From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only. 2012 119: 1107-1116 Prepublished online November 17, 2011; doi:10.1182/blood-2011-09-349993Hematopoietic stem cell engineering at a crossroadsIsabelle Rivière, Cynthia E. Dunbar and Michel SadelainUpdated information and services can be found at:http://bloodjournal.hematologylibrary.org/content/119/5/1107.full.htmlArticles on similar topics can be found in the following Blood collections Free Research Articles (1345 articles) Hematopoiesis and Stem Cells (2969 articles) Review Articles (374 articles)Information about reproducing this article in parts or in its entirety may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about ordering reprints may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about subscriptions and ASH membership may be found online at:http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published weeklyby the American Society of Hematology, 2021 L St, NW, Suite 900,Washington DC 20036.Copyright 2011 by The American Society of Hematology; all rights reserved.
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.Review articleHematopoietic stem cell engineering at a crossroadsIsabelle Riviere,1 Cynthia E. Dunbar,2 and Michel Sadelain1 `1Center for Cell Engineering, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY; and 2HematologyBranch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MDThe genetic engineering of hematopoietic technologies used to date, which have on cell therapies that may transform medicalstem cells is the basis for potentially occasion resulted in clonal expansion, practice. In this review, we place thesetreating a large array of hereditary and myelodysplasia, or leukemogenesis. New recent advances in perspective, empha-acquired diseases, and stands as the research directions, predicated on im- sizing the solutions emerging from a waveparadigm for stem cell engineering in proved vector designs, targeted gene de- of new technologies and highlighting thegeneral. Recent clinical reports support livery or the therapeutic use of pluripo- challenges that lie ahead. (Blood. 2012;the formidable promise of this approach tent stem cells, herald the advent of safer 119(5):1107-1116)but also highlight the limitations of the and more effective hematopoietic stemIntroductionThe safe engineering and engraftment of hematopoietic stem cells made possible by the advent of patient-speciﬁc pluripotent stem(HSCs) are the keys to treating a vast spectrum of genetic and cells. Some of these have recently entered the clinical arena andacquired disorders that affect hematopoietic and other tissues. others are soon to follow. This review brieﬂy summarizes the ﬁrstThese include disorders of the immune system, such as severe 2 decades of HSC gene therapy, based on the use of ﬁrst-generationcombined immunodeﬁciency (SCID) syndromes and AIDS, the (ie, LTR-driven) ␥-retroviral vectors, and critically assesses futurethalassemias, sickle cell anemia, metabolic disorders, including directions from the perspective of genetic engineering, examiningcentral nervous system pathologies, autoimmune diseases, and an the prospects, and challenges that lie ahead.array of hematologic malignancies, which could be treated withcancer-free autologous cells or prevented (eg, in the case ofFanconi anemia).1,2 The safe and effective engineering of HSCsthus represents one of the central goals of stem cell and gene LTR-driven vectorstherapies. Since the pioneering studies in adenosine deaminase(ADA) deﬁciency initiated at the National Institutes of Health in Successes in HSC gene therapy have slowly but steadily accumu-the early 1990s, nearly 100 patients have been treated with lated over the past decade. Most early trials focused on severegenetically modiﬁed CD34ϩ hematopoietic progenitors worldwide monogenic immune deﬁciencies, including ADA deﬁciency,(Table 1). This slow adaptation reﬂects both the complexity of the X-SCID, chronic granulomatous deﬁciency (CGD), and, morebiologic challenges posed by the ex vivo manipulation and genetic recently, Wiskott-Aldrich syndrome (WAS). These severe disor-engineering of HSCs as well as the chilling impact of the ﬁrst report ders were chosen in part because ubiquitous expression of theof a leukemic transformation caused by a ␥-retroviral vector in a therapeutic protein (the ADA enzyme for ADA deﬁciency, theboy with X-linked SCID (X-SCID).3 The series of leukemias that interleukin receptor common ␥-chain for X-SCID, the gp91phoxensued (in 5 of 20 patients with X-SCID), which were later oxidase complex protein for CGD, and the WAS signalingfollowed by similar adverse events in trials for chronic granuloma- integrator protein for WAS), in all hematopoietic cells, not justtous disease and Wiscott-Aldrich syndrome, raised serious doubtsas to the merits of this approach and undermined human and the defective cell types, was deemed to be acceptable, thusﬁnancial investments in this ﬁeld over the past decade. However, justifying the use of the “ﬁrst-generation” vectors available inthese serious adverse events also spurred an enormous, collectiveinvestigation into the genotoxicity of gene delivery methodologies,resulting in tremendous progress in our understanding of retroviral Table 1. Patients treated with engineered HSCsvector integration and its impact on endogenous gene structure and Disease Vector type LTR-driven No. of treated patientsfunction.4-6 Although these serious adverse events have resulted in ADA ␥RV ϩ 40discontinuation of the use of long terminal repeat (LTR)–driven Gaucher ␣RV ϩ 3␥-retroviral vectors for the genetic modiﬁcation of HSCs, they X-SCID ␥RV/SIN-␥RV ϩ/Ϫ 20/3provided a major impetus for developing novel approaches to CGD ␥RV ϩ 6genetically modify human cells. ALD SIN-LV ϩ 4 WAS ␥RV/SIN-LV ϩ/Ϫ 10/3 These new trends in HSC engineering broadly fall into 3 catego- ␤-thal SIN-LV Ϫ 2ries: improvements in the design of retroviral vectors, development MLD SIN-LV Ϫ 4of technologies for targeted gene delivery, and novel approachesSubmitted September 1, 2011; accepted November 8, 2011. Prepublished online asBlood First Edition paper, November 17, 2011; DOI 10.1182/blood-2011-09-349993.BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 1107
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.1108 ` RIVIERE et al BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5Figure 1. Retroviral vector designs under clinical evaluation. (A) LTR-driven ␥-RV, exempliﬁed by the MFG/SFG vector design used in X-SCID and WAS clinical trials.(B) SIN-␥-RV, exempliﬁed by the SRS11 EFS vector design used in the X-SCID consortium trial. (C) Nonspeciﬁc SIN-LV, exempliﬁed by the MND-ALD vector design used in theALD trial. (D) Lineage-restricted SIN-LV, exempliﬁed by the TNS9.3 vector for the treatment of ␤-thalassemia major. U3 E/P indicates retroviral enhancer/promoter from the LTRU3 region; PRE/WPRE (woodchuck hepatitis), posttranscriptional regulatory element; SIN, self-inactivating vector design (ƒ represents U3 deletion); speciﬁcity: Ϫ indicatesubiquitous; and ϩ, lineage-speciﬁc; LCR, locus control region; and HBB, human ␤-globin gene. Green represents retroviral enhancer/promoter elements; and red, mammalianenhancer/promoter elements.the early 1990s. These consist of recombinant replication-incompetent ␥-retroviral genomes derived from murine leuke- Current technologies show their limitsmia viruses (MLVs), termed LXSN,7 MFG/SFG,8 and FMEV,9which provide constitutive expression of the therapeutic cDNA Just as the beneﬁts of retroviral therapies were starting to bedriven by the viral enhancer/promoter present in the vector’s 5Ј- revealed, so were the shortcomings of gene transfer into stem cells.and 3Ј-LTRs (Figure 1). These include limitations of therapeutic efﬁcacy and toxicities, In terms of therapeutic efﬁcacy, the overall clinical results have especially genotoxicity. Some of these problems are inherent tobeen compelling. The majority of patients with SCID, ADA, and stem cell harvest and cell culture, whereas others are disease- orWAS showed dramatic improvements in their immune function, vector-speciﬁc. A successful therapy requires polyclonal hematopoi-including improved T- and B-cell immunity, as well as restored etic reconstitution by self-renewing HSCs, with a sufﬁcient fractionnatural killer cell function in X-SCID and regression of eczema and of engrafting HSCs that harbor the vector and expression of thethrombocytopenia in WAS.10-14 Quality of life was improved for corrective genetic material on a per-cell basis that reaches over thethe majority of these patients.15 The outcome in CGD, a myeloid thresholds required to therapeutically impact on the underlyingdisorder in contrast to the aforementioned lymphoid syndromes, disease over the long term.and where adults rather than children were treated, was less Several obstacles of a quantitative nature can interfere with thisdramatic but still resulted in short-term regression or stabilization objective. Effective retroviral transduction of HSCs requires hav-of intractable infections in the ﬁrst 2 treated subjects.16 ing enough patient CD34ϩ cells and adequate vector stocks for Using LTR-driven expression in another class of retroviral their transduction. Although CD34ϩ cell collection from bone marrow or mobilized blood is usually satisfactory, it may bevectors derived from HIV-1, promising clinical results were challenging in some conditions, such as Fanconi anemia19 andrecently obtained in 2 children with adrenoleukodystrophy (ALD), sickle cell anemia.20a disease characterized by multifocal brain demyelination. The Current approaches to vector transduction require ex vivo2 ﬁrst patients showed neuroradiologic improvement and stabiliza- culture of CD34ϩ cells in the presence of pro-survival cytokines,tion of their declining cognitive functions.17 Although the observa- and even short-term culture results in decreased engraftment andtion period is still limited (ϳ 3 years), these results bode very well ability to compete with endogenous HSCs, perhaps resulting fromfor this class of vectors, which are currently entering the clinic for loss of self-renewal capacity or defects in homing.21,22 As a result,WAS and other metabolic disorders, including metachromatic some degree of potentially toxic conditioning with chemotherapyleukodystrophy, and ␤-thalassemia (Table 1). Like the ﬁrst- or irradiation is required in clinical settings where corrected HSCsgeneration ␥-retroviral vectors, the lentiviral vectors used in the and their progeny do not have a competitive advantage.ALD study express the protein nonspeciﬁcally in all hematopoi- Vector production to reach a sufﬁcient titer may pose aetic lineages, including the myeloid cells that eventually recon- challenge. Although adequate for most phase 1 or 2 studies, robuststitute the brain microglia.18 Signiﬁcantly, these vectors lack the manufacturing to support larger trials has yet to be developed.duplicated full-length LTR that is characteristic of the early Some speciﬁc vectors, such as the complex globin vectors23-25 or␥-RVs, although they still encode an LTR as their internal vectors containing the cHS4 insulator element,26,27 exhibit lowerpromoter (Figure 1). titers that pose a manufacturing challenge. More research on vector Altogether, these studies support the feasibility of geneti- production is needed to advance the ﬁeld and eventually meetcally engineering HSCs for use in an autologous setting and the commercial goals.notion that genetically engineered HSCs could provide substan- Sufﬁcient and stable expression of vector-encoded transgenes istial beneﬁts to patients with a broad range of inherited and another category of concern because of the vagaries of positionacquired disorders. effects and the risk of transcriptional inactivation.28-30 Although
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 HSC ENGINEERING AT A CROSSROADS 1109Table 2. Clonal expansion, myelodysplasias, and transformation Relevant vectorPatient/disease/ sequences Secondary effect (mo after Genomic insertion sites Other genetic alterations (motransgene (references) treatment) (transcript status) after treatment) Reference(s)P4/SCID-X1/␥C MFG(B2), Moloney-MLV T-ALL, mature T cell (30) LMO2 (1) Translocation(6,13); CDKN2A 3,139 LTR (8,138) deletionP5/SCID-X1/␥C T-ALL, late cortical T cell (34) LMO2 (1) SIL-TAL microdeletion, trisomy 10, 139 Notch mutation (1593F/S)P7/SCID-X1/␥C T-ALL, late cortical T cell (68) CCND2 (1) CDKN2A deletion 42,139P10/SCID-X1/␥C T-ALL, late cortical T cell (33) LMO2 (1), BMI1 (1) Notch mutation (1707A/P) 42,139P1/X-CGD/gp91phox SFFV LTR (9,16) Multiple predominant progenitor MDS1-EVI1 (1), CpG methylation in promoter of 16,32 cell clones (5), subsequent PRDM16 (ϭ), the viral LTR (9); CDKN2B and oligoclonal hematopoiesis, SETBP1 (1) p15INK4B hypermethylation; monosomy 7 (21), MDS (27) phosphorylation of H2AX and DNA double-strand breaks (27)P2/X-CGD/gp91phox Multiple predominant progenitor MDS1-EVI1 (1), CpG methylation in promoter of 16,32 cell clones (5), subsequent PRDM16 (1) the viral LTR (15); CDKN2B and oligoclonal hematopoiesis, p15INK4B hypermethylation; monosomy 7 (33), MDS (43) phosphorylation of H2AX and DNA double-strand breaks (43)P8/SCID-X1/␥C MFG, Moloney-MLV LTR T-ALL (24) LMO2 (1) Notch1 mutation (gain-of-function, 41 (8,140) 1559R/P), CDKN2A deletion, TCRb/STIL-TAL1 translocationP2/Thalassemia/ ⌬U3 HIV LTR ϩ 2xcHS4 Dominant, myeloid-biased cell HMGA2 (1) Vector rearrangement; 24 ␤(T87Q)-globin insulators (24,141) clone transcriptional activation of HMGA2 in erythroid cells with increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 micro-RNAswell documented and extensively studied in murine models,30,31 unless the LTR prevents this from occurring,43 which may result invector silencing has been less investigated in clinical studies. It is the activation of an HSC-like transcription proﬁle in thymocytesnoteworthy that the vast collections of integration sites documented and clonal expansion, eventually resulting in full malignantin many trials10,11 do not provide information on vector expression. transformation with the acquisition over time of additional chromo-Furthermore, studies in SCID and WAS may blunt such an analysis somal abnormalities or point mutations in genes, such as Notch.44because of the selective outgrowth of transgene-expressing cells. In Just as striking is the absence of such transformations in patientsthe case of CGD, where such selective pressure on transgene with ADA-SCID, who also harbor LTR-containing vectors in theirexpression does not apply, silencing and methylation of the T-cell precursors, including occasional integrations in the vicinityvector’s retroviral promoter were found in several clones as early of LMO2.10 Marked expansion of a single corrected clone, persist-as 5 months after therapy.32 On the other hand, very prolonged ing without malignant transformation for many years, has beenexpression of marker genes, with no evidence for signiﬁcant documented in an early ADA-SCID gene therapy trial, suggestingsilencing, has been documented in early human clinical and that clonal expansion does not irrevocably progress to malig-nonhuman primate studies using both ␥-retroviral and lentiviral nancy.45 The reason for such contrasting outcomes is still unclearvectors.33-35 Silencing may be more problematic in murine cells, and may have to do the nature of the disease, with less profoundwhich have evolved mechanisms for inactivation of the huge and rapid expansion of corrected T-cell precursors, or a uniqueproviral load integrated into the murine endogenous genome, but interaction between the transgene and activated LMO2.46occurs in human cells as well. The anticipation that malignant transformation after HSC gene The toxicities associated with the ␥-retroviral transduction of therapy would be limited to SCID patients receiving cells engi-HSCs are the consequence of the semirandom pattern of retroviral neered to overexpression, a growth-promoting gene, such as theintegration and the presence of strong enhancers in the proviral common ␥-cytokine receptor and resulting from a unique interac-LTR,36-38 resulting in obligatory insertional mutagenesis. Activa- tion with insertions activating LMO2, was quashed by subsequenttion of proto-oncogenes in the genomic neighborhood is the most reports indicating that insertional mutagenesis after HSC genedreaded consequence. The direst outcome, frank leukemic transfor- transfer using ␥-retroviral was a universal risk. The lack of eventsmation, has been dramatically illustrated in X-SCID and WAS, in the earlier clinical trials and large animal studies may havewhere so far 5 of 20 and 1 of 10 patients, respectively, have resulted from exceedingly low HSC gene transfer efﬁciency anddeveloped clonal T-cell leukemias.39,40 A surprising feature of these lack of prolonged follow-up. Nonhuman primates and seriallyclonal transformations, all linked to the integration of an LTR- transplanted mice receiving HSCs transduced with ␥-retroviraldriven ␥-retroviral vector in the vicinity of an oncogene, is the vectors carrying only marker genes developed clonal over-striking involvement in all but one case of the LMO-2 gene (Table representation or overt myeloid and lymphoid leukemias, with LTR2).41,42 This gene is expressed in early hematopoietic progenitors activation of a stereotypical group of proto-oncogenes, most(where it is therefore accessible to the retroviral pre-integration strikingly MDS1/EVI1.47-50 Two patients with CGD receivingcomplex) and normally silenced on hematopoietic differentiation, corrected CD34ϩ cells developed ﬁrst clonal expansion of myeloid
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.1110 ` RIVIERE et al BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5cells with vector insertions activating the MDS1/EVI1 or the that enhancers located within a vector are less prone to activation ofrelated PRDM16 gene loci, and then clonal myelodysplasia and neighboring genes than the same enhancer contained within anmarrow failure, with acquisition of an additional monosomy 7 LTR.65 Expression of miRNAs and shRNAs also require robustabnormality in the malignant clone in both patients16,32 (Table 2). expression levels,66,67 but the polIII promoters they use may pose a The use of LTR-driven ␥-retroviral vectors to modify HSCs is lesser risk of deregulating endogenous gene expression than polIIthus all but over (with the possible exception of ADA deﬁciency, promoters. RNA-based anti-HIV moieties composing an shRNA towhich has been remarkably devoid of malignant complications to tat/rev, a TAR decoy, and an anti-CCR5 ribozyme were recentlydate). New approaches are needed. The next paths to HSC gene evaluated after lentiviral-mediated gene transfer to autologoustherapy point in 3 directions, pursuing either new designs of CD34ϩ cells in subjects with AIDS-related lymphoma.68 Whereasrandomly integrating viral vectors, targeted gene delivery strate- the gene-marking levels were approximately 2 logs lower than ingies, or the use of reprogrammed stem cells. Each one of these the ALD study, well below levels expected to provide a therapeuticavenues shows great promise but also distinctive, real challenges. beneﬁt, sustained expression of the shRNA was detected for up to 24 months in one of the patients, without discernable toxicity. For the most common inherited blood disorders, including the thalasse- mias and sickle cell anemia, high-level globin chain expression (onNew retroviral vector designs the order of picograms per cell)64 requires the vectors to incorpo-New retroviral vector types and vector designs, using a “self- rate powerful erythroid-speciﬁc enhancers (Figure 1D),23 reviewedinactivating” (SIN) vector modiﬁcation of ␥-retroviral vectors,51 elsewhere.69 Here the safety concern posed by inclusion oflentiviral vectors,52 and lineage-restricted vectors,23 are now enter- powerful elements is in part mitigated by their tissue speciﬁcity,ing the clinic (Table 1). The ﬁrst fundamental alteration to the limiting the probability that an adjacent oncogene would beﬁrst-generation design (Figure 1A) was the elimination of strong trans-activated in nonerythroid cells.43,64 A single patient treatedpromoter/enhancer elements in integrated proviral LTRs via dele- with such a vector has been followed for more than 3 years.24 Thistion of the LTR enhancer/promoter region from the 3Ј end of the subject, afﬂicted with HbE thalassemia, showed sustained expres-vector, which on proviral integration replaces the 5Ј LTR. This SIN sion of the vector-encoded globin starting 5 to 6 months afterdesign then requires the incorporation of an internal promoter to transplantation, most of which could be attributed to a singledrive transgene expression (Figure 1B). Hardly a new technique,51 expanded clone. This patient is currently leukemia-free despite thethis ␥-retroviral vector design is now in use in an X-SCID clinical prolonged clonal expansion and continues to produce an additionaltrial (Table 1). Because of concerns regarding recombination with 2 to 3 grams per dL of hemoglobin comprising the vector-encodedendogenous HIV, this SIN vector design has been adopted from the ␤(T37Q)–globin chain. The proviral insertion in this clone resultedget-go in later vectors derived from HIV-152 and foamy viruses53 in aberrant splicing and dysregulated expression of the HMGA2(Figure 1C). gene. A recent murine study links HMGA2 overexpression to In addition, HIV-derived vectors may possess a safety advan- clonal myeloid expansion, without leukemic transformation.70 Thistage over those derived from MLV because of their natural example serves as a cautionary note regarding the risk for clonalpropensity to integrate all along transcription units without prefer- expansion with any integrating vector, even those without strongence for promoter regions, in contrast to LTR-driven MLV-derived constitutive enhancers.vectors, as documented both in cell lines and in predictive large Powerful enhancers, especially nonspeciﬁc ones, would prob-animal models.36-38,54-56 These differences in integration patterns ably require to be ﬂanked by genetic elements with enhancer-have now been veriﬁed in human clinical trials via large-scale promoter blocking activity.71,72 The optimization use of suchinsertion site analyses.11,17,57,58 New studies using SIN ␥-retroviral elements, however, still remains elusive,73,74 their utility is un-vectors will be interesting to compare in this regard. Common proven in human or relevant large animal models, and inclusion inintegration sites detected after HSC lentiviral transduction and vectors may even precipitate mutagenic events, as demonstrated bytransplantation are located throughout large genomic regions and the alternative splicing from HMGA2 to the cHS4 chicken insulatorappear to result from integration biases associated with the core element in the aforementioned expanded clone.24 Additionaladditional factor of in vivo clonal selection and expansion via genetic switches and posttranscriptional regulatory mechanisms75proto-oncogene activation documented with ␥-retroviral HSC gene may add a further layer of control to these various vector designs.transfer.57,59 A number of reviews discuss recent insights into Several other types of integrating retroviruses are being devel-proviral integration into the genome, made possible by high oped as potential gene therapy vectors for HSCs but are much lessthroughput retrieval of integration sites and next-generation far along in development and have not yet been used in clinicalsequencing.60-63 The most commonly used lentiviral vectors typi- trials. The human foamy virus has not been associated with diseasecally harbor ubiquitous internal promoters to drive transgene in any species, has a broad target cell range, and efﬁcientlyexpression, such as that of human phosphoglycerate kinase or transduces hematopoietic cells.76 Its integration proﬁle is remark-elongation factor-1␣ (EF-1␣). These are not strong promoters, but ably random, and it has been used to phenotypically correct CD18they appear adequate for correction of enzymopathies, for which integrin deﬁciency in a canine model of leukocyte adhesionmodest amounts of transgene product are therapeutic (on the order deﬁciency.77 The avian sarcoma leucosis virus has also beenof femtograms protein per cell).64 Whether phosphoglycerate developed and tested in a nonhuman primate HSC transplantationkinase, EF-1␣, or WAS promoter-driven vectors will prove to be model. It also has a relatively random integration pattern and hassufﬁcient to redress defects in structural proteins or signaling the advantage that the LTR promoter/enhancer is completelymolecules or receptors is yet to be determined. A concern is that the inactive in mammalian cells.78use of stronger, ubiquitous polII promoter/enhancers will increase The inclusion of suicide genes in vectors, which would allowthe risk of trans-activating neighboring genes and malignant ablation of vector-containing cells in the context of an adversetransformation back toward the level of genotoxicity encountered event, has been little investigated in HSCs compared with other cellwith intact LTR regulatory elements. However, there is evidence types, but new studies are exploring their efﬁcacy in preclinical
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 HSC ENGINEERING AT A CROSSROADS 1111models. To this end, a number of new highly effective suicide genes occult genotoxicity.96 The ability to accurately predict and monitorhave been reported,79,80 including the human-derived, dimerizable the off-target effects of modiﬁed endonucleases is still an opencaspase-9 gene.81 In sum, all of the vector designs shown in Figure question.97,981B through D are expected to reduce the risk of insertional Gene targeting via HR in HSCs has lagged behind other targetoncogenesis relative to that of LTR-driven ␥-retroviral vectors. A cell types because of the challenges inherent in efﬁciently andnumber of preclinical models have been developed to try to assess nontoxically introducing the endonuclease and corrective targetinggenotoxic risk qualitatively or quantitatively before new vectors constructs into these fragile and rare cells unable to be cloned orare used clinically. These include in vitro immortalization of effectively expanded ex vivo. However, there has been recentmurine myeloid progenitor cells,82-84 serial transplantation in a progress using nonintegrating lentiviral vectors or optimizedmurine model,50 transplantation of transduced HSCs from geneti- nucleofection.95,99 Off-target genotoxicity and efﬁciency of long-cally tumor-prone mouse strains,85 or long-term follow-up of term HSC correction will be difﬁcult to assess in xenograft modelsnonhuman primates.56 It is reassuring that most of these models and will require both large-animals studies and pilot clinical trialscome to similar conclusions on the relative genotoxicity of different in a patient population with sufﬁciently serious disease complica-vector backbones. However, the proof can only come from clinical tions to justify introduction of these potentially risky approaches.studies, which will take several more years to come to fruition. It isnoteworthy that the vector conﬁgurations shown in Figure 1B through Dare already undergoing clinical testing (Table 1). New cell types for HSC engineering Current strategies to genetically engineer HSCs are conﬁned by ourTargeted gene delivery inability to expand or subclone genetically modiﬁed HSCs, whetherThe aforementioned strategies all continue to rely on semirandom adult or cord blood-derived. Whether this is the result of propertiesintegration of the vector provirus into the HSC genome and, as inherent to HSCs or lack of knowledge regarding appropriatesuch, will never be free of genotoxic risk. An alternative goal is the culture conditions, any strategy for mitigation of genotoxicitytargeting of transgene delivery to a predetermined chromosomal dependent on screening of vector insertion sites before celllocation, or the repair of a mutated locus, greatly decreasing the administration is not feasible at this time. Recent advances inrisk of insertional mutagenesis. Targeted gene delivery and gene pluripotent stem cell technology may, however, transform the facerepair would be optimal if clinically relevant targeting efﬁciencies of stem cell engineering, allowing much better characterization ofcan be achieved without off-target genotoxicity or immediate corrected cells before clinical use. The ground-breaking discoverytoxicity to transduced cells. of Yamanaka, who successfully reprogrammed mouse ﬁbroblasts to The gold standard for targeted gene delivery is homologous a pluripotent state similar to that of embryonic stem cells afterrecombination (HR). HR is a DNA repair mechanism that has been ␥RV-mediated introduction of the transcription factors Oct4, Sox2,successfully used to repair mutated genes and is therefore appli- Klf4, and c-myc,100 opens up new prospects for therapeutic stemcable in principle to cell-based therapies of monogenic diseases.86 cell engineering. The feasibility of expanding pluripotent stem cellsGene targeting by HR requires the use of homologous DNA without compromising their stem cell properties makes it possiblesurrounding the targeted site, usually delivered as plasmid DNA. to subclone and select genetically modiﬁed cells, as well as toIntroducing large amounts of plasmid DNA into target cells is perform extensive efﬁcacy and safety testing in the selected clonalinefﬁcient and toxic and has thus posed a major challenge in HSCs. derivatives (Figure 2). Thus, relatively inefﬁcient techniques, suchThe efﬁciency of DNA entry and of HR can be increased with the as classic HR, which are inapplicable to HSCs because of theiruse of adenoviral87 and adeno-associated virus vectors,88,89 but inefﬁciency, now become relevant. These concepts were dramati-these vector types are not well suited for use in HSCs. Another cally illustrated by Hanna et al in a mouse model of sickle celltechnique to promote speciﬁc HR uses triplex-forming oligonucle- anemia.101 In this study, the ␤S-globin gene was corrected by HR inotides that bind the major groove of duplex DNA,90 which are a ﬁbroblast obtained from a humanized sickle cell transgeniccoupled to a donor DNA sequence. Using nanoparticles for mouse, which was then reprogrammed to a pluripotent state byintracellular oligonucleotide delivery, this approach has been retroviral transduction100 and subjected to in vitro directed hemato-shown to target the endogenous ␤-globin locus in human CD34ϩ poietic differentiation in the presence of HoxB4 protein. Transplan-cells, resulting in levels of globin gene modiﬁcation in the range of tation of the speciﬁed hematopoietic cells did not achieve full0.5% to 1.0%.91 hematopoietic reconstitution but effectively blunted the sickle cell The efﬁciency of HR versus nonhomologous recombination can syndrome.101be increased by the introduction of DNA double-strand breaks at Patient-speciﬁc induced pluripotent stem (iPS) cells can bethe targeted site using an endonuclease.92 This requires the transient generated from various cell types obtained from patients withexpression of an endonuclease, which can be directed to a speciﬁc inherited or acquired disorders, using a range of techniques.102,103sequence using modular zinc ﬁnger proteins,86 homing endonu- Use of nonintegrating or excisable vectors for generation of iPScleases,93 or transcription activator-like effectors derived from cells may avoid issues of insertional mutagenesis and incompletephytopathogenic bacteria.94 Zinc ﬁnger nucleases were recently silencing of reprogramming factors. However, new genetic mate-shown to afford remarkable targeting frequencies at the CCR5 rial must be permanently introduced to correct the underlyinglocus, disrupting an average 17% of all CCR5 loci in CD34ϩ cord disease mutation. Recognizing that iPS-like cells can arise byblood cells, with retained ability to engraft immunodeﬁcient mice LV-mediated insertional mutagenesis alone,104 one approach is toand demonstration of engrafted human CCR5-disrupted cells pursue targeted correction, via engineered zinc ﬁnger nucleases,105resistant to HIV infection.95 Signiﬁcant questions remain regarding bacterial artiﬁcial chromosomes,106 and adeno-associated virus-the efﬁciency of targeting in bona ﬁde HSCs, and the risk of mediated HR.88 An alternative to HR and its enhanced variations isinﬂicting off-target effects, which may result in translocations or to screen iPS clones for the integration of lentiviral or other vectors
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.1112 ` RIVIERE et al BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 Figure 2. Evolving paradigms in HSC engineering. (A) Current strategies are restricted by the use of nonclon- able adult HSCs. (B) The advent of patient-speciﬁc pluripotent stem cells may open new strategies for ge- netic engineering and biosafety testing.into putative genomic “safe harbors” (ie, sites that sustain trans- able even if reprogramming were to be induced by chemical orgene expression without interfering with endogenous gene expres- other means that avoid the use of integrating vectors. Thesion).107,108 This capitalizes on the high efﬁciency of lentiviral immunogenicity of iPS-derived cells may also cause concern,transduction and their lack of nonspeciﬁc occult genotoxicity but is although currently available information is limited to the pluripo-potentially constrained by imperfect knowledge of all possible tent stem cells themselves.119mechanisms by which integrated foreign DNA can dysregulate The generation of engraftable adult HSCs from human ES andgene expression. iPS cells remains elusive to date and probably represents the single The ability to generate patient-speciﬁc iPS cells and correct largest hurdle to use of pluripotent or reprogrammed cells fortheir abnormalities via genetic repair or transgene delivery at hematologic applications. The ﬁrst hematopoietic cells arise in thewell-characterized “safe harbor” sites108 does not imply that the use primitive streak of the embryo, yielding a distinctive “primitive”of pluripotent stem cells as a source of HSCs is ready for hematopoiesis120 that cannot reconstitute adult hosts, and produces,implementation or that it may ever become suitable for human for example, erythroid cells with embryonic hemoglobins thatapplication. Several major questions need to be further investi- would be unable to function optimally for oxygen delivery ingated: how to best generate iPS cells efﬁciently with minimal postnatal life. The immediate precursors of “deﬁnitive” HSCs aregenotoxicity imparted through the reprogramming process; how to arterial endothelial cells, which generate HSCs capable of long-identify and qualify iPS clones that are suitable for clinical term multilineage repopulation of adult hosts beginning in theinvestigation, which addresses the genetic, epigenetic, tumori- dorsal aorta of the aorta-gonad-mesonephros region and thegenic, and differentiation potential of individual iPS clones; how to chorioallantoic vessels of the placenta.121-126 It is these earlygenetically engineer iPS clones effectively but without adding any CD34ϩ, c-kitϩ, CD41ϩ HSCs that migrate to the yolk sac and thegenotoxic insults through the repair process; how to generate fetal liver, where they vastly expand before relocating to the boneengraftable HSC-like cells capable of full and durable hematopoi- marrow around birth.120 To date, candidate HSCs derived frometic reconstitution in transplanted recipients; and how to scale up human ES and iPS cells by and large fail to engraft and reconstitutethe differentiation culture processes and ensure the depletion of irradiated adult recipients.127-130 Even production of engraftingcells with teratoma formation potential. Some of these concerns HSCs from murine ES cells, studied intensively for more thanapply to the use of pluripotent stem cells or reprogrammed cells in 20 years, is extremely inefﬁcient. Only the ectopic expression ofgeneral, whereas others speciﬁcally relate to their potential use for the transcription factor HoxB4 in the hematopoietic progeny ofhematopoietic applications. Recent studies have documented frequent genetic alterations in murine ESCs has resulted in long-term efﬁcient in vivo engraft-human ES and iPS cells, including point mutations (some affecting ment.131 Ectopic expression of HoxB4 has been shown to result inoncogenes), deletions, and gene duplications.109-111 These occur abnormal myeloid/lymphoid ratios in mice, and leukemogenesis inindependently of the reprogramming vectors and are thus distinct dogs and monkeys, suggesting that this approach to drivingfrom the problems related to insertional mutagenesis. These events hematopoiesis from pluripotent ES or IPS cells is not clinicallymay be in part linked to the reprogramming phase, which is known relevant.84,132,133to be enhanced in the absence of p53.112-114 Successful reprogram- Another recent approach, bypassing the need for iPS cells andming could even require accumulation of genetic and/or epigenetic the obstacles to generating HSCs from embryonic-type cells,alterations in cells undergoing extended cell culture, including consists of direct reprogramming of skin cells to a multipotentpluripotent stem cells, and the low efﬁciency of reprogramming progenitor stage via introduction of a single transcription factor,may result from a requirement for rare permissive mutations.115-117 Oct4.134 Unlike ES and iPS-derived hematopoietic cells, Oct4-In one recent report comparing the genome sequence of ﬁbroblasts reprogrammed progenitor cells possess desirable traits, such as thebefore and after reprogramming,118 several mutations found in the expression of adult globin genes on erythroid differentiation, andiPS cells could be traced back to the original ﬁbroblast; however, robust albeit short-term myeloid engraftment potential in immuno-others arose during the reprogramming period. No further genomic deﬁcient mice. The exact nature and therapeutic potential of thesealterations were detected after further clonal expansion, suggesting cells are presently unknown, but these ﬁndings point to tantalizingthe unique susceptibility or requirement for genomic changes discoveries that may come out of reprogramming and trans-during reprogramming. More studies are needed to better gauge the differentiation research.135 Another game-changing advance wouldintrinsic genotoxicity of reprogramming, which may be unavoid- entail the ability to reprogram human adult HSCs to an expandable
From bloodjournal.hematologylibrary.org at CAPES CONSORTIUM on February 7, 2012. For personal use only.BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 HSC ENGINEERING AT A CROSSROADS 1113state without diminishing their long-term self-renewal properties Genotoxicities remain a fundamental concern in all the aforemen-and their safety, a goal that has remained elusive to date. tioned approaches: insertional mutagenesis in the case of retroviral- mediated gene transfer, off-target effects when using nucleases to induce double-strand breaks to enhance targeted gene delivery, genetic alterations incurred during reprogramming to a pluripotentHSC engineering at a crossroads state, and genetic alterations arising throughout extended cell culture. Inclusion of suicide genes137 allowing in vivo ablation ofHSC engineering is now at a crossroads. HSC gene therapy has dangerous clones may be worthwhile and is under preclinicalproven beneﬁts in patients with severe immunodeﬁciencies, but the development.MLV-derived LTR-driven vectors used in initial clinical trials posetoo great a risk of genotoxicity for further clinical development. Astheir clinical use comes to an end, with the possible exception of ProspectsADA deﬁciency, the ﬁrst chapter of HSC gene therapy is nowclosed. New approaches are needed, and their development will The ﬁrst 2 decades of HSC gene therapy have been rich in lessons,greatly beneﬁt from important lessons learned in the early age of providing both strong support for the therapeutic potential of thisgene therapy. Clinical experience with LTR enhancer-deleted MLV approach and sobering lessons on the shortcomings of the geneticvector- or lentiviral vector-transduced HSCs is not mature enough engineering of stem cells. As the chapter on LTR-driven vectorsfor meaningful risk assessment, although results from animal comes to a close, several new chapters are already being written.models and in vitro transformation assays suggest that either of The immediate future will evaluate SIN-␥RVs, SIN-LVs, andthese vector classes will be signiﬁcantly less genotoxic and thus lineage-restricted vectors, all of which should reduce the risk ofsafer. Any integrating vector can integrate near and activate trans-activating proto-oncogenes after semirandom integrations.oncogenes, but removal of strong ubiquitous enhancers from both Next, targeted gene delivery systems have the potential to furtherself-inactivating MLV vectors and lentiviral vectors will greatly reduce the risk of integrating vectors at undesirable chromosomaldecrease the latter risk. However, other genetic effects, such as locations. Later, if pluripotent stem cells fulﬁll their promise for theabnormal splicing events,24 or inactivation of tumor suppressor generation of HSCs and if the genotoxicity issues of their owngenes, may still occur. Lentiviral vectors, as well as several novel types prove not to be prohibitive, genetically corrected cells in whichof vectors in preclinical development, including those derived from vector integration or gene repair can be fully ascertained before cellhuman foamy virus or avian sarcoma leucosis virus, have integration infusion will become available. Despite the signiﬁcant challenges,proﬁles that differ subtly in the targeting of genes or their promoter at least one of these new directions will eventually lead to safe andregion, which may result in signiﬁcantly different safety proﬁles.36,78,136 effective HSC therapies for hereditary and acquired disorders. HSCLentiviral vectors encoding ubiquitous promoters of moderate strength engineering remains one of the most tantalizing medical researchafford adequate titers and express transgenes at levels that seem to be objectives for the 21st century.appropriate for enzymatic deﬁciencies. The design of vectors that arebetter suited for conditions requiring higher protein expression is at thepresent less well deﬁned. Acknowledgments Targeted gene delivery approaches, including the use of zinc The authors thank Jason Plotkin for help with ﬁgures.ﬁnger nucleases, meganucleases, transcription activator-like effec- I.R. and M.S. were supported by the National Institutes oftor nucleases, and triplex-forming oligonucleotides, have made Health (grants HL053750, CA59350, and CA08748), the Experi-great advances. Their targeting frequencies, albeit 1 to 2 logs lower mental Therapeutics Center at Memorial Sloan-Kettering Cancerthan retroviral-mediated gene transfer efﬁciency, are on the way to Center, the Niarchos Foundation, the Leonardo Giambrone Founda-reaching clinically relevant values. tion, the Cooley’s Anemia Foundation, and NYSTEM. Somatic cell reprogramming opens the door to many geneticengineering approaches, including screening for retroviral integra-tions in potential genomic safe harbors108 and targeted gene Authorshipdelivery, including nuclease-based approaches and adeno-associated virus-mediated homologous recombination. The advent Contribution: I.R., C.E.D., and M.S. wrote the manuscript.of iPS cells is far from clinically relevant and poses a number of Conﬂict-of-interest disclosure: M.S. holds patents on globinfascinating biologic questions, spanning a broad range of issues gene transfer and chimeric antigen receptors for immune engineer-that concern the epigenetic and genetic status of iPS cells, their ing. The remaining authors declare no competing ﬁnancial interests.differentiation potential, and their propensity to transform, whether Correspondence: Michel Sadelain, Center for Cell Engineering,they have been genetically modiﬁed or not. In particular, the Molecular Pharmacology and Chemistry Program, Memorial Sloan-generation of human HSCs from ES or iPS cells remains an Kettering Cancer Center, Box 182, 1275 York Ave, New York, NYenigma, one that will hopefully soon be resolved. 10065; e-mail: firstname.lastname@example.org.References 1. Dunbar CE. 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