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Prepublished online as a Blood First Edition Paper on January 2, 2003; DOI 10.1182/blood-2002-07-2314.
REVIEW IN TRANSLATIONAL HEMATOLOGY
From the Department of Hematology and Oncology,
Hannover Medical School, Hannover, Germany; the Division
of Experimental Hematology, Cincinnati Children's Research Foundation,
Cincinnati, OH; the Departments of Neuroanatomy and Bone Marrow
Transplantation, University Hospital Eppendorf, Hamburg,
Germany; and the Department of Cell and Virus Genetics,
Heinrich Pette Institute, Hamburg, Germany.
Recent conceptual and technical improvements have resulted in
clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects,
based on preclinical and clinical data. To assess the therapeutic
potential of genetic interventions in hematopoietic cells, it will be
important to derive a classification of side effects, to obtain
insights into their underlying mechanisms, and to use rigorous
statistical approaches in comparing data. We here review side effects
related to target cell manipulation; vector production; transgene
insertion and expression; selection procedures for transgenic cells;
and immune surveillance. We also address some inherent differences
between hematopoiesis in the most commonly used animal model, the
laboratory mouse, and in humans. It is our intention to emphasize the
need for a critical and hypothesis-driven analysis of "transgene
toxicology," in order to improve safety, efficiency, and
prognosis for the yet small but expanding group of patients that could
benefit from gene therapy.
(Blood. 2003;101:2099-2113) "This is a strange drop in my blood" (Goethe).
"It is the dose that makes the poison" (Paracelsus).
"What can go wrong, will go wrong" (Murphy).
Hematopoietic stem cells (HSCs) are important targets for somatic gene
therapy, considering their availability for in vitro manipulation and
their enormous biologic capacity.1,2 In selected entities,
gene therapy involving manipulation of HSCs has now clearly shown
clinical efficiency, opening up new perspectives for the entire
field.3,4 However, it is a principle in pharmacology that
no true effect is possible without inducing side effects. Prognosticating the type and incidence of side effects is an important step toward predicting the overall therapeutic benefit for a new modality.
The genetic modification of HSCs generates special concerns:
1. These cells are long-lived and might represent a reservoir for the
accumulation of proto-oncogenic lesions.5
2. Current technology requires that HSCs have to be enriched and
cultured in vitro to become accessible to genetic manipulation.
3. This also implies that the engineered graft represents only a small
fraction (probably about 1%-10%) of the hematopoietic cell pool of a
healthy individual. Infused cells may therefore be altered not only in
terms of quality, but will also be heavily diluted by unmodified
counterparts residing in the body. This may result in the establishment
of a "strange drop in the blood," which could correct diseases only
if it were strongly enriched in vivo.
4. Therefore, achieving targeted amplification or preferential survival
of engineered cells is one important key to success in hematopoietic
gene therapy.2-4 However, clonal expansion, while limited
by cellular senescence and exhaustion,6 has also been
suggested as a risk factor contributing to cellular transformation, at
least when occurring under nonphysiologic conditions of
growth.7
5. HSCs, or at least the cell preparations enriched for HSCs,
may not only reconstitute the entire myeloerythroid and lymphoid spectrum, but they may also differentiate into or fuse with other cell
types, including endothelial; skeletal and heart muscle cells; hepatocytes; neurons; and epithelial of gut and lungs. However, the
frequency of such events is controversial.8-12 The
developmental potential of HSCs generates a huge repertoire of
conceivable biologic conditions and anatomic sites where side effects
may manifest. However, the likelihood of manifestations outside the
hematopoietic system appears to be relatively low unless special
triggers exist that drive fate-switching.11,12
6. Because of the high proliferative potential of HSCs, stable,
heritable gene transfer is required for successful genetic modification. In the current "state-of-the-art" only viral
vectors on the basis of retroviruses (including lentiviruses) mediate a
predictable efficiency of stable transgene insertion with a predefined
copy number.13 Chromosomal insertion guarantees transgene maintenance during clonal amplification. Episomally persisting viral
vector systems such as those based on Epstein-Barr virus are still
suboptimal14 because efficient gene transfer into HSCs is
either not yet available or maintenance and expression of transgene
copies are insufficiently investigated. Physicochemical methods result
in a low probability for stable transgene insertion (< 10 7. The use of retroviral (including lentiviral) vectors implies that
engineered cells of the same graft will vary with respect to transgene
insertion sites (which are unpredictable and can affect both transgene
and cellular gene expression), copy number per cell (which can be
controlled more easily, but not entirely), and sequence (which can be
modified in the error-prone process of reverse transcription). This
produces a mixed chimerism of genetic modification in different stem
cell clones, each with a theoretically distinct potential for eliciting
side effects.
To facilitate the evaluation and discussion of side effects, we
introduce a classification system at this point (Table
1).
As the whole process of genetic manipulation of transplantable HSCs is
complex (Figure
1), problems may be encountered at different levels: (1) enrichment and
culture of target cells (toxicity of cell manipulation); (2) vector
production (vector toxicity); (3) insertion of foreign sequences or
other alterations of the cellular genome (genotoxicity); (4) expression
of transgenes (for which we would like to introduce the term
"phenotoxicity"); (5) conditioning or selective drugs for
enrichment of gene-manipulated cells (selection toxicity); (6) immune
responses evoked by vector components or the transgene product
(immunogenicity); and (7) aggravating interactions of some of these
events.
Depending on the type, severity, and kinetics of side effects, patients
may be asymptomatic or present with unclear symptoms, such as fever of
unknown origin, signs of hemolysis, cytopenia of any lineage,
immunodeficiency, autoimmune disorders, myelodysplasia, or, at worst,
lymphoma, leukemia, or other types of malignancy. Some of these
disorders, most of which are of only theoretical signficance at
present, will occur only after prolonged periods of
time18,19 and may be missed in preclinical studies with limited follow-up after genetic manipulation of HSCs. However, increasing the potency of the methods and the numbers of treatments may
confront us with a growing number of reports.
Indeed, this review was prompted by our observation of a leukemia in a
mouse study with prolonged follow-up after retroviral gene transfer
into hematopoietic cells.20 Unfortunately, the first case
of a malignant disorder following clinical retroviral vector-mediated
gene transfer into human hematopoietic cells was observed shortly
thereafter, manifesting 3 years after the infusion of retrovirally
modified cells21,22 so that a once theoretical risk has become a real one. The uncertainty observed in the scientific and regulatory community following these reports23,24
reflects a considerable need for systematic toxicology of genetic cell modifications.
Paracelsus, a founder of toxicology, has provided 3 golden rules for
the assessment of side effects. The first is that poison is a question
of dose.25 Dose issues are encountered at several levels
in hematopoietic gene therapy (Figure
2): the number of gene transfer particles to which the cells are exposed,
the transgene copy number per cell, transcription rates, efficiency of
RNA processing, protein features such as activity or stability of
enzymes, the size of the target cell pool (generating a clonal
repertoire due to the variations in transgene processing and
integration), the life span of transplanted cells, and the number of
patients treated.
Paracelsus' second rule is that a compound has a specific site (within
the body) where it exerts the greatest effect.25 Applied
to gene therapy, this indicates that cell type and its developmental
plasticity really matter. The third rule is to use animal models for
preclinical dose finding.25 Therefore, the limitations of
animal models also have to be considered. Cell specificity and animal
testing have been central items in gene therapy from the beginning.
However, most studies focused on efficiency and were not designed to
measure unexpected effects.
The present review summarizes recent insights into molecular mechanisms
underlying side effects of genetic interventions in HSCs, following the
classification of issues listed above (Table 1), and discusses
consequences for the most commonly used animal model, the laboratory mouse.
Under steady-state conditions (normal hematopoietic turnover and
an intact bone marrow niche), the majority of HSCs cycles slowly, yet
continuously.26-28 For genetic modification, HSCs are either harvested from peripheral blood or bone marrow.29
The yield and biologic features of cells from these sources differ depending on the use of mechanical harvest versus cytokines (typically granulocyte colony-stimulating factor [G-CSF]) and/or
chemotherapy, which may have direct implications for the efficiency of
retroviral transduction and engraftment.29-31 Exposure to
cytotoxic agents may compromise the engraftment potential of
HSCs.32 Umbilical cord blood is a promising resource of
stem cells, but the limited numbers of HSCs contained in cord blood may
restrict a wider use in adults.33,34
Target cells of genetic manipulation usually have to be enriched to
facilitate physical interaction with vector particles (Figure 1).
Enrichment of HSCs for clinical use is most frequently achieved by
immunoaffinity selection for the CD34 antigen. Developed for
"mainstream" clinical applications, these processes for cell harvesting and enrichment have an excellent safety profile, and the
engraftment potential of CD34-enriched cells is very
good.35 However, according to our current understanding,
long-term repopulating HSCs probably represent less than 1%
of the CD34+ cell pool. Thus, the target pool size
currently used for gene transfer is probably about 100-fold greater
than actually required.
In theory, manipulating 10 000 HSCs (or maybe even much smaller
numbers) should be sufficient to achieve a polyclonal transgenic hematopoiesis.27,36,37 This would reduce significantly the numbers of vector particles required for cell manipulation, the risk of
random mutagenic events that are related to the number of transgene
insertions (below), and probably also the costs of the
procedure. However, methods required for further enrichment of HSCs,
such as isolation of the CD34+CD38 Although short-term reconstitution may be promoted following cell
expansion in vitro,41 current culture conditions may
induce a selective loss of long-term HSCs.29 Several
underlying mechanisms have been identified: commitment to
differentiation (loss of pluripotency) or even apoptosis, a
cell-cycle-associated loss of engraftment/homing properties, and
differential susceptibility to natural killer cell-mediated
rejection.29,42-44 Although engraftment with cultured cells alone has been rapid and sustained in clinical gene therapy studies,45,46 extended manipulations, such as prolonged
culture or enrichment of cells expressing the transgene prior to
infusion, may promote deficits in long-term
reconstitution.29,41,47 Similar considerations apply for
lymphocyte cultures.48 Long-term follow-up, which in
humans encompasses many years, will be required to draw firm
conclusions that HSC exhaustion is not triggered by the procedures used
during HSC manipulation in vitro.29 Therefore, all efforts invested to maintain stem cell properties during in vitro
culture are important. Improvements of HSC culture can be achieved by
(1) the use of serum-free culture conditions,49 (2) the
definition of appropriate cytokine combinations,50 (3) the
manipulation of transcription factor levels such as
HOXB4,51 (4) the introduction of other (such as
extracellular matrix) molecules52-54 or appropriate stroma
components,55,56 and (5) protocols allowing a return to
cell-cycle quiescence prior to infusion.57,58 Moreover, new vector systems are being developed to reduce the need for stem
cell proliferation prior to gene transfer.13,59-61
It may also be interesting to expand engineered cells in vitro
following gene transfer. However, in at least one case, this attempt
has been associated with an increased risk of malignant transformation
of transduced murine hematopoietic cells.62 Although it is
possible that the expansion culture promoted a specific side effect of
the vector or packaging cells used in this study, further work is
required to address the extent to which culture conditions support a
preferential growth of mutants with proto-oncogenic lesions.
In summary, new procedures for HSC harvest, enrichment, gene transfer,
and expansion culture need to be studied intensively before clinical
application. Besides "conventional" mouse models,32 immunodeficient mice38,63 or fetal sheep47
supporting engraftment of primitive human hematopoietic cells and
supporting studies in nonhuman primates64 serve as
valuable models for this purpose.
Conventional retroviral vectors based on mouse leukemia virus
(MLV) and the more recently developed lentiviral vectors (such as those
based on HIV-1) differ in many respects, particularly in their nuclear
import strategies.13,60,61 While MLV vectors require cell
division for chromosomal insertion, lentiviral vectors may also
transduce nonproliferating cells. However, lentiviral transduction
efficiency also declines according to cell-cycle stages in the order
M > G1 > G0. Another feature of
HIV-based lentiviral vectors is that complex transgene cassettes
containing cryptic splice sites are more reliably
transferred,65-68 which may be related to regulatory
functions of the viral REV protein expressed during the packaging
process. Because of the significant differences in the biologic
properties of the viral proteins involved in the generation of
replication-defective vectors, MLV and HIV vectors have distinct
requirements for the design and culture of their respective
producer cells.
Stable producer clones are more easily established with long
terminal repeat (LTR)-driven vectors
Replication-competent retrovirus Contamination of vector stocks with RCR can be detected by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), or cell biologic assays. While the sensitivity of these methods can be very high, residual contamination of a clinical vector preparation as a matter of principle cannot be fully excluded. Important improvements in the design of vectors and packaging cells have greatly reduced the risk of generating RCR.69The risk of developing a disease following accidental exposure to an MLV-related RCR depends heavily on the genetic background of the recipient and the integrity of the immune system. Replication-competent MLV with an amphotropic envelope protein was not found to represent a significant pathogen for immunocompetent or transiently immunosuppressed nonhuman primates.19 However, when CD34+-enriched cells were exposed to high titers of RCR-contaminated vector preparations in vitro and infused under conditions of strong immunosuppression, rhesus monkeys developed lymphomas within one year.72 This required the absence of an immune response against retroviral particles or infected cells, and was likely driven by insertional mutagenesis (below) due to massive virus replication within susceptible lymphoid cells.19,72 When inoculated into newborn mice, amphotropic MLV may also induce a spongiform encephalopathy, whose kinetics and anatomic distribution depend on the type of the envelope protein.73 Potential RCR originating from lentiviral packaging cells has not been described to date. Accordingly, the potential pathogenicity of such recombinants is unknown, yet expected to be unlike that of wild-type HIV as a result of the anticipated differences in Env proteins, regulatory elements for gene expression, and the absence of many HIV accessory genes. An established limitation of currently used stable lentivirus packaging lines is genetic instability, because they may undergo multiple superinfection events when cultured (due to the lack of subgroup interference with the VSV-G pseudotype used).74-76 Although no side effects have been reported in more than a decade of clinical experience even with early generations of retroviral producer cells,77 stringent safety testing and further technological improvements are still desirable for both retro- and lentivirus production systems. In the unlikely event of accidental exposure to RCR and their escape from immune control, it may be possible to suppress viral replication in patients using clinically approved inhibitors, unless resistance develops.78 Mobilization In the absence of an RCR originating from the packaging cells, spread of a retroviral vector could be possible when naturally occurring viruses exist that can package the vector RNA and are transmitted in the human population. This concern appears to be irrelevant for MLV vectors,79 but needs to be considered for vectors developed on the basis of HIV or other lentiviruses.80 To prevent this problem, lentiviral vectors are typically designed with a so-called self-inactivating (SIN) LTR. This is achieved by placing the enhancer-promoter into an internal position between defective LTRs, eliminating transcription of the packaging signal required for incorporation of the vector RNA in virus particles.60,61Transient transfection for vector production So far, both lentiviral and previously investigated retroviral SIN vectors cannot be produced at sufficient titers from cloned packaging cell lines.74 Efficient production of SIN vectors has been achieved only following transient transfection of plasmid vector constructs into packaging cells.61,74,81 Although significant amounts of vector particles can be produced using this procedure,81 concerns remain unresolved regarding the type and incidence of plasmid recombinations, accidental transfer of plasmid DNA with vector particles,70 and the identity of the product obtained in independent production batches.The infidelity of reverse transcription A limitation common to all types of retroviral vectors is the possibility for transgene recombination or mutation occurring during the obligate step of reverse transcription. The retroviral enzyme reverse transcriptase converts RNA to double-stranded DNA with an infidelity of about 10 4, suggesting that mutations are
introduced once per 10 kb of a retroviral RNA template.82
This may reflect an evolutionary pressure to produce about one mutation
per replication cycle, given a genome size of natural retroviruses in
the range of 8 to 11 kb. The misincorporation rate is similar for
vectors based on MLV and HIV.83 If we consider as a worst
case scenario a proto-oncogene such as N-RAS with a size of
570 bp, one mutation could occur per 18 retrovirally transduced copies.
For N-RAS, at least 3 activating mutations are known from a
total of 1710 (3 × 570) possibilities for single-point mutations
(http://www.expasy.ch/cgi-bin/niceprot.pl?p01111). Thus, about one
oncogenic N-RAS mutant would be formed per 104
reverse transcriptions. In a clinical setting, about
108 to 109 infectious particles are
required per CD34+ cell preparation. Therefore, it may
be important to define the oncogenic mutation frequency for a given
cDNA, especially when dealing with transgenes encoding
"signaling" molecules.
Much more frequent errors in transgene replication may result from sequence deletions or recombinations before or during reverse transcription.82,83 Regulatory genome sequences that can be required to achieve cell-type-specific gene expression84 and some clinically relevant cDNAs such as MDR1 or HSV-TK may contain cryptic splice sites that give rise to pregenomic splicing of the vector RNA in packaging cells.85,86 Interestingly, the frequency of these cryptic splicing events also depends on the packaging cell line.87 Also, intrastrand or interstrand recombinations are not uncommon during reverse transcription (retroviruses typically package 2 copies of a pregenomic RNA). These can be triggered by direct sequence repeats within the transgene,83,88 and again occur with similar frequency in vectors based on MLV or HIV.83 The vast majority of such events will simply reduce the efficiency of the gene transfer. However, it may be worthwhile to address potential hazards induced by aberrations of a given transgene prior to clinical testing. Attempts to reduce sequence repetitions, to eliminate unwanted splice sites, and to choose appropriate packaging cell clones greatly improve the fidelity of transferring intact transgene sequences.84,87,89,90 Another concern related to vector production is the accidental incorporation of cellular RNA in the retroviral particle. Acutely transforming retroviruses encoding cellular oncogenes have evolved through such events, again requiring recombination during reverse transcription.82,91 However, to create such an unwanted oncogene vector, further mutations triggered by multiple rounds of replication in virus/vector spread are typically required. Therefore, this risk appears extremely low with a replication-defective vector.
Complications resulting from transgene insertion (insertional
mutagenesis) are a concern for all stable gene transfer methods. Retroviral insertion has some unique properties. The first resides in
the fact that insertion is a default event in the retroviral life
cycle,82,91 implying that the frequency of transgene
insertion per cell can be predetermined by adjusting the multiplicity
of infection.92 The second is that insertion tends to take
place in euchromatin, possibly because of its improved
accessibility.93 Consequently, the risk for insertion in
transcriptionally active regions of chromosomal DNA is increased, as
recently also demonstrated for HIV and derived vectors.94
This implies a possibility for a cell-type-specific distribution, also
assisted by host factors that participate in the preintegration
complex.94 Retroviral integrases are not sequence-specific
with respect to transgene insertion, yet prefer specific structural
features (bended DNA).95 Thus, some yet unknown genetic
loci may be at increased risk for retroviral insertion.94
The third important feature of retroviral insertion is that it
typically does not create subsequent recombinations within or outside
the affected locus, although exceptions to this rule have been
reported. Postintegration deletions may occur within repeats
present in a single retroviral transgene, but these events appear to be
rare.96 Mutations within and surrounding a retroviral genome during expansion or malignant transformation of a transduced cell have also been described.97,98 Finally,
recombinations may occur between sequence-related, yet independently
inserted, retroviral alleles.99 However, the incidence of
such events in nontransformed cells is assumed to be low (although
probably not as low as the error rate of the cellular replication
machinery, which is in the range of 10 Incidence of recessive and dominant oncogenic insertions With improving sequence information available from the murine and human genome projects, retroviral insertion events become increasingly mappable with regard to their exact to-the-base chromosomal location, relation to neighboring sequences, and potential interference with coding and regulatory regions.20-22,94Previous assessments of the risk of untoward side effects from
retrovirus insertion have been estimated to be rather low (between 10 Based on the hypothesis of semirandom choice of target sequence,
proto-oncogenic activation by a transgene insertion event would be
expected to be more frequent. Considering that the entire human genome
consists of approximately 3 × 109 base pairs (bp), a
transforming insertion event frequency of 10 Restricting the area of retrovirus insertion interference to a diameter
of about 10 kbp around a given gene, the chance of a single insertion
interfering with a defined allele is roughly 10 At least 3 layers of safety, however, prevent such insertion events from being directly cancerogenic: first, retrovirus vector insertion is almost uniformly monoallelic,107 reducing the relevance of most recessive mutational events. This restricts the influence of insertional disturbance to the much more rare setting of dominant effects that are biologically active even if just one locus has been changed. Second, some signal alterations may trigger differentiation or apoptosis, impede engraftment, or otherwise reduce the survival probability of the affected cell clone. Third, and foremost, a single insertional mutation is, to our current knowledge, not sufficient to develop a malignant phenotype by itself.108 In the vector-associated incidents of murine and human leukemia that have recently been described,21,22 the insertional oncogene activation has at best contributed to a premalignant expansion of cells later developing the malignant clone because of additional genetic events. This underlines the need to screen for potential cooperation of insertional mutagenesis with side effects of the transgene or other circumstances contributing to clonal expansion of gene-modified cells (below). An issue of unknown significance is whether multiple insertions in single cells will lead to a disproportionate increase in the risk for insertional mutagenesis, although the few available data suggest a linear relationship between insertion frequency and mutagenesis.97,102 It cannot be excluded that a high copy number of largely identical retrovirus transgenes distributed all over the genome may trigger chromosomal instability. In general, side effects observed under conditions of a high multiplicity of infection62,109 may not be relevant for a more carefully controlled transduction procedure.110 Considering these uncertainties, it appears reasonable to opt for the transfer of not more than 1 or 2 transgenes per cell. This represents the efficiency of currently available methods,36,111 but may in the future be more of an issue in vector systems with a higher efficiency of integration or high multiplicity of infection.112 In conclusion, the likelihood of oncogenic lesions induced by insertional mutagenesis alone would be expected to be relatively small when compared with some other established medical treatments, such as irradiation or chemotherapy with DNA-damaging agents.103 Transformation of non-stem cells initiated by insertional mutagenesis does not seem to occur frequently: before 2002, no severe side effects related to insertional mutagenesis had been reported in more than a decade of clinical experience with retrovirus gene transfer into more committed hematopoietic cells and mature lymphocytes,1,2 probably involving the manipulation of more than 1012 cells. The number of cases in which these observations were made long-term is substantial, although the number of repopulating stem cells engrafted altogether probably did not exceed 10 to 100 per patient, putting the overall number of transgene insertion events in HSCs under long-term observation at approximately 104 to 105 worldwide. Impact of vector design The LTR configuration of conventional retroviral vectors comes with an increased risk to activate neighboring cellular sequences. The LTR establishes the enhancer-promoter regulating initiation of transcription and the polyadenylation signal giving rise to its termination on both ends of the transgene (Figure 3). Although MLV enhancer-promoters are strongly active in most hematopoietic cells (albeit with pronounced differentiation dependence),71,113 the polyadenylation signal is relatively weak.114 Moreover, the major retroviral splice donor or related motifs in the transgene may interact with downstream splice acceptors of cellular genes. Combined with insufficient termination, these features generate a number of possibilities for activation of cellular sequences located downstream of the transgene insertion site (Figure 3).
Some of the mechanisms giving rise to activation of a cellular gene also apply to lentiviral or MLV vectors with a SIN architecture.115 However, the most frequent mechanism involved in retroviral insertional oncogene activation appears to be enhancer related, possibly working orientation independent and over large distances. Such a risk applies to almost any type of transgene configuration. Considering the molecular mechanisms underlying activation of cellular genes, one could design vectors of improved safety. Such a vector should have a strong RNA termination/polyadenylation signal (serving as an "RNA insulator")114,115; an internal position of the enhancer and promoter sequences that are excluded from functional interactions with neighboring sequences through the inclusion of dominant DNA insulators116; and a strong internal splice acceptor that largely prevents interaction of the retroviral splice donor with downstream sequences. If functioning as predicted, such a (hypothetical) construct depicted in Figure 3B would reduce the risk of insertional mutagenesis to the residual risk of disrupting genes. The latter may often be irrelevant unless haploinsufficiency becomes phenotypically relevant or loss of heterozygosity occurs through independent hits. Another strategy to avoid insertional mutagenesis would be to achieve targeted insertion of transgenes into predefined "benign" cellular loci. Although conceptual progress has been achieved in the manipulation of retroviral integrase, experimental evidence for a stringent, sequence-specific targeting strategy is limited.117 A recent report indicates that physicochemical transfection procedures may be developed for targeted transgene insertion into defined genome loci in vivo (murine hepatocytes).16 It remains to be seen whether such technologies are free from genotoxic side effects and how they can be adapted to HSCs. Similar considerations apply to targeted transgene insertion technologies developed on the basis of AAV.118 Besides these primary prevention strategies, vectors could also be equipped with selectable marker genes to generate options for secondary prevention strategies. A drug-resistance marker could be used to reduce the clonal repertoire in vivo (Figure 3C) by ablating cells with low expression levels.119 However, if insertional oncogene activation enhances the fitness of cells during selection, this strategy may be counterproductive. Experiments addressing this issue have not been reported to our knowledge. Another option would be to include a negative selection marker in the transgene cassette. A conditional suicide gene (such as HSV-TK)90 may help to eliminate a malignant clone (Figure 3D), especially when combined with other antineoplastic treatments. However, this would also result in the loss of nontransformed transgenic cells. Before such an approach can be recommended, the potential immunogenicity of many suicide gene products and the limited preclinical experience with introducing suicide genes into HSCs has to be overcome.
The ultimate goal of genetic therapy is to replace in situ a defective gene sequence, ideally by homologous recombination repair of the original locus. However, using available vectors and HSCs as targets, somatic gene transfer typically results in ectopic and nonregulated expression of the transgene, both with respect to the cell type affected and the level of expression achieved. Depending on the type and assembly of cis elements used, expression levels generated by different vectors may differ by up to 3 orders of magnitude. Different variants of MLV enhancer-promoters and some cellular promoters have shown a great potential for multilineage and persistent transgene expression in hematopoietic cells in vivo, typically accounting for less than 1% of the total cellular protein content.71,113,119-123 Cellular control elements have been modified to provide lineage-specific expression with promising potency,65-67,124 and inducible expression has been achieved with designer promoters.68 The insertion site modulates all aspects of transgene expression, including duration, level, and differentiation dependence. With LTR-driven retroviral vectors, the majority of unselected clones shows fairly similar transgene expression levels. However, interclonal variability of transgene expression may be as high as 50-fold, and complete silencing can be observed in some HSCs and their progeny.113,120,122 Unless targeted insertion into the correct cellular allele or specific regulation is achieved, transgene expression will hardly ever be physiologic in every transduced cell. According to Paracelsus' first rule (poison is a question of dose), it can be predicted that any transgene product has a defined therapeutic window compatible with the desired function and without the predominance of unwanted effects. Toxicity related to transgene expression may most frequently manifest in a competitive disadvantage, leading to the extinction of the affected cell (clone) and thus to a loss of efficiency. However, transgene interference with cellular decisions related to homing, proliferation, or differentiation may eventually result in the manifestation of new types of diseases. Currently, few observations are available that support these concerns. However, we have to be aware that up to now far less than 1% of the human cDNA pool and a necessarily minute fraction of all artificial sequences possible have been introduced into gene therapy research. Moreover, gene delivery systems have and will continue to become increasingly potent, also allowing the simultaneous transfer of 2 or more cDNAs with a single vector. To support these considerations, 4 examples may be sufficient. Of the 4, 3 deal with the use of selectable marker genes, a key technology in hematopoietic gene therapy. These examples provide evidence for dose-dependent toxicity (HOXB4), an as yet uncertain contribution to a severe side effect (MDR1), and evidence for context-dependent side effects (dLNGFR). These and a final example (CD40L) highlight the importance of developing vectors for spatially or temporally controlled expression of transgenes. Ectopic expression of HOXB4: dose-dependent side effects? Retroviral vector-mediated expression of HOXB4, encoding a homeodomain transcription factor involved in the regulation of hematopoietic pool size, has been shown to promote polyclonal and regulated expansion of engineered HSCs.51,125 In contrast to many other homeobox genes, ectopic expression of HOXB4 in hematopoietic cells did not lead to overt alterations of differentiation or uncontrolled expansion of gene-modified cells in mice.126 The interest in HOXB4 gene transfer for cell therapy has been reinforced by the finding that murine embryonic stem (ES) cell-derived hematopoiesis can be partially converted to repopulation competence in adult hosts upon transient or stable activation of HOXB4 expression.127In human HSCs transplanted into immunodeficient mice, ectopic expression of HOXB4 promoted the expansion of primitive hematopoietic cells.128,129 However, high levels of HOXB4 expressed from "stronger" vectors impeded myeloid and lymphoid differentiation of human hematopoietic cells.129 In line with these data, impaired repopulation of lymphatic tissues was observed in a study using HOXB4-engineered hematopoietic cells derived from a somatic cloning procedure.130 These studies taken together argue that the effects of HOXB4 are highly dependent on the dose and the kinetics of its ectopic expression. Importantly, activation of HOXB4-interacting partners such as PBX1 (possibly by insertional mutagenesis) may be sufficient to promote transformation of HSCs with constitutive ectopic expression of HOXB4.131 Thus, a potential therapeutic use of HOXB4 may require an exact definition of a therapeutic window and may depend on the ability of regulated expression. Murine leukemia following MDR1 gene transfer: phenotoxicity, genotoxicity, or both? Adenosine triphosphate binding cassette (ABC) transporter pumps encoded by multidrug resistance 1 (MDR1) or ABCG2 are naturally expressed in primitive hematopoietic cells, explaining their inherent competence for extruding some fluorescent dyes and other amphiphilic compounds.132,133 Increasing expression levels of such pumps may promote a survival advantage in the presence of high doses of some chemotherapeutic agents,134,135 and independently antagonize some proapoptic signals, as shown for MDR1.132,136,137Interestingly, ectopic expression of ABCG2 was associated with impaired differentiation of myeloid cells in mice.138 It is yet unclear to what extent this effect is dose related. The results with MDR1 have been controversial. Numerous studies, including a transgenic mouse model, have shown the ability to overexpress MDR1 in hematopoietic cells without overt alterations of cell functions (other than the acquired drug-resistance phenotype).85,134,135,139,140 Applications in dogs,141 nonhuman primates,110 and clinical trials45,46,142 have been safe, with occasional evidence for increased pump activity, although gene transfer efficiency was likely very low. However, myeloproliferative disorders have also been observed in different strains of mice using retroviral vector-mediated transfer of MDR1 into hematopoietic cells,62,109 and disease induction was promoted by prolonged expansion of cells in vitro prior to transplantation.62 Interestingly, this disease was associated not only with ectopic expression of MDR1, but also with an unusually high transgene copy number (in many cases exceeding 10 copies per clone, which is quite unusual even in mouse studies). Therefore, the most straightforward explanation is that excess MDR1 expression in this study may have been pathogenic. Besides, sequences other than MDR1 could have been expressed from insertion of intact or rearranged vectors. This aspect needs to be clarified, as the genetic integrity of the inserted transgenes has not been investigated, and the disease was so far observed only with a specific vector backbone (based on a first generation vector derived from Harvey murine sarcoma virus containing, in addition to an engineered "splice corrected" cDNA, considerable amounts of viral gene remnants that are not required for proper vector function).62,109 Moreover, it has not been reported whether the otherwise well-designed control vectors used had a similar high copy number in the producer and target cells.62,109 Thus, it remains formally unclear whether the disease was dependent on side effects of very high MDR1 expression (driven from multiple transgenic alleles in the mouse model);137 the expression of vector sequences other than MDR1 (potentially driven from rearranged vectors); or an increased risk for insertional mutagenesis or genomic instability under conditions of high copy numbers per genome. It is also quite possible that some or all of these factors acted together to produce the myeloproliferation. Another open question is whether MDR1 overexpression may
promote engraftment of gene-modified HSCs,45 although
MDR1 expression alone would not be sufficient to overcome a
culture-dependent loss of engraftment capacity.143 Taken
collectively, these data indicate that defining a therapeutic window
for ectopic expression of MDR1 or other efflux pumps in
hematopoietic cells may be difficult. If future research will not
facilitate the definition of safe conditions of transgenic
MDR1 expression, alternative metabolic selection markers may
be more promising (Table 2 and references therein).
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Abstract
Effects and side effects...
