2009 Hematopoietic Stem Cell Gene Transfer for the Treatment of Hemoglobin Disorders

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NEWER PROGRESS IN GENE THERAPY ________________________________________________________________________ Hematopoietic stem cell gene transfer for the treatment of hemoglobin disorders Derek A. Persons1 1 Division of Experimental Hematology, Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN Hematopoietic stem cell (HSC)–targeted gene transfer is an attractive approach for the treatment of a number of hematopoietic disorders caused by single gene defects. Indeed, in a
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  690American Society of Hematology Hematopoietic stem cell gene transfer for thetreatment of hemoglobin disorders Derek A. Persons 1 1 Division of Experimental Hematology, Department of Hematology, St. Jude Children’s Research Hospital,Memphis, TN  Hematopoietic stem cell (HSC)–targeted gene transfer is an attractive approach for the treatment of anumber of hematopoietic disorders caused by single gene defects. Indeed, in a series of gene transfertrials for two different primary immunodeficiencies beginning early in this decade, outstanding successhas been achieved. Despite generally low levels of engrafted, genetically modified HSCs, these trials weresuccessful because of the marked selective advantage of gene-corrected lymphoid precursors thatallowed reconstitution of the immune system. Unlike the immunodeficiencies, this robust level of in vivoselection is not available to hematopoietic repopulating cells or early progenitor cells following genetransfer of a therapeutic globin gene in the setting of β -thalassemia and sickle cell disease. Both preclini-cal and clinical transplant studies involving bone marrow chimeras suggest that 20% or higher levels ofengraftment of genetically modified HSCs will be needed for clinical success in the most severe of thesedisorders. Encouragingly, gene transfer levels in this range have recently been reported in a lentiviralvector gene transfer clinical trial for children with adrenoleukodystrophy. A clinical gene transfer trial for β -thalassemia has begun in France, and one patient with transfusion-dependent HbE/ β -thalassemia hasdemonstrated a therapeutic effect after transplantation with autologous CD34 + cells genetically modifiedwith a β -globin lentiviral vector. Here, the development and recent progress of gene therapy for thehemoglobin disorders is reviewed. T he hemoglobin disorders are highly prevalent,recessive genetic diseases in which co-inheritanceof two defective globin alleles results in severehematological disease. Patients with the β -globin disorders,sickle cell disease (SCD) and β -thalassemia suffer severeanemia and a myriad of other problems affecting numerousorgan systems. In patients with SCD, the beta chaincontains a substitution of valine for glutamic acid atposition 6. 1 This substitution results in a change in surfacecharge that predisposes deoxygenated HbS to polymerize,causing red cells to assume rigid sickled shapes, leading tosevere anemia, vaso-occlusion, painful crises and organdamage. Although drug and transfusion therapies improvethe quality and duration of life for many individuals,developing curative treatments for SCD remains the desiredgoal. The only curative therapy, hematopoietic stem cell(HSC) transplantation, has been performed on about 200patients with SCD worldwide. 2,3 Most patients havereceived bone marrow (BM) transplantation from HLA-matched siblings, with a highly successful outcome.However, this approach is available only for a minorityof patients due to lack of available, matched donors.Unfortunately, a 5% to 10% mortality rate due toregimen-related mortality (infection) and graft-versus-host disease 3,4 continues to be problematic.Unlike SCD, in which there are normal levels of an abnor-mal β -globin protein that causes the pathophysiology, β -thalassemia results from deficient β -globin chain synthesisdue to a variety of deletions and mutations in the β -globingene or its upstream regulatory elements. Patients withmarkedly reduced or no β -globin expression rely on routinered cell transfusion therapy for survival and quality of life.Advances in chelation therapy, including the recentavailability of oral chelators, have improved the quality of life and potential for longevity in these patients. 5 However,iron overload, particularly in the heart, remains a significantissue, despite chelation that is adequate to unload the liver. 6 In comparison with stem cell transplantation for SCD,almost 1600 stem cell transplants have been performedworldwide for β -thalassemia. 7 Great success has beenobtained with matched-sibling allogeneic transplants forpatients with early stage disease. However, as for SCDpatients, early mortality can occur in 5% to 10% of patients.For patients with later stage disease, the outcome is lessfavorable. Similarly, transplantation using matched, N EWER P ROGRESS   IN G ENE T HERAPY  ________________________________________________________________________   Hematology 2009 691 unrelated donors has been problematic. 8 Thus, substantialefforts have been invested in the development of genetransfer into autologous HSC as an alternative therapy withcurative intent. Murine Leukemia Virus as a Vector toTransfer Genes into Blood Stem Cells:Success in Two Immunodeficiency Disorders A critical element in developing gene therapy for blooddisorders was the research focusing on identifying aneffective vector system for gene transfer into hematopoieticcells. In the early 1980s, gene transfer vectors based on themurine leukemia retrovirus (MLV) were developed thatcould successfully and stably transfer a marker gene intothe genome of HSCs in murine models. 9,10 These MLV-basedvectors utilize the powerful enhancer and promoter ele-ments within the long-terminal repeat (LTR) at the 5’ end of the viral genome to drive transgene expression. Replica-tion-incompetent, recombinant vectors were derived byreplacing the viral genes with a marker gene or other geneof interest. Fibroblast cell lines, stably expressing both therecombinant vector genome and the required componentsof the viral particle, were used to produce the vector. Yearslater, these efforts culminated in the success of humanclinical trials for two severe combined immunodeficiencies(SCID). To date, more than 30 patients with SCID secondaryto deficiency of the common gamma chain (X-SCID1) ordue to adenosine deaminase (ADA) deficiency have beensuccessfully treated using MLV-based vectors encodingeither the common gamma chain or the ADA enzyme,respectively. 11-14 Despite this success, vector-mediatedinsertional activation of nearby proto-oncogenes initiatedthe development of leukemia in five patients treated forcommon gamma chain deficiency, highlighting theimportance of the role of vector design in the safety of genetherapy. 11,15 These trials provided encouragement forcontinued effort to develop stem cell–targeted gene therapyfor hemoglobin disorders, while at the same time focusingnew attention to vector safety. Lentiviral Vectors Emerge as an EffectiveSystem to Mediate Gene Transfer and High-Level Expression of Globin Genes MLV-based globin gene vectors, as opposed to the rela-tively simple MLV vectors used in the X-SCID1 and ADAtrials, proved challenging to design and produce due to therequirement to include specific endogenous regulatoryelements from the β -globin locus that were obligatory toachieve adequate expression. Despite more than a decade of intensive efforts by several laboratories to develop MLV-based globin retroviral vectors, a potentially therapeuti-cally useful vector design was never identified. 16 In the mid-1990s, the development of lentiviral vectorsbased on the human immunodeficiency virus (HIV) 17 is nowrecognized as a key milestone that made possible thesubsequent development of globin lentiviral vectors.Indeed, a significant breakthrough in the globin genetherapy field occurred in 2000 when an HIV-based, globinlentiviral vector was used to cure a mouse model of  β -thalassemia intermedia. 18 The lentiviral vector backbone,coupled with the nuclear to cytoplasmic RNA exportsystem utilized by HIV, made possible the transmission of a β -globin expression cassette containing a large constella-tion of regulatory elements. Unlike the MLV-based globinvectors, this lentiviral vector transmitted the globinregulatory and coding sequences without rearrangementand could be produced in sufficient titer. Since then,additional reports describing use of globin lentiviralvectors to correct murine β -thalassemia and SCD modelshave emerged (see below).Recently, it has been reported that relatively high levels of lentiviral vector-mediated HSC gene transfer were obtainedin a clinical trial for children with cerebral X-linkedadrenoleukodystrophy (ALD), a demyelinating disease dueto deficiency of an enzyme that breaks down very longchain fatty acids. Three children enrolled in this trial inFrance have been described. 19 After pre-transplant condi-tioning with myeloablative doses of busulfan and cyclo-phosphamide, the patients received autologous, cytokine-mobilized peripheral blood CD34 + cells transduced with alentiviral vector encoding the ALD enzyme. As reported byDr. Natalie Cartier at the 2009 American Society of GeneTherapy Annual Meeting, 10% to 20% of the peripheralblood cells in these patients contain the vector, with follow-up ranging from 9 to 30 months. There may be sometherapeutic effect as measured by the slowing or cessationof central nervous system disease. Notably, these resultsdemonstrate that lentiviral vectors can mediate significantlevels of gene transfer into human HSCs. Correction of Murine and Human Models of βββββ -thalassemia and SCD Sadelain and colleagues were the first to demonstrate that alentiviral vector encoding human β -globin could be used tocure a murine model severe β -thalassemia. 18 In this and afollow-up study, they showed significant long-termhematologic and pathologic correction using a β -globinvector containing a somewhat large constellation of regulatory elements from the β -globin locus control region(LCR) totaling 3.2 kb in size. 20 Approximately 80% of theHSCs were genetically modified, resulting in chimerichemoglobin molecules incorporating human β -globincomprising about 21% of total hemoglobin. In anotherstudy, at least one copy of a lentiviral β -globin vector in  692American Society of Hematology every stem cell was required to correct severe murine β -thalassemia. 21 The fact that correction in these experimentsrequired most or all of the HSCs to be modified suggestedthat inconsistent globin expression could be occurring, as itwas well known that location in the genome could affectexpression of transgenes. In a different study and furthersupporting this possibility, only 1 of 6 animals in a modelof thalassemia major demonstrated a relatively high level of hemoglobin following the gene transfer procedure despitenearly all the HSCs being genetically modified. Theremaining animals remained severely anemic with athalassemia intermedia phenotype. 22 Leboulch and colleagues were the first to demonstratehematologic correction and diminished end organ damagein murine SCD using lentiviral-mediated HSC gene transferof an anti-sickling variant of the human β -globin chain. 23 An average vector copy number of 3 in the HSCs wasobserved with dependence of globin transgene expressionon genomic position. Subsequently, others confirmed thisresult by using a different SCD model and a slightlydifferent anti-sickling β -globin variant. Phenotypicimprovement with an average vector copy of 2.2 resulted inhemoglobin tetramers incorporating the transgene globinchain at a level of 20% of the total hemoglobin. 24 Because increased fetal hemoglobin (HbF; α 2 γ  2 ) levelsnaturally ameliorate the clinical severity of both β -thalas-semia and sickle cell anemia, γ  -globin MLV-based andlentiviral vectors have been developed and tested as analternative to β -globin vectors. 25-27 HbF acts as a naturalanti-sickling hemoglobin in part because of formation of mixed tetramers, α 2 γ  β S , which do not participate in polymerformation. 28 A γ  -globin lentiviral vector containing 1.7 kbof LCR elements to drive expression of the human A γ  -globin gene showed therapeutic efficacy in a murine modelof severe β -thalassemia, with significant disease correctionachieved with a copy number of 2 to 2.5 in the HSCs. 27 Position-dependent expression of the globin vector wasalso observed and was a significant factor that affectedefficacy. Hanawa et al subsequently developed a secondgeneration γ  -globin lentiviral vector, having more extensiveLCR-derived regulatory sequences. 29 This vector, shown tobe less susceptible to position effects, was able to producehigher and more consistent levels of HbF, and one vectorcopy per cell was curative. 29 Globin lentiviral vectors have also been used to transfer a β -globin gene into normal human hematopoietic cellscapable of establishing hematopoiesis in immunodeficientmice. 30 Correction of the β -thalassemia phenotype inpatient cells was demonstrated by the establishment of effective erythropoiesis in erythroid cultures, both inmarrow cells from patients cultured in vitro and in cellsobtained from immunodeficient mice several months aftertransplant with the patients’ genetically modified primitivecells. 31 Preclinical Studies of Globin Lentiviral Vectors: What We Have Learned From the above studies, we can conclude that therapeuti-cally adequate levels of globin protein expression can beobtained following HSC gene transfer by using lentiviralvectors containing a relative large and complex set of endogenous β -globin regulatory elements. However, thegenomic site of integration can affect the level of globinexpression. One potential solution to this problem has beento use DNA elements called “insulators,” which are DNAelements in the genome that can function both as barrierelements to dampen position effects and enhancer blockersto prevent nearby genes from interacting with one another 32 .Inclusion of such elements in globin vectors may reduce,but not eliminate, the problem of variable expression due toposition of the vector in the genome. 33 However, addition of insulator elements to the vector design can hamper vectorproduction and may prevent successful scale up for clinicaltrials. 34,35 Thus, many laboratories are now focusing onidentifying functional “insulator” elements that will notdramatically affect vector titer. 34,36 Highly efficient transduction of HSCs has been obtained withglobin lentiviral vectors in mice (almost every HSC can betransduced) and this facilitated the ability to “cure” thevarious globin disease models. High concentrations of vectorparticles are required during the ex vivo transduction process.Thus, it has remained unclear whether significant levels of lentiviral vector-mediated gene transfer could be obtained forhuman HSCs. Encouragingly, the lentiviral vector trialsdescribed above and below suggest that HSC gene transferrates of 10% or higher can be obtained in humans. However,these rates remain much lower than those obtained in miceand suggest that further research aimed at improving lentiviralvector gene transfer into human HSCs is worthwhile. Side Effects of Retroviral Gene Transfer:Insertional Gene Activation Despite the enormous success of the X-SCID1 gene therapytrials, with many patients demonstrating substantialimprovements in their clinical and immunological status,four patients in the trial in France and one in the UnitedKingdom subsequently developed lymphoid leukemia due,in part, to vector-mediated insertional mutagenesis. 15,37-39 Inanother trial for chronic granulomatous disease (CGD),insertional gene dysregulation resulted in the outgrowth of myeloid clones. 40 In these cases, the aberrant gene expres-sion caused by the vector integration was due to the effects  Hematology 2009 693 of the enhancer elements present in the LTR of the vector( Figure 1 ). This information is detailed elsewhere in thisvolume. These results prompted investigators to developnew in vitro and in vivo assays to measure vectorgenotoxicity. 41-44 Subsequent studies using these new assays indicated thatlentiviral vectors may be a safer alternative to the LTR-containing, MLV-based vectors used in the X-SCID1 andCGD trials. 42,43 Similarly, MLV-based vectors that have beenmodified to delete the enhancer and promoter elementswithin the LTR also show less genotoxicity in theseassays. 45 In both the lentiviral and modified MLV vectors,an internal promoter is used to direct transgene expression.Less powerful cellular promoters seem to be the best choicefor driving transgene expression while minimizing poten-tial effects on genes surrounding the insertion site. 45 For thisapproach to be successful, such weaker promoters bynecessity must be capable of directing therapeutic expres-sion levels of the particular transgene in the appropriatecellular context. An effective vector design for a particulardisorder will need to be identified empirically throughstudies in mice and human cells. Globin Gene Transfer Clinical Trials One clinical gene transfer trial for patients with β -thalas-semia and SCD using globin lentiviral vectors is underwayin France, 46 while other groups, including our own, areplanning to begin trials in the United States in the nearfuture. 47 As reported at the American Society of GeneTherapy Annual Meeting (May 2009) and detailed by theFrench Medicine Agency 48 and the NIH Office of BiologicalActivities, 49 Leboulch and colleagues, in a clinical trialsponsored by Genetix France, have treated two patientswith β -thalassemia in Paris. The patients underwent bonemarrow harvest and enriched CD34 + cells were transducedwith a lentiviral vector containing β -globin locus regula-tory elements driving expression of a β -globin protein witha mutation at amino acid 87. Initially developed as an anti-sickling β -globin variant, 23 the use of the variant in thissetting allowed vector-encoded β -globin to be distin-guished from low levels of endogenous β -globin.The first patient, with transfusion-dependent β -thalassemiamajor, received ex vivo transduced autologous CD34 + cellsfollowing myeloablative pre-transplant conditioning withhigh-dose busulfan. Unfortunately, the patient had pro-longed post-transplant cytopenia and eventually receivedfrozen, backup CD34 + cells for hematopoietic rescue. Thesecond patient, a 19-year-old male with HbE/  β -thalassemia,also received autologous, vector-transduced CD34 + cells(~4 × 10 6 cells per kg) following a myeloablative dose of busulfan in June 2007. The patient had hematologicreconstitution about five weeks post-transplant. In mid-2008, the patient became transfusion independent with astable hemoglobin level above 9 g/dL. DNA analysisshowed that about 10% of peripheral blood myeloid cellswere genetically modified with the vector. Interestingly, of the ~9 g/dL of hemoglobin, one third was found to becomposed of endogenous HbE, one third composed of vector-encoded β -globin, and, somewhat surprisingly, onethird composed of HbF. Regarding this last observation, it isnotable that several β -thalassemic and sickle cell patientswho underwent allogeneic transplantation but subsequentlyrejected the donor grafts have been reported to havetherapeutic levels of HbF following reconstitution withendogenous HSCs. 50,51 Thus, it appears that the vector-encoded β -globin and “reactivation” of HbF expressionboth contributed to the therapeutic efficacy in this case.Despite the clinical improvement of the patient, it wasreported on May 27, 2009 by the French Medicine Agencythat the patient was found to have a “relative clonaldominance.” Of the 10% or so of genetically modified cells,one clone, identified as having integration in the  HMGA2 gene, was present in excessive proportion relative to thecontributions of other clones, as identified by their genomic Figure 1. Mechanism of vector insertional geneactivation. A genomic integration site of a MLV-basedretroviral vector in a target cell is depicted. With this MLV vectordesign, the enhancer and promoter within the U3 region (bluerectangle) of the long terminal repeat (LTR; white rectangles)drive transcription of the transgene (indicated by the parallelarrow arising from the blue rectangle). At top is shown a vectorintegration near Gene X. The enhancer elements located in theU3 region (blue rectangle) of the vector can interact with theregulatory elements upstream of Gene X to increase the basallevel of transcription to inappropriately high levels, potentiallyaltering the growth of the cell. Two alternatives to eliminate theuse of the powerful enhancer in the U3 include 1) middle panel:use of a self-inactivating (SIN) MLV-based vector in which theU3 region has been deleted (noted by X) and which utilizes aninternal cellular promoter to drive transgene expression (parallelarrow) and 2) bottom panel: use of a SIN lentiviral vector inwhich the U3 (yellow rectangle) has also been eliminated and,like the SIN MLV vector, uses an internal cellular promoter todrive transgene expression.
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