Celulas tronco hematopoieticas

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-2011-09-349993

Hematopoietic stem cell engineering at a crossroads
Isabelle Rivière, Cynthia E. Dunbar and Michel Sadelain

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Review article

Hematopoietic stem cell engineering at a crossroads
Isabelle 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 2Hematology

Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD

The genetic engineering of hematopoietic technologies used to date, which have on cell therapies that may transform medical
stem cells is the basis for potentially occasion resulted in clonal expansion, practice. In this review, we place these
treating 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 wave
paradigm for stem cell engineering in proved vector designs, targeted gene de- of new technologies and highlighting the
general. 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 stem

Introduction
The 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 and
acquired disorders that affect hematopoietic and other tissues. others are soon to follow. This review briefly summarizes the first
These include disorders of the immune system, such as severe 2 decades of HSC gene therapy, based on the use of first-generation
combined immunodeficiency (SCID) syndromes and AIDS, the (ie, LTR-driven) ␥-retroviral vectors, and critically assesses future
thalassemias, sickle cell anemia, metabolic disorders, including directions from the perspective of genetic engineering, examining
central nervous system pathologies, autoimmune diseases, and an the prospects, and challenges that lie ahead.
array of hematologic malignancies, which could be treated with
cancer-free autologous cells or prevented (eg, in the case of
Fanconi anemia).1,2 The safe and effective engineering of HSCs
thus represents one of the central goals of stem cell and gene LTR-driven vectors
therapies. 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 severe
genetically 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, more
biologic 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 the
of a leukemic transformation caused by a ␥-retroviral vector in a
therapeutic protein (the ADA enzyme for ADA deficiency, the
boy with X-linked SCID (X-SCID).3 The series of leukemias that
interleukin receptor common ␥-chain for X-SCID, the gp91phox
ensued (in 5 of 20 patients with X-SCID), which were later
oxidase complex protein for CGD, and the WAS signaling
followed by similar adverse events in trials for chronic granuloma-
integrator protein for WAS), in all hematopoietic cells, not just
tous disease and Wiscott-Aldrich syndrome, raised serious doubts
as to the merits of this approach and undermined human and the defective cell types, was deemed to be acceptable, thus
financial investments in this field over the past decade. However, justifying the use of the “first-generation” vectors available in
these serious adverse events also spurred an enormous, collective
investigation into the genotoxicity of gene delivery methodologies,
resulting in tremendous progress in our understanding of retroviral Table 1. Patients treated with engineered HSCs
vector integration and its impact on endogenous gene structure and Disease Vector type LTR-driven No. of treated patients
function.4-6 Although these serious adverse events have resulted in ADA ␥RV ϩ 40
discontinuation 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 20/3
provided a major impetus for developing novel approaches to CGD ␥RV ϩ 6
genetically 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 2
ries: improvements in the design of retroviral vectors, development
MLD SIN-LV Ϫ 4
of technologies for targeted gene delivery, and novel approaches

Submitted September 1, 2011; accepted November 8, 2011. Prepublished online as
Blood First Edition paper, November 17, 2011; DOI 10.1182/blood-2011-09-349993.

BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 1107

1108 `
RIVIERE et al BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5

Figure 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 the
ALD 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 LTR
U3 region; PRE/WPRE (woodchuck hepatitis), posttranscriptional regulatory element; SIN, self-inactivating vector design (ƒ represents U3 deletion); specificity: Ϫ indicates
ubiquitous; and ϩ, lineage-specific; LCR, locus control region; and HBB, human ␤-globin gene. Green represents retroviral enhancer/promoter elements; and red, mammalian
enhancer/promoter elements.

the early 1990s. These consist of recombinant replication-
incompetent ␥-retroviral genomes derived from murine leuke- Current technologies show their limits
mia viruses (MLVs), termed LXSN,7 MFG/SFG,8 and FMEV,9
which provide constitutive expression of the therapeutic cDNA Just as the benefits of retroviral therapies were starting to be
driven 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 to
been compelling. The majority of patients with SCID, ADA, and stem cell harvest and cell culture, whereas others are disease- or
WAS 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 fraction
natural killer cell function in X-SCID and regression of eczema and of engrafting HSCs that harbor the vector and expression of the
thrombocytopenia in WAS.10-14 Quality of life was improved for corrective genetic material on a per-cell basis that reaches over the
the majority of these patients.15 The outcome in CGD, a myeloid thresholds required to therapeutically impact on the underlying
disorder 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 this
dramatic 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 be
vectors derived from HIV-1, promising clinical results were
challenging in some conditions, such as Fanconi anemia19 and
recently obtained in 2 children with adrenoleukodystrophy (ALD),
sickle cell anemia.20
a disease characterized by multifocal brain demyelination. The
Current approaches to vector transduction require ex vivo
2 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 and
tion period is still limited (ϳ 3 years), these results bode very well
ability to compete with endogenous HSCs, perhaps resulting from
for 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 chemotherapy
leukodystrophy, and ␤-thalassemia (Table 1). Like the first- or irradiation is required in clinical settings where corrected HSCs
generation ␥-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 a
etic lineages, including the myeloid cells that eventually recon- challenge. Although adequate for most phase 1 or 2 studies, robust
stitute 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 lower
promoter (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 meet
cally 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 is
tial benefits to patients with a broad range of inherited and another category of concern because of the vagaries of position
acquired disorders. effects and the risk of transcriptional inactivation.28-30 Although

BLOOD, 2 FEBRUARY 2012 ⅐ VOLUME 119, NUMBER 5 HSC ENGINEERING AT A CROSSROADS 1109

Table 2. Clonal expansion, myelodysplasias, and transformation
Relevant vector
Patient/disease/ sequences Secondary effect (mo after Genomic insertion sites Other genetic alterations (mo
transgene (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) deletion
P5/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 42,139
P10/SCID-X1/␥C T-ALL, late cortical T cell (33) LMO2 (1), BMI1 (1) Notch mutation (1707A/P) 42,139
P1/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 translocation
P2/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-RNAs

well documented and extensively studied in murine models,30,31 unless the LTR prevents this from occurring,43 which may result in
vector silencing has been less investigated in clinical studies. It is the activation of an HSC-like transcription profile in thymocytes
noteworthy that the vast collections of integration sites documented and clonal expansion, eventually resulting in full malignant
in 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.44
because of the selective outgrowth of transgene-expressing cells. In Just as striking is the absence of such transformations in patients
the case of CGD, where such selective pressure on transgene with ADA-SCID, who also harbor LTR-containing vectors in their
expression does not apply, silencing and methylation of the T-cell precursors, including occasional integrations in the vicinity
vector’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 been
expression of marker genes, with no evidence for significant documented in an early ADA-SCID gene therapy trial, suggesting
silencing, 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 unclear
vectors.33-35 Silencing may be more problematic in murine cells, and may have to do the nature of the disease, with less profound
which have evolved mechanisms for inactivation of the huge and rapid expansion of corrected T-cell precursors, or a unique
proviral load integrated into the murine endogenous genome, but interaction between the transgene and activated LMO2.46
occurs 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 the
integration 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 subsequent
tion of proto-oncogenes in the genomic neighborhood is the most reports indicating that insertional mutagenesis after HSC gene
dreaded consequence. The direst outcome, frank leukemic transfor- transfer using ␥-retroviral was a universal risk. The lack of events
mation, has been dramatically illustrated in X-SCID and WAS, in the earlier clinical trials and large animal studies may have
where so far 5 of 20 and 1 of 10 patients, respectively, have resulted from exceedingly low HSC gene transfer efficiency and
developed clonal T-cell leukemias.39,40 A surprising feature of these lack of prolonged follow-up. Nonhuman primates and serially
clonal transformations, all linked to the integration of an LTR- transplanted mice receiving HSCs transduced with ␥-retroviral
driven ␥-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 LTR
2).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 receiving
complex) and normally silenced on hematopoietic differentiation, corrected CD34ϩ cells developed first clonal expansion of myeloid

1110 `

cells with vector insertions activating the MDS1/EVI1 or the that enhancers located within a vector are less prone to activation of
related PRDM16 gene loci, and then clonal myelodysplasia and neighboring genes than the same enhancer contained within an
marrow failure, with acquisition of an additional monosomy 7 LTR.65 Expression of miRNAs and shRNAs also require robust
abnormality 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 polII
thus all but over (with the possible exception of ADA deficiency, promoters. RNA-based anti-HIV moieties composing an shRNA to
which has been remarkably devoid of malignant complications to tat/rev, a TAR decoy, and an anti-CCR5 ribozyme were recently
date). New approaches are needed. The next paths to HSC gene evaluated after lentiviral-mediated gene transfer to autologous
therapy point in 3 directions, pursuing either new designs of CD34ϩ cells in subjects with AIDS-related lymphoma.68 Whereas
randomly integrating viral vectors, targeted gene delivery strate- the gene-marking levels were approximately 2 logs lower than in
gies, or the use of reprogrammed stem cells. Each one of these the ALD study, well below levels expected to provide a therapeutic
avenues 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 (on
New 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 reviewed
inactivating” (SIN) vector modification of ␥-retroviral vectors,51 elsewhere.69 Here the safety concern posed by inclusion of
lentiviral 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 be
first-generation design (Figure 1A) was the elimination of strong trans-activated in nonerythroid cells.43,64 A single patient treated
promoter/enhancer elements in integrated proviral LTRs via dele- with such a vector has been followed for more than 3 years.24 This
tion 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 after
design then requires the incorporation of an internal promoter to transplantation, most of which could be attributed to a single
drive transgene expression (Figure 1B). Hardly a new technique,51 expanded clone. This patient is currently leukemia-free despite the
this ␥-retroviral vector design is now in use in an X-SCID clinical prolonged clonal expansion and continues to produce an additional
trial (Table 1). Because of concerns regarding recombination with 2 to 3 grams per dL of hemoglobin comprising the vector-encoded
endogenous HIV, this SIN vector design has been adopted from the ␤(T37Q)–globin chain. The proviral insertion in this clone resulted
get-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 This
tage over those derived from MLV because of their natural example serves as a cautionary note regarding the risk for clonal
propensity to integrate all along transcription units without prefer- expansion with any integrating vector, even those without strong
ence 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 such
insertion 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 in
integration sites detected after HSC lentiviral transduction and vectors may even precipitate mutagenic events, as demonstrated by
transplantation are located throughout large genomic regions and the alternative splicing from HMGA2 to the cHS4 chicken insulator
appear to result from integration biases associated with the core element in the aforementioned expanded clone.24 Additional
additional factor of in vivo clonal selection and expansion via genetic switches and posttranscriptional regulatory mechanisms75
proto-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 less
throughput retrieval of integration sites and next-generation far along in development and have not yet been used in clinical
sequencing.60-63 The most commonly used lentiviral vectors typi- trials. The human foamy virus has not been associated with disease
cally harbor ubiquitous internal promoters to drive transgene in any species, has a broad target cell range, and efficiently
expression, 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 CD18
they appear adequate for correction of enzymopathies, for which integrin deficiency in a canine model of leukocyte adhesion
modest amounts of transgene product are therapeutic (on the order deficiency.77 The avian sarcoma leucosis virus has also been
of femtograms protein per cell).64 Whether phosphoglycerate developed and tested in a nonhuman primate HSC transplantation
kinase, EF-1␣, or WAS promoter-driven vectors will prove to be model. It also has a relatively random integration pattern and has
sufficient to redress defects in structural proteins or signaling the advantage that the LTR promoter/enhancer is completely
molecules or receptors is yet to be determined. A concern is that the inactive in mammalian cells.78
use of stronger, ubiquitous polII promoter/enhancers will increase The inclusion of suicide genes in vectors, which would allow
the risk of trans-activating neighboring genes and malignant ablation of vector-containing cells in the context of an adverse
transformation back toward the level of genotoxicity encountered event, has been little investigated in HSCs compared with other cell
with intact LTR regulatory elements. However, there is evidence types, but new studies are exploring their efficacy in preclinical


models. To this end, a number of new highly effective suicide genes occult genotoxicity.96 The ability to accurately predict and monitor
have been reported,79,80 including the human-derived, dimerizable the off-target effects of modified endonucleases is still an open
caspase-9 gene.81 In sum, all of the vector designs shown in Figure question.97,98
1B through D are expected to reduce the risk of insertional Gene targeting via HR in HSCs has lagged behind other target
oncogenesis relative to that of LTR-driven ␥-retroviral vectors. A cell types because of the challenges inherent in efficiently and
number of preclinical models have been developed to try to assess nontoxically introducing the endonuclease and corrective targeting
genotoxic risk qualitatively or quantitatively before new vectors constructs into these fragile and rare cells unable to be cloned or
are used clinically. These include in vitro immortalization of effectively expanded ex vivo. However, there has been recent
murine myeloid progenitor cells,82-84 serial transplantation in a progress using nonintegrating lentiviral vectors or optimized
murine 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 models
nonhuman primates.56 It is reassuring that most of these models and will require both large-animals studies and pilot clinical trials
come 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 is
noteworthy that the vector configurations shown in Figure 1B through D
are already undergoing clinical testing (Table 1).
New cell types for HSC engineering
Current strategies to genetically engineer HSCs are confined by our
Targeted gene delivery inability to expand or subclone genetically modified HSCs, whether
The aforementioned strategies all continue to rely on semirandom adult or cord blood-derived. Whether this is the result of properties
integration of the vector provirus into the HSC genome and, as inherent to HSCs or lack of knowledge regarding appropriate
such, will never be free of genotoxic risk. An alternative goal is the culture conditions, any strategy for mitigation of genotoxicity
targeting of transgene delivery to a predetermined chromosomal dependent on screening of vector insertion sites before cell
location, or the repair of a mutated locus, greatly decreasing the administration is not feasible at this time. Recent advances in
risk of insertional mutagenesis. Targeted gene delivery and gene pluripotent stem cell technology may, however, transform the face
repair would be optimal if clinically relevant targeting efficiencies of stem cell engineering, allowing much better characterization of
can be achieved without off-target genotoxicity or immediate corrected cells before clinical use. The ground-breaking discovery
toxicity 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 after
recombination (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 stem
cable in principle to cell-based therapies of monogenic diseases.86 cell engineering. The feasibility of expanding pluripotent stem cells
Gene targeting by HR requires the use of homologous DNA without compromising their stem cell properties makes it possible
surrounding the targeted site, usually delivered as plasmid DNA. to subclone and select genetically modified cells, as well as to
Introducing large amounts of plasmid DNA into target cells is perform extensive efficacy and safety testing in the selected clonal
inefficient and toxic and has thus posed a major challenge in HSCs. derivatives (Figure 2). Thus, relatively inefficient techniques, such
The efficiency of DNA entry and of HR can be increased with the as classic HR, which are inapplicable to HSCs because of their
use 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 cell
technique to promote specific HR uses triplex-forming oligonucle- anemia.101 In this study, the ␤S-globin gene was corrected by HR in
otides that bind the major groove of duplex DNA,90 which are a fibroblast obtained from a humanized sickle cell transgenic
coupled to a donor DNA sequence. Using nanoparticles for mouse, which was then reprogrammed to a pluripotent state by
intracellular 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 full
0.5% to 1.0%.91 hematopoietic reconstitution but effectively blunted the sickle cell
The efficiency of HR versus nonhomologous recombination can syndrome.101
be increased by the introduction of DNA double-strand breaks at Patient-specific induced pluripotent stem (iPS) cells can be
the targeted site using an endonuclease.92 This requires the transient generated from various cell types obtained from patients with
expression of an endonuclease, which can be directed to a specific inherited or acquired disorders, using a range of techniques.102,103
sequence using modular zinc finger proteins,86 homing endonu- Use of nonintegrating or excisable vectors for generation of iPS
cleases,93 or transcription activator-like effectors derived from cells may avoid issues of insertional mutagenesis and incomplete
phytopathogenic 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 underlying
locus, disrupting an average 17% of all CCR5 loci in CD34ϩ cord disease mutation. Recognizing that iPS-like cells can arise by
blood cells, with retained ability to engraft immunodeficient mice LV-mediated insertional mutagenesis alone,104 one approach is to
and demonstration of engrafted human CCR5-disrupted cells pursue targeted correction, via engineered zinc finger nucleases,105
resistant 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 is
inflicting off-target effects, which may result in translocations or to screen iPS clones for the integration of lentiviral or other vectors

1112 `

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 or
gene expression without interfering with endogenous gene expres- other means that avoid the use of integrating vectors. The
sion).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.119
mechanisms by which integrated foreign DNA can dysregulate The generation of engraftable adult HSCs from human ES and
gene 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 for
their abnormalities via genetic repair or transgene delivery at hematologic applications. The first hematopoietic cells arise in the
well-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 that
application. Several major questions need to be further investi- would be unable to function optimally for oxygen delivery in
gated: how to best generate iPS cells efficiently with minimal postnatal life. The immediate precursors of “definitive” HSCs are
genotoxicity 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 the
investigation, which addresses the genetic, epigenetic, tumori- dorsal aorta of the aorta-gonad-mesonephros region and the
genic, and differentiation potential of individual iPS clones; how to chorioallantoic vessels of the placenta.121-126 It is these early
genetically engineer iPS clones effectively but without adding any
CD34ϩ, c-kitϩ, CD41ϩ HSCs that migrate to the yolk sac and the
genotoxic insults through the repair process; how to generate
fetal liver, where they vastly expand before relocating to the bone
engraftable HSC-like cells capable of full and durable hematopoi-
marrow around birth.120 To date, candidate HSCs derived from
etic reconstitution in transplanted recipients; and how to scale up
human ES and iPS cells by and large fail to engraft and reconstitute
the differentiation culture processes and ensure the depletion of
irradiated adult recipients.127-130 Even production of engrafting
cells with teratoma formation potential. Some of these concerns
HSCs from murine ES cells, studied intensively for more than
apply to the use of pluripotent stem cells or reprogrammed cells in
20 years, is extremely inefficient. Only the ectopic expression of
general, whereas others specifically relate to their potential use for
the transcription factor HoxB4 in the hematopoietic progeny of
hematopoietic 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 in
oncogenes), deletions, and gene duplications.109-111 These occur abnormal myeloid/lymphoid ratios in mice, and leukemogenesis in
independently of the reprogramming vectors and are thus distinct dogs and monkeys, suggesting that this approach to driving
from the problems related to insertional mutagenesis. These events hematopoiesis from pluripotent ES or IPS cells is not clinically
may be in part linked to the reprogramming phase, which is known relevant.84,132,133
to be enhanced in the absence of p53.112-114 Successful reprogram- Another recent approach, bypassing the need for iPS cells and
ming 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 multipotent
pluripotent 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 the
before and after reprogramming,118 several mutations found in the expression of adult globin genes on erythroid differentiation, and
iPS 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 these
alterations were detected after further clonal expansion, suggesting cells are presently unknown, but these findings point to tantalizing
the 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 would
intrinsic genotoxicity of reprogramming, which may be unavoid- entail the ability to reprogram human adult HSCs to an expandable


state 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 pluripotent
HSC engineering at a crossroads state, and genetic alterations arising throughout extended cell
culture. Inclusion of suicide genes137 allowing in vivo ablation of
HSC engineering is now at a crossroads. HSC gene therapy has dangerous clones may be worthwhile and is under preclinical
proven benefits in patients with severe immunodeficiencies, but the development.
MLV-derived LTR-driven vectors used in initial clinical trials pose
too great a risk of genotoxicity for further clinical development. As
their clinical use comes to an end, with the possible exception of Prospects
ADA deficiency, the first chapter of HSC gene therapy is now
closed. 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 this
gene therapy. Clinical experience with LTR enhancer-deleted MLV approach and sobering lessons on the shortcomings of the genetic
vector- or lentiviral vector-transduced HSCs is not mature enough engineering of stem cells. As the chapter on LTR-driven vectors
for 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, and
these vector classes will be significantly less genotoxic and thus lineage-restricted vectors, all of which should reduce the risk of
safer. 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 further
self-inactivating MLV vectors and lentiviral vectors will greatly reduce the risk of integrating vectors at undesirable chromosomal
decrease the latter risk. However, other genetic effects, such as locations. Later, if pluripotent stem cells fulfill their promise for the
abnormal splicing events,24 or inactivation of tumor suppressor generation of HSCs and if the genotoxicity issues of their own
genes, may still occur. Lentiviral vectors, as well as several novel types prove not to be prohibitive, genetically corrected cells in which
of vectors in preclinical development, including those derived from vector integration or gene repair can be fully ascertained before cell
human 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 and
region, which may result in significantly different safety profiles.36,78,136 effective HSC therapies for hereditary and acquired disorders. HSC
Lentiviral vectors encoding ubiquitous promoters of moderate strength engineering remains one of the most tantalizing medical research
afford 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 are
better suited for conditions requiring higher protein expression is at the
present 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 of
tor 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 Cancer
than 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 genetic
engineering approaches, including screening for retroviral integra-
tions in potential genomic safe harbors108 and targeted gene Authorship
delivery, 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 globin
fascinating 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, NY
enigma, one that will hopefully soon be resolved. 10065; e-mail: m-sadelain@ski.mskcc.org.

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