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.

Effects and side effects in hematopoietic gene therapy

 “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 (< 10−4).15 Their efficiency may be increased when combined with endonucleases from retrotransposons or site-specific integrases.16 Adeno-associated viruses (AAVs) also have a low and variable rate of stable insertion.13 Recent advances in adenoviral vector technology may increase their potential for stable gene delivery.17 However, the utility of all of these alternative methods for transduction of HSCs with a defined and persisting transgene copy number is still unknown, as is the genetic risk associated with transgene insertion through these modalities.

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 (Table1).

Table 1.

Categories of side effects in the genetic manipulation of hematopoietic stem cells

LevelCategoryExample
Culture Loss of homing potential  
Vector Fusiogenic properties of viral envelope proteins  
Genotoxicity Insertional mutagenesis 
Phenotoxicity Interference of transgene product with cellular signaling networks  
Selection toxicity Side effects of regimens used to selectively engraft or expand manipulated cells 
Immune response Elimination of modified transgenic cells by antibodies or cytotoxic T cells directed against transgene-encoded antigens  
Interactions of 1-6 Cooperation of 3, 4, and 5 
LevelCategoryExample
Culture Loss of homing potential  
Vector Fusiogenic properties of viral envelope proteins  
Genotoxicity Insertional mutagenesis 
Phenotoxicity Interference of transgene product with cellular signaling networks  
Selection toxicity Side effects of regimens used to selectively engraft or expand manipulated cells 
Immune response Elimination of modified transgenic cells by antibodies or cytotoxic T cells directed against transgene-encoded antigens  
Interactions of 1-6 Cooperation of 3, 4, and 5 

As the whole process of genetic manipulation of transplantable HSCs is complex (Figure1), 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.

Fig. 1.

Schematic overview of the procedures involved in the ex vivo manipulation of hematopoietic stem cells for gene therapy and preclinical approaches.

Fig. 1.

Schematic overview of the procedures involved in the ex vivo manipulation of hematopoietic stem cells for gene therapy and preclinical approaches.

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 reports23,24reflects 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 (Figure2): 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.

Fig. 2.

Overview of dose issues in stem cell gene therapy.

The following items are indicated in the lettered circles: A, particle dosage; B, number of inserted genes; C, transcription; D, RNA processing; E, protein features; F, target pool size; G, cellular life span and plasticity; and H, number of patients.

Fig. 2.

Overview of dose issues in stem cell gene therapy.

The following items are indicated in the lettered circles: A, particle dosage; B, number of inserted genes; C, transcription; D, RNA processing; E, protein features; F, target pool size; G, cellular life span and plasticity; and H, number of patients.

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.

Toxicity of cell manipulation

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.29The 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 population or their more primitive precursors,29,38-40 have not yet been established for routine clinical use. High-grade purification of HSCs based on flow cytometry sorting has been shown to be feasible, but concerns remain regarding the risk of contamination, fitness of the sorted cells, selective interference with short-term engraftment, and risks associated with cell expansion.39 

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,32immunodeficient mice38,63 or fetal sheep47supporting engraftment of primitive human hematopoietic cells and supporting studies in nonhuman primates64 serve as valuable models for this purpose.

Risks related to vector production

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

Progress in the design of retroviral vectors developed on the basis of MLV has improved their performance with respect to vector production, gene transfer efficiency, and transgene expression.19,69-71 This vector system is the only one currently used in clinical trials for stable gene transfer into HSCs.2 One important advantage of MLV packaging components is their lack of cytotoxic effects, resulting in the ability to derive defined cloned producer cell lines with only 1 or 2 transgenes (“provector”) inserted to encode the vector. This ensures the highest degree of transgene stability that can be achieved with retroviral packaging cell technology. However, the establishment of stable cloned producers with a limited and defined transgene copy number is greatly alleviated when retroviral gene transfer is used to establish the provector. Thus, transcriptional control needs to originate from the long terminal repeat (LTR), as in wild-type retroviruses. This configuration implies an increased risk of activating neighboring cellular sequences in target cells (below) and for recombination with viral coding sequences in the packaging cells, potentially facilitating the accidental generation of replication-competent retrovirus (RCR) recombinants.19 

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.69 

The 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.60,61 

Transient 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.82This 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 104reverse 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 asMDR1 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.

Risks related to transgene insertion

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.94This 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.94The 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 10−9 per base and replication). Compared with retroviral gene transfer, physicochemically transfected DNA, especially when forming transgene concatamers,15,100 as well as AAV gene transfer101 may be associated with increased risk of genetic instability, also involving flanking cellular sequences.

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.20-22,94 

Previous assessments of the risk of untoward side effects from retrovirus insertion have been estimated to be rather low (between 10−6 and 10−8 per insertion event).97,102,103 In an experiment involving retrovirus infection of mouse embryonic carcinoma cells with a high copy number, the risk for inactivating a single gene locus (usually a recessive mutation) comprising 0.001% of a murine genome was determined to be in the range of 1 to 4 × 10−8 per insertion.97 The risk for producing a phenotype that could also be induced by dominant activation of oncogenes (growth-factor independence in TF-1 human leukemia cells) was in the range of 2 × 10−7 per insertion.102 However, these experiments focused on specific transforming events or mutation of specific target genes and involved cloning procedures to identify mutants, possibly reducing the sensitivity of the detection systems.

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 10−7 would mean that only a few hundred base pairs in the entire genome would allow oncogene activation. In the light of the fact that wild-type retroviruses have been demonstrated to interfere with genetic regulation from distances as far as 90 kbp upstream,104such numbers appear unrealistic.

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 10−5. Between 100 and 200 proto-oncogenes or oncogenes have been “fished” from the murine genome by retrovirus insertional mutagenesis studies.105,106 With a margin of safety, the number of potential proto-oncogenes in the human genome is therefore probably not higher than 1000. The risk of an insertional event within 10 kbp of a potential proto-oncogene can therefore be estimated to range between 10−2 and 10−3.

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.103Transformation 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,1,2probably involving the manipulation of more than 1012cells. 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 (Figure3). 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).

Fig. 3.

Impact of vector design on the potential activation of a cellular gene located downstream of the transcriptional direction of the vector.

The gray boxes in panel A show a randomly inserted retroviral transgene with a conventional LTR architecture. Here, the enhancer (E) and promoter (P) are terminally repeated, and the polyadenylation [p(A)] signal is weak; a splice donor is present in the retroviral untranslated region and another one within the transgene cDNA. The desired vector transcript is shown as line D; potential aberrant transcripts are numbered and shown as dotted lines. Either of the vector's SDs may interact with a splice acceptor (SA) of a downstream-located cellular gene to generate alternative splice products 1 and 2. Aberrant transcripts 1 to 3 result from lack of termination; transcript 4, from activation of the 3′ promoter of the vector; and transcript 5, from a distant action of the vector's enhancer on a cellular promoter (which may also be located upstream and/or in inverse orientation to the insertion). Transcripts similar to 1 and 4 have been detected in the case of leukemia following retroviral gene marking in mice.20 The hypothetical vector shown in panel B was designed to prevent aberrant transcripts by deleting the enhancer-promoter from the LTR, inserting a strong splice acceptor upstream of the vectors coding sequences, deleting SD of the cDNA, utilizing a strong p(A) signal, and flanking the transgene cassette with insulator (INS) sequences that prevent long-distance enhancer interactions. Secondary prevention strategies shown in panels C and D make use of coexpressed selectable marker genes (“Impact of vector design”).

Fig. 3.

Impact of vector design on the potential activation of a cellular gene located downstream of the transcriptional direction of the vector.

The gray boxes in panel A show a randomly inserted retroviral transgene with a conventional LTR architecture. Here, the enhancer (E) and promoter (P) are terminally repeated, and the polyadenylation [p(A)] signal is weak; a splice donor is present in the retroviral untranslated region and another one within the transgene cDNA. The desired vector transcript is shown as line D; potential aberrant transcripts are numbered and shown as dotted lines. Either of the vector's SDs may interact with a splice acceptor (SA) of a downstream-located cellular gene to generate alternative splice products 1 and 2. Aberrant transcripts 1 to 3 result from lack of termination; transcript 4, from activation of the 3′ promoter of the vector; and transcript 5, from a distant action of the vector's enhancer on a cellular promoter (which may also be located upstream and/or in inverse orientation to the insertion). Transcripts similar to 1 and 4 have been detected in the case of leukemia following retroviral gene marking in mice.20 The hypothetical vector shown in panel B was designed to prevent aberrant transcripts by deleting the enhancer-promoter from the LTR, inserting a strong splice acceptor upstream of the vectors coding sequences, deleting SD of the cDNA, utilizing a strong p(A) signal, and flanking the transgene cassette with insulator (INS) sequences that prevent long-distance enhancer interactions. Secondary prevention strategies shown in panels C and D make use of coexpressed selectable marker genes (“Impact of vector design”).

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.

Risks related to transgene expression

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 HOXB4expression.127 

In 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) orABCG2 are naturally expressed in primitive hematopoietic cells, explaining their inherent competence for extruding some fluorescent dyes and other amphiphilic compounds.132,133Increasing 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 forMDR1.132,136,137 

Interestingly, ectopic expression of ABCG2 was associated with impaired differentiation of myeloid cells in mice.138It 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 ofMDR1 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.62,109Thus, 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 althoughMDR1 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 transgenicMDR1 expression, alternative metabolic selection markers may be more promising (Table 2 and references therein).

Table 2.

Classes of selectable marker genes and corresponding drugs

PrincipleUtilityExample for geneDrug/agent categoryAgentReference no.
Surface tag In vitro dLNGFR Monoclonal antibody NA 120,121,123, 144-149  
Drug resistance In vitro and in vivo MGMT Cytotoxic agent Temozolomide, BCNU 137, 150-152  
  DHFR Cytotoxic agent MTX 137  
  MDR1 Cytotoxic agent Paclitaxel, etoposide 45,46,134,135,139-142  
Growth promoting In vitro and in vivo FK-mpl Chemical dimerizer AP1903 153,154  
  SAG Inducer of protein function Estrogens 155  
  HOXB4 Controlled expression required NA 51,125-131  
PrincipleUtilityExample for geneDrug/agent categoryAgentReference no.
Surface tag In vitro dLNGFR Monoclonal antibody NA 120,121,123, 144-149  
Drug resistance In vitro and in vivo MGMT Cytotoxic agent Temozolomide, BCNU 137, 150-152  
  DHFR Cytotoxic agent MTX 137  
  MDR1 Cytotoxic agent Paclitaxel, etoposide 45,46,134,135,139-142  
Growth promoting In vitro and in vivo FK-mpl Chemical dimerizer AP1903 153,154  
  SAG Inducer of protein function Estrogens 155  
  HOXB4 Controlled expression required NA 51,125-131  

This table does not provide a comprehensive overview of the genes developed as selectable markers. Its purpose is rather to show the different principles with a few selected examples. NA indicates currently not available as good manufacturing practice product.

Context-dependent toxicity of a cell-surface marker:dLNGFR

The cytoplasmically deleted low-affinity nerve growth factor receptor (dLNGFR, also abbreviated ΔLNGFR, LNGFR, tNGFR, or NGFR) was derived from p75 neurotrophin receptor (p75NTR) to develop a clinically applicable cell-surface marker for hematopoietic cells.144 Although dLNGFR has been used by several laboratories to tag gene-modified cells,145 few data have been published regarding the ability for long-term marking (> 1 year) of HSCs and their progeny.146 A nonhuman primate study reported a failure to mark long-term repopulating HSCs with dLNGFR, however without investigating potential mechanisms.147 On the other hand, use of dLNGFRin clinical trials with gene-modified T cells has been shown feasible and safe.148,149 However, a recent mouse experiment20 in conjunction with an earlier study in fibroblasts156 offered the hypothesis thatdLNGFR expression in myeloid cells may promote their transformation in an unusual, highly context-dependent manner. It is this proposed context dependence that renders the discussion of this issue interesting.

p75NTR is a member of the tumor necrosis factor (TNF) receptor superfamily that can bind all known neurotrophins (NTs) including nerve growth factor (NGF).157,p75NTR is usually not expressed in hematopoietic cells, with the exception of some B-cell subsets.158 The cytoplasmic domain of p75NTR contains a proapoptotic juxtamembrane region and a death domain.157 These sequences were deleted in dLNGFR before their precise function was known in an attempt to create an inert surface marker.144 The deletion may weaken the anchoring in the cell membrane, and therefore the shedding of dLNGFR,145 which is still able to bind NTs in vivo,159 may affect the local extracellular cytokine milieu. Moreover, deletion of the intracellular domain renders dLNGFR structurally similar to naturally occurring antiapoptotic decoy receptors of the TNF-receptor family, which can act as dominant-negative inhibitors of proapoptotic intracellular pathways.160 

In cells expressing TrkA, TrkB, orTrkC, which encode tyrosine kinase receptors for different NTs, association of p75NTR creates a heterodimeric receptor complex with increased ligand affinity that is not dependent on the presence of the cytoplasmic residues.157 It is noteworthy that coexpression of either one of the Trk receptors with ap75NTR mutant that lacked most of the intracellular domain, a construct basically identical with dLNGFR, resulted in transformation of fibroblasts when NTs were added to the culture.156 This growth-promoting role dLNGFR is clearly dependent on the coexpression of a Trk gene and the presence of NTs (Figure4). The same configuration occurred in the murine monocytic leukemia originating in association with retroviral insertional up-regulation ofEvi1 in hematopoietic cells, which provides circumstantial evidence but no formal proof of a contributing role ofdLNGFR.20 

Fig. 4.

Proposed context-dependence of side effects elicited by dLNGFR.

Situation A represents a physiologic situation that can be observed in neuronal and some other cell types.157 Situation B was shown to promote the transformation of fibroblasts in vitro.156 Situation C represents the ideal context for cell marking.144 

Fig. 4.

Proposed context-dependence of side effects elicited by dLNGFR.

Situation A represents a physiologic situation that can be observed in neuronal and some other cell types.157 Situation B was shown to promote the transformation of fibroblasts in vitro.156 Situation C represents the ideal context for cell marking.144 

Evi1 encodes a Zinc-finger transcription factor that has been implicated in the pathogenesis of human myelodysplastic syndromes and acute myeloid leukemia (AML). Ectopic expression of Evi1impairs granulocytic differentiation, but leads to only mild alterations of hematopoiesis in transgenic mouse models.161 We proposed a specific interaction of dLNGFR and Evi1 in the induction of the leukemic clone, possibly reflecting a bias for a lineage (ie, monocytic) in whichTrkA expression and NGF signaling were also present and functionally relevant.20,158 

If this hypothesis can be confirmed, it would represent an example for cooperation of random insertional mutagenesis (genotoxicity) and transgene-related side effects (phenotoxicity) in the induction of leukemia. Alternatively or in addition, Evi1 may have induced expression of TrkA,162 and the interaction with dLNGFR may have promoted the transformation of a monocytic precursor. Also, a protein related to Evi1 has been shown to play a role in Trk-signaling of C elegans,163 opening further possibilities for transforming loops.

The potential risk associated with the use of dLNGFRin HSCs is underlined by observations that signals generated through oncogenic versions of Trk receptors may contribute to the pathogenesis of human AML.164-166 Therefore, dLNGFR does not appear to be a perfect choice for the manipulation of cells with a broad plasticity such as HSCs. However, as side effects of dLNGFR are proposed to be context dependent, its use in restricted cell lineages lacking cooperating signal transducers can be justified (considering Paracelsus' second rule). Interestingly, variants of dLNGFR have been developed that are deficient in ligand binding167 in order to reduce the probability of side effects. Similar concerns of context-dependent side effects and potential for cooperation with randomly activated oncogenes apply to many other therapeutic or marker genes.

Problems resulting from unregulated expression:CD40L

Finally, the mode of transgene expression is an important determinant of potential toxicity. This has been exemplified in an attempt to develop gene therapy for inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome). Ectopic constitutive, but not naturally regulated, expression of CD40L, although at low level, produced abnormal proliferative responses in developing murine T lymphocytes, apparently through dysregulated intercellular interactions during thymic maturation and selection.168 For many applications of hematopoietic gene therapy it is worth repeating the conclusion of this study: “Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.”168 

These 4 examples should be sufficient to underline the importance of a systematic risk assessment of the transgenes under consideration. Special attention should be paid to molecules that are involved in cellular signaling networks, such as those required for correction of some inherited disorders3,168,169 or those generated as surface tags120,123,144 or artificially inducible proteins that promote cellular proliferation or differentiation decisions.153-155 We would propose that such transgenes should be tested under conditions of high, intermediate, and low constitutive expression,129 preferentially achieved with vector design and not with variation of transgene dosage. Preclinical assay systems available for such work range from cell-culture–based model systems to animal studies and functional genomics or proteomics (Figure5).

Fig. 5.

Schematic overview of potential interactions between genotoxicity (alteration of cellular genes by vector insertion) and phenotoxicity (side effects of transgene expression), and experimental approaches allowing their detection.

Interaction may either occur in cis (on the same DNA molecule) or in trans (through mobile factors).

Fig. 5.

Schematic overview of potential interactions between genotoxicity (alteration of cellular genes by vector insertion) and phenotoxicity (side effects of transgene expression), and experimental approaches allowing their detection.

Interaction may either occur in cis (on the same DNA molecule) or in trans (through mobile factors).

Risks related to conditioning, use of selective drugs, and cell amplification

Following genetic modification of HSCs in vitro, their engraftment and contribution to hematopoiesis in vivo are dependent upon the methods used for conditioning or selective amplification. Conditioning eliminates host cells prior to infusion of gene-modified cells. Irradiation or cytotoxic agents induce a moderate to severe (myeloablative) lymphohematotoxicity. However, these regimens can be complicated by severe long-term toxicity. Nonmyeloablative regimens with sublethal toxicity have become increasingly well investigated170 and begin to show great promise for HSC-mediated gene therapy.4,171 In animal models, high doses of donor cells32 and application of G-CSF to the recipient before nonmyeloablative conditioning172 have been shown to promote engraftment. However, it is unclear whether chimerism will be maintained in a stable manner in the long term when nonmyeloablative protocols are performed in an autologous clinical setting. Here, donor-dependent immune functions have no facilitating role to promote engraftment of the transplant; tolerance may be incomplete and engineered cells usually do not have a spontaneous selective advantage. An alternative, potentially more specific and thus less toxic approach to conditioning is the use of monoclonal antibodies directed against stem cell antigens or more common leukocyte antigens.173 Although it is likely that side effects associated with conditioning regimens will be reduced significantly in the near future, this issue will continue to be an important aspect of the risk-benefit evaluation for stem cell–based gene therapy.

Importantly, several diseases could be successfully treated with a moderate rate of chimerism (5%-30%). A selective survival advantage of engineered HSCs can be promoted upon transfer and expression of appropriate selectable marker genes. Table 2 summarizes 3 different categories of such genes that have a well-documented efficiency in animal models. Potential side effects resulting from the expression of selectable marker genes have been reviewed above (“Risks related to transgene expression”).

For most of these selectable marker genes, drugs are required to trigger their function. Therefore, side effects associated with these drugs represent another important aspect of the preclinical and clinical evaluation. Some of these agents have a well-documented toxicity profile in humans; others represent experimental agents with limited clinical experience. In this context, it is interesting to note that the most powerful selection system currently available for gene-modified hematopoietic cells requires the use of DNA-damaging agents.137,150-152 Although potentially less toxic alternatives for selective amplification of gene-modified cells have been proposed,153-155 expansion of hematopoietic cells promoted by these gene functions may be incomplete, lineage restricted, and unstable, suggesting preferential action at the level of progenitor cells as opposed to HSCs. This implies a need for repetitive use of the corresponding drugs over prolonged periods of time, or induction of a distorted hematopoiesis with unclear long-term consequences.

Clonal amplification of transgenic cells is another important variable.7 The risk for accumulating mutations that are not related to gene transfer increases with the life span and the number of generations of the engineered cell. In most conditions of human bone marrow transplantation, the size of the graft's stem cell dose implies a modest pressure for expansion and a high likelihood for polyclonal reconstitution. This is underlined by results from nonhuman primate studies of gene-marking.36,111 With the advent of genetic selection strategies, a risk related to forced expansion of individual clones may become more relevant. On the other hand, single clones of transduced HSCs may provide a perfectly normal hematopoiesis with persistence of transgene expression in all hematopoietic lineages, at least in mice.120,123 This supports the idea that clones with favorable insertion sites and “neutral” transgenes are not necessarily at increased risk for transformation, even when undergoing massive expansion. The minimal number of HSCs that stably support primate hematopoiesis remains to be defined.

Risks related to immune surveillance

A further category of side effects is related to innate or acquired immunity against vector components or immune surveillance of engineered cells. A recent review proposed that certain gene transfer procedures may set “danger” signals that result in an increased likelihood of an immune reaction.174 However, severe inflammatory reactions elicited by viral proteins in the vector preparation, as observed with early generations of adenoviral vectors administered in vivo,175 are unlikely following a single administration of ex vivo–manipulated hematopoietic cells. In principle, a transient exposure to antigens may be caused by remnants of vector particles or culture media components on infused cells even if the transgene does not encode viral antigens.176 This risk appears small with conventional retroviral transduction protocols in which cells are cultured for at least a day following the final exposure to vector particles. However, with the use of adenovirus177 or lentivirus vectors178 the time in culture after the final round of vector exposure may be shortened, which could increase the probability of contamination with viral antigens.

Repetitive infusion of engineered cells may be complicated by sensitization to antigens originating from components of culture media, vector particles, or transgene expression, potentially resulting in clearance of transgenic cells179 or even severe acute adverse reactions. This potential problem could be solved by appropriate preparation of cells and recipient prior to infusion or simply by single use of engineered cells (given that the vector system does not generate antigens to which pre-existing immunity exists). Also, sensitization could be diagnosed prior to repeated infusion of cells.

Moreover, it will be interesting to determine whether some clinical settings, such as those resulting from repeated infections, may trigger cellular or humoral innate immune functions to clear incoming gene-modified cells or create an unfavorable cytokine milieu. If so, a transient blockade of these mechanisms, as proposed in the context of preclinical adenoviral gene therapy,180 may improve the “take” of gene-modified cells.

Immune responses mounted against transgene antigens may develop with some latency. This concern is of particular relevance when introducing artificial or xenogenic sequences (as in the use of some selectable marker genes) and when correcting inherited genetic disorders in so-called CRIM (cross-reactive immunologic material)-negative patients. Although bone marrow transplantation may promote tolerance to multiple or individual antigens,181-183 this does not necessarily occur following nonmyeloablative conditioning regimens. Immune-mediated rejection of transgenic cells expressing the xenogenic marker enhanced green fluorescent protein occurred in a study with nonhuman primates.184 Disturbingly, one affected animal developed hemolytic anemia after rejecting the transgenic cells.184Further investigations are needed to determine whether autoimmunity can be induced as a side effect of sensitization against transgenic cells. For more advanced applications of gene therapy as well as for allogeneic transplantation, tolerance induction is a key issue of future research.

Combination and interactions of risk factors

After listing this collection of potential problems, it is important to mention that combinatorial side effects of transgene insertion (genotoxicity), transgene expression (phenotoxicity), and cell expansion (selection toxicity) may be required to produce malignant transformation. Monocytic leukemia observed afterdLNGFR marking in mice and serial bone marrow transplantation may serve as a paradigm.20 It is possible that at least some clones observed in the CD40L-induced lymphoproliferation168 or in theMDR1-associated myeloproliferative disease62,109 had a similar history involving insertional mutagenesis in addition to transgene side effects and/or forced cell expansion. Insertional activation (in cis) of an oncogene or an otherwise “innocent” transcription factor may change the cellular program (in trans), which in turn may cooperate with the transgene product to induce an undesired phenotype (Figure 5). Such program alterations may also influence expression levels of the transgene, jointly acting to promote the initial survival of a premalignant clone.

Similar considerations apply for the serious adverse event recently observed in a clinical gene therapy trial.21,22 Ten children with X-linked severe combined immunodeficiency (X-SCID) were successfully treated by retroviral transfer of the interleukin 2 receptor common γ-chain into CD34+ cells and reinfusion of cells without conditioning. This transgene was required to correct the underlying genetic deficiency and provides (in X-SCID patients with this deficiency) a powerful selective advantage during T-cell maturation.3 The initial outcome of gene therapy was better than that typically achieved with allogeneic bone marrow transplantation.3 At 3 years after cell infusion, one patient presented with clinical signs of an acute lymphoblastic leukemia (ALL) caused by a monoclonal proliferation of γδ T cells. The clone had one insertion of an intact vector copy that occurred in one LMO2 allele (readily identified by LAM-PCR)21,36 inducing ectopic expression of this proto-oncogene. LMO2 is known to be involved in the pathogenesis of ALL, but probably not sufficient to cause malignancy (for which 4 to 6 independent genetic “hits” seem to be required).21,22,108,185 Although no evidence for aberrant signaling through the transgenic common γ-chain has been reported, its physiologic function obviously was sufficient to promote a strong expansion of the clone. Although no detailed analysis is available at this point, it can be postulated that additional genetic hits could have subsequently promoted malignant progression: the malignant clone (expressing a mature T-cell phenotype) was more mature than the initially transduced cell population (which must have lacked T cells), and a severe varizella zoster virus infection was reported to have coincided with first clinical symptoms of hematologic abnormalities. Moreover, a cytogenetic abnormality was observed that did not involve the insertion site. Another level of concern was a genetic background of childhood cancer.21,22 

Given that an unfavorable concert of oncogenic factors is required for tumor manifestation (as in the model presented in Figure6), such a serious adverse event is not expected to be found in any clinical scenario for retroviral gene therapy, nor can we predict its frequency in gene therapy for X-SCID patients. Besides the specific clinical setting and features of the transgene (cDNA and regulatory regions), target cell features represent an important variable. These determine the overall susceptibility to gene transfer, which loci are open for transgene insertion, and how many of these may contribute to malignant transformation.103 Further points to consider include the number of cell generations following gene transfer,103 the expansion conditions that may sometimes suppress balancing proapoptotic signals, the exposure to mutagenic hazards that are independent of the genetic manipulation, and the endogenous capacity for DNA repair and proliferation control. Finally, systemic responses to transformed cell clones add another level of complexity (Table3).

Fig. 6.

Not every insertional oncogene activation is expected to result in clinical manifestation of malignancy (hypothetical curve).

Transformation-promoting events are symbolized by flashes; the one marked with i represents insertional mutagenesis; the one labeled t indicates transgene side effects; u1 and u2 indicate subsequent unrelated hits. Clone size, time course, and the probability of extinction represent arbitrary values.

Fig. 6.

Not every insertional oncogene activation is expected to result in clinical manifestation of malignancy (hypothetical curve).

Transformation-promoting events are symbolized by flashes; the one marked with i represents insertional mutagenesis; the one labeled t indicates transgene side effects; u1 and u2 indicate subsequent unrelated hits. Clone size, time course, and the probability of extinction represent arbitrary values.

Table 3.

How cell type may determine the risk of insertional mutagenesis

LevelBefore insertionAfter insertion
Genome Number of pre-existing mutations Potential for secondary mutations 
 Number and size of activated, potentially disruptible “dangerous” loci susceptible to transgene insertion Proliferation capacity/propensity  
Cell-transgene interactions, direct Host factors participating in preintegration complex Recognition of transgene regulatory elements and expression level of transgene  
 Cell-cycle status Type and number of mechanisms supporting transgene side effect  
Cellular response, indirect  Response balance to “danger” signals 
  Potential for escape from senescence  
  Number of generations following genetic modification  
  Level of differentiation 
LevelBefore insertionAfter insertion
Genome Number of pre-existing mutations Potential for secondary mutations 
 Number and size of activated, potentially disruptible “dangerous” loci susceptible to transgene insertion Proliferation capacity/propensity  
Cell-transgene interactions, direct Host factors participating in preintegration complex Recognition of transgene regulatory elements and expression level of transgene  
 Cell-cycle status Type and number of mechanisms supporting transgene side effect  
Cellular response, indirect  Response balance to “danger” signals 
  Potential for escape from senescence  
  Number of generations following genetic modification  
  Level of differentiation 

Bearing this in mind, we need to consider appropriate preclinical models, including animal experiments, in order to derive clear statistics indicating the importance of individual risk factors and the probability of their combinations. For scientific, economical, and ethical reasons, studies will often rely on work with cell lines and laboratory mice. In this respect, it is important to discuss features of the mouse model that distinguish it from human hematopoiesis.

The mouse model compared with human hematopoiesis

The outstanding role of the laboratory mouse for modeling human development and disease has received further support by the recent findings of the mouse genome project.186 Nevertheless, the differences between the hematopoietic systems of mice and humans must be carefully evaluated to diagnose with certainty reactive and neoplastic blood cell disorders and to improve the predictive value of the animal model.

Even in humans, the classification of preleukemic states such as the myelodysplastic syndromes is still controversial.187Very recently, preleukemia and its progression to leukemia have been classified in mice,188,189 in analogy to the French-American-British (FAB) scheme developed for human leukemia.190 Murine leukemias may be experimentally induced with specific genetic alterations.191 Examples for preleukemic alterations are ectopic expression ofBCR-ABL,192,N-RAS,193,BCL2,194 or Evi1,161and the ICSBP-knock-out mouse.195 The latency period between leukemia induction by application of x-rays and/or inoculation of MLVs and leukemia manifestation could be regarded as a preleukemic condition.

The susceptibility of mice to develop leukemia varies according to strain and its contamination with MLVs. Unless a genetic predisposition is involved (such as endogenous RCRs), spontaneous leukemia occurs only sporadically in older animals. It can be induced with high incidence by irradiation and inoculation of newborn or immunodeficient animals with MLVs. RCRs transform cells by insertional mutagenesis, which has been useful in the identification of tumor-associated genes.82,91,104-106,196,197 In some cases acute leukemia can be induced following a rapid polyclonal expansion of progenitor cells when a mouse is infected with a retrovirus complex that cotransfers a replication-defective retrovirus encoding an oncogene.82,91 

The mouse has a significantly higher daily hematopoietic cell turnover (especially of red blood cells and platelets) compared with humans. Accordingly, the complete bone cavities are used for hematopoiesis, and there are very few or no fat cells interspersed.198,199Reactive or malignant increases of hematopoietic tissue rapidly lead to extramedullary hematopoiesis, typically starting in the spleen.200-202 Thus, splenomegaly with expansion of red pulp caused by leukemic infiltrations (> 20% blasts) and regression of the white pulp (periarteriolar lymphatic sheath and lymph follicles) are characteristic and early findings in murine leukemia.188,199 In advanced disease, normal architecture of spleen is totally abolished and widely replaced by masses of blasts. Liver involvement in AML is characterized by leukemic infiltrates in periportal areas. Therefore, unlike human leukemia, bone marrow is only variably, and spleen is constantly, involved in murine leukemia.200 

The mouse model may not be fully predictive for human leukemia development when considering differences in HSC turnover. Leukemia development often involves genetic alterations of true HSCs.5 However, it is unclear whether this also applies to oncogenesis related to retroviral manipulations. The pool size of murine HSCs is tightly regulated, although with considerable genetic and age-dependent variability.203 Abkowitz et al postulate a similar size of the HSC pool in mice and cats (approximately 12 000 per animal), and possibly also in humans.27 The study also suggests a conservation of the replicative activity per lifetime between murine and human HSCs.27 Thus, mice would present with a higher density and shortened cycling times of HSCs within the bone marrow.

However, if the burden of insertional mutagenesis also involves less primitive progenitor and precursor cells, typical mouse experiments performed with a relatively small total number of hematopoietic cells would underestimate the risk (about 106transplanted per mouse, compared with at least 108 cells in a clinical trial). Moreover, the life span of this animal is short (2 years), and for practical reasons, observation periods rarely exceed 6 to 12 months, further reducing the chance to detect slowly developing dysplasias. Bone marrow transplantation and inappropriate cell culture strongly reduce the pool size of HSCs.51 Serial bone marrow transplantations generate monoclonal or oligoclonal hematopoiesis in mice, suggesting an enormous pressure for massive amplification of individual HSCs.51,120,123,204,205 Such a forced expansion may promote the manifestation of dysplastic or overt leukemic clones.7,206,207 Thus, although the mouse model suffers from a poor sensitivity to detect rare mutagenic events related to integrating, nonreplicating vectors, in case such events do occur, experimental conditions can be adjusted to promote their manifestation.

It has been demonstrated that rodent cells can be more easily transformed than human cells, and several functional differences of signaling pathways involved in human and rodent models of transformation have been identified.108 One has been linked to a stronger activity of the telomerase function in mature rodent cells. Thus, only 2 to 3 hits are typically required for transformation of a rodent cell: 1 that dysregulates apoptosis and 1 or 2 further hits that alter cell-cycle control and provide proliferative stimuli. In humans, yet another event promoting telomerase activity is required for malignant transformation of mature epithelial and fibroblastic cells, but it is still unclear whether this would be needed to transform a human HSC.5,108 If so, human cells would be more resistant to transformation by random vector insertions and transgene side effects.

Summarizing these considerations, it appears justifiable to develop mouse models in which the sensitivity for detecting side effects related to genetic manipulations is increased by the choice of the experimental conditions, such as in vitro expansion62; hemolysis202 or bleeding206; serial bone marrow transplantation20,207; or introduction of a proto-oncogenic genetic lesion.161,191-195 Also, “humanization” of the mouse genome may be helpful to obtain models of increased predictive value.108,186 Currently available “large”-animal models would be even more relevant for human gene therapy, but obviously do not allow broader genotoxicity screening.64 Moreover, tumor manifestations usually take many years in nonhuman primates. It may be better to design oversensitive mouse models as a “worst case” scenario, thereby generating clear statistics that reflect the impact of defined risk factors.

Conclusions

Successfully exploiting the enormous potential of gene therapy targeting hematopoietic cells requires an open eye for side effects. Proof of principle in animal models may be spectacular, but is not all that is relevant for developing safe clinical applications. As with the use of drugs or irradiation, dose finding is a required next step. One realistic option to improve safety without loss of efficiency is to translate better procedures for stem cell purification into clinical use. The resulting reduction in the target cell pool likely represents a straightforward way to reduce the risk of insertional mutagenesis. The associated long-term goal is to reconstitute hematopoiesis with just a few clones of genetically characterized transgenic stem cells. Beyond the issue of target cells, dose finding takes into account the interaction of multiple features in vectors, transgenes, and clinical scenarios, which can be reflected in the design of preclinical models. Improved vector design may result in greater target site specificity of insertion and a transgene composition that has a reduced risk of activating cellular genes. Another important goal is to derive a risk classification of transgenes and clinical scenarios, considering all conditions potentially contributing to side effects. This may help to avoid premature generalization of side effects that occurred under specific circumstances. A database of vector insertion sites and statistical analyses on the clonality of reconstitution will be key to understanding the dynamics of these processes. Results obtained in long-term follow-up, especially, will allow us to interpret the impact of the combined action of the transgene and the vector insertion site on cell biology, a factor that until now, because of lack of accessibility, has not been studied in detail.

Individual institutions may provide significant contributions, but a combined international effort will probably be needed to accumulate the amount of data required. Revisions of the existing regulatory guidelines may be helpful only if they promote reasonable standards of comparison and agreements on basic experimental approaches in preclinical research. We propose to consider the collaborative development of preclinical proto-oncogenic worst case models as one basis for dose finding. Thus, developing tools for the best feasible genetic treatment will profit from a dialectic approach that anticipates adverse events by active, hypothesis-driven investigation. While doing so, we need to continue with many of the current clinical trials on the basis of the risk evaluation at hand. It should not be forgotten that for a number of patients, after carefully weighing risks and benefits, the worst case scenario of gene therapy may be to not receive it.

We thank the reviewers and the editors for important suggestions.

Prepublished online as Blood First Edition Paper, January 2, 2003; DOI 10.1182/blood-2002-07-2314.

Supported by grants from the Deutsche Forschungsgemeinschaft (KFO 110; WI1955/1-1); the European Union (QLK3-2001-01265, QLRT-2001-00427); the VolkswagenStiftung; and the National Institutes of Health (D.A.W.; grant no. HL 53586).

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

1
Anderson
WF
Gene therapy: the best of times, the worst of times.
Science
288
2000
627
629
2
Williams DA, Nienhuis AW, Hawley RG, Smith FO. Gene Therapy 2000. In: Hematology. American Society of Hematology Education Program Book. American Society of Hematology: Washington, DC; 2000:376-393.
3
Hacein-Bey-Abina
S
Le Deist
F
Carlier
F
et al
Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.
N Engl J Med.
346
2002
1185
1193
4
Aiuti
A
Slavin
S
Aker
M
et al
Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.
Science.
296
2002
2410
2413
5
Reya
T
Morrison
SJ
Clarke
MF
Weissman
IL
Stem cells, cancer, and cancer stem cells.
Nature.
414
2001
105
111
6
Allsopp
RC
Weissman
IL
Replicative senescence of hematopoietic stem cells during serial transplantation: does telomere shortening play a role?
Oncogene.
21
2002
3270
3273
7
Chow
M
Rubin
H
Clonal selection versus genetic instability as the driving force in neoplastic transformation.
Cancer Res.
60
2000
6510
6518
8
Blau
HM
Brazelton
TR
Weimann
JM
The evolving concept of a stem cell: entity or function?
Cell.
105
2001
829
841
9
Orkin
SH
Zou
LI
Hematopoiesis and stem cells: plasticity versus developmental heterogeneity.
Nat Immunol.
3
2002
323
328
10
Ying
QL
Nichols
J
Evans
EP
Smith
AG
Changing potency by spontaneous fusion.
Nature.
416
2002
545
548
11
Wagers
AJ
Sherwood
RI
Christensen
JL
Weissman
IL
Little evidence for developmental plasticity of adult hematopoietic stem cells.
Science.
297
2002
2256
2259
12
Morshead
CM
Benveniste
P
Iscove
NN
van der Kooy
D
Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations.
Nat Med.
8
2002
268
273
13
Kay
MA
Glorioso
JC
Naldini
L
Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.
Nat Med.
7
2001
33
40
14
Delecluse
H-J
Pich
D
Hilsendegen
T
Baum
C
Hammerschmidt
W
A first generation packaging cell line for Epstein-Barr virus derived vectors.
Proc Natl Acad Sci U S A.
96
1999
5188
5193
15
Baum
C
Transfection.
Encyclopedia of Molecular Biology.
Creighton
TE
1999
2596
2600
Wiley & Sons
New York, NY
16
Olivares
EC
Hollis
RP
Chalberg
TW
Meuse
L
Kay
MA
Calos
MP
Site-specific genomic integration produces therapeutic Factor IX levels in mice.
Nat Biotechnol.
20
2002
1124
1128
17
Mitani
K
Kubo
S
Adenovirus as an integrating vector.
Curr Gene Ther.
2
2002
135
144
18
U.S. Food and Drug Administration, Center for Biologics Evaluation and Research. Guidance for Industry. Supplemental guidance on testing for replication competent retrovirus in retroviral vector based gene therapy products and during follow-up of patients in clinical trials using retroviral vectors.http://www.fda.gov/cber/gdlns/retrogt1000.pdf
19
Cornetta
K
Morgan
RA
Anderson
WF
Safety issues related to retroviral-mediated gene transfer in humans.
Hum Gene Ther.
2
1991
5
14
20
Li
Z
Düllmann
J
Schiedlmeier
B
et al
Murine leukemia induced by retroviral gene marking.
Science.
296
2002
497
21
Hacein-Bey-Abina
S
von Kalle
C
Schmidt
M
et al
A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.
N Engl J Med.
348
2003
255
256
22
Check
E
A tragic setback.
Nature.
420
2002
116
118
23
Check
E
Regulators split on gene therapy as patient shows signs of cancer.
Nature.
419
2002
545
546
24
European Society of Gene Therapy. Annual meeting of the ESGT: French gene therapy group reports on the adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID). Position of the ESGT. European Society of Gene Therapy: Munich, Germany. 2002. Available at:http://213.80.3.170/esgt/downloads/ESGTXSCID2.pdf
25
Borzelleca
JF
Paracelsus: herald of modern toxicology.
Toxicol Sci.
53
2000
2
4
26
Cheshier
SH
Morrison
SJ
Liao
X
Weissman
IL
In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells.
Proc Natl Acad Sci U S A .
96
1999
3120
3125
27
Abkowitz
JL
Catlin
SN
McCallie
MT
Guttorp
P
Evidence that the number of hematopoietic stem cells per animal is conserved in mammals.
Blood.
100
2002
2665
2667
28
Bradford
GB
Williams
B
Rossi
R
Bertoncello
I
Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment.
Exp Hematol.
25
1997
445
453
29
Verfaillie
CM
Hematopoietic stem cells for transplantation.
Nature Immunol.
3
2002
314
317
30
Hematti P, Sellers SE, Agricola BA, et al. Retroviral transduction efficiency of G-CSF+SCF mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L mobilized cells in nonhuman primates. Blood. Prepublished online November 7, 2002, as DOI 10.1182/blood-2002-08-2663.
31
Lapidot
T
Petit
I
Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells.
Exp Hematol.
30
2002
973
981
32
Stewart
FM
Crittenden
RB
Lowry
PA
Pearson-White
S
Quesenberry
PJ
Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice.
Blood.
81
1993
2566
2571
33
Moritz
T
Keller
DC
Williams
DA
Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease.
J Exp Med.
178
1993
529
536
34
Kohn
DB
Parkman
R
Gene therapy for newborns.
FASEB J.
11
1997
635
639
35
Vogel
W
Scheding
S
Kanz
L
Brugger
W
Clinical applications of CD34(+) peripheral blood progenitor cells (PBPC).
Stem Cells.
18
2000
87
92
36
Schmidt
M
Zickler
P
Hoffmann
G
et al
Polyclonal long-term repopulating stem cell clones in a primate model.
Blood.
100
2002
2737
2743
37
Kim
HJ
Tisdale
JF
Wu
T
et al
Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates.
Blood
96
2000
1
8
38
Larochelle
A
Vormoor
J
Hanenberg
H
et al
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.
Nat Med.
2
1996
1329
1337
39
Michallet
M
Philip
T
Philip
I
et al
Transplantation with selected autologous peripheral blood CD34+Thy1+ hematopoietic stem cells (HSCs) in multiple myeloma: impact of HSC dose on engraftment, safety, and immune reconstitution.
Exp Hematol.
28
2000
858
870
40
Bhatia
M
Bonnet
D
Murdoch
B
Gan
OI
Dick
JE
A newly discovered class of human hematopoietic cells with SCID-repopulating activity.
Nat Med.
4
1998
1038
1045
41
McNiece
I
Bridell
R
Ex vivo expansion of hematopoietic progenitor cells and mature cells.
Exp Hematol.
29
2001
3
11
42
Glimm
H
Eisterer
W
Lee
K
et al
Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice.
J Clin Invest.
107
2001
199
206
43
Berrios
VM
Dooner
GJ
Nowakowski
G
et al
The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic stem cells.
Exp Hematol.
29
2001
1326
1335
44
Jetmore
A
Plett
PA
Tong
X
et al
Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients.
Blood.
99
2002
1585
1593
45
Moscow
JA
Huang
H
Carter
C
et al
Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.
Blood.
94
1999
52
61
46
Abonour
R
Williams
DA
Einhorn
L
et al
Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.
Nat Med.
6
2000
652
658
47
McNiece
IK
Almeida-Porada
G
Shpall
EJ
Zanjani
E
Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential.
Exp Hematol.
30
2002
612
616
48
Sauce
D
Bodinier
M
Garin
M
et al
Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms.
Blood.
99
2002
1165
1173
49
Glimm
H
Flugge
K
Mobest
D
et al
Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.
Hum Gene Ther.
9
1998
771
778
50
Zandstra
PW
Lauffenburger
DA
Eaves
CJ
A ligand-receptor signaling threshold model of stem cell differentiation control: a biologically conserved mechanism applicable to hematopoiesis.
Blood.
96
2000
1215
1222
51
Antonchuk
J
Sauvageau
G
Humphries
RK
HOXB4-induced expansion of adult hematopoietic stem cells ex vivo.
Cell.
109
2002
39
45
52
Moritz
T
Patel
VP
Williams
DA
Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.
J Clin Invest.
93
1994
1451
1457
53
Hanenberg
H
Xiao
XL
Dilloo
D
Hashino
K
Kato
I
Williams
DA
Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.
Nat Med.
2
1996
876
882
54
Donahue
RE
Sorrentino
BP
Hawley
RG
et al
Fibronectin fragment CH-296 inhibits apoptosis and enhances ex vivo gene transfer by murine retrovirus and human lentivirus vectors independent of viral tropism in nonhuman primate CD34+ cells.
Mol Ther.
3
2001
359
367
55
Schiedlmeier
B
Buss
EC
Veldwijk
MR
Zeller
WJ
Fruehauf
S
Soluble bone marrow stroma factors improve the efficiency of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells.
Hum Gene Ther.
10
1999
1443
1452
56
Nolta
JA
Thiemann
FT
Arakawa-Hoyt
J
et al
The AFT024 stromal cell line supports long-term ex vivo maintenance of engrafting multipotent human hematopoietic progenitors.
Leukemia.
16
2002
352
361
57
Takatoku
M
Sellers
S
Agricola
BA
et al
Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.
J Clin Invest.
108
2001
447
455
58
Dao
MA
Hwa
J
Nolta
JA
Molecular mechanism of transforming growth factor beta-mediated cell-cycle modulation in primary human CD34(+) progenitors.
Blood.
99
2002
499
506
59
Williams
DA
Smith
FO
Progress in the use of gene transfer methods to treat genetic blood diseases.
Hum Gene Ther.
11
2000
2059
2066
60
Buchschacher
GL
Jr
Wong-Staal
F
Development of lentiviral vectors for gene therapy for human diseases.
Blood.
95
2000
2499
2504
61
Ailles
LE
Naldini
L
HIV-1-derived lentiviral vectors.
Curr Top Microbiol Immunol.
261
2002
31
52
62
Bunting
KD
Galipeau
J
Topham
D
Benaim
E
Sorrentino
BP
Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice.
Blood.
92
1998
2269
2279
63
Dao
MA
Tsark
E
Nolta
JA
Animal xenograft models for evaluation of gene transfer into human hematopoietic stem cells.
Curr Opin Mol Ther.
1
1999
553
557
64
Donahue
RE
Dunbar
CE
Update on the use of nonhuman primate models for preclinical testing of gene therapy approaches targeting hematopoietic cells.
Hum Gene Ther.
12
2001
607
617
65
May
C
Rivella
S
Callegari
J
et al
Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin.
Nature.
406
2000
82
86
66
May
C
Rivella
S
Chadburn
A
Sadelain
M
Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene.
Blood.
99
2002
1902
1908
67
Pawliuk
R
Westerman
KA
Fabry
ME
et al
Correction of sickle cell disease in transgenic mouse models by gene therapy.
Science.
294
2001
2368
2371
68
Vigna
E
Cavalieri
S
Ailles
L
et al
Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors.
Mol Ther.
5
2002
252
261
69
Wilson
CA
Ng
TH
Miller
AE
Evaluation of recommendations for replication-competent retrovirus testing associated with use of retroviral vectors.
Hum Gene Ther.
8
1997
869
874
70
Chen
J
Reeves
L
Sanburn
N
et al
Packaging cell line DNA contamination of vector supernatants: implication for laboratory and clinical research.
Virology.
282
2001
186
197
71
Baum
C
Richters
A
Ostertag
W
Retroviral vector-mediated gene expression in hematopoietic cells.
Curr Opin Mol Ther.
1
1999
605
612
72
Donahue
RE
Kessler
SW
Bodine
D
et al
Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.
J Exp Med.
176
1992
1125
1135
73
Münk
C
Löhler
J
Prassolov
V
et al
Amphotropic murine leukemia viruses induce spongiform encephalomyelopathy.
Proc Natl Acad Sci U S A.
94
1997
5837
5842
74
Farson
D
Witt
R
McGuinness
R
et al
A new-generation stable inducible packaging cell line for lentiviral vectors.
Hum Gene Ther.
12
2001
981
997
75
Klages
N
Zufferey
R
Trono
D
A stable system for the high-titer production of multiply attenuated lentiviral vectors.
Mol Ther.
2
2000
170
176
76
Vogt
B
Roscher
S
Abel
B
et al
Lack of superinfection interference in retroviral vector producer cells.
Hum Gene Ther.
12
2001
359
365
77
Miller
AD
PA317 retrovirus packaging cells.
Mol Ther.
6
2002
572
575
78
Powell
SK
Artlip
M
Kaloss
M
et al
Efficacy of antiretroviral agents against murine replication-competent retrovirus infection in human cells.
J Virol.
73
1999
8813
8816
79
Patience
C
Takeuchi
Y
Cosset
FL
Weiss
RA
Packaging of endogenous retroviral sequences in retroviral vectors produced by murine and human packaging cells.
J Virol.
72
1998
2671
2676
80
Browning
MT
Schmidt
RD
Lew
KA
Rizvi
TA
Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions.
J Virol.
75
2001
5129
5140
81
Yang
S
Delgado
R
King
SR
et al
Generation of retroviral vector for clinical studies using transient transfection.
Hum Gene Ther.
10
1999
123
132
82
Coffin
JM
Retroviridae: the viruses and their replication.
Fundamental Virology.
Fields
BN
Knipe
DM
Howley
PM
1996
763
844
Lippincott Raven
Philadelphia, PA
83
An
W
Telesnitsky
A
Frequency of direct repeat deletion in a human immunodeficiency virus type 1 vector during reverse transcription in human cells.
Virology.
286
2001
475
482
84
Leboulch
P
Huang
GM
Humphries
RK
et al
Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.
EMBO J.
13
1994
3065
3076
85
Sorrentino
BP
McDonagh
KT
Woods
D
Orlic
D
Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice.
Blood.
86
1995
491
501
86
Garin
MI
Garrett
E
Tiberghien
P
et al
Molecular mechanism for ganciclovir resistance in human T lymphocytes transduced with retroviral vectors carrying the herpes simplex virus thymidine kinase gene.
Blood.
97
2001
122
129
87
Cmejlova J, Hildinger M, Cmejla R, et al. Impact of splice-site mutations of the human MDR1 cDNA on its stability and expression following retroviral gene transfer. Gene Ther. In press.
88
Zhang
J
Temin
HM
Retrovirus recombination depends on the length of sequence identity and is not error prone.
J Virol.
68
1994
2409
2414
89
Galipeau
J
Benaim
E
Spencer
HT
Blakley
RL
Sorrentino
BP
A bicistronic retroviral vector for protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs.
Hum Gene Ther.
8
1997
1773
1783
90
Chalmers
D
Ferrand
C
Apperley
JF
et al
Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene.
Mol Ther.
4
2001
146
148
91
Rosenberg
N
Joelicoer
P
Retroviral pathogenesis.
Retroviruses.
1st ed.
Coffin
JM
Hughes
SH
Varmus
HE
1997
475
586
Cold Spring Harbor Laboratory Press
Cold Spring Harbor, NY
92
Wahlers
A
Schwieger
M
Li
Z
et al
Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells.
Gene Ther.
8
2001
477
486
93
Rohdewohld
H
Weiher
H
Reik
W
Jaenisch
R
Breindl
M
Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites.
J Virol.
61
1987
336
343
94
Schroder
AR
Shinn
P
Chen
H
Berry
C
Ecker
JR
Bushman
F
HIV-1 integration in the human genome favors active genes and local hotspots.
Cell.
110
2002
521
529
95
Muller
HP
Varmus
HE
DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes.
EMBO J.
13
1994
4704
4714
96
Copeland
NG
Hutchison
KW
Jenkins
NA
Excision of the DBA ecotropic provirus in dilute coat-color revertants of mice occurs by homologous recombination involving the viral LTRs.
Cell.
33
1983
379
387
97
King
W
Patel
MD
Lobel
LI
Goff
SP
Nguyen-Huu
MC
Insertion mutagenesis of embryonal carcinoma cells by retroviruses.
Science.
228
1985
554
558
98
Mortreux
F
Leclercq
I
Gabet
AS
et al
Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo.
J Natl Cancer Inst.
93
2001
367
377
99
Stoye
JP
Endogenous retroviruses: still active after all these years?
Curr Biol.
11
2001
R914
R916
100
Zhou
ZH
Akgun
E
Jasin
M
Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences.
Proc Natl Acad Sci U S A.
98
2001
8326
8333
101
Miller
DG
Rutledge
EA
Russell
DW
Chromosomal effects of adeno-associated virus vector integration.
Nat Genet.
30
2002
147
148
102
Stocking
C
Bergholz
U
Friel
J
et al
Distinct classes of factor-independent mutants can be isolated after retroviral mutagenesis of a human myeloid stem cell line.
Growth Factors.
8
1993
197
209
103
Moolten
FL
Cupples
LA
A model for predicting the risk of cancer consequent to retroviral gene therapy.
Hum Gene Ther.
3
1992
479
486
104
Bartholomew
C
Ihle
JN
Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter.
Mol Cell Biol.
11
1991
1820
1828
105
Joosten
M
Vankan-Berkhoudt
Y
Tas
M
et al
Large-scale identification of novel potential disease loci in mouse leukemia applying an improved strategy for cloning common virus integration sites.
Oncogene.
21
2002
7247
7255
106
Lund
AH
Turner
G
Trubetskoy
A
et al
Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.
Nat Genet.
32
2002
160
165
107
Roe
T
Reynolds
TC
Yu
G
Brown
PO
Integration of murine leukemia virus DNA depends on mitosis.
EMBO J.
12
1993
2099
2108
108
Hahn
WC
Weinberg
RA
Modelling the molecular circuitry of cancer.
Nature Rev Cancer.
2
2002
331
341
109
Bunting
KD
Zhou
S
Lu
T
Sorrentino
BP
Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo.
Blood.
96
2000
902
909
110
Sellers
SE
Tisdale
JF
Agricola
BA
et al
The effect of multidrug-resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells.
Blood.
97
2001
1888
1891
111
Shi
PA
Hematti
P
Von Kalle
C
Dunbar
CE
Genetic marking as an approach to studying in vivo hematopoiesis: progress in the non-human primate model.
Oncogene.
21
2002
3274
3283
112
Woods NB, Muessig A, Schmidt M, et al. Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood. Prepublished online October 17, 2002, as DOI 10.1182/blood-2002-07-2238.
113
Riviere
I
Brose
K
Mulligan
RC
Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.
Proc Natl Acad Sci U S A.
92
1995
6733
6737
114
Furger
A
Monks
J
Proudfoot
NJ
The retroviruses human immunodeficiency virus type 1 and Moloney murine leukemia virus adopt radically different strategies to regulate promoter-proximal polyadenylation.
J Virol.
75
2001
11735
11746
115
Zaiss
AK
Son
S
Chang
LJ
RNA 3′ readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy.
J Virol.
76
2002
7209
7219
116
Emery
DW
Yannaki
E
Tubb
J
Nishino
T
Li
Q
Stamatoyannopoulos
G
Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo.
Blood.
100
2002
2012
2019
117
Bushman
F
Targeting retroviral integration?
Mol Ther.
6
2002
570
571
118
Hirata
R
Chamberlain
J
Dong
R
Russell
DW
Targeted transgene insertion into human chromosomes by adeno-associated virus vectors.
Nat Biotechnol.
20
2002
735
738
119
Eckert
HG
Stockschlader
M
Just
U
et al
High-dose multidrug resistance in primary human hematopoietic progenitor cells transduced with optimized retroviral vectors.
Blood.
88
1996
3407
3415
120
Pawliuk
R
Eaves
CJ
Humphries
RK
Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen.
Hum Gene Ther.
8
1997
1595
1604
121
Persons
DA
Allay
JA
Riberdy
JM
et al
Use of the green fluorescent protein as a marker to identify and track genetically modified hematopoietic cells.
Nat Med.
4
1998
1201
1205
122
Halene
S
Wang
L
Cooper
RM
et al
Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector.
Blood.
94
1999
3349
3357
123
Li
Z
Fehse
B
Schiedlmeier
B
et al
Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.
Leukemia.
16
2002
1655
1663
124
Richard
E
Mendez
M
Mazurier
F
et al
Gene therapy of a mouse model of protoporphyria with a self-inactivating erythroid-specific lentiviral vector without preselection.
Mol Ther.
4
2001
331
338
125
Sauvageau
G
Thorsteinsdottir
U
Eaves
CJ
et al
Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.
Genes Dev.
9
1995
1753
1765
126
Buske
C
Humphries
RK
Homeobox genes in leukemogenesis.
Int J Hematol.
71
2000
301
308
127
Kyba
M
Perlingeiro
RC
Daley
GQ
HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.
Cell.
109
2002
29
37
128
Buske
C
Feuring-Buske
M
Abramovich
C
et al
Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells.
Blood.
100
2002
862
868
129
Schiedlmeier B, Klump H, Will E, et al. High level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage to human cord blood CD34+ cells, but impairs lymphomyeloid differentiation. Blood. Prepublished online October 24, 2002, as DOI 10.1182/blood-2002-03-07672002.
130
Rideout
WM
III
Hochedlinger
K
Kyba
M
Daley
GQ
Jaenisch
R
Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy.
Cell.
109
2002
17
27
131
Krosl
J
Baban
S
Krosl
G
Rozenfeld
S
Largman
C
Sauvageau
G
Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with PBX1.
Oncogene.
16
1998
3403
3412
132
Bunting
KD
ABC transporters as phenotypic markers and functional regulators of stem cells.
Stem Cells.
20
2002
11
20
133
Scharenberg
CW
Harkey
MA
Torok-Storb
B
The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.
Blood.
99
2002
507
512
134
Sorrentino
BP
Brandt
SJ
Bodine
D
et al
Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1.
Science.
257
1992
99
103
135
Mickisch
GH
Aksentijevich
I
Schoenlein
PV
et al
Transplantation of bone marrow cells from transgenic mice expressing the human MDR1 gene results in long-term protection against the myelosuppressive effect of chemotherapy in mice.
Blood.
79
1992
1087
1093
136
Pallis
M
Russell
N
P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin-ceramide apoptotic pathway.
Blood.
95
2000
2897
2904
137
Sorrentino
BP
Gene therapy to protect haematopoietic cells from cytotoxic cancer drugs.
Nat Rev Cancer.
2
2002
431
441
138
Zhou
S
Schuetz
JD
Bunting
KD
et al
The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.
Nat Med.
7
2001
1028
1034
139
Carpinteiro
A
Peinert
S
Ostertag
W
et al
Genetic protection of repopulating hematopoietic cells with an improved MDR1-retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice.
Int J Cancer.
98
2002
785
792
140
Licht
T
Aksentijevich
I
Gottesman
MM
Pastan
I
Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells.
Blood.
86
1995
111
121
141
Licht
T
Haskins
M
Henthorn
P
et al
Drug selection with paclitaxel restores expression of linked IL-2 receptor gamma-chain and multidrug resistance (MDR1) transgenes in canine bone marrow.
Proc Natl Acad Sci U S A.
99
2002
3123
3128
142
Cowan
KH
Moscow
JA
Huang
H
et al
Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.
Clin Cancer Res.
5
1999
1619
1628
143
Qin
S
Ward
M
Raftopoulos
H
et al
Competitive repopulation of retrovirally transduced haemopoietic stem cells.
Br J Haematol.
107
1999
162
168
144
Mavilio
F
Ferrari
G
Rossini
S
et al
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood.
83
1994
1988
1997
145
Comoli
P
Dilloo
D
Hutchings
M
Hoffma
T
Heslop
HE
Measuring gene-transfer efficiency.
Nat Med.
2
1996
1280
1281
146
Austin
TW
Salimi
S
Veres
G
et al
Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells.
Blood.
95
2000
829
836
147
Rosenzweig
M
MacVittie
TJ
Harper
D
et al
Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning.
Blood.
94
1999
2271
2286
148
Bonini
C
Ferrari
G
Verzelleti
S
et al
HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.
Science.
276
1997
1719
1724
149
Bonini
C
et al
Infusion of donor lymphocytes expressing a suicide gene for controlled immune reconstitution and anti-tumor reactivity after stem cell transplantation.
Mol Ther.
3,5-2/2
2001
S144
150
Gerson
SL
Clinical relevance of MGMT in the treatment of cancer.
J Clin Oncol.
20
2002
2388
2399
151
Ragg
S
Xu-Welliver
M
Bailey
J
et al
Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells.
Cancer Res.
60
2000
5187
5195
152
Hobin
DA
Fairbairn
LJ
Genetic chemoprotection with mutant O6-alkylguanine-DNA-alkyltransferases.
Curr Gene Ther.
2
2002
1
8
153
Jin
L
Zeng
H
Chien
S
et al
In vivo selection using a cell-growth switch.
Nat Genet.
26
2000
64
66
154
Neff
T
Horn
PA
Valli
VE
et al
Pharmacologically regulated in vivo selection in a large animal.
Blood.
100
2002
2026
2031
155
Matsuda
KM
Kume
A
Ueda
Y
et al
Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy.
Gene Ther.
6
1999
1038
1044
156
Hantzopoulos
PA
Suri
C
Glass
DJ
Goldfarb
MP
Yancopoulos
GD
The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins.
Neuron.
13
1994
187
201
157
Lee
FS
Kim
AH
Khursigara
G
Chao
MV
The uniqueness of being a neurotrophin receptor.
Curr Opin Neurobiol.
11
2001
281
286
158
Labouyrie
E
Dubus
P
Groppi
A
et al
Expression of neurotrophins and their receptors in human bone marrow.
Am J Pathol.
154
1999
405
415
159
Lauer
UM
Staehler
P
Lambrecht
RM
et al
A prototype transduction tag system (delta LNGFR/NGF) for noninvasive clinical gene therapy monitoring.
Cancer Gene Ther.
7
2000
430
437
160
Ashkenazi
A
Dixit
VM
Apoptosis control by death and decoy receptors.
Curr Opin Cell Biol.
11
1999
255
260
161
Louz
D
van den Broek
M
Verbakel
S
et al
Erythroid defects and increased retrovirally induced tumor formation in Evi1 transgenic mice.
Leukemia.
14
2000
1876
1884
162
Kazama
H
Kodera
T
Shimizu
S
Mizoguchi
H
Morishita
K
Ecotropic viral integration site-1 is activated during, and is sufficient for, neuroectodermal P19 cell differentiation.
Cell Growth Differ.
10
1999
565
573
163
Chittka
A
Chao
MV
Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor.
Proc Natl Acad Sci U S A.
96
1999
10705
10710
164
Katzav
S
Martin-Zanca
B
Barbacid
M
et al
The trk oncogene abrogates growth factor requirements and transforms hematopoietic cells.
Oncogene.
4
1989
1129
1135
165
Kaebisch
A
Brokt
S
Seay
U
et al
Expression of the nerve growth factor receptor c-TRK in human myeloid leukaemia cells.
Br J Haematol.
95
1996
102
109
166
Reuther
GW
Lambert
QT
Caligiuri
MA
Der
CJ
Identification and characterization of an activating TrkA deletion mutation in acute myeloid leukemia.
Mol Cell Biol.
20
2000
8655
8666
167
Sadelain
M
Riviere
I
Sturm und Drang over Suicidal Lymphocytes.
Mol Ther.
5
2002
655
657
168
Brown
MP
Topham
DJ
Sangster
MY
et al
Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.
Nat Med.
4
1998
1253
1260
169
Bunting
KD
Sangster
MY
Ihle
JN
Sorrentino
BP
Restoration of lymphocyte function in Janus kinase 3-deficient mice by retroviral-mediated gene transfer.
Nat Med.
4
1998
58
64
170
Sandmaier
BM
McSweeney
P
Yu
C
Storb
R
Nonmyeloablative transplants: preclinical and clinical results.
Semin Oncol.
27
2000
78
81
171
Kohn
DB
Adenosine deaminase gene therapy protocol revisited.
Mol Ther.
5
2002
96
97
172
Mardiney
M
III
Malech
HL
Enhanced engraftment of hematopoietic progenitor cells in mice treated with granulocyte colony-stimulating factor before low-dose irradiation: implications for gene therapy.
Blood.
87
1996
4049
4056
173
Dahlke
MH
Lauth
OS
Jager
MD
et al
In vivo depletion of hematopoietic stem cells in the rat by an anti-CD45 (RT7) antibody.
Blood.
99
2002
3566
3572
174
Brown
BD
Lillicrap
D
Dangerous liaisons: the role of “danger” signals in the immune response to gene therapy.
Blood.
100
2002
1133
1140
175
Raper
SE
Yudkoff
M
Chirmule
N
et al
A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency.
Hum Gene Ther.
13
2002
163
175
176
Balague
C
Zhou
J
Dai
Y
et al
Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector.
Blood.
95
2000
820
828
177
Knaan-Shanzer
S
Van Der Velde
I
Havenga
MJ
et al
Highly efficient targeted transduction of undifferentiated human hematopoietic cells by adenoviral vectors displaying fiber knobs of subgroup B.
Hum Gene Ther.
12
2001
1989
2005
178
Scherr
M
Battmer
K
Blomer
U
et al
Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.
Blood.
99
2002
709
712
179
Tuschong
L
Soenen
SL
Blaese
RM
Candotti
F
Muul
LM
Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy.
Hum Gene Ther.
13
2002
1605
1610
180
Chuah MK, Schiedner G, Thorrez L, et al. Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia A following gene therapy with high-capacity adenoviral vectors. Blood. Prepublished online October 24, 2002, as DOI 10.1182/blood-2002-03-0823.
181
Heim
DA
Hanazono
Y
Giri
N
et al
Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.
Mol Ther.
1
2000
533
544
182
Kang
E
Giri
N
Wu
T
et al
In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fully or minimally myeloablated setting.
Hum Gene Ther.
12
2001
1663
1672
183
Wekerle
T
Sykes
M
Mixed chimerism and transplantation tolerance.
Annu Rev Med.
52
2001
353
370
184
Rosenzweig
M
Connole
M
Glickman
R
et al
Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells.
Blood.
97
2001
1951
1959
185
Larson
RC
Lavenir
I
Larson
TA
et al
Protein dimerization between Lmo2 (Rbtn2) and Tal1 alters thymocyte development and potentiates T cell tumorigenesis in transgenic mice.
EMBO J.
15
1996
1021
1027
186
Waterston
RH
Lindblad-Toh
K
Birney
E
et al
Initial sequencing and comparative analysis of the mouse genome.
Nature.
420
2002
520
562
187
Cheson
BD
Bennett
JM
Kantarjian
H
et al
Report of an international working group to standardize response criteria for myelodysplastic syndromes.
Blood.
96
2000
3671
3674
188
Kogan
SC
Ward
JM
Anver
MR
et al
Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.
Blood.
100
2002
238
245
189
Morse
HC
III
Anver
MR
Fredrickson
TN
et al
Bethesda proposals for classification of lymphoid neoplasms in mice.
Blood.
100
2002
246
258
190
Bennett
JM
Catovsky
D
Daniel
MT
et al
Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group.
Ann Intern Med.
103
1985
620
625
191
Bernardi
R
Grisendi
S
Pandolfi
PP
Modelling haematopoietic malignancies in the mouse and therapeutical implications.
Oncogene.
21
2002
3445
3458
192
Wong
S
Witte
ON
Modeling Philadelphia chromosome positive leukemias.
Oncogene.
20
2001
5644
5659
193
MacKenzie
KL
Dolnikov
A
Millington
M
Shounan
Y
Symonds
G
Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice.
Blood.
93
1999
2043
2056
194
Traver
D
Akashi
K
Weissman
IL
Lagasse
E
Mice defective in two apoptosis pathways in the myeloid lineage develop acute myeloblastic leukemia.
Immunity.
9
1998
47
57
195
Holtschke
T
Löhler
J
Kanno
Y
et al
Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene.
Cell.
87
1996
307
317
196
Hayward
WS
Neel
BG
Astrin
SM
Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis.
Nature.
290
1981
475
480
197
Jonkers
J
Berns
A
Retroviral insertional mutagenesis as a strategy to identify cancer genes.
Biochim Biophys Acta.
1287
1996
29
57
198
Endicott
KM
Gump
H
Hemograms and myelograms of healthy female mice C-57 brown and CFW strains.
Blood.
1
1947
165
167
199
Brecher
G
Endicott
KM
Gump
H
Brawner
HP
Effects of x-ray on lymphoid and hemopoietic tissues of albino mice.
Blood.
3
1948
1259
1274
200
Perkins
AS
The pathology of murine myelogenous leukemias.
Curr Top Microbiol Immunol.
149
1989
3
21
201
Fredrickson
TN
Harris
AW
Atlas of mouse hematopathology.
2000
Harwood Academic Publishers
Newark, NJ
202
Vannucchi
AM
Paoletti
F
Linari
S
et al
Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.
Blood.
95
2000
2559
2568
203
Geiger
H
Van Zant
G
The aging of lympho-hematopoietic stem cells.
Nat Immunol.
3
2002
329
333
204
Keller
G
Clonal analysis of hematopoietic stem cell development in vivo.
Curr Top Microbiol Immunol.
177
1992
41
58
205
Lemischka
IR
What have we learned from retroviral marking of hematopoietic stem cells?
Curr Top Microbiol Immunol.
177
1992
59
71
206
Gong
JK
Braunschweiger
PG
Glomski
CA
Anemic stress as a trigger of myelogenous leukemia in the unirradiated RF mouse.
Science.
177
1972
274
276
207
Holyoake
TL
Freshney
MG
Samuel
K
et al
In vivo expansion of the endogenous B-cell compartment stimulated by radiation and serial bone marrow transplantation induces B-cell leukaemia in mice.
Br J Haematol.
114
2001
49
56

Author notes

Christopher Baum, Experimental Cell Therapy, Department of Hematology and Oncology, Hannover Medical School, Carl-Neuberg-Straße 1, D-30625 Hannover, Germany; e-mail:baum.christopher@mh-hannover.de.