Biomedical

Biomedical

Promising and delivering non-viral vector gene therapies for sickle cell disease
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Promising and delivering non-viral vector gene therapies for sickle cell disease
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For personal use. Only reproduce with permission from The Lancet Publishing Group. RAPID REVIEW Lancet 2002; 360: 629–31 Department of Hematology/Oncology, Children’s Hospital Oakland, Oakland, CA 94609, USA (Elliott Vichinsky MD) Correspondence to: Dr Elliott Vichinsky (e-mail: evichinsky@mail.cho.org) In the past decade, understanding of the pathophysiology of sickle cell disease (SCD) and its treatment has progressed. Infections, brain injury, renal disease, pain, and priapism can now be prevented. Complications from pulmonary injury, surgery, and transfusion can be minimised (panel 1). Vaso-occlusion and tissue ischaemia in SCD involve not only the polymerisation of the sickle haemoglobin (Hb S) but also interactions between red cells, endothelium, platelets, leucocytes, and plasma factors.1–3 Polymerisation of Hb S is the most important factor in the sickling cycle, and a rise in fetal haemoglobin (Hb F) decreases intracellular polymerisation of Hb S. Hb F concentrations are inversely related to morbidity in SCD.4 Increasing Hb F is the most clinically studied approach against sickling. Management Sickling can be interrupted at several key pathways (panel 2). Hydroxyurea, the most prescribed therapy for SCD, causes myelosuppressive-induced Hb F synthesis by decreasing terminal differentiation of erythroid stem cells, resulting in improved red-cell survival and decreased sickling. Hydroxyurea is orally active, safe in the short term, and beneficial in most patients.5 Although hydroxyurea halves pain episodes, pulmonary events, and hospitalisations, 40% of treated patients do not respond or have progressive organ failure.3,4,6 Individual reports of responses to short-chain fatty acids (eg, valproic acid) have been dramatic, with almost complete correction of anaemia. These drugs increase Hb F by affecting globin gene-expression, possibly by inhibition of histone deacetylases.4 They are not myelosuppressive or cross-resistant with hydroxyurea. But existing compounds inhibit stem-cell proliferation and are thus unpredictable clinically. Newly developed orally active short-chain fatty acids and pulse therapy of existing agents may not inhibit stem-cell growth. Another agent that modulates Hb F is 5-azacytidine, which may increase gamma-gene expression by hypomethylation of the gamma promoter, but there are tumorigenic concerns about this drug. Decitabine, a safer analogue of azacytidine, increased total Hb and Hb F in patients resistant to hydroxyurea.7 The agents that modulate Hb F (hydroxyurea, short-chain fatty acids, erythropoietin) may be synergistic. Another therapeutic approach aims to prevent polymerisation-induced damage of the red-cell membrane.3 Membrane injury causes cell dehydration and the formation of the rigid dense cells associated with anaemia and vasoocclusion. Several of the transport systems for cell dehydration have been successfully modulated, including the calcium-activated K+ channel, described by Gardos. Sickling-induced increased cytosolic Ca2+ leads to cell dehydration. Clotrimazole, a Gardos-channel blocker, led to decreased sickling events and improved laboratory variables in pilot work.8 Magnesium pidolate, an inhibitor of the pH sensitive K:Cl co-transport system support, is another anticell-dehydration approach with promise.1,3,9,10–12 New therapies in sickle cell disease Elliott Vichinsky Rapid review THE LANCET • Vol 360 • August 24, 2002 • www.thelancet.com 629 Context New therapies have evolved from our improved understanding of the biology of sickle cell disease (SCD) and the availability of a useful transgenic animal model. Several therapeutic options are available that interrupt the sickling process at various key pathways. Nitric oxide (NO) is a critical factor in the pathophysiology of SCD and is a promising antisickling agent with vasodilation properties. NO regulates blood vessel tone, endothelial adhesion, and the severity of ischaemia-reperfusion injury and anaemia in SCD. Although NO is difficult to administer, its precursor, L-arginine, is an oral supplement. Starting point J R Romero and colleagues recently demonstrated in sickle transgenic mice that oral arginine supplementation induced NO production and reduced red-cell density by inhibiting the Gardos channel, which modulates cell hydration and polymerisation of haemoglobin S (Blood 2002; 99: 1103–08). Haemoglobinopathies can be cured by stem-cell transplantation. This therapy is now accepted treatment in symptomatic children. However, most patients lack a genotypically identical family donor. G La Nasa and colleagues demonstrated unrelated-donor stem-cell transplantation may give similar results to related-donor stem-cell transplantation when extended phenotypic matching is used (Blood 2002; 99: 4350–56). This pilot study offers the possibility of cure to patients without a family donor. Where next Although potential opportunities to prevent morbidity in SCD through new therapies are exciting, most patients do not have access to standard multidisciplinary specialty care. Patients require both. For personal use. Only reproduce with permission from The Lancet Publishing Group. Ex-vivo microcirculation studies show that endothelial adherence of sickle cells activates vaso-occlusion. Any adhesive interaction that impairs flow, delaying the transit time of sickle cells, can initiate or amplify intravascular sickling. Many cellular contact sites and plasma factors are now being targeted.2,3,13–16 Sulphasalazine, an inhibitor of nuclear factor kappa-b modifies endothelial activation,1 and in pilot studies reduced the expression of adhesion molecules and E selectin. Antithrombotic therapy is a growing area,2,3 with increasing evidence that hypercoagulation plays an important role in the pathophysiology of SCD. Sickle cells stimulate the coagulation system by increasing platelet activation, thrombin generation, and fibrinolysis. Pilot studies with anticoagulants (acenocoumarol, n3 fatty acids, heparin) showed improvement in coagulation markers, and, anecdotally, pain.17 Several new drugs alter multiple pathological processes in SCD. These drugs are promising, but require extensive studies to determine the most effective method of use. Poloxamer 188 is a nonionic surfactant copolymer that improves microvascular blood flow,18 decreasing viscosity, thrombosis, frictional forces, and inflammation. Its antiadhesive properties result from blocking of hydrophobic adhesive interactions between erythrocytes and vascular endothelium. In a randomised double-blind placebocontrolled phase 2 study, a 48-h infusion of poloxamer 188 reduced pain intensity and analgesic use, which led to a larger placebo-controlled trial of 255 patients with painful sickle-cell crisis.18 There was significant improvement in the treatment group, but this improvement was associated with only a limited reduction in the duration of painful crises. Two groups of patients have an amplified response to poloxamer 188, including children receiving hydroxyurea. These observations are consistent with previous antisickling drug studies, suggesting that children are more responsive to intervention because they have less irreversible organ injury and that combination therapy is better than monotherapy. Nitric oxide (NO) is a critical factor in the pathophysiology of SCD and is a potentially powerful treatment modality.3,19,20 NO regulates blood vessel tone, endothelial adhesion, leucocytes, and platelet activity, important factors in ischaemia-reperfusion injury and sickle-cell-induced ischaemia. In SCD, more adhesion molecules are produced due to decreased availability of NO. In sickle transgenic mice, oral arginine supplementation induced NO production and reduced red-cell density by inhibiting the Gardos channel.21 Whether the low NO concnetrations are because of decreased endothelial production or increased use is unknown. In SCD, low NO concentrations are associated with low L-arginine, the precursor of NO. Pilot studies treating sickle-cell patients with NO have shown promising antisickling activity with vasodilator properties. Clinical trials with L-arginine supp
lementation appear to correct the NO deficiency and improve pulmonary hypertension. In addition, red-cell adherence to pulmonary endothelium appears to decrease with NO.19,20 NO or arginine supplementation may be synergistic with hydroxyurea, and seems to further increase NO release and decrease adhesives molecules. Transplantation and transfusion Stem-cell transplantation and chronic transfusion can cure or dramatically lessen the severity of SCD.3 The overall survival rate for HLA-identical sibling-donor stem-cell transplantation is 93%, and event-free survival is 82%. Allogeneic bone-marrow transplantation (BMT) is now accepted therapy for symptomatic children. Initially, RAPID REVIEW 630 THE LANCET • Vol 360 • August 24, 2002 • www.thelancet.com Panel 2: Emerging therapeutic agents in SCD Category Mechanism Agent 1) Red-cell Gardos channel inhibition Clotrimazole, rehydration ICA–17403* K:CL co-transport inhibition Mg pidolate Chloride movement blockage NS 1652*, NS 3623* 2) Antiadhesion Red-cell endothelial adhesion RGD peptide Antiadhesion antibodies PAF-induced adhesion Anti-von-Willebrand factor Anti-white-cell adhesion Anti-integrin receptors Endothelial activation Sulphasalazine (inhibitor of NF-kb) 3) Hb F Ribonucleotide reductase Hydroxyurea augmentation Histone deacetylase Short-chain fatty acids DNA hypomethylation 2-deoxy-5-azacytidine Stress erythropoiesis Erythropoietin 4) Antioxidative Chelate membrane iron Deferiprone therapy Glutathione metabolism Glutamine N-acetyl-cysteine 5) Antithrombotic Decrease thrombin Acenocoumarol, heparin therapy Inhibition of platelet activation N3 fatty acids 6) Antisickling Vasodilation Nitric oxide through multiple Decreased adhesion, Arginine pathways polymerisation Flocor (non-ionic Antiadhesion, surfactant) anti-inflammation, increased flow 7) Transfusion Decrease Hb S cells Pheresis therapy Simple transfusion 8) Transplantation Haemopoietic stem cell Allogeneic, nonmyeloablative 9) Gene therapy Direct gene replacement Viral delivery of -globin gene Indirect gene therapy Erythropoietin delivery DNA, RNA repair Cord blood *Anti-cell dehydration research drugs undergoing investigation in SCD. Panel 1: Clinical advances for treatment of SCD Category Intervention 1) Newborn screening Family education Counselling Comprehensive care 2) Infection Prophylactic penicillin Pneumococcal vaccine 3) Brain-injury prevention* Screening with transcranial doppler, MRI, neurocognitive testing 4) Transfusion safety and Phenotypically matched red-cells iron-overload prevention†4 Red-cell pheresis 5) Lung-injury prevention Incentive spirometry Antibiotics, including macrolides Transfusion Prevention with hydroxyurea Screening for pulmonary hypertension 6) Surgery/anaesthesia safety Preoperative transfusion 7) Avascular necrosis of hip‡ Decompression coring procedures 8) Priapism Adrenergic agonist Antiandrogen therapy 9) Pain§ Prevention with hydroxyurea Patient-controlled analgesic devices New non-steroidal anti-inflammatory drugs Sickle day–unit 10) Renal¶ ACE inhibitors for proteinuria Improved renal transplantation 11) Gallbladder disease|| Laproscopic cholecystectomy 12) Severe disease** Allogenic BMT (<16 yr) Chronic transfusions and/or hydroxyurea *Detection by transcrannial doppler and subsequent transfusion of high-risk patients is now recommended. †Routine transfusion with C, E, and Kell red-cells minimises alloimmunisation. Pheresis minimises iron overload. ‡Decompression coring may prevent progression of avascular necrosis of hip. Randomised trial is in process. §Multidisciplinary pain-management decreases hospitalisation rate. ¶Treating use of ACE inhibitors in patients with proteinuria may prevent renal disease. ||Laproscopic cholecystectomy decreases perioperative morbidity. **Recurrent acute chest syndrome, pain crises, or CNS disease are indications of severe disease eligible for transplantation. References available at: http://image.thelancet.com/extras/02art7443webreferences.pdf For personal use. Only reproduce with permission from The Lancet Publishing Group. eligible candidates for BMT were restricted to patients with overt brain injury, recurrent acute chest syndrome, or pain crises. Given its successful outcomes, BMT is increasingly being used for patients with less severe, but symptomatic, diseases.22 This success rate is increasing due to several factors, including better perioperative management, immune suppression, and stem-cell collection. New developments in BMT include mixed donor-host chimerism transplants, unrelated stem-cell transplantation, and cord-blood donors. Non-myeloablative regimens can induce stable mixed chimerism, are ameliorative, and significantly reduce the toxicity of transplantation. This largely outpatient procedure, often referred to as a minitransplant, may have wide applicability. In particular, older patients who have severe organ injury and are excluded from standard allogenic transplantation are good potential candidates for future trials.22 However, the long-term stability of the donor graft is unknown.23 Matched unrelated BMT offers the possibility of cure to patients without genotypically identical family donors. Pilot data showed as great as a 70% success rate. Unfortunately the mortality rate of about 20% remains too high. Improvements in immunosuppression and extended phenotyping should increase the safety of these procedures. Since the 1940s, it has been accepted that chronic transfusions ameliorate or prevent sickle-cell complications. Several recent studies have shown that chronic transfusions can prevent serious sickle-cell events, including central nervous system ischaemia, acute chest syndrome, pain crises, growth failure, and splenic dysfunction.24 Although efficacious, the side-effects of the treatment, such as iron overload and alloimmunisation, prevent widespread use. However, red-cell pheresis can prevent the iron overload, and the routine use of extended red-cell matching limits transfusion reactions. As a result of these improvements, a recent quality-of-life study found similar results in transfusion and BMT patients.25 Gene therapy Gene therapy offers enormous promise, but progress has been slow, due to inadequate gene transfer and efficacy and low gene-expression.26 The identification of a locus control region (LCR), essential for transcription activity of the globin cluster, has accelerated the field. In addition, advances in vector research have resulted in recombinant lentiviruses, with large fragments of the human -globin gene and needed regulatory elements now being available. Recently, Pawliuk and colleagues demonstrated in transgenic sickle models the ability of gene therapy to correct the disease’s pathophysiology.6 To transfer large LCR globin gene sequences, RNA splicing and export control elements of HIV and a lentiviral vector were used. The gene responsible for the antisickling effect of gamma globin was inserted to prevent sickling. Studies on transplants in SAD and Berkeley mice proved transduction with the antisickling gene. Half the total Hb consisted of the new mutant and there was a marked decrease in Hb S polymerisation. Furthermore, irreversible sickle cells, dense cells, Hb concentrations, and reticulocyte counts improved. Clinically there was correction of splenomegaly and urinary deficit. Although several steps are still needed, including improvement in the quantity and safety of lentiviral production, and the development of safer myeloablation regimens, clinical trials with gene therapy are likely. References 1 Solovey AA, Solovey AN, Harkness J, Hebbel RP. Modulation of endothelial cell activation in sickle cell disease: a pilot study. Blood 2001; 97: 1937–41. 2 Tomer A, Harker LA, Kasey S, Eckman JR. Thrombogenesis in sickle cell disease. J Lab Clin Med 2001; 137: 398–407. 3 Mankad VN, ed. Pediatric pathology and molecular medicine. Jan–Feb edn. London: Taylor & Francis, 2001. 4 Atweh GF, Loukopoulos D. Pharmacological induction of fetal hemoglobin in sickle cell disease and beta-thalassemia. Semin Hematol 2001; 38: 367–73. 5 Charache S, Terrin ML, Moore RD, et
al. Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N Engl J Med 1995; 332: 1317–22. 6 Pawliuk R, Westerman KA, Fabry ME, et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001; 294: 2368–71. 7 Koshy M, Dorn L, Bressler L, et al. 2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia. Blood 2000; 96: 2379–84. 8 Brugnara C, Gee B, Armsby CC, et al. Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest 1996; 97: 1227–34. 9 De Franceschi L, Bachir D, Galacteros F, et al. Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease. J Clin Invest 1997; 100: 1847–52. 10 Brugnara C, De Franceschi L, Beuzard Y. Erythrocyte-active agents and treatment of sickle cell disease. Semin Hematol 2001; 38: 324–32. 11 Hebbel RP. Blockade of adhesion of sickle cells to endothelium by monoclonal antibodies. N Engl J Med 2000; 342: 1910–12. 12 Hebbel RP, Boogaerts MA, Eaton JW, Steinberg MH. Erythrocyte adherence to endothelium in sickle-cell anemia. A possible determinant of disease severity. N Engl J Med 1980; 302: 992–95. 13 Kumar A, Eckman JR, Wick TM. Inhibition of plasma-mediated adherence of sickle erythrocytes to microvascular endothelium by conformationally constrained RGD- containing peptides. Am J Hematol 1996; 53: 92–108. 14 Lefkovits J, Topol EJ. Platelet glycoprotein IIb/IIIa receptor inhibitors in ischemic heart disease. Curr Opin Cardiol 1995; 10: 420–26. 15 Udani M, Zen Q, Cottman M, et al. Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin. J Clin Invest 1998; 101: 2550–58. 16 Hillery CA, Du MC, Montgomery RR, Scott JP. Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin. Blood 1996; 87: 4879–86. 17 Tomer A, Kasey S, Connor WE, Clark S, Harker LA, Eckman JR. Reduction of pain episodes and prothrombotic activity in sickle cell disease by dietary n-3 fatty acids. Thromb Haemost 2001; 85: 966–74. 18 Orringer EP, Casella JF, Ataga KI, et al. Purified poloxamer 188 for treatment of acute vaso-occlusive crisis of sickle cell disease: a randomized controlled trial. JAMA 2001; 286: 2099–106. 19 Morris CR. Arginine therapy: a new treatment for pulmonary hypertension in sickle cell disease? Blood 2000; 96 (suppl, part 1): 485a. 20 Morris CR, Kuypers FA, Larkin S, et al. Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease. Br J Haematol 2000; 111: 498–500. 21 Romero JR, Suzuka SM, Nagel RL, Fabry ME. Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity. Blood 2002; 99: 1103–08. 22 Walters MC, Patience M, Leisenring W, et al. Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia. Biol Blood Marrow Transplant 2001; 7: 665–73. 23 La Nasa G, Giardini C, Argiolu F, et al. Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes. Blood 2002; 99: 4350–56. 24 Styles LA, Vichinsky E. Effects of a long-term transfusion regimen on sickle cell-related illnesses. J Pediatrics 1994; 125: 909–11. 25 Nietert PJ, Abboud MR, Silverstein MD, Jackson SM. Bone marrow transplantation versus periodic prophylactic blood transfusion in sickle cell patients at high risk of ischemic stroke: a decision analysis. Blood 2000; 95: 3057–64. 26 Tisdale J, Sadelain M. Toward gene therapy for disorders of globin synthesis. Semin Hematol 2001; 38: 382–92. RAPID REVIEW THE LANCET • Vol 360 • August 24, 2002 • www.thelancet.com 631
Research review paper Taking the good out of the bad: lentiviral-based gene therapy of the hemoglobinopathies Peter B. Stathopulos * Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 Received 12 May 2003; accepted 26 May 2003 Abstract Sickle cell disease and h-thalassemia are excellent candidates for gene therapy since transfer of a single gene into hematopoietic stem cells should theoretically elicit a therapeutic response. Initial attempts at gene therapy of these hemoglobinopathies have proved unsuccessful due to limitations of available gene transfer vectors. With the extensive research on human immunodeficiency virus-1 due to the acquired immune deficiency syndrome pandemic, researchers have realized that this lentivirus, engineered to be devoid of any pathogenic elements, can be an effective gene transfer vector. This review discusses the gene therapy strategy for the hemoglobinopathies and outlines why lentiviralderived vectors are particularly suited for this type of application, keeping past failures at gene therapy of these hemoglobinopathies in mind. Development, improvement, and methods for preparation of lentiviral-derived vectors are examined. Recently published results of successful gene therapy treatment of h-thalassemic and sickle cell diseased mice using lentiviral-derived vectors are described. Finally, criticisms and future directions of lentiviral-based biotechnology are considered. D 2003 Elsevier Inc. All rights reserved. Keywords: Lentiviral vector; Gene therapy; h-Thalassemia; Sickle cell disease; Hemoglobin; Hemoglobinopathy 0734-9750/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0734-9750(03)00102-2 Abbreviations: AIDS, acquired immune deficiency syndrome; CMV, cytomegalovirus; cPT, central polypurine tract; Hb, hemoglobin; HIV-1, human immunodeficiency virus-1; HS, DNase I hypersensitivity sites; LTR, long terminal repeat; PRE, post-transcriptional regulatory element; RCL, replication competent lentivirus; RRE, rev responsive element; SCD, sickle cell disease; snRNA, small nuclear ribonucleic acid; VSV-G, vesicular stomatitis virus glycoprotein G. * Tel.: +1-519-888-4567×6730; fax: +1-519-746-0435. E-mail address: pbstatho@sciborg.uwaterloo.ca (P.B. Stathopulos). www.elsevier.com/locate/biotechadv Biotechnology Advances 21 (2003) 513 – 526 1. Hemoglobinopathies Hemoglobin (Hb) is a large transport protein. In adult vertebrates, two a-globin chains and two h-globin chains form this tetrameric protein following the dimerization of ah protomers. The bright red colour of this macromolecule is a result of four heme complexes associated with the a2h2 quarternary structure. Heme is required for the binding of diatomic oxygen. Hb moves the bound oxygen from the lungs to distal tissues where it is necessary for cellular respiration. In humans, two slightly different a-globin genes are encoded on chromosome 16; however, these genes translate into identical a-globin polypeptide chains. Chromosome 11 contains the h-globin gene. Since humans inherit one maternal set as well as one paternal set of chromosomes during sexual reproduction, offspring acquire a total of four a-globin and two h-globin genes (Karlsson and Nienhuis, 1985). Over 500 globin variants have been described to date, where the vast majority of these variants are the result of single amino acid substitutions that do not manifest disease. The most common group of autosomally inheritable human diseases are the thalassemias. The thalassemias are a class of anemic diseases where there is impaired formation of functional Hb due to reduced or abolished globin synthesis. Alpha-thalassemia is a result of deleted a-globin genes; moreover, the co-inheritance of two deleted genes from each parent results in the most severe form of a-thalassemia where the fetus usually only survives until birth. An individual that inherits 1, 2, or 3 deleted a-globin genes shows an increasing level of anemia, respectively; however, even individuals with three of four missing a-globin genes generally show only mild to moderate anemia. Inherited hthalassemia (Cooley’s anemia) is caused by 1 of more than 200 different mutations affecting the h-globin gene. There are about 80,000,000 carriers of h-thalassemic traits worldwide and in some areas mutant gene frequencies are as high as 10% (Weatherall and Clegg, 1996). Heterozygotes for one h-globin gene mutation are usually asymptomatic; however, compound heterozygotes, where each h-globin gene contains a different mutation, and homozygotes are severely anemic as a result of a large imbalance between a- and h-globin chains. Excess a-chains aggregate and precipitate in red blood cell precursors resulting in abnormal cell maturation and apoptosis (Olivieri, 1999). Red blood cell a-chain inclusions cause hemolytic anemia. The resultant anemia stimulates the synthesis of erythropoietin and propagation of the ineffective marrow. Persistent erythroid cell proliferation in the marrow leads to severe skeletal deformities (Cooley and Lee, 1925). Since erythropoiesis is ineffective, patients must undergo lifelong blood transfusions to survive past the first few years of life (Cooley and Lee, 1925). Although regular blood transfusions are very effective at retarding the progression of thalassemia, patients that receive chronic transfusions have an increased risk of contracting blood-borne infections, have high rates of allo-immunization, and most significantly, develop iron overload which is highly toxic if untreated. Iron chelators such as desferrioxamine are widely used in developed nations to combat iron overload, but the high price excludes them from practical use in underdeveloped nations. Also, since the most effective chelating agents must be parenterally applied, poor compliance results. Hb is the first protein where a point mutation was identified to cause a single amino acid substitution. In sickle cell disease (SCD), a polar glutamate residue at codon 6 of the h-globin gene is substituted with a hydrophobic valine residue (Ingram, 1957). Individuals 514 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 must inherit two copies of the mutant gene for disease manifestation. Up to 25% of West Africans are heterozygous for the glutamate-6-valine mutation, and in the United States alone there are 50,000 homozygotes for SCD. SCD is characterized by sickling of erythrocytes due to polymerization of Hb containing the mutant h-globin. Aggregation is a result of Hb pairing between the mutant valine-6 in the h2 chain of one macromolecule and a hydrophobic pocket (formed by phenylalanine-85 and leucine-88) in the h1 chain of another Hb molecule. Ultimately, fibres with 22 nm diameters, which are readily identifiable by transmission electron microscopy, form in mature erythrocytes (Wishner et al., 1975). Hemolytic anemia results due to these Hb fibres which also cause microcirculatory occlusions and infarctions. Organ damage also occurs due to the deformed, rigid, and adhesive sickle-shaped erythrocytes (Ley et al., 1982). Fetal Hb is the predominant Hb produced prior to birth. In the fetal form, two g-globin chains come together with two a-chains to form an a2g2 tetramer. Fetal Hb is an excellent inhibitor of SCD Hb polymerization; consequently, recent treatment strategies for SCD patients have focused on drugs such as hydroxyurea and 5-azacytidine that increase g-globin expression to fetal levels (Anderson, 1984; Ley, 1991). In the landmark study that first related structural alteration of Hb with symptoms of anemia, Perutz and Lehman (1968) mentioned the possibility of replacing hematopoietic stem cell-containing bone marrow in homozygotes with normal marrow to facilitate a cure for the inherited anemias. Thirty-five years later, allogenic bone marrow transplantation stands as the only cure for the hemoglobinopathies. Allogenic bone marrow transplantation is only available to patients with human leukocyte antigen-matched donors. Potential complications include premature mortality and chronic graft-versus-host disease even in perfect human leukoc
yte antigen sibling-matched grafts (Lucarelli et al., 1990). Also, high transplantation success rates are limited to children since adults are usually in progressive pathophysiological states. Interestingly, Perutz and Lehman (1968) also suggested that repairing the proteins at the genetic level by transduction with wild-type genes was a better strategy, but represented only an imaginable and Utopian view as a cure for the hemoglobinopathies. Thirty-five years ago, repair of proteins at the genetic level was indeed only a vision; however, with advancements in the understanding of molecular biology and the emergence of biotechnology, therapy of the hemoglobinopathies through gene transfer is a very practical notion. 2. Gene therapy strategy for hemoglobinopathies Today, gene transfer into autologous hematopoietic stem cells is a keenly investigated approach that holds great promise and has already demonstrated efficacy in animal models. Since the patient’s own hematopoietic stem cells are used, the need for a histocompatible donor and the immunological complications associated with allogenic bone marrow transplantation are eliminated. Hemoglobinopathies were the first diseases considered for gene therapy since the transfer of a single gene could theoretically elicit a therapeutic effect. The large database of information on the pathophysiology of anemias and the extensive knowledge on globin gene expression make these diseases excellent candidates for correction by gene therapy. The gene therapy strategy for the hemoglobinP.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 515 opathies has four objectives: (1) transfer of a single gene into lineage specific hematopoietic stem cells, (2) endogenous expression of the transgene at high levels, (3) permanent maintenance of expression of the transferred gene, and (4) utilization of a safe nonpathogenic vector. Most available gene delivery techniques do not conform to all four expectations. For example, nonviral vectors, which can be in the form of naked DNA, cationic lipids that bind DNA tightly by electrostatic interactions, or condensed DNA particles, all demonstrate inefficient transduction in vivo; furthermore, gene transfer using nonviral vectors results in only transient expression of genes of interest (Crystal, 1995). More efficient gene transfer is achievable using adenoviral vectors; however, expression is also only transient due to immune responses against cells which express low levels of viral proteins (Knowles et al., 1995). The immune response to adenoviral proteins also restricts this vector to a single dosing. 3. Initial attempts at gene therapy of hemoglobinopathies Onco-retroviral vectors such as those derived from Moloney murine leukemia virus are beneficial for gene therapy since they integrate the gene of interest into the target cells without transferring any viral genes (Miller et al., 1993). In fact, the first optimistic results for transfer of h-globin into hematopoietic stem cells using an onco-retroviral vector appeared back in 1988 (Dzierzak et al., 1988; Karlsson et al., 1988). Gene transfer as well as lineage specific expression was demonstrated, but levels of expression were too low and variable (position-dependent) to be of therapeutic benefit; further, permanency was not demonstrated. Since 1988, studies aimed at increasing expression levels of transferred hglobin genes have focused on including locus control region (LCR) elements of the human h-globin gene cluster as the LCR contains cis-acting DNase I hypersensitivity sites (HS) that are critical for high-level, long-term, position-independent, and erythroid-specific expression (Grosveld et al., 1987; Sadelain et al., 1995). Four of these HS (HS1, HS2, HS3, and HS4) are DNase I insensitive in non-erythroid cells and are normally found upstream of the h-globin gene cluster on chromosome 11 in humans. These four HS are made up of several DNA-binding motifs for transcriptional factors. The binding of transcriptional factors to HS facilitates chromatin opening by displacement or perturbing of nucleosomes. The open chromatin structure accounts for position-independent transgene expression as an open domain can be maintained by the DNA-binding proteins regardless of chromosomal location. Also, this open structure allows for binding of other regulatory elements required for high-level expression of the h-globin gene (Fu et al., 2002). After incorporation of small ( < 1 kb) minimal core HS2, HS3, and HS4 elements into Moloney murine leukemia virus-derived vectors failed to alleviate the h-globin expression problems, the use of larger HS2, HS3, and HS4 spanning elements was considered (Raftopoulos et al., 1997). Unfortunately, incorporation of larger HS spanning elements into onco-retroviral vectors is problematic due to the inability of the vector to incorporate large quantities of genetic material in a stable manner. When this integration is attempted vector rearrangement frequently occurs due to splicing of the retroviral RNA within sites of the larger LCR sequences (Leboulch et al., 1994; Sadelain et al., 1995). Another major limitation of onco-retroviral vectors is the obligate requirement of these 516 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 vectors to transduce cells that divide shortly after infection because the vector RNA cannot migrate into the nucleus due to the presence of a nuclear membrane, and thus, must wait for mitosis to make the transition (Case et al., 1999). Since most hematopoietic stem cells are in a quiescent state, they must be induced to divide in order to achieve higher transduction efficiencies and overall expression levels. The problem with stimulating quiescent hematopoietic stem cells is that they tend to lose long-term repopulating capacities when treated with cell division stimulants such as cytokines (Case et al., 1999). 4. Lentiviral-derived vectors One of the greatest pandemics in medical history is the acquired immune deficiency syndrome (AIDS) crisis. There are currently over 70,000,000 affected individuals and 20,000,000 documented deaths (Gallo, 2002). The virus responsible for this illness, human immunodeficiency virus (HIV-1), was identified in 1983 (Barre-Sinoussi et al., 1983; Zagury et al., 1984). Search for AIDS treatment has sparked intensive research on HIV-1 biology; consequently, much has been elucidated regarding the life cycle and molecular biochemistry of this virus. HIV-1 belongs to a group of retroviruses known as the lentiviruses. Other primate lentiviruses include HIV-2 which is prevalent in West Africa, and simian immunodeficiency virus. Nonprimate lentiviruses exist as well, namely feline immunodeficiency virus, bovine immunodeficiency virus, and equine infectious anemia virus. Lentiviruses require host cells for their propagation and the life cycle is similar to most retroviruses, as depicted in Fig. 1. Initial interest for lentiviral vectors arose with the observation that HIV-1 is able to transduce nondividing cells, as the pre-integration complex can translocate through a fully intact nuclear membrane (Lewis and Emerman, 1994). However, researchers soon realized that the greatest asset of lentiviral vectors in biotechnology is the ability to package fulllength, unspliced RNA. The instability of recombinant viral genomes due to RNA splicing during the preparation of onco-retroviral vectors created for hemoglobinopathy gene therapy is not a concern in lentiviral vectors since HIV-1 expresses machinery which suppresses processing of RNA and allows for nucleocytoplasmic export of long, unspliced RNA (Naldini and Verma, 2000). Hence, HIV-1-derived vectors have a greater capacity for the incorporation of larger LCR sequences required for high h-globin expression. All retroviruses contain the prototypical gag, pol, and env genes, where gag encodes matrix and core proteins, pol encodes reverse transcriptase, integrase, and a protease, and env encodes associated envelope proteins. The HIV-1 genome contains six additional open reading frames. Along with critical splicing control of virion RNA, the rev protein regulates expressi
on levels of gag, pol, and env. The tat regulatory protein is involved in transcriptional control of RNA, and the accessory proteins vif, vpr, vpu, and nef are virulence factors involved in host cell recognition and infection (Trono, 1995). Fig. 2 illustrates the wild-type HIV-1 genome. Due to safety concerns, first generation lentiviral vectors for gene transfer employed a three-plasmid expression system and generated a pseudotyped vector through transient transfection of producer cells (Naldini et al., 1996). The first generation system included a packaging construct that had a deleted env fragment (1.4 kb) and vpu gene, a partially P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 517 Fig. 1. The lentivirus life cycle. [1] Binding of envelope proteins to the plasma membrane of the host cell leads to internalization of the virus and loss of the envelope. [2] Viral reverse transcriptase (RT) synthesizes doublestranded (ds)DNA from the single-stranded (ss)RNA viral genome. [3] The pre-integration complex (dsDNA and associated viral proteins) moves through the nucleus during mitosis or interphase of the host cell cycle. [4] Random and permanent integration of viral dsDNA into the host cell genome forms the provirus. [5] Transcription of the integrated proviral DNA by host cell RNA polymerase forms multiple unspliced, full-length viral ssRNA copies. [6] Processing of full-length viral RNA into spliced mRNA occurs. [7] Viral proteins are translated from the spliced mRNA. [8] Packing of viral proteins and unspliced viral RNA into capsids ensues. [9] Packaged capsids associate with the host cell membrane and budding of new virus particles results. 518 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 deleted packaging sequence (DC), replacement of the 5Vand 3Vlong terminal repeat (LTR) regions responsible for the regulation of viral gene expression, with a strong cytomegalovirus (CMV) promoter and polyadenylation signal of the insulin gene, respectively. A second plasmid encoding an alternative envelope protein, the G glycoprotein of vesicular stomatitis virus (VSV-G) was controlled by the CMV promoter. The VSV-G envelope protein is advantageous due to its higher stability that allows for concentration by ultracentrifugation and since VSV-G pseudotyped viral particles show broad host cell infectivity (Burns et al., 1993). Finally, a third plasmid encoding the expression cassette for the transgene contained all the cis-acting sequences required for encapsidation, reverse transcription, and integration. The transgene plasmid also contained the CMV promoter, intact 5Vas well as 3VLTRs, the portion of the gag gene containing the packaging sequence (C) and a sequence known as the rev responsive element (RRE) that is involved in the control of splicing. By using a multiple plasmid expression system for the construction of the vector and minimizing the number of overlapping sequences between plasmids, the possibility of producing a replication competent lentivirus (RCL) by recombination was greatly reduced. The rev and tat regulatory proteins and the remaining accessory proteins critically involved in pathogenesis from HIV-1 were present in first generation vectors (Naldini et al., 1996). Second generation HIV-1-derived vectors improved on biosafety by deleting HIV-1 genes not required for generating functional vector particles. Second generation vectors used multiple plasmids as described for first generation; however, the packaging plasmid had the vif, vpr, vpu, and nef genes, as well as the entire env gene deleted (Zufferey et al., 1997). In total, five of the nine genes found in wild-type HIV-1 were deleted in these second generation vectors. Third generation vectors deleted tat as well as rev genes from the packaging construct. In wild-type HIV-1 infection, tat activates the promoter in upstream LTR sequences in order to transcribe viral RNA efficiently; however, replacement of these upstream LTR sequences with active CMV promoter sequences made tat redundant (Dull et al., 1998). The trans-acting rev protein was expressed from a fourth plasmid, further decreasing the probability of the emergence of an RCL through recombination. The safety further increased by making the vector self-inactivating. Self-inactivation was achieved by deleting a portion of the U3 region of the 3V LTR. This U3 region contained the TATA box and binding sites for specificity protein-1 and nuclear factor-nB host cell transcriptional factors. During reverse transcription of the viral RNA, this U3 deletion is transferred to the 5V LTR, resulting in transcriptional inactivation of the LTR in integrated proviruses Fig. 2. Structure of the wild-type HIV-1 genome. The gag, pol, and env genes, common to all retrovirus are shaded in grey. Six additional open reading frames encode virulence factors vif, vpr, vpu, and nef, as well as tat and rev trans-acting regulatory proteins. The packaging sequence (C), the rev responsive element (RRE), and the long terminal repeats (LTR)s are indicated. Modified from Follenzi and Naldini (2002). P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 519 (Miyoshi et al., 1998). The possibility of generating an RCL is remote in third generation vectors, and any RCL that does form lacks all factors essential for HIV-1 virulence as well as replication in vivo. Fig. 3 illustrates the constructs used in third generation lentiviral vectors. Further enhancements in the performance of lentiviral vectors have been achieved by incorporating a post-transcriptional regulatory element (PRE) derived from the woodchuck hepatitis virus, into the 3Vend of the vector containing the transgene (Zufferey et al., 1999). The PRE enhances nuclear export of transcribed RNA and increases the polyadenylation efficiency of the transcript, thereby augmenting the amount of mRNA in cells. Transduction efficiency of the vector was increased to wild-type HIV-1 levels by placing a copy of the central polypurine tract (cPT) in the transgene vector. This cPT is a cis-acting Fig. 3. Third generation lentiviral vector for therapy of the hemoglobinopathies. [A] The packaging construct contains the trans-acting gag and pol genes while tat and env have been eliminated. Further, 5Vand 3VLTR regions have been replaced and only a partial packaging sequence remains (DC) flanked by splice acceptor (SA) and donor (SD) sites. [B] The transgene construct contains cis-acting sequences. A full packaging sequence (C), partial gag gene (Dgag), and rev responsive element (RRE) are flanked by SA and SD sites. Human h-globin expression is under the control of the h-globin promoter (P) and the h-globin locus control region (LCR) present with large DNase I hypersensitivity (HS)2, HS3, and HS4 elements. A woodchuck post-transcriptional regulatory element (PRE) enhances nuclear export of transcribed RNA and the central polypurine tract (cPT) is added to increase transduction efficiency. The 3VLTR is partially deleted to make the construct self-inactivating (SIN). The 5VLTR is replaced by a strong cytomegalovirus (CMV) promoter. [C] The envelope construct encodes vesicular stomatitis glycoprotein G (VSV-G) under the control of CMV promoter for pseudotyping of lentiviral particles. [D] The trans-acting rev gene is encoded on a separate plasmid to reduce the possibility of creating a replication competent lentivirus (RCL). Wild-type HIV-1 elements are shaded in grey. Modified from Brenner and Malech (2003) and Imren et al. (2002). 520 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 sequence that is located in the pol gene of wild-type HIV-1 and is an element required for nuclear transport of the pre-integration complex through the nuclear membrane of quiescent cells (Follenzi et al., 2000). 5. Methods for preparation of lentiviral vectors Constructs used for lentiviral vector production are maintained as bacterial plasmids that are propagated in Escherichia coli cells. It is critical to ensure purified constructs grown in E. coli are free of endotoxins. Third generation lentiviral vectors are prepared by transient co-transfection of human embryonic kidney 293T
cells with the multiple plasmids; moreover, transfection is routinely done using calcium phosphate precipitation or lipid-based methods (Follenzi and Naldini, 2002). The 293T cells are plated in Iscove’s modified Dulbecco’s medium, containing 10% fetal bovine serum, 25 U/ml of penicillin, and 25 U/ml of streptomycin; moreover, the medium is replaced 14– 16 h after DNA treatment. The cell supernatant (medium) is exchanged and collected at 24, 48, and 72 h (Follenzi and Naldini, 2002). VSV-G pseudotyped lentiviral particles are concentrated by ultracentrifugation at 50,000 g for 140 min. Viral pellets that are resuspended in 1:500 of the starting volume of medium in sterile phosphate-buffered saline containing 0.5% of bovine serum albumin can yield titres of more than 1010 transducing units/ml (Follenzi and Naldini, 2002; Reiser et al., 2000). 6. Applying lentiviral vectors to treatment of hemoglobinopathies In the hallmark study using this lentiviral vector-mediated gene therapy strategy, hglobin transduced bone marrow cells not subjected to selection, were transplanted into lethally irradiated normal C57Bl/6 mice (May et al., 2000). Cells transduced with vectors containing minimal HS2 (423 bp), HS3 (280 bp), and HS4 (283 bp) elements demonstrated an average vector copy number of 1.8 per peripheral blood cell with h-globin transcript levels dropping from 7% to about 2.8% after 24 weeks; however, cells that were transduced with vectors containing larger encompassing HS2 (840 bp), HS3 (1308 bp), and HS4 (1069 bp) elements maintained h-globin transcript levels in the 10– 20% range during the same time period even though vector copy number per peripheral blood cell was estimated at 0.8 (May et al., 2000). These in vivo results suggested that cells transduced with the larger LCR elements are more resistant to transcriptional silencing, as previously predicted in onco-retroviral vector studies. Therapeutic efficacy was also tested in this same study using heterozygous mice that lacked a copy of both murine h-globin genes (Hbbth3/ +) and showed clinical characteristics similar to human h-thalassemia (Yang et al., 1995). Hbbth3/ + mice underwent lethal irradiation followed by transplantation of unselected bone marrow cells transduced with lentiviral vectors containing the human hglobin gene and associated large LCR elements. Fifteen weeks post-transplantation, five out of five investigated Hbbth3/ + mice showed marked improvements in hematocrit level, red blood cell count, reticulocyte count, and Hb levels, while control mice transplanted with Hbbth3/ + cells that were transduced with a vector encoding enhanced green P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 521 fluorescent protein remained severely anemic. May et al. (2000) were the first to demonstrate high efficiency h-globin gene transfer and expression in the therapeutic range using a lentiviral vector containing a large human LCR configuration and h-globin gene. While wild-type human h-globin inhibits SCD Hb polymerization only at very high concentrations, g-globin is effective at much lower concentrations (Bookchin and Nagel, 1971). The glutamine codon believed responsible for the anti-sickling activity of g-globin aligns with threonine at codon 87 of the human h-globin gene (Nagel et al., 1979). In another very significant paper, researchers applied the lentiviral approach introduced by May et al. (2000) to SCD by generating vectors containing mutant threonine-87-glutamine h-globin genes (Pawliuk et al., 2001). Mutant threonine-87-glutamine h-globin was almost as potent as wild-type g-globin in inhibition of SCD Hb polymerization (Pawliuk et al., 2001). The threonine-87-glutamine h-globin gene was introduced into lethally irradiated C57Bl/6 recipient mice via transduced, nonselected bone marrow cells. Proviral copy number was about 3 per peripheral blood cell genome and was stable for at least 3 months; further, long-term secondary transplants were generated using marrow of a primary recipient mouse killed 5 months after initial transplantation (Pawliuk et al., 2001). The therapeutic efficacy of this lentiviral vector carrying the mutant h-globin gene was tested in two SCD models. The SAD SCD mouse model expresses human a-globin together with a human mutant h-globin chain containing two mutations that are very prone to inducing Hb polymerization (Trudel et al., 1991). In BERK transgenic mice, human aglobin along with the human mutant h-globin that is responsible for SCD is expressed, while all endogenous murine a- and h-globin expression is disrupted (Paszty et al., 1997). The BERK model demonstrates a more severe anemia than the SAD model due to suboptimal expression of the human h-globin gene resulting in thalassemia (Nagel and Fabry, 2001). Transduction of the lentiviral vector containing the threonine-87-glutamine h-globin gene into bone marrow cells and transplantation into lethally irradiated mice resulted in pancellular, erythroid-specific expression sufficient to ameliorate the pathological features characteristic of SCD in both models (Pawliuk et al., 2001). Erythrocyte counts, Hb levels, reticulocyte counts, the number of irreversibly sickled cells and urine concentrations all approached C57Bl/6 control values in both models. Successful gene therapy of SCD requires expression of the therapeutic gene in most red blood cells to prevent vaso-occlusive events that can be caused by a very small fraction of erythrocytes. Pawliuk et al. (2001) showed balanced expression of the variant h-globin that was sufficiently high and homogeneous to cure SCD. Most recent hemoglobinopathy gene therapy studies have focused on treatment of hthalassemia. In a follow-up study, May et al. (2002) showed that a sustained increase of 3– 4 g/dl of Hb is sufficient to correct ineffective erythropoiesis associated with hthalassemia. The third generation lentiviral vector employed persistently augmented Hb levels without any signs of transcriptional silencing for over 40 weeks. Extramedullary hematopoiesis was abolished and hematological parameters were normalized in Hbbth3/ + h-thalassemic mice (May et al., 2002). In an independent study, researchers demonstrated permanent and panerythroid correction of h-thalassemia in mice that were homozygous for a deletion in the major murine h-globin producing gene (Hbbth1/th1) (Imren et al., 2002). Lentiviral vector transduction of hematopoietic stem cells resulted in about 3 integrated 522 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 proviral copies per genome and expression was sustained for greater than 7 months in both primary and secondary transplant recipients. After 7 months, about 95% of erythrocytes contained the human h-globin protein (Imren et al., 2002). Of special interest was the complete correction of all disease manifestations in both primary as well as secondary Hbbth1/th1 recipient mice, prompting the suggestion that in light of therapeutic permanency, human clinical trials should be initiated shortly. Imren et al. (2002) achieved better transduction efficiencies than May et al. (2000) (3 versus 0.8 proviral copies per genome), by incorporating the cPT into the transgene vector. The multiple proviral integration alleviated position effect variegation. A few h-thalassemic mutations cause aberrant splicing in introns within the h-globin gene, leading to incorrectly spliced mRNAs and improper translation. Very recently, a lentiviral vector delivery system has been employed for the transfer and expression of an antisense small nuclear (sn)RNA directed to a splicing enhancer sequence located between aberrant h-globin splice sites (Vacek et al., 2003). SnRNAs were selected since they are expressed in high levels independent of the cell cycle, concentrated in the nucleus, and nuclease resistant, while the lentiviral system was selected due to the ability to transduce quiescent cells. Genes modified to express the appropriate antisense snRNA were inserted in the transgene vector without the need for bulky LCR elements. Upon transduction of cervical carcinoma HeLa cells modelling h-thalassemic intron mutations
, expression of full-length h-globin protein was observed. Transduction of the same vector into erythroid progenitor cells from actual h-thalassemic patients resulted in expression of normal Hb in vitro (Vacek et al., 2003). In this approach, globin expression occurs from the natural chromosomal environment and is properly controlled by a native, full-sized LCR. Considering that up to 15% of point mutations contributing to genetically inherited diseases are due to aberrant splicing, this approach is broadly applicable (Krawczak et al., 1995). Another recently examined approach for treatment of thalassemia that is applicable to SCD is lentiviral-mediated transfer of a human g-globin gene. Using a g-globin gene containing third generation lentiviral vector under the control of the h-globin LCR elements previously described, researchers found that Hbbth3/ + thalassemic mice transplanted with transduced bone marrow expressed high levels of Hb with incorporated gglobin (Persons et al., 2003). An increase of 2.5 g/dl of Hb and normalization in erythrocyte morphology relative to control animals was observed. Unfortunately, the efficacy of the g-globin containing lentiviral vector was not tested in SCD models where it would have much more practical use considering that g-globin is a much better inhibitor of SCD Hb polymerization than h-globin. 7. Criticisms and future considerations The greatest concern with lentiviral vectors is safety against the pathogenicity of HIV-1. To address this issue, the newest vector systems contain only three of the nine wild-type HIV-1 genes and over 60% of the viral genome has been removed. Also, it is often argued that by using up to four separate plasmids for the generation of the vector, the probability of generating an RCL is very low. The separation of the lentiviral rudiments on distinct plasmids does not completely eliminate the possibility of recombination events from P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 523 occurring between plasmids. However, multiple events are required to produce an RCL using third generation vectors. In general, recombinant events are kept to a minimum by diminishing the number of overlapping sequences within and between plasmids. Modifications such as the creation of self-inactivating vectors further reduce the probability of spawning an RCL. The use of lentiviral vectors derived from nonprimates, such as equine infectious anemia virus, is an alternative approach. Equine infectious anemia virus-derived vectors may be safer because they have not shown any pathogenicity in humans (Sellon et al., 1994). Nonprimate lentiviral vector use is limited by the fact that much less is known about the molecular biology of these vectors versus HIV-1. The potential for the generation of an RCL is not the only safety issue in the lentiviral-based approach. As with any retrovirus for the transfer of genetic material into a host cell genome, there exists the possibility of an insertional mutageneic event. Since current lentiviral transduction technique and vector design make no provisions for directed insertion into the genome of interest, activation of an oncogene or the deletion of a critical tumour suppressor gene are unavoidable risks. Considering that permanent and panerythroid expression of h-globin has been observed only when there are multiple proviral insertions, the mutagenesis risk is amplified further due to the need for multiple gene insertions. The development of lentiviral vectors that can insert transgenes into directed chromosomal locations, diminishing the probability of insertional mutagenesis, is an important direction for the lentiviral approach. Currently, there is no literature describing this type of focus with respect to lentiviral gene therapy of the hemoglobinopathies. Another criticism of the current lentiviral system is the viral reverse transcriptase (RT). The RT encoded in pol of HIV- 1 has a low fidelity; therefore, replacement of the RT portion of pol with a higher fidelity RT from another retrovirus may increase the efficiency of the lentiviral gene therapy system. There are no current examples of such a modification in the literature. The very promising results in mouse models of SCD and thalassemia have prompted some researchers to suggest that human clinical trials commence immediately. Although mammalian, mouse physiology is distinct from human physiology; therefore, it would be prudent to test this therapeutic approach in primate models of the hemoglobinopathies. Studies using lentiviral gene therapy to correct hemoglobinopathies in primates have not appeared in the literature, but this research is likely ongoing and results are expected shortly. If this approach shows correction, permanency, and high efficacy in primates, human trials will be around the corner. Safety concerns will not all be solved by the time clinical trials start, but the relative risk will be low and the benefit of successful therapy will be high. A cure for the hemoglobinopathies using a gene transfer approach that was once believed to be a Utopian idea by Perutz and Lehman (1968) is viable today due to advances in the biotechnology of lentiviral vectors. References Anderson WF. Prospects for human gene therapy. Science 1984;226:401 – 9. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983;220:868 – 71. Bookchin RM, Nagel RL. Ligand-induced conformational dependence of hemoglobin in sickling interactios. J Mol Biol 1971;60:263 – 70. 524 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 Brenner S, Malech HL. Current developments in the design of onco-retrovirus and lentivirus vector systems for hematopoietic cell gene therapy. Biochim Biophys Acta 2003;1640:1 – 24. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 1993;90:8033 – 7. Case SS, Price MA, Jordan CT, Yu XJ, Wang L, Bauer G, et al. Stable transduction of quiescent CD34(+)CD38( ) human hematopoietic cells by HIV-1-based lentiviral vectors. Proc Natl Acad Sci U S A 1999;96:2988 – 93. Cooley TB, Lee P. Series of cases of splenomegaly in children with anemia and peculiar bone changes. Trans Am Pediatr Soc 1925;37:29. Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science 1995;270:404 – 10. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998;72:8463 – 71. Dzierzak EA, Papayannopoulou T, Mulligan RC. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature 1988;331:35 – 41. Follenzi A, Naldini L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol 2002;346:454 – 65. Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000;25:217 – 22. Fu XH, Liu DP, Liang CC. Chromatin structure and transcriptional regulation of the beta-globin locus. Exp Cell Res 2002;278:1 – 11. Gallo RC. Historical essay. The early years of HIV/AIDS. Science 2002;298:1728 – 30. Grosveld F, van Assendelft GB, Greaves DR, Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 1987;51:975 – 85. Imren S, Payen E, Westerman KA, Pawliuk R, Fabry ME, Eaves CJ, et al. Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc Natl Acad Sci U S A 2002;99:14380 – 5. Ingram VM. Gene mutations in human humoglobin: the chemical difference between normal and sickle humoglobin. Nature 1957;180:326 – 8. Karlsson S, Bodine DM, Perry L, Papayannopoulou T, Nienhuis AW. Expressi
on of the human beta-globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice. Proc Natl Acad Sci U S A 1988;85:6062 – 6. Karlsson S, Nienhuis AW. Developmental regulation of human globin genes. Ann Rev Biochem 1985;54: 1071 – 108. Knowles MR, Hohneker KW, Zhou Z, Olsen JC, Noah TL, Hu PC, et al. A controlled study of adenoviralvector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 1995; 333:823 – 31. Krawczak M, Smith-Sorensen B, Schmidtke J, Kakkar VV, Cooper DN, Hovig E. Somatic spectrum of cancerassociated single basepair substitutions in the TP53 gene is determined mainly by endogenous mechanisms of mutation and by selection. Human Mutat 1995;5:48 – 57. Leboulch P, Huang GM, Humphries RK, Oh YH, Eaves CJ, Tuan DY, et al. Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure. EMBO J 1994;13:3065 – 76. Lewis PF, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994;68:510 – 6. Ley TJ. The pharmacology of hemoglobin switching: of mice and men. Blood 1991;77:1146 – 52. Ley TJ, DeSimone J, Anagnou NP, Keller GH, Humphries RK, Turner PH, et al. 5-Azacytidine selectively increases gamma-globin synthesis in a patient with beta+ thalassemia. N Engl J Med 1982;307: 1469 – 75. Lucarelli G, Galimberti M, Polchi P, Angelucci E, Baronciani D, Giardini C, et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990;322:417 – 21. May C, Rivella S, Callegari J, Heller G, Gaensler KM, Luzzatto L, et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000;406:82 – 6. P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526 525 May C, Rivella S, Chadburn A, Sadelain M. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene. Blood 2002;99:1902 – 8. Miller AR, McBride WH, Dubinett SM, Dougherty GJ, Thacker JD, Shau H, et al. Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: comparison of immunologic properties with interleukin-2. Blood 1993;82:3686 – 94. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol 1998;72:8150 – 7. Nagel RL, Fabry ME. The panoply of animal models for sickle cell anaemia. Br J Haematol 2001;112:19 – 25. Nagel RL, Bookchin RM, Johnson J, Labie D, Wajcman H, Isaac-Sodeye WA, et al. Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S. Proc Natl Acad Sci U S A 1979;76:670 – 2. Naldini L, Verma IM. Lentiviral vectors. Adv Virus Res 2000;55:599 – 609. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263 – 7. Olivieri NF. The beta-thalassemias. N Engl J Med 1999;341:99 – 109. Paszty C, Brion CM, Manci E, Witkowska HE, Stevens ME, Mohandas N, et al. Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 1997;278:876 – 8. Pawliuk R, Westerman KA, Fabry ME, Payen E, Tighe R, Bouhassira EE, et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001;294:2368 – 71. Persons DA, Hargrove PW, Allay ER, Hanawa H, Nienhuis AW. The degree of phenotypic correction of murine beta-thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number. Blood 2003;101:2175 – 83. Perutz MF, Lehmann H. Molecular pathology of human haemoglobin. Nature 1968;219:902 – 9. Raftopoulos H, Ward M, Leboulch P, Bank A. Long-term transfer and expression of the human beta-globin gene in a mouse transplant model. Blood 1997;90:3414 – 22. Reiser J, Lai Z, Zhang XY, Brady RO. Development of multigene and regulated lentivirus vectors. J Virol 2000;74:10589 – 99. Sadelain M, Wang CH, Antoniou M, Grosveld F, Mulligan RC. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene. Proc Natl Acad Sci U S A 1995;92:6728 – 32. Sellon DC, Fuller FJ, McGuire TC. The immunopathogenesis of equine infectious anemia virus. Virus Res 1994;32:111 – 38. Trono D. HIV accessory proteins: leading roles for the supporting cast. Cell 1995;82:189 – 92. Trudel M, Saadane N, Garel MC, Bardakdjian-Michau J, Blouquit Y, Guerquin-Kern JL, et al. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO J 1991;10:3157 – 65. Vacek MM, Ma H, Gemignani F, Lacerra G, Kafri T, Kole R. High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA. Blood 2003;101:104 – 11. Weatherall DJ, Clegg JB. Thalassemia—a global public health problem. Nat Med 1996;2:847 – 9. Wishner BC, Ward KB, Lattman EE, Love WE. Crystal structure of sickle-cell deoxyhemoglobin at 5 A resolution. J Mol Biol 1975;98:179 – 94. Yang B, Kirby S, Lewis J, Detloff PJ, Maeda N, Smithies O. A mouse model for beta 0-thalassemia. Proc Natl Acad Sci U S A 1995;92:11608 – 12. Zagury D, Bernard J, Leibowitch J, Safai B, Groopman JE, Feldman M, et al. HTLV-III in cells cultured from semen of two patients with AIDS. Science 1984;226:449 – 51. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997;15:871 – 5. Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999;73:2886 – 92. 526 P.B. Stathopulos / Biotechnology Advances 21 (2003) 513–526
12 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease Michel Sadelain* MD, PhD Stefano Rivella 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. 1521-6926/$ – see front matter Q 2004 Elsevier Ltd. All rights reserved. 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 * Corresponding author. Tel.: C1-212-639-6190. E-mail address: m-sadelain@ski.mskcc.org (M. Sadelain). 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. 518 M. Sadelain et al 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 50 end and the adult bglobin gene at the 30 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 sugges
ted 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 50 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 519 β-globin gene LCR βe+ βe+ 1 kb SA RRE SD Ψ Ψ Ψ Ψ Ψ Ψ 5´LTR βA-87Thr GFP PGK ∆L cPPT HS2 HS3 HS4 I8 H WPRE K GFP p HS2 HS3 HS4 p 3´LTR HS2, 3, 4 p G A : RNS1 B : TNS9 C : β87 E : HS40/I8/K F : G.W/P WPRE * D : HS40/I8/K ± γRE/βe+ s.n. HS2 HS3 HS4s.n. βp 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 30 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) d432bA g113 was generated using the b-globin promoter (K130), fusing the b-globin 50 promoter untranslated sequences to the Ag-globin coding sequence at position 3 and C 1 relative to the endogenous translational start site. Also, d432bAg113 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 d432bA g113, 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: d432bAg113, d432bAg113 b-30 enhancer (b 30 Enh) and d432bAg113 g-globin 30 regulatory element (g 30 RE). The b 30 Enh or the g 30 RE were placed downstream of the g-globin coding sequences. Moreover, d432bAg113 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. 520 M. Sadelain et al 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 50 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 521 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 s
ubfragments 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 30 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, 522 M. Sadelain et al 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 30 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 d432bAg113 (Figure 1) and tested in mice affected by b-thalassemia intermedia. The vector d432bAg was generated using the b-globin promoter (K130), fusing the b-globin 50 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 d432bA g is smaller (2.0 kb) than TNS9 and the promoter is 130 bp long. Persons et al113 generated three globin vectors: d432bAg113, d432bAg113$b-30 enhancer (b 30 Enh) and d432bAg113 g-globin 30 regulatory element (g 30 RE). The b 30 Enh or the g 30 RE were placed downstream of the g-globin coding sequences. Moreover, d432bAg113 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 523 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-nul
l 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 524 M. Sadelain et al 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 525 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 d432bAg (Fig. 1), d432bAg b-30 or d432bAg g-30 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 d432bAg 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.123 526 M. Sadelain et al 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 d432bAg 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, Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 527 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 30 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 T
halassemia and the Cooley’s Anemia Foundation. REFERENCES 1. Weatherall DJ & Clegg JB. The Thalassemia Syndrome. Oxford: Blackwell Scientific; 1981. 2. Stamatoyannopoulos G, Nienhuis AW, Majerus P & Varmus H. The Molecular Basis of Blood Disease. Philadelphia: WB Saunders; 1994. 3. Weatherall DJ. Phenotype–genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet 2001; 2: 245–255. 4. Steinberg MH, Forget BG, Higgs DR & Nagel RL. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge: Cambridge University Press; 2001. 5. Cooley TB & Lee P. A series of cases of splenomegaly in children with anemia and peculiar bone changes. Trans Am Pediatr Soc 1925; 37: 29. 6. Giardina PJ & Grady RW. Chelation therapy in beta-thalassemia: an optimistic update. Semin Hematol 2001; 38: 360–366. 7. Giardini C & Lucarelli G. Bone marrow transplantation in the treatment of thalassemia. Curr Opin Hematol 1994; 1: 170–176. 8. Boulad F et al. Bone marrow transplantation for homozygous beta-thalassemia. The Memorial SloanKettering Cancer Center experience. Ann NY Acad Sci 1998; 850: 498–502. 528 M. Sadelain et al 9. Lucarelli G et al. Bone marrow transplantation in adult thalassemic patients. Blood 1999; 93: 1164–1167. 10. Tisdale J & Sadelain M. Toward gene therapy for disorders of globin synthesis. Semin Hematol 2001; 38: 382–392. 11. Steinberg MH, Forget BG, Higgs DR & Nagel RL. Molecular Mechanism of b Thalassemia. Cambridge: Cambridge University Press; 2001. 12. Pauling L, Itano HA, Singer SJ & Wells IC. Sickle cell anemia, a molecular disease. Science 1949; 110: 543–546. 13. Swank RA & Stamatoyannopoulos G. Fetal gene reactivation. Curr Opin Genet Dev 1998; 8: 366–370. 14. Platt OS et al. Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia. J Clin Invest 1984; 74: 652–656. 15. Charache S et al. Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia. Blood 1992; 79: 2555–2565. 16. Atweh GF & Loukopoulos D. Pharmacological induction of fetal hemoglobin in sickle cell disease and beta-thalassemia. Semin Hematol 2001; 38: 367–373. 17. Vermylen C et al. Haematopoietic stem cell transplantation for sickle cell anaemia: the first 50 patients transplanted in Belgium. Bone Marrow Transplant 1998; 22: 1–6. 18. Luzzatto L & Goodfellow P. Sickle cell anaemia. A simple disease with no cure. Nature 1989; 337: 17–18. 19. Sadelain M. Genetic treatment of the haemoglobinopathies: recombinations and new combinations. Br J Haematol 1997; 98: 247–253. 20. Leboulch P et al. Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure. EMBO J 1994; 13: 3065–3076. 21. Sadelain M, Wang CH, Antoniou M et al. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene. Proc Natl Acad Sci USA 1995; 92: 6728–6732. *22. May C et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000; 406: 82–86. 23. Higgs DR. Do LCRs open chromatin domains? Cell 1998; 95: 299–302. 24. Bulger M & Groudine M. Looping versus linking: toward a model for long-distance gene activation. Genes Dev 1999; 13: 2465–2477. 25. Grosveld F. Activation by locus control regions? Curr Opin Genet Dev 1999; 9: 152–157. 26. Engel JD & Tanimoto K. Looping, linking, and chromatin activity: new insights into beta-globin locus regulation. Cell 2000; 100: 499–502. 27. Navas PA et al. Activation of the beta-like globin genes in transgenic mice is dependent on the presence of the beta-locus control region. Hum Mol Genet 2002; 11: 893–903. 28. de Krom M, van de Corput M, von Lindern M et al & Strouboulis J. Stochastic patterns in globin gene expression are established prior to transcriptional activation and are clonally inherited. Mol Cell 2002; 9: 1319–1326. 29. Bulger M, Sawado T, Schubeler D & Groudine M. ChIPs of the beta-globin locus: unraveling gene regulation within an active domain. Curr Opin Genet Dev 2002; 12: 170–177. 30. Levings PP & Bungert J. The human beta-globin locus control region. Eur J Biochem 2002; 269: 1589–1599. 31. Trudel M & Costantini F. A 30 enhancer contributes to the stage-specific expression of the human beta-globin gene. Genes Dev 1987; 1: 954–961. 32. Trudel M, Magram J, Bruckner L & Costantini F. Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice. Mol Cell Biol 1987; 7: 4024–4029. 33. Antoniou M, deBoer E, Habets G & Grosveld F. The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers. EMBO J 1988; 7: 377–384. *34. Grosveld F, van Assendelft GB, Greaves DR & Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 1987; 51: 975–985. 35. Forrester WC et al. A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus. Genes Dev 1990; 4: 1637–1649. 36. Li Q, Zhang M, Duan Z & Stamatoyannopoulos G. Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region. Genomics 1999; 61: 183–193. Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 529 37. Philipsen S, Talbot D, Fraser P & Grosveld F. The beta-globin dominant control region: hypersensitive site 2. EMBO J 1990; 9: 2159–2167. 38. Talbot D, Philipsen S, Fraser P & Grosveld F. Detailed analysis of the site 3 region of the human beta-globin dominant control region. EMBO J 1990; 9: 2169–2177. 39. Li Q, Harju S & Peterson KR. Locus control regions: coming of age at a decade plus. Trends Genet 1999; 15: 403–408. 40. Talbot D & Grosveld F. The 50 HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites. EMBO J 1991; 10: 1391–1398. 41. Jackson JD, Petrykowska H, Philipsen S et al. Role of DNA sequences outside the cores of DNase hypersensitive sites (HSs) in functions of the beta-globin locus control region. Domain opening and synergism between HS2 and HS3. J Biol Chem 1996; 271: 11871–11878. 42. Bungert J et al. Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. Genes Dev 1995; 9: 3083–3096. 43. Ney PA et al. Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner. Mol Cell Biol 1993; 13: 5604–5612. 44. Fraser P, Pruzina S, Antoniou M & Grosveld F. Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev 1993; 7: 106–113. 45. Johnson KD et al. Cooperative activities of hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin domain. Proc Natl Acad Sci USA 2002; 99: 11760–11765. 46. Li Q & Stamatoyannopoulos G. Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator. Blood 1994; 84: 1399–1401. 47. Li Q, Zhang M, Han H, Rohde A & Stamatoyannopoulos G. Evidence that DNase I hypersensitive site 5 of the human beta-globin locus control region functions as a chromosomal insulator in transgenic mice. Nucleic Acids Res 2002; 30: 2484–2491. 48. Talbot D et al. A dominant control region from the human beta-globin locus conferring integration siteindependent gene expression. Nature 1989; 338: 352–355. 49. Pasceri P, Pannell D, Wu X & Ellis J. Full activity from human beta-globin locus control region transgenes requires 5(HS1, distal beta-globin promoter, and 30 beta-globin sequences. Blood 1998; 92: 653–663. 50. Sharpe JA et al. Role of upstream
DNase I hypersensitive sites in the regulation of human alpha globin gene expression. Blood 1993; 82: 1666–1671. 51. Molete JM et al. Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement. Mol Cell Biol 2001; 21: 2969–2980. 52. Bender MA et al. Targeted deletion of 50 HS1 and 50 HS4 of the beta-globin locus control region reveals additive activity of the DNaseI hypersensitive sites. Blood 2001; 98: 2022–2027. 53. Bender MA et al. Description and targeted deletion of 50 hypersensitive site 5 and 6 of the mouse betaglobin locus control region. Blood 1998; 92: 4394–4403. 54. Bender MA, Bulger M, Close J & Groudine M. Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region. Mol Cell 2000; 5: 387–393. 55. Ley TJ et al. Reduced beta-globin gene expression in adult mice containing deletions of locus control region 50 HS-2 or 50 HS-3. Ann NY Acad Sci 1998; 850: 45–53. 56. Reik A et al. The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells. Mol Cell Biol 1998; 18: 5992–6000. 57. Li Q, Peterson KR, Fang X & Stamatoyannopoulos G. Locus control regions. Blood 2002; 100: 3077–3086. 58. Grosveld F et al. The dynamics of globin gene expression and position effects. Novartis Found Symp 1998; 214: 67–79. 59. Milot E et al. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell 1996; 87: 105–114. 60. Wijgerde M, Grosveld F & Fraser P. Transcription complex stability and chromatin dynamics in vivo. Nature 1995; 377: 209–213. 530 M. Sadelain et al 61. Dillon N, Trimborn T, Strouboulis J et al. The effect of distance on long-range chromatin interactions. Mol Cell 1997; 1: 131–139. 62. Peterson KR & Stamatoyannopoulos G. Role of gene order in developmental control of human gammaand beta-globin gene expression. Mol Cell Biol 1993; 13: 4836–4843. 63. Carter D, Chakalova L, Osborne CS et al. Long-range chromatin regulatory interactions in vivo. Nat Genet 2002; 32: 623–626. 64. Dekker J, Rippe K, Dekker M & Kleckner N. Capturing chromosome conformation. Science 2002; 295: 1306–1311. 65. Tolhuis B, Palstra RJ, Splinter E et al. Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol Cell 2002; 10: 1453–1465. 66. Hu X et al. Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region. Proc Natl Acad Sci USA 2003; 100: 1111–1115. 67. Tuan D, Kong S & Hu K. Transcription of the hypersensitive site HS2 enhancer in erythroid cells. Proc Natl Acad Sci USA 1992; 89: 11219–11223. 68. Blackwood EM & Kadonaga JT. Going the distance: a current view of enhancer action. Science 1998; 281: 61–63. 69. Ashe HL, Monks J, Wijgerde M et al. Intergenic transcription and transinduction of the human betaglobin locus. Genes Dev 1997; 11: 2494–2509. 70. Kiekhaefer CM, Grass JA, Johnson KD, Boyer ME & Bresnick EH. Hematopoietic-specific activators establish an overlapping pattern of histone acetylation and methylation within a mammalian chromatin domain. Proc Natl Acad Sci USA 2002; 99: 14309–14314. 71. Cone RD, Weber-Benarous A, Baorto D & Mulligan RC. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol 1987; 7: 887–897. 72. Karlsson S, Papayannopoulou T, Schweiger SG, Stamatoyannopoulos G & Nienhuis AW. Retroviralmediated transfer of genomic globin genes leads to regulated production of RNA and protein. Proc Natl Acad Sci USA 1987; 84: 2411–2415. 73. Dzierzak EA, Papayannopoulou T & Mulligan RC. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature 1988; 331: 35–41. 74. Bender MA, Gelinas RE & Miller AD. A majority of mice show long-term expression of a human betaglobin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol 1989; 9: 1426–1434. 75. Bodine DM, Karlsson S & Nienhuis AW. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci USA 1989; 86: 8897–8901. 76. Plavec I, Papayannopoulou T, Maury C & Meyer F. A human beta-globin gene fused to the human betaglobin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells. Blood 1993; 81: 1384–1392. 77. Chang JC, Liu D & Kan YW. A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression. Proc Natl Acad Sci USA 1992; 89: 3107–3110. 78. Novak U, Harris EA, Forrester W et al. High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells. Proc Natl Acad Sci USA 1990; 87: 3386–3390. 79. Ellis J et al. Evaluation of beta-globin gene therapy constructs in single copy transgenic mice. Nucleic Acids Res 1997; 25: 1296–1302. 80. Ren S et al. Production of genetically stable high-titer retroviral vectors that carry a human gammaglobin gene under the control of the alpha-globin locus control region. Blood 1996; 87: 2518–2524. 81. Lung HY, Meeus IS, Weinberg RS & Atweh GF. In vivo silencing of the human gamma-globin gene in murine erythroid cells following retroviral transduction. Blood Cells Mol Dis 2000; 26: 613–619. 82. Emery DW, Morrish F, Li Q & Stamatoyannopoulos G. Analysis of gamma-globin expression cassettes in retrovirus vectors. Hum Gene Ther 1999; 10: 877–888. 83. Sabatino DE et al. A minimal ankyrin promoter linked to a human gamma-globin gene demonstrates erythroid specific copy number dependent expression with minimal position or enhancer dependence in transgenic mice. J Biol Chem 2000; 275: 28549–28554. Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 531 84. Sabatino DE et al. Long-term expression of gamma-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma-globin gene fused to the ankyrin-1 promoter. Proc Natl Acad Sci USA 2000; 97: 13294–13299. 85. Emery DW, Yannaki E, Tubb J & Stamatoyannopoulos G. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA 2000; 97: 9150–9155. 86. Yannaki E, Tubb J, Aker M et al. Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors. Mol Ther 2002; 5: 589–598. 87. Rivella S, Callegari JA, May C et al. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J Virol 2000; 74: 4679–4687. 88. Emery DW et al. Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo. Blood 2002; 100: 2012–2019. 89. Kalberer CP et al. Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice. PG—5411-5. Proc Natl Acad Sci USA 2000; 97: 5411–5415. 90. Nicolini FE et al. Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells. PG—1257-64. Blood 2002; 100: 1257–1264. 91. Salmon P & Trono D. Lentiviral vectors for the gene therapy of lympho-hematological disorders. Curr Top Microbiol Immunol 2002; 261: 211–227. 92. Follenzi A & Naldini L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol 2002; 346: 454–465. 93. Lewis PF & Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994; 68:
510–516. 94. Goff SP. Intracellular trafficking of retroviral genomes during the early phase of infection: viral exploitation of cellular pathways. J Gene Med 2001; 3: 517–528. 95. Fischer U, Huber J, Boelens WC et al. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 1995; 82: 475–483. 96. Wen W, Meinkoth JL, Tsien RY & Taylor SS. Identification of a signal for rapid export of proteins from the nucleus. Cell 1995; 82: 463–473. 97. Fornerod M, Ohno M, Yoshida M & Mattaj IW. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 1997; 90: 1051–1060. 98. Yi R, Bogerd HP, Wiegand HL & Cullen BR. Both ran and importins have the ability to function as nuclear mRNA export factors. RNA 2002; 8: 180–187. 99. Yi R, Bogerd HP & Cullen BR. Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mRNAs. J Virol 2002; 76: 2036–2042. 100. Miyoshi H, Blomer U, Takahashi M et al. Development of a self-inactivating lentivirus vector. J Virol 1998; 72: 8150–8157. 101. Miyoshi H, Smith KA, Mosier DE, Verma IM & Torbett BE. Transduction of human CD34C cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 1999; 283: 682–686. 102. Guenechea G et al. Transduction of human CD34C CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol Ther 2000; 1: 566–573. 103. Mangeot PE et al. High levels of transduction of human dendritic cells with optimized SIV vectors. Mol Ther 2002; 5: 283–290. 104. Follenzi A, Sabatino G, Lombardo A et al. Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors. Hum Gene Ther 2002; 13: 243–260. 105. Dyall J, Latouche JB, Schnell S & Sadelain M. Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes. Blood 2001; 97: 114–121. 106. Piacibello W et al. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient. Blood 2002; 100: 4391–4400. 107. Sutton RE, Reitsma MJ, Uchida N & Brown PO. Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J Virol 1999; 73: 3649–3660. 532 M. Sadelain et al 108. Sadelain M, Frassoni F & Riviere I. Issues in the manufacture and transplantation of genetically modified hematopoietic stem cells. Curr Opin Hematol 2000; 7: 364–377. 109. Kumar M, Keller B, Makalou N & Sutton RE. Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther 2001; 12: 1893–1905. *110. Rivella S, May C, Chadburn A et al. A novel murine model of Cooley anemia and its rescue by lentiviralmediated human beta -globin gene transfer. Blood 2003; 101: 2932–2939. *111. Pawliuk R et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001; 294: 2368–2371. 112. Nagel RL et al. Structural bases of the inhibitory effects of hemoglobin F, hemoglobin A2 on the polymerization of hemoglobin S. Proc Natl Acad Sci USA 1979; 76: 670–672. *113. Persons DA, Hargrove PW, Allay ER et al. The degree of phenotypic correction of murine {beta}- thalassemia intermedia following lentiviral-mediated transfer of a human {gamma}-globin gene is influenced by chromosomal position effects and vector copy number. Blood 2002;. 114. Sirven A et al. The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 2000; 96: 4103–4110. 115. Follenzi A, Ailles LE, Bakovic S, Geuna M & Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25: 217–222. 116. Rivella S & Sadelain M. Therapeutic globin gene delivery using lentiviral vectors. Curr Opin Mol Ther 2002; 4: 505–514. 117. Zufferey R, Donello JE, Trono D & Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999; 73: 2886–2892. 118. Huang ZM & Yen TS. Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts. Mol Cell Biol 1995; 15: 3864–3869. 119. Skow LC et al. A mouse model for beta-thalassemia. Cell 1983; 34: 1043–1052. 120. Shehee WR, Oliver P & Smithies O. Lethal thalassemia after insertional disruption of the mouse major adult beta-globin gene. Proc Natl Acad Sci USA 1993; 90: 3177–3181. 121. Yang B et al. A mouse model for beta 0-thalassemia. Proc Natl Acad Sci USA 1995; 92: 11608–11612. 122. Ciavatta DJ, Ryan TM, Farmer SC & Townes TM. Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells. Proc Natl Acad Sci USA 1995; 92: 9259–9263. 123. Rivella S, May C, Chadburn A et al. A novel murine model of Cooley’s anemia and its rescue by lentiviral mediated human {beta}-globin gene transfer. Blood 2002;. 124. Zhang D et al. An optimized system for studies of EPO-dependent murine pro-erythroblast development. Exp Hematol 2001; 29: 1278–1288. 125. Fabry ME et al. Magnetic resonance evidence of hypoxia in a homozygous alpha-knockout of a transgenic mouse model for sickle cell disease. J Clin Invest 1996; 98: 2450–2455. 126. Fabry ME et al. A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity. Blood 1995; 86: 2419–2428. 127. Ryan TM et al. Human sickle hemoglobin in transgenic mice. Science 1990; 247: 566–568. 128. Greaves DR et al. A transgenic mouse model of sickle cell disorder. Nature 1990; 343: 183–185. 129. Fabry ME et al. High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia. Proc Natl Acad Sci USA 1992; 89: 12155–12159. 130. Fabry ME, Nagel RL, Pachnis A et al. High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences. Proc Natl Acad Sci USA 1992; 89: 12150–12154. 131. Popp RA et al. A transgenic mouse model of hemoglobin S Antilles disease. Blood 1997; 89: 4204–4212. 132. Rhoda MD et al. Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains. Biochim Biophys Acta 1988; 952: 208–212. 133. D’Surney SJ & Popp RA. Oxygen association-dissociation and stability analysis on mouse hemoglobins with mutant alpha- and beta-globins. Genetics 1992; 132: 545–551. 134. Trudel M et al. Sickle cell disease of transgenic SAD mice. Blood 1994; 84: 3189–3197. 135. Paszty C et al. Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 1997; 278: 876–878. Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 533 136. Ryan TM, Ciavatta DJ & Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science 1997; 278: 873–876. 137. Chang JC et al. Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb betas-globin yeast artificial chromosome: a mouse model of sickle cell anemia. Proc Natl Acad Sci USA 1998; 95: 14886–14890. *138. May C, Rivella S, Chadburn A & Sadelain M. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene. Blood 2002; 99: 1902–1908. 139. Trudel M et al. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO J 1991; 10: 3157–3165. 140. Imren S et al. Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc Natl Acad Sci USA 2002; 9
9: 14380–14385. 141. Kohn DB. Gene therapy for genetic haematological disorders and immunodeficiencies. J Intern Med 2001; 249: 379–390. 142. Sadelain M & Riviere I. Sturm und drang over suicidal lymphocytes. Mol Ther 2002; 5: 655–657. 143. Jolicoeur P & Lamontagne L. Impaired Tand B cell subpopulations involved in a chronic disease induced by mouse hepatitis virus type 3. J Immunol 1994; 153: 1317–1318. 144. Baum C et al. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003; 101: 2099–2114. 145. Kohn DB, Sadelain M & Glorioso JC. Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer 2003; 3: 477–488. 146. Li Z et al. Murine leukemia induced by retroviral gene marking. Science 2002; 296: 497. 147. Stocking C et al. Distinct classes of factor-independent mutants can be isolated after retroviral mutagenesis of a human myeloid stem cell line. Growth Factors 1993; 8: 197–209. 148. Hantzopoulos PA, Suri C, Glass DJ et al. The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins. Neuron 1994; 13: 187–201. 149. Hacein-Bey-Abina S et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346: 1185–1193. 150. Kohn DB et al. American Society of Gene Therapy (ASGT) Ad Hoc Subcommittee on retroviralmediated gene transfer to hematopoietic stem cells. Mol Ther 2003; 8: 180–187. 151. Leonard WJ. Cytokines and immunodeficiency diseases. Nat Rev Immunol 2001; 1: 200–208. 152. Rivella S & Sadelain M. Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing. Semin Hematol 1998; 35: 112–125. 153. May C & Sadelain M. A promising genetic approach to the treatment of beta-thalassemia. Trends Cardiovasc Med 2001; 11: 276–280. 154. Sadelain M. Insertional oncogenesis in gene therapy: how much of a risk? Gene Ther 2004; 11: 569–573. 534 M. Sadelain et al
12 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease Michel Sadelain* MD, PhD Stefano Rivella 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. 1521-6926/$ – see front matter Q 2004 Elsevier Ltd. All rights reserved. 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 * Corresponding author. Tel.: C1-212-639-6190. E-mail address: m-sadelain@ski.mskcc.org (M. Sadelain). 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. 518 M. Sadelain et al 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 50 end and the adult bglobin gene at the 30 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 sugges
ted 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 50 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 519 β-globin gene LCR βe+ βe+ 1 kb SA RRE SD Ψ Ψ Ψ Ψ Ψ Ψ 5´LTR βA-87Thr GFP PGK ∆L cPPT HS2 HS3 HS4 I8 H WPRE K GFP p HS2 HS3 HS4 p 3´LTR HS2, 3, 4 p G A : RNS1 B : TNS9 C : β87 E : HS40/I8/K F : G.W/P WPRE * D : HS40/I8/K ± γRE/βe+ s.n. HS2 HS3 HS4s.n. βp 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 30 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) d432bA g113 was generated using the b-globin promoter (K130), fusing the b-globin 50 promoter untranslated sequences to the Ag-globin coding sequence at position 3 and C 1 relative to the endogenous translational start site. Also, d432bAg113 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 d432bA g113, 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: d432bAg113, d432bAg113 b-30 enhancer (b 30 Enh) and d432bAg113 g-globin 30 regulatory element (g 30 RE). The b 30 Enh or the g 30 RE were placed downstream of the g-globin coding sequences. Moreover, d432bAg113 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. 520 M. Sadelain et al 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 50 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 521 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 s
ubfragments 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 30 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, 522 M. Sadelain et al 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 30 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 d432bAg113 (Figure 1) and tested in mice affected by b-thalassemia intermedia. The vector d432bAg was generated using the b-globin promoter (K130), fusing the b-globin 50 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 d432bA g is smaller (2.0 kb) than TNS9 and the promoter is 130 bp long. Persons et al113 generated three globin vectors: d432bAg113, d432bAg113$b-30 enhancer (b 30 Enh) and d432bAg113 g-globin 30 regulatory element (g 30 RE). The b 30 Enh or the g 30 RE were placed downstream of the g-globin coding sequences. Moreover, d432bAg113 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 523 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-nul
l 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 524 M. Sadelain et al 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 Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 525 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 d432bAg (Fig. 1), d432bAg b-30 or d432bAg g-30 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 d432bAg 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.123 526 M. Sadelain et al 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 d432bAg 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, Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 527 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 30 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 T
halassemia and the Cooley’s Anemia Foundation. REFERENCES 1. Weatherall DJ & Clegg JB. The Thalassemia Syndrome. Oxford: Blackwell Scientific; 1981. 2. Stamatoyannopoulos G, Nienhuis AW, Majerus P & Varmus H. The Molecular Basis of Blood Disease. Philadelphia: WB Saunders; 1994. 3. Weatherall DJ. Phenotype–genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet 2001; 2: 245–255. 4. Steinberg MH, Forget BG, Higgs DR & Nagel RL. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge: Cambridge University Press; 2001. 5. Cooley TB & Lee P. A series of cases of splenomegaly in children with anemia and peculiar bone changes. Trans Am Pediatr Soc 1925; 37: 29. 6. Giardina PJ & Grady RW. Chelation therapy in beta-thalassemia: an optimistic update. Semin Hematol 2001; 38: 360–366. 7. Giardini C & Lucarelli G. Bone marrow transplantation in the treatment of thalassemia. Curr Opin Hematol 1994; 1: 170–176. 8. Boulad F et al. Bone marrow transplantation for homozygous beta-thalassemia. The Memorial SloanKettering Cancer Center experience. Ann NY Acad Sci 1998; 850: 498–502. 528 M. Sadelain et al 9. Lucarelli G et al. Bone marrow transplantation in adult thalassemic patients. Blood 1999; 93: 1164–1167. 10. Tisdale J & Sadelain M. Toward gene therapy for disorders of globin synthesis. Semin Hematol 2001; 38: 382–392. 11. Steinberg MH, Forget BG, Higgs DR & Nagel RL. Molecular Mechanism of b Thalassemia. Cambridge: Cambridge University Press; 2001. 12. Pauling L, Itano HA, Singer SJ & Wells IC. Sickle cell anemia, a molecular disease. Science 1949; 110: 543–546. 13. Swank RA & Stamatoyannopoulos G. Fetal gene reactivation. Curr Opin Genet Dev 1998; 8: 366–370. 14. Platt OS et al. Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia. J Clin Invest 1984; 74: 652–656. 15. Charache S et al. Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia. Blood 1992; 79: 2555–2565. 16. Atweh GF & Loukopoulos D. Pharmacological induction of fetal hemoglobin in sickle cell disease and beta-thalassemia. Semin Hematol 2001; 38: 367–373. 17. Vermylen C et al. Haematopoietic stem cell transplantation for sickle cell anaemia: the first 50 patients transplanted in Belgium. Bone Marrow Transplant 1998; 22: 1–6. 18. Luzzatto L & Goodfellow P. Sickle cell anaemia. A simple disease with no cure. Nature 1989; 337: 17–18. 19. Sadelain M. Genetic treatment of the haemoglobinopathies: recombinations and new combinations. Br J Haematol 1997; 98: 247–253. 20. Leboulch P et al. Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure. EMBO J 1994; 13: 3065–3076. 21. Sadelain M, Wang CH, Antoniou M et al. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene. Proc Natl Acad Sci USA 1995; 92: 6728–6732. *22. May C et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000; 406: 82–86. 23. Higgs DR. Do LCRs open chromatin domains? Cell 1998; 95: 299–302. 24. Bulger M & Groudine M. Looping versus linking: toward a model for long-distance gene activation. Genes Dev 1999; 13: 2465–2477. 25. Grosveld F. Activation by locus control regions? Curr Opin Genet Dev 1999; 9: 152–157. 26. Engel JD & Tanimoto K. Looping, linking, and chromatin activity: new insights into beta-globin locus regulation. Cell 2000; 100: 499–502. 27. Navas PA et al. Activation of the beta-like globin genes in transgenic mice is dependent on the presence of the beta-locus control region. Hum Mol Genet 2002; 11: 893–903. 28. de Krom M, van de Corput M, von Lindern M et al & Strouboulis J. Stochastic patterns in globin gene expression are established prior to transcriptional activation and are clonally inherited. Mol Cell 2002; 9: 1319–1326. 29. Bulger M, Sawado T, Schubeler D & Groudine M. ChIPs of the beta-globin locus: unraveling gene regulation within an active domain. Curr Opin Genet Dev 2002; 12: 170–177. 30. Levings PP & Bungert J. The human beta-globin locus control region. Eur J Biochem 2002; 269: 1589–1599. 31. Trudel M & Costantini F. A 30 enhancer contributes to the stage-specific expression of the human beta-globin gene. Genes Dev 1987; 1: 954–961. 32. Trudel M, Magram J, Bruckner L & Costantini F. Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice. Mol Cell Biol 1987; 7: 4024–4029. 33. Antoniou M, deBoer E, Habets G & Grosveld F. The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers. EMBO J 1988; 7: 377–384. *34. Grosveld F, van Assendelft GB, Greaves DR & Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 1987; 51: 975–985. 35. Forrester WC et al. A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus. Genes Dev 1990; 4: 1637–1649. 36. Li Q, Zhang M, Duan Z & Stamatoyannopoulos G. Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region. Genomics 1999; 61: 183–193. Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 529 37. Philipsen S, Talbot D, Fraser P & Grosveld F. The beta-globin dominant control region: hypersensitive site 2. EMBO J 1990; 9: 2159–2167. 38. Talbot D, Philipsen S, Fraser P & Grosveld F. Detailed analysis of the site 3 region of the human beta-globin dominant control region. EMBO J 1990; 9: 2169–2177. 39. Li Q, Harju S & Peterson KR. Locus control regions: coming of age at a decade plus. Trends Genet 1999; 15: 403–408. 40. Talbot D & Grosveld F. The 50 HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites. EMBO J 1991; 10: 1391–1398. 41. Jackson JD, Petrykowska H, Philipsen S et al. Role of DNA sequences outside the cores of DNase hypersensitive sites (HSs) in functions of the beta-globin locus control region. Domain opening and synergism between HS2 and HS3. J Biol Chem 1996; 271: 11871–11878. 42. Bungert J et al. Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. Genes Dev 1995; 9: 3083–3096. 43. Ney PA et al. Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner. Mol Cell Biol 1993; 13: 5604–5612. 44. Fraser P, Pruzina S, Antoniou M & Grosveld F. Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev 1993; 7: 106–113. 45. Johnson KD et al. Cooperative activities of hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin domain. Proc Natl Acad Sci USA 2002; 99: 11760–11765. 46. Li Q & Stamatoyannopoulos G. Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator. Blood 1994; 84: 1399–1401. 47. Li Q, Zhang M, Han H, Rohde A & Stamatoyannopoulos G. Evidence that DNase I hypersensitive site 5 of the human beta-globin locus control region functions as a chromosomal insulator in transgenic mice. Nucleic Acids Res 2002; 30: 2484–2491. 48. Talbot D et al. A dominant control region from the human beta-globin locus conferring integration siteindependent gene expression. Nature 1989; 338: 352–355. 49. Pasceri P, Pannell D, Wu X & Ellis J. Full activity from human beta-globin locus control region transgenes requires 5(HS1, distal beta-globin promoter, and 30 beta-globin sequences. Blood 1998; 92: 653–663. 50. Sharpe JA et al. Role of upstream
DNase I hypersensitive sites in the regulation of human alpha globin gene expression. Blood 1993; 82: 1666–1671. 51. Molete JM et al. Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement. Mol Cell Biol 2001; 21: 2969–2980. 52. Bender MA et al. Targeted deletion of 50 HS1 and 50 HS4 of the beta-globin locus control region reveals additive activity of the DNaseI hypersensitive sites. Blood 2001; 98: 2022–2027. 53. Bender MA et al. Description and targeted deletion of 50 hypersensitive site 5 and 6 of the mouse betaglobin locus control region. Blood 1998; 92: 4394–4403. 54. Bender MA, Bulger M, Close J & Groudine M. Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region. Mol Cell 2000; 5: 387–393. 55. Ley TJ et al. Reduced beta-globin gene expression in adult mice containing deletions of locus control region 50 HS-2 or 50 HS-3. Ann NY Acad Sci 1998; 850: 45–53. 56. Reik A et al. The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells. Mol Cell Biol 1998; 18: 5992–6000. 57. Li Q, Peterson KR, Fang X & Stamatoyannopoulos G. Locus control regions. Blood 2002; 100: 3077–3086. 58. Grosveld F et al. The dynamics of globin gene expression and position effects. Novartis Found Symp 1998; 214: 67–79. 59. Milot E et al. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell 1996; 87: 105–114. 60. Wijgerde M, Grosveld F & Fraser P. Transcription complex stability and chromatin dynamics in vivo. Nature 1995; 377: 209–213. 530 M. Sadelain et al 61. Dillon N, Trimborn T, Strouboulis J et al. The effect of distance on long-range chromatin interactions. Mol Cell 1997; 1: 131–139. 62. Peterson KR & Stamatoyannopoulos G. Role of gene order in developmental control of human gammaand beta-globin gene expression. Mol Cell Biol 1993; 13: 4836–4843. 63. Carter D, Chakalova L, Osborne CS et al. Long-range chromatin regulatory interactions in vivo. Nat Genet 2002; 32: 623–626. 64. Dekker J, Rippe K, Dekker M & Kleckner N. Capturing chromosome conformation. Science 2002; 295: 1306–1311. 65. Tolhuis B, Palstra RJ, Splinter E et al. Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol Cell 2002; 10: 1453–1465. 66. Hu X et al. Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region. Proc Natl Acad Sci USA 2003; 100: 1111–1115. 67. Tuan D, Kong S & Hu K. Transcription of the hypersensitive site HS2 enhancer in erythroid cells. Proc Natl Acad Sci USA 1992; 89: 11219–11223. 68. Blackwood EM & Kadonaga JT. Going the distance: a current view of enhancer action. Science 1998; 281: 61–63. 69. Ashe HL, Monks J, Wijgerde M et al. Intergenic transcription and transinduction of the human betaglobin locus. Genes Dev 1997; 11: 2494–2509. 70. Kiekhaefer CM, Grass JA, Johnson KD, Boyer ME & Bresnick EH. Hematopoietic-specific activators establish an overlapping pattern of histone acetylation and methylation within a mammalian chromatin domain. Proc Natl Acad Sci USA 2002; 99: 14309–14314. 71. Cone RD, Weber-Benarous A, Baorto D & Mulligan RC. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol 1987; 7: 887–897. 72. Karlsson S, Papayannopoulou T, Schweiger SG, Stamatoyannopoulos G & Nienhuis AW. Retroviralmediated transfer of genomic globin genes leads to regulated production of RNA and protein. Proc Natl Acad Sci USA 1987; 84: 2411–2415. 73. Dzierzak EA, Papayannopoulou T & Mulligan RC. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature 1988; 331: 35–41. 74. Bender MA, Gelinas RE & Miller AD. A majority of mice show long-term expression of a human betaglobin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol 1989; 9: 1426–1434. 75. Bodine DM, Karlsson S & Nienhuis AW. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci USA 1989; 86: 8897–8901. 76. Plavec I, Papayannopoulou T, Maury C & Meyer F. A human beta-globin gene fused to the human betaglobin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells. Blood 1993; 81: 1384–1392. 77. Chang JC, Liu D & Kan YW. A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression. Proc Natl Acad Sci USA 1992; 89: 3107–3110. 78. Novak U, Harris EA, Forrester W et al. High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells. Proc Natl Acad Sci USA 1990; 87: 3386–3390. 79. Ellis J et al. Evaluation of beta-globin gene therapy constructs in single copy transgenic mice. Nucleic Acids Res 1997; 25: 1296–1302. 80. Ren S et al. Production of genetically stable high-titer retroviral vectors that carry a human gammaglobin gene under the control of the alpha-globin locus control region. Blood 1996; 87: 2518–2524. 81. Lung HY, Meeus IS, Weinberg RS & Atweh GF. In vivo silencing of the human gamma-globin gene in murine erythroid cells following retroviral transduction. Blood Cells Mol Dis 2000; 26: 613–619. 82. Emery DW, Morrish F, Li Q & Stamatoyannopoulos G. Analysis of gamma-globin expression cassettes in retrovirus vectors. Hum Gene Ther 1999; 10: 877–888. 83. Sabatino DE et al. A minimal ankyrin promoter linked to a human gamma-globin gene demonstrates erythroid specific copy number dependent expression with minimal position or enhancer dependence in transgenic mice. J Biol Chem 2000; 275: 28549–28554. Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 531 84. Sabatino DE et al. Long-term expression of gamma-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma-globin gene fused to the ankyrin-1 promoter. Proc Natl Acad Sci USA 2000; 97: 13294–13299. 85. Emery DW, Yannaki E, Tubb J & Stamatoyannopoulos G. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA 2000; 97: 9150–9155. 86. Yannaki E, Tubb J, Aker M et al. Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors. Mol Ther 2002; 5: 589–598. 87. Rivella S, Callegari JA, May C et al. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J Virol 2000; 74: 4679–4687. 88. Emery DW et al. Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo. Blood 2002; 100: 2012–2019. 89. Kalberer CP et al. Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice. PG—5411-5. Proc Natl Acad Sci USA 2000; 97: 5411–5415. 90. Nicolini FE et al. Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells. PG—1257-64. Blood 2002; 100: 1257–1264. 91. Salmon P & Trono D. Lentiviral vectors for the gene therapy of lympho-hematological disorders. Curr Top Microbiol Immunol 2002; 261: 211–227. 92. Follenzi A & Naldini L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol 2002; 346: 454–465. 93. Lewis PF & Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994; 68:
510–516. 94. Goff SP. Intracellular trafficking of retroviral genomes during the early phase of infection: viral exploitation of cellular pathways. J Gene Med 2001; 3: 517–528. 95. Fischer U, Huber J, Boelens WC et al. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 1995; 82: 475–483. 96. Wen W, Meinkoth JL, Tsien RY & Taylor SS. Identification of a signal for rapid export of proteins from the nucleus. Cell 1995; 82: 463–473. 97. Fornerod M, Ohno M, Yoshida M & Mattaj IW. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 1997; 90: 1051–1060. 98. Yi R, Bogerd HP, Wiegand HL & Cullen BR. Both ran and importins have the ability to function as nuclear mRNA export factors. RNA 2002; 8: 180–187. 99. Yi R, Bogerd HP & Cullen BR. Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mRNAs. J Virol 2002; 76: 2036–2042. 100. Miyoshi H, Blomer U, Takahashi M et al. Development of a self-inactivating lentivirus vector. J Virol 1998; 72: 8150–8157. 101. Miyoshi H, Smith KA, Mosier DE, Verma IM & Torbett BE. Transduction of human CD34C cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 1999; 283: 682–686. 102. Guenechea G et al. Transduction of human CD34C CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol Ther 2000; 1: 566–573. 103. Mangeot PE et al. High levels of transduction of human dendritic cells with optimized SIV vectors. Mol Ther 2002; 5: 283–290. 104. Follenzi A, Sabatino G, Lombardo A et al. Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors. Hum Gene Ther 2002; 13: 243–260. 105. Dyall J, Latouche JB, Schnell S & Sadelain M. Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes. Blood 2001; 97: 114–121. 106. Piacibello W et al. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient. Blood 2002; 100: 4391–4400. 107. Sutton RE, Reitsma MJ, Uchida N & Brown PO. Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J Virol 1999; 73: 3649–3660. 532 M. Sadelain et al 108. Sadelain M, Frassoni F & Riviere I. Issues in the manufacture and transplantation of genetically modified hematopoietic stem cells. Curr Opin Hematol 2000; 7: 364–377. 109. Kumar M, Keller B, Makalou N & Sutton RE. Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther 2001; 12: 1893–1905. *110. Rivella S, May C, Chadburn A et al. A novel murine model of Cooley anemia and its rescue by lentiviralmediated human beta -globin gene transfer. Blood 2003; 101: 2932–2939. *111. Pawliuk R et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001; 294: 2368–2371. 112. Nagel RL et al. Structural bases of the inhibitory effects of hemoglobin F, hemoglobin A2 on the polymerization of hemoglobin S. Proc Natl Acad Sci USA 1979; 76: 670–672. *113. Persons DA, Hargrove PW, Allay ER et al. The degree of phenotypic correction of murine {beta}- thalassemia intermedia following lentiviral-mediated transfer of a human {gamma}-globin gene is influenced by chromosomal position effects and vector copy number. Blood 2002;. 114. Sirven A et al. The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 2000; 96: 4103–4110. 115. Follenzi A, Ailles LE, Bakovic S, Geuna M & Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25: 217–222. 116. Rivella S & Sadelain M. Therapeutic globin gene delivery using lentiviral vectors. Curr Opin Mol Ther 2002; 4: 505–514. 117. Zufferey R, Donello JE, Trono D & Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999; 73: 2886–2892. 118. Huang ZM & Yen TS. Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts. Mol Cell Biol 1995; 15: 3864–3869. 119. Skow LC et al. A mouse model for beta-thalassemia. Cell 1983; 34: 1043–1052. 120. Shehee WR, Oliver P & Smithies O. Lethal thalassemia after insertional disruption of the mouse major adult beta-globin gene. Proc Natl Acad Sci USA 1993; 90: 3177–3181. 121. Yang B et al. A mouse model for beta 0-thalassemia. Proc Natl Acad Sci USA 1995; 92: 11608–11612. 122. Ciavatta DJ, Ryan TM, Farmer SC & Townes TM. Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells. Proc Natl Acad Sci USA 1995; 92: 9259–9263. 123. Rivella S, May C, Chadburn A et al. A novel murine model of Cooley’s anemia and its rescue by lentiviral mediated human {beta}-globin gene transfer. Blood 2002;. 124. Zhang D et al. An optimized system for studies of EPO-dependent murine pro-erythroblast development. Exp Hematol 2001; 29: 1278–1288. 125. Fabry ME et al. Magnetic resonance evidence of hypoxia in a homozygous alpha-knockout of a transgenic mouse model for sickle cell disease. J Clin Invest 1996; 98: 2450–2455. 126. Fabry ME et al. A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity. Blood 1995; 86: 2419–2428. 127. Ryan TM et al. Human sickle hemoglobin in transgenic mice. Science 1990; 247: 566–568. 128. Greaves DR et al. A transgenic mouse model of sickle cell disorder. Nature 1990; 343: 183–185. 129. Fabry ME et al. High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia. Proc Natl Acad Sci USA 1992; 89: 12155–12159. 130. Fabry ME, Nagel RL, Pachnis A et al. High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences. Proc Natl Acad Sci USA 1992; 89: 12150–12154. 131. Popp RA et al. A transgenic mouse model of hemoglobin S Antilles disease. Blood 1997; 89: 4204–4212. 132. Rhoda MD et al. Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains. Biochim Biophys Acta 1988; 952: 208–212. 133. D’Surney SJ & Popp RA. Oxygen association-dissociation and stability analysis on mouse hemoglobins with mutant alpha- and beta-globins. Genetics 1992; 132: 545–551. 134. Trudel M et al. Sickle cell disease of transgenic SAD mice. Blood 1994; 84: 3189–3197. 135. Paszty C et al. Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 1997; 278: 876–878. Globin gene transfer for treatment of the b-thalassemias and sickle cell disease 533 136. Ryan TM, Ciavatta DJ & Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science 1997; 278: 873–876. 137. Chang JC et al. Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb betas-globin yeast artificial chromosome: a mouse model of sickle cell anemia. Proc Natl Acad Sci USA 1998; 95: 14886–14890. *138. May C, Rivella S, Chadburn A & Sadelain M. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene. Blood 2002; 99: 1902–1908. 139. Trudel M et al. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO J 1991; 10: 3157–3165. 140. Imren S et al. Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc Natl Acad Sci USA 2002; 9
9: 14380–14385. 141. Kohn DB. Gene therapy for genetic haematological disorders and immunodeficiencies. J Intern Med 2001; 249: 379–390. 142. Sadelain M & Riviere I. Sturm und drang over suicidal lymphocytes. Mol Ther 2002; 5: 655–657. 143. Jolicoeur P & Lamontagne L. Impaired Tand B cell subpopulations involved in a chronic disease induced by mouse hepatitis virus type 3. J Immunol 1994; 153: 1317–1318. 144. Baum C et al. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003; 101: 2099–2114. 145. Kohn DB, Sadelain M & Glorioso JC. Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer 2003; 3: 477–488. 146. Li Z et al. Murine leukemia induced by retroviral gene marking. Science 2002; 296: 497. 147. Stocking C et al. Distinct classes of factor-independent mutants can be isolated after retroviral mutagenesis of a human myeloid stem cell line. Growth Factors 1993; 8: 197–209. 148. Hantzopoulos PA, Suri C, Glass DJ et al. The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins. Neuron 1994; 13: 187–201. 149. Hacein-Bey-Abina S et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346: 1185–1193. 150. Kohn DB et al. American Society of Gene Therapy (ASGT) Ad Hoc Subcommittee on retroviralmediated gene transfer to hematopoietic stem cells. Mol Ther 2003; 8: 180–187. 151. Leonard WJ. Cytokines and immunodeficiency diseases. Nat Rev Immunol 2001; 1: 200–208. 152. Rivella S & Sadelain M. Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing. Semin Hematol 1998; 35: 112–125. 153. May C & Sadelain M. A promising genetic approach to the treatment of beta-thalassemia. Trends Cardiovasc Med 2001; 11: 276–280. 154. Sadelain M. Insertional oncogenesis in gene therapy: how much of a risk? Gene Ther 2004; 11: 569–573. 534 M. Sadelain et al

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