marrow transplantation (BMT).7–10 In SCD, the b chain is mutated at the sixth amino acid, leading to the synthesis of bS instead of the normal bA.11,12 The abnormal hemoglobin, HbS, causes accelerated red cell destruction, erythroid hyperplasia and painful vaso-occlusive ’crises’.4 Vasoocclusion can damage various organs, eventually causing long-term disabilities (e.g. following stroke or bone necrosis), and sometimes sudden death. While a very serious disorder, the course of SCD is typically unpredictable.4 By increasing production of fetal hemoglobin13 and suppressing hematopoiesis, hydroxyurea can produce a measurable clinical benefit.14–16 Since hydroxyurea is a cytotoxic agent, there is a great need for alternative, less toxic drugs to induce g-globin gene expression. As for the b-thalassemias, allogeneic BMT is the only curative therapy at present.10–18 However, while potentially curative, allogeneic BMT is not devoid of complications. Safe transplantation requires the identification of a histocompatible donor to minimise the risks of graft rejection and graft-vs-host disease (GVHD).10–18 In the absence of a suitable donor, the genetic correction of autologous haematopoietic stem cells (HSCs) represents a highly attractive alternative treatment because it is potentially curative.19 This approach could resolve the search for a donor and eliminate the risk of GVHD and graft rejection associated with allogeneic BMT. While filled with promise, a genetic approach raises a number of challenging biological questions regarding the isolation and transduction of HSCs, the design of vectors that provide therapeutic levels of transgene expression and a minimal risk of insertional oncogenesis, and the implementation of non-toxic transplant conditions that permit host repopulation with minimal conditioning. Many of these issues are common to all stem-cell-based gene therapies. However, the b-globin gene is particular in its stringent transcriptional requirements. Transgene expression has to be erythroid specific and differentiation stage-specific, and expression has to be extremely elevated compared with most other genes. Achieving regulated b-globin expression has represented a tremendous obstacle in the past decade.20,21 Four years ago, a breakthrough was reported using a lentiviral vector that harbored an optimised combination of proximal and distal b-globin transcription control elements, demonstrating for the first time that therapeutic levels of globin expression could be achieved in thalassemic mice.22 Several groups have confirmed these results, and extended them to various animal models of severe hemoglobinopathies. Collectively, these data support the conclusion that transgene expression can be reasonably regulated in the progeny of virally transduced stem cells, although position effects and gene variegation are not fully overcome. These results provide important general lessons for vector design, vector function and vector safety.

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GLOBIN GENE STRUCTURE AND EXPRESSION The human b-globin locus The human b-globin locus has been studied extensively as a model system for understanding tissue- and developmental-stage-specific expression of mammalian gene families.23–30 The human b-globin locus is located on chromosome 11p15.5 and spans 80 kb encompassing both the five expressed b-like globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny.11 The genes are in the same transcriptional orientation and are arranged in the order of their expression during development, with the embryonic 3 gene located at the 5 0 end and the adult bglobin gene at the 3 0 end of the locus2 (Figure 1A). Developmental-stage-specific expression is controlled mainly at the transcriptional level by a variety of proximal or distal cis-element and transcriptional factors that bind to these regions. In the case of the b-globin gene, proximal regulatory elements comprise the b-globin promoter and two downstream enhancers, one located in the second intron and one approximately 800 bp downstream of the gene.31–33 The most prominent distal regulatory element is the b-globin locus control region (LCR), located 8–22 kb upstream of the 3-globin gene and composed of several subregions that exhibit heightened sensitivity to digestion with exogenous DNaseI in erythroid cells.11,34 The functional significance of the region upstream of the b-cluster was first inferred from rare thalassemic patients that bore deletions far upstream of the b-globin locus rather than in or near the b-globin gene itself. These deletions cause the classical hematological features of b-thalassemia. In one such deletion, referred to as Hispanic deletion b-thalassemia, a 35-kb region located upstream of the HS1 site and the 3-gene was found to be deleted, which suggested that this region contained cisacting elements required for expression of the b-globin gene.35 In the human genome, five HS sites (HS1-HS5) have been identified. HSs 1–4 are DNAse I hypersensitive in erythroid cells only, while 5 0 HS5 forms in multiple cell lineages.36 The human b-globin transgene in mice Direct evidence of the importance of the LCR in the expression of the b-globin genes first came from transgenic mouse studies.34 Linkage of a 20-kb fragment from this region, spanning HS1-HS4, to the b-globin gene resulted in high-level, copy-numberdependent expression of the transgene, at levels similar to that of endogenous mouse b-globin genes.34 Individual HSs 2–4 have enhancer activity in stable assays.37–39 HS2 behaves as a classical enhancer, showing enhancer activity in transient transfection assays.40 The activity of HS3 or HS4 is only apparent when they are integrated into chromatin.41,42 The enhancer activity of HSs 2–4 resides in 200–300-bp core elements, which contain an array of binding sites for ubiquitous and erythroid-specific trans-acting factors 30, including GATA-1 and NF-E2.40,43–45 HS1 and HS5 alone do not have enhancer activity. HS5 has properties characteristic of an insulator element.46,47 Together, HSs 1–4 are sufficient to direct high-level expression in transgenic mice48, especially when combined with an extended human b-globin promoter.49 The sequences flanking the core elements are likely to be involved in the activation of the b-like globin genes, as suggested by the presence of long segments of high similarity in the b-locus domain of several species50, and by functional analyses which indicate that the core elements alone

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β-globin gene 5´LTR A : RNS1

Ψ

B : TNS9

p HS2, 3, 4 3´LTR

RRE

SD

Ψ

LCR 1 kb

SA p

βe+

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βA-87Thr cPPT C : β87

D : HS40/I8/K

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βp HS2 HS3 HS4s.n.

± γRE/βe+ s.n.

WPRE E : HS40/I8/K

Ψ

I8 H K GFP

WPRE

F : G.W/P

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GFP

PGK ∆L

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Figure 1. Erythroid-specific lentiviral vectors. (A) RNS1 harbors the entire human b-globin gene along with its minimal promoter (p, from K265) and the HS2, 3 and 4 core sites (HS2: 423 bp; HS3: 280 bp; HS4: 283 bp), as described for the vector Mb6L.21,22,152 Other elements of the vector are indicated. RRE, rev response element; SD, splice donor; SA, splice acceptor; LTR, long terminal repeat; j, packaging region). (B) TNS9, compared with RNS1, has an extended promoter sequence (p, from K615), the b-globin 3 0 proximal enhancer (eC) and large LCR elements (3.2 kb) spanning HSs 2–422 and two additional tandem GATA-1 sites (*). TNS9 was generated with an 840-bp HS2 fragment, a 1308-bp HS3 fragment and a 1069-bp HS4 fragment. The function of TNS9 has been described elsewhere.22,110,138,153 (C) b87 closely replicates the structure of TNS9 with the following modifications: codon 87 is mutated (bA-Thr87Gln) to generate a variant b chain, the promoter is from position K265, as in RNS1, and the size of the LCR is slightly smaller (3.2 kb in TNS9 vs 2.7 kb in b87, 840 bp vs 644 bp in HS2, 1308 bp vs 845 bp in HS3, 1069 bp vs 1153 bp in HS4). b87 also contains the HIV-1 cPPTelement.114,115 (D) d432b-Ag113 was generated using the b-globin promoter (K130), fusing the b-globin 5 0 promoter untranslated sequences to the Ag-globin coding sequence at position 3 and C 1 relative to the endogenous translational start site. Also, d432b-Ag113 replicates the structure of the regulatory elements present in TNS9 with the following modifications: the size of the LCR is smaller (3.2 kb in TNS9 vs 2.0 kb in d432b-Ag113, 840 bp vs 374 bp in HS2, 1308 bp vs 898 bp in HS3, 1069 bp vs 756 bp in HS4 with a 311-bp deletion in HS4 outside of the ‘core’ element) and the promoter is 130-bp long. In the paper by Persons et al113, three globin vectors were generated: d432b-Ag113, d432b-Ag113 b-3 0 enhancer (b 3 0 Enh) and d432b-Ag113 g-globin 3 0 regulatory element (g 3 0 RE). The b 3 0 Enh or the g 3 0 RE were placed downstream of the g-globin coding sequences. Moreover, d432b-Ag113 vectors contain the HIV-1 cPPT element.114,115 (E) HS40/I8/K vector contains the ankyrin-1 promoter (K), the HS-40 (H) and I8 (I8) enhancers, the green fluorescent protein (GFP) and the woodchuck hepatitis virus post-regulatory element (WPRE) element. (F) G.W/P vector has a replacement of the long terminal repeat enhancer (in the U3 region) with the upstream enhancer (HS2) of the erythroid-specific GATA-1 gene (G). The vector contains GFP, a truncated form of the p75 nerve growth factor receptor (DL), an internal promoter (PGK) and the WPRE element.

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are not sufficient to express the human b-globin gene51,22 consistently and at a high level. It is now appreciated that full activity of the LCR ultimately requires co-ordinated interaction of many of its components.51,52 Lessons from the mouse locus Originally, five HS sites (HS1–HS5) were associated with LCR function; recently, an additional site was mapped 5 0 to the LCR. This site, HS6, is associated with a minor HS53 and contains a high density of potential binding sites for the erythroid transcription factors GATA-1 and NF-E2, consistent with other b-globin LCR HSs. Surprisingly, when the entire mouse b-globin LCR (HS1-6) was deleted by homologous recombination, the formation of the general DNAse I sensitivity throughout the b-like globin domain was not affected. However, transcription of all b-like globin genes was reduced strikingly.54 The deletion of mouse HS2 and HS3 reduces the expression of the endogenous b-globin genes by 41 and 29%, respectively.55 Similar results were obtained with individual deletion of the endogenous murine HS1 and HS4, reducing expression of the endogenous b-globin genes by 22 and 24%, respectively. In all these transgenic animals, no change in the ontological activation of all the b-like globin genes or tissue specificity of expression was noted.52 Deletion of HS5 and HS6 had a minimal effect on transcription and did not prevent formation of the remaining LCR HSs.53 Together, these data indicate that the mouse LCR HSs form independently and appear to contribute additively to the overall expression from the b-globin locus. Models of LCR functions It was originally suggested that the LCR possessed a dominant chromatin-opening activity, essential for the transcription of the b-like globin genes.48 However, when most of the human LCR (HS2-5) was deleted in the context of its normal chromosomal location in cell lines, the formation of the remaining HSs along the entire locus and the presence of general DNase I sensitivity associated with the b-globin domain were not affected56, thus paving the way for various hypotheses and models on the role and mechanism of action of the LCR. The LCR acts over a long chromosomal distance. The mechanisms proposed for long-range enhancer action fall into two basic categories. Contact models assert that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that ‘loop out’ the intervening sequences. Non-contact models contend that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately reach the gene. According to these two general mechanisms, four models of LCR function have been proposed: looping, tracking, facilitated tracking, and linking.24,26,28,57–70

GLOBIN GENE TRANSFER IN HSCS Oncoretroviral-mediated globin gene transfer Retroviral vectors generally provide an efficient method for the transduction of murine HSCs. Recombinant oncoretroviruses were the first viral vectors used to transfer the human b-globin gene in mouse HSCs. Early experimentation with vectors harboring

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the b-globin gene resulted in tissue-specific but low and variable human b-globin expression in bone marrow chimeras, usually varying between 0 and 2% of endogenous mouse bmajor RNA levels.71–75 Initial efforts to incorporate LCR subfragments into oncoretroviral vectors resulted in low titers76, low expression77 or unstable vectors prone to sequence re-arrangements.78 Incorporation of the core elements of HS2, HS3 and HS4 of the human b-globin LCR significantly increased expression levels in MEL cells20,21, but failed to abolish positional variability of expression.21 This suggested that a minimal LCR comprising juxtaposed core elements did not provide full LCR function but rather acted like an erythroid-specific enhancer. These findings, arguing against the effectiveness of minimal core elements, were consistent with contemporary transgenic studies establishing that data obtained in mice bearing multiple core copies cannot be extrapolated to the single copy context79, and thus further confirmed that data obtained in multicopy transgenic animals can be misleading for guiding vector design for gene therapy. The incorporation of larger LCRs into oncoretroviral vectors proved to be problematic, leading to vector instability and considerable genomic re-arrangements. In view of these difficulties, some investigators began exploring alternative transcriptional control elements. At present, several erythroid-specific transcriptional elements are under investigation within oncoretroviral vectors, including the HS40 regulatory region from the human a-locus80–82 and alternative promoters. Thus, the promoter of ankyrin, a red cell membrane protein, has shown some promise in transgenic mice and in transduced mouse erythro leukemia cells.83 In mice, the ankyrin promoter has been used to drive expression of the human g-globin gene resulting, at double copy, in an average expression of 8% of that of the endogenous a-globin genes.84 Additional elements have been incorporated into the vector to stabilise or increase the levels of globin expression. We and others have demonstrated that integration of the cHS4 insulator element into the 3 0 LTR of recombinant murine leukemia virus increases the probability that randomly integrated proviruses will express the transgene.85–87 Using the cHS4 element in conjunction with globin elements88, Emery et al substantially increased globin expression from a vector harboring the b-globin promoter, a modified g-globin gene, and the aglobin enhancer. It is not yet clear if the level of expression achieved in red blood cells would be therapeutically relevant.88 Selection of transduced cells prior to transplantation has also been used to reduce the frequency of transgene silencing in vivo. Using this strategy, stable expression of the human b-globin gene was obtained in the red cells of mice engrafted with a murine-stem-cell-virus-based oncoretroviral vector containing the core sequence of HS2 and the green fluorescence protein (GFP).89 Primary human hematopoietic cells were transduced with this vector, resulting in b-globin gene expression in human cells in mice after their differentiation into erythroid cells.90 Lentiviral-mediated gene transfer Lentiviral vectors are replication-defective retroviral particles containing lentiviral core proteins and enzymes, which are pseudotyped with a heterologous retroviral envelope or equivalent. Lentiviral vectors derived from HIV-1 and other lentiviruses have elicited great interest for their ability to transduce non-dividing cells.91,92 While oncoretroviral vectors are restricted to cells proceeding through mitosis, the pre-integration complex of lentiviral vectors has the ability to translocate to the nucleus and successfully integrate in the absence of cell division.93,94 Lentiviral vectors have the capacity to transduce a broad spectrum of target cells, including neurons, retinal photoreceptors,

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dendritic cells, macrophages, hepatocytes and HSCs.95–106 It is important here to make the distinction between non-dividing cells and quiescent, G0 cells, as the latter seem to be refractory to lentiviral vector transduction.107,108 Another fundamental attribute of lentiviral vectors is their relative genomic stability, as shown with globin vectors.22 Furthermore, lentiviral vectors may provide an additional advantage in terms of their packaging capacity.109 Erythroid-specific lentiviral vectors The ultimate goal of globin gene transfer is to achieve erythroid-specific, regulated, high-level, sustained transgene expression. The disappointing results obtained with the minimal 1.0-kb LCR suggested that larger LCR sequences and their optimised combination with promoter and enhancer elements would be needed to achieve this goal. As we have shown, lentiviral vectors enable the stable transfer of large genomic regions and thus successful regulation of globin transgenes.22 The TNS9 vector encodes the human b-globin gene, deleted of a cryptic polyadenylation site within intron 221, flanked by an extended promoter sequence and the b-globin 3 0 proximal enhancer, as well as large LCR elements (3.2 kb) spanning HSs 2–4 (TNS9, Figure 1). The combination of these proximal and distal control elements was the best amongst several. Using the lentiviral-based-vector system described by Zufferey et al117, we succeeded in stably transmitting TNS9, which is w9 kb in size, and correcting the hematological features in mice affected by b-thalassamia intermedia and major.22,110 As expected, similar results have been achieved in mouse models of SCD.111 In this case, a vector that reproduced the structure of TNS9 closely was generated (bA-T87Q_globin lentivirus or b87, Figure 1). The vector b87 harbors a variant globin gene mutated at codon 87 to encode the amino acid residue believed to account for the greater antisickling activity of g-globin and antagonise bs.112 The g-globin gene has also been cloned into a vector of the TNS9 type called d432b-Ag113 (Figure 1) and tested in mice affected by b-thalassemia intermedia. The vector d432b-Ag was generated using the b-globin promoter (K130), fusing the b-globin 5 0 promoter untranslated sequences to the Ag-globin coding sequence at position 3 and C1 relative to the endogenous translational start site. The size of the LCR in d432b-Ag is smaller (2.0 kb) than TNS9 and the promoter is 130 bp long. Persons et al113 generated three globin vectors: d432b-Ag113, d432b-Ag113$b-3 0 enhancer (b 3 0 Enh) and d432b-Ag113 g-globin 3 0 regulatory element (g 3 0 RE). The b 3 0 Enh or the g 3 0 RE were placed downstream of the g-globin coding sequences. Moreover, d432b-Ag113 vectors contain the HIV-1 cPPT element.114,115 Vectors based on alternative erythroid elements have been reviewed recently.116 Their design is based on the combination of various non-globin erythroid promoters and enhancers, and the woodchuck hepatitis virus post-regulatory element (WPRE)117,118 (Figure 1). While lineage restricted, these vectors do not appear to express at the levels required for hemoglobin chains. STUDIES OF b-THALASSEMIA AND SCD IN MOUSE MODELS Models of b-thalassemia Three mouse models of b-thalassemia intermedia are available. The th1 model results from the deletion of the bmajor gene; th1/th1 homozygotes exhibit a moderate form of

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thalassemia.119 A second model (th2) was generated by insertional disruption of the bmajor gene, causing lethality in th2/th2 homozygotes but only a very mild phenotype in the heterozygotes.120 The third model, th3, was generated by deletion of both the bmajor and bminor genes.121,122 Mice homozygous for this deletion die late in gestation, while heterozygotes are viable and thalassemic. Adult th1/th1 and th3/Cmice exhibit anemia, slightly more severe in the latter, abnormal red cell morphology, splenomegaly and develop spontaneous hepatic iron deposition similar to that found in humans with b-thalassemia intermedia. The lack of an adult animal model for Cooley’s anemia has limited the full investigation of the pathophysiology underlying this disease and hampered the evaluation of both pharmacological and genetic treatments. For this reason, we established an adult mouse model of b0-thalassemia.123 To generate this model, we engrafted myelo-ablated wild-type animals with b-globin-null fetal liver cells (FLCs) harvested from Hbbth3/th3embryos which lack both bmajor and bminor genes. Unlike mice engrafted with HbbC/C or Hbbth3/C FLCs, which survived for at least 8 months (nZ11 and 24, respectively), recipients of Hbbth3/th3 cells died 7–9 weeks after transplantation (T50Z50 days, nZ31), significantly later than radiation controls (T50Z15 days, nZ10, P!0.01).123 Control thalassemic animals (mice engrafted with eGFP-transduced Hbbth3/th3 FLCs) revealed severe anemia (2.8G0.8 g/dl of Hb, vs 13.2G1.0 in 11 HbbC/C chimeras and 11.1G2.1 in 23 HbbC/th3 chimeras) 6 weeks post transplantation. Low red blood cell counts, hematocrit values and reticulocyte counts, together with very high levels of serum erythropoietin, further confirmed the development of a profound erythroid deficiency. Moreover, these mice presented with massive splenomegaly due to major erythroid hyperplasia, and the profound anemia settled in after 50 days, consistent with the clearance rate of the recipient’s normal red blood cells.124 These mice succumb to ineffective erythropoiesis within 60 days, showing massive splenomegaly and diffuse iron deposition, especially in the liver.123 Models of SCD Advances in the treatment of SCD have, in part, been hampered by the lack of an animal model that accurately reproduces the pathophysiology and genetics of this disorder. There is no mouse model that adequately recapitulates the disease found in patients with SCD. Initial efforts focused on the generation of transgenic animals expressing the mutant bS-globin gene. However, chimeric hemoglobin consisting of murine a-globin and human bS-globin did not polymerise efficiently. Even with the addition of a human a-globin transgene, only a small fraction of the cells sickled in vivo because of the disruption of HbS by murine a- and b-globins.125–132 Under hypoxic conditions, there is more extensive de-oxygenation of murine Hb than HbS, as mouse hemoglobin has a lower O2 affinity than HbS.133 To produce a hemoglobin that would polymerise more readily, two additional mutations were introduced into bS transgenes; a second mutation in codon 23 to reproduce the bS Antilles allele, and a third mutation to yield bS AntillesD-Punjab or HbSAD.134 While the acronym designating one mouse model of sickle cell disease (SAD) mice exhibited a greater propensity for red cell sickling under hypoxic conditions, this model did not fully recapitulate the features of SCD. Three other models have been generated in which the human a and bS globin genes are expressed exclusively. To create a mouse model expressing sickle human hemoglobin exclusively, Paszty et al

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co-injected three fragments of human DNA into fertilised mouse eggs to generate transgenic founders expressing human a- and bS-globin (Berkeley or BERK mouse model135). As g-globin has antisickling properties, they included the Gg- and Ag-globin genes to decrease the likelihood that erythrocytes would sickle during gestation and cause fetal death. Ryan et al created transgenic animals carrying a 22-kb DNA fragment encompassing the human b-globin LCR and a 9.7-kb DNA fragment containing the Agglobin and bS-globin genes. The LCR was also linked to a 3.8-kb DNA fragment containing the human a1-globin gene.136 A third murine model of SCD, created by Chang et al, used a 240-kb bS-globin YAC in which members of the human b-globin gene cluster are present in their native genomic context.137 To exclude expression of mouse globin chains, mice heterozygous for the constructs described above and heterozygous for the mouse b and a knockout loci were interbred to produce transgenic animals that were homozygous for the mouse knockout globin loci and expressed human HbS exclusively. These animals are viable, show irreversibly sickled cells in their peripheral blood smears, and have hemolytic anemia. However, while their phenotype is more severe than that of SAD mice, the severity of anemia is compounded by the suboptimal expression of these genes, thus causing thalassemia. This state not only complicates the pathophysiology in these mice, but also confounds the interpretation of therapy. Expression of normal b- or g-globin chains in these mice could ameliorate the anemia by correcting the thalassemic features rather than sickling, and would not lead to an overall excess of non-a chains as would be the case in SAD mice or in patients with SCD. Therapeutic achievements in mouse disease models The first severe hemoglobinopathy to be treated in mice was b-thalassemia intermedia.22 Following integration in mouse HSCs, human b-globin expression was erythroid specific and elevated. Four months after transplantation, mice harboring on average 0.5–1.0 vector copies in peripheral blood cells showed Hb levels of 11–13 g/dl (compared with 8.0–8.5 g/dl in age-matched controls), a hematocrit of 39–45 (compared with 29–32 in controls), and decreased reticulocyte counts from 19 to 23% in control mice to 5–10% in the TNS9-treated cohort.22 As a control, we used the original combination of short LCR core and promoter elements into a lentiviral vector that we called RNS1.22 This vector appeared to silence over time.22 In long-term primary transplant recipients, TNS9 maintained stable levels of human b-globin gene over a 40-week period22 with an average of one copy of the vector per cell. This expression remained stable in secondary transplant recipients of TNS9-transduced bone marrow up to 40 weeks after transplant, with no indication of loss of expression, and was sufficient to durably improve anemia, correct extramedullary hematopoiesis, and markedly reduce hepatic iron accumulation.138 These findings demonstrated that viral-mediated transfer of a b-like globin gene could achieve major therapeutic benefit in a severe hemoglobinopathy, providing the first critical demonstration of the medical relevance of globin gene transfer. The second hemoglobinopathy to be treated in mice was SCD/thalassemia. As no transgenic mouse model perfectly recapitulates the exact disease characteristics of human SCD patients, Pawliuk et al111 investigated the efficacy of a lentiviral vector that closely recapitulates the features of TNS9 called b87 (Figure 1). This vector was tested in two different SCD transgenic mouse models: SAD134 and BERK.135 Three months

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after transplantation, mice harbored on average 3G0.5 vector copies in peripheral blood cells. In particular, in mice transplanted with b87 lentivirus-transduced BERK marrow, red blood cell and reticulocyte counts were corrected with amelioration of Hb concentration, anisocytosis and poikilocytosis. The b87 lentivirus-transduced recipient mice showed Hb levels of 13.0G0.4 and 13.7G0.2 g/dl, compared with 9.4G 0.9 and 13.0G0.6 g/dl, respectively, in SAD and BERK age-matched controls. The reticulocyte counts decreased from 17.8G0.6 to 5.8G1.8 in BERK mice, whereas in SAD mice, the reticulocyte counts were 3.4G1.2 in control mice and 2.8G0.1 in the b87-treated cohort. Moreover, the proportion of irreversibly sickled cells, SCDassociated splenomegaly and characteristic urine concentration defect in SAD and BERK mice were vastly improved or corrected by b87. Persons et al113 investigated a lentiviral vector based on the TNS9 design but encoding the human g-globin gene. In this study, the chimeras consisted of normal C57Bl/6J mice transplanted with transduced thalassemic (th3/C) BM cells. The authors did not observe any difference in the level of g-globin produced in animals transplanted with either d432b-Ag (Fig. 1), d432b-Ag b-3 0 or d432b-Ag g-3 0 e (using fluorescence-activated cell sorter (FACS) analysis to detect HbF).113 Fifteen weeks after transplantation, the engrafted mice showed 7–90% HbF-containing red blood cells by FACS analysis. In mice with an average vector copy number of 0.8G 0.2, HbF accounted for about 4% of total Hb, with no improvement of the anemia (9.4G0.1 g/dl Hb compared with 9.4G0.1 g/dl in age-matched controls). In mice with a vector copy number of (VCN) 2.1G0.2, HbF represented 10–15% of total Hb, which increased to 10.1G0.1 g/dl. Mice with high VCNs (2.4G0.7) showed a marked amelioration of their anemia, with Hb levels of 11.6G0.3 g/dl, associated with a reticulocyte count of 12.6G2.8%. It is noteworthy that correction of anemia in SAD139, BERK and th3/C animals, using the b87 and d432b-Ag vectors, required an average of 2.5–3.5 vector copies/cell. This suggests that a high fraction of cells need to be genetically modified to achieve a therapeutic benefit in SCD and/or that multiple vector copies/cells140 are required to express sufficient levels of b-chain expression. This is to be contrasted with the results obtained with TNS9 in th3/C mice.22,138 In order to better evaluate the therapeutic efficiency of TNS9 and its potential clinical applicability to the most severe hemoglobinopathies, we investigated its efficacy in the context of a fatal anemia. Whereas all mice engrafted with Hbbth3/th3 FLCs succumb within 60 days, mice engrafted with TNS9-transduced Hbbth3/th3 FLCs survived for at least 4 months. Our long-term studies focused on chimeras with less than 5% murine Hb (produced by residual host hematopoiesis) in the 8 months following transplantation (nZ6, excluding five mice with endogenous repopulation O5% and two mice with very low vector copy number that died after 4–5 months). Over this period, Hb levels in TNS9-treated animals averaged 6.5G2.9 g/dl. Southern blot analyses showed a mean vector copy number of 1.6G0.6 in bone marrow and 1.2G0.5 in blood (nZ6), thus indicating that TNS9 could generate 4 g/dl Hb/vector copy in this in-vivo setting, an amount approximating half of hemizygous Hb production (8.1G0.3 g/dl in Hbbth3/C chimeras). Pathologic examination performed between 5 and 8 months after transplantation showed variable degrees of ineffective erythropoiesis, commensurate to the degree of anemia.110 In conclusion, after establishing a novel mouse model for the most severe form of b-thalassemia (Cooley’s anemia), we demonstrated that these mice can be rescued and eventually cured by lentivirusmediated transfer of the human b-globin gene.123

Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 527

SAFETY AND EFFICACY ARE LINKED TO IMPROVED TRANSGENE REGULATION Lentiviral vectors offer exciting new prospects for gene transfer in stem cells. In our studies, the use of recombinant HIV-1-derived vectors was instrumental in facilitating the investigation of different promoters, enhancers and chromatin structure determinants to achieve tissue-specific, elevated, and sustained transgene expression. The impressive results obtained with the TNS9, b87 and d432b-Ag vectors make recombinant lentiviruses the most effective vector system to date for gene therapy of the severe hemoglobinopathies. These findings, obtained in murine disease models, are yet to be extended to large animal models. While lentiviral vectors have generated much interest for their ability to transduce non-dividing cells, they provide another major advantage over oncoretroviral vectors, their genomic stability, which makes them more dependable for regulating transgene expression. Lentiviral vectors are thus likely to emerge as the vectors of choice for the stable delivery of regulated transgenes in stem-cell-based gene therapy. Their safe use will entail the development of safe packaging systems. The investigation of lentiviral vectors has greatly benefited from two decades of experience with oncoretroviral vectors, enabling rapid progress and insightful comparisons between the two vector systems. Several third-generation lentiviral systems are presently under development. It is reasonable to expect that some of these will achieve a degree of safety as satisfactory as that already achieved with oncoretroviral vectors.141 The validation of these systems will depend on the establishment of methods to reliably detect replication-competent genomes as well as the intermediates that lead to their formation.142 This task is well underway in several laboratories and companies, warranting optimism with regard to the future availability of clinically acceptable vectors and biosafety testing methodologies. Another safety concern relates to the risk of insertional mutagenesis. This risk is inherent to the random integration of foreign genetic material, whether of viral or nonviral origin. The risk of transformation is major with replication-competent oncoretroviruses.143–145 It is also present with replication-defective oncoretroviral vectors144–147, although it appears to be very rare.145 In one mouse study where myeloid leukemia was shown to be caused by insertional oncogenesis, the transgene was a truncated but not fully disabled form of the human low-affinity nerve growth factor receptor.142,144,146,148 The risk of insertional oncogenesis has also been established in humans, in the context of gene therapy for X-linked severe combined immunodeficiency disease.149 This therapy was remarkably successful in 10 of 11 treated patients, but two patients developed a lymphoid leukemia that could be linked to the integration of the retroviral vector in or near the LMO-2oncogene. In both instances, the LMO-2 oncogene, which is normally silent in T lymphocytes, was transcribed as a direct consequence of the neighboring vector insertion.145,150 The therapeutic molecule encoded by the vector is a component of a multichain receptor that controls lymphocyte proliferation in response to cytokines151, raising the possibility that it may have played a role in transformation, despite the lack of experimental evidence to support this hypothesis.144,145 While the mechanisms underlying transformation in these murine and human cases of insertional oncogenesis remain unclear, these reports indicate that transcriptional activation of neighboring oncogenes by retroviral vectors is a possible, although rare, occurrence. In both instances of insertional oncogenesis, the vector relied on the use of powerful,

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non-tissue-specific enhancer/promoters located in the vector’s LTRs, which resulted in transcriptional activity in progenitor cells and all hematopietic lineages. The design of lineage- and differentiation-stage-restricted vectors represents one major step towards reducing the risk of spuriously transactivating oncogenes. To this end, the incorporation of tissue-restricted promoters and enhancers, internal polyadenylation signals and transcriptional attenuation sites into vectors with deleted 3 0 U3 regions will represent a major advance in gene therapy.145 As reviewed in this article, the globin vectors represent a paradigm for this next generation of retroviral vectors. Also of interest are genetic elements with enhancer-blocking properties, such as insulators. So far, these elements have been investigated to shelter the vector from the repressive influence of flanking chromatin by blocking interactions between regulatory elements within the vector and chromosomal elements.87 This property of insulators might also be harnessed to diminish the risk of the vector activating a neighboring oncogene.145 Medical interventions are weighed in terms of relative risk and benefit. These have to be evaluated in relevant animal models before proceeding to exploratory clinical trials.145 As for the severe hemoglobinopathies, recent studies from several laboratories attest to the efficacy of globin gene transfer in mouse models. As argued here and elsewhere154, the concerns regarding the use of recombinant lentiviral vectors derived from HIV-1 and the risk of insertional oncogenesis should be amenable to effective prevention through rational vector design. The anticipated benefits from tightly regulating expression of the vector-encoded transgene and minimising interactions between vector elements, flanking chromatin and adjacent genes are fundamental. In this respect, the globin vectors provide an excellent model for stemcell-based gene therapy.

ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health (Grants HL-57612, HL 66952, CA-59350 and CA-08748), the Leonardo Giambrone Foundation for the Cure of Thalassemia and the Cooley’s Anemia Foundation.

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