Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hanazono, Y.
Right arrow Articles by Ozawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hanazono, Y.
Right arrow Articles by Ozawa, K.
Stem Cells, Vol. 19, No. 1, 12-23, January 2001
© 2001 AlphaMed Press

CONCISE REVIEW

Gene Transfer into Nonhuman Primate Hematopoietic Stem Cells: Implications for Gene Therapy

Yutaka Hanazonoa, Keiji Teraob, Keiya Ozawaa

a Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan;
b Tsukuba Primate Center, National Institute of Infectious Diseases, Ibaraki, Japan

Key Words. Gene therapy • Hematopoietic stem cells • Retroviral vectors • Nonhuman primate models

Yutaka Hanazono, M.D., Ph.D., Assistant Professor, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Telephone: 81-285-58-7402; Fax: 81-285-44-8675; e-mail: hanazono{at}jichi.ac.jp


   ABSTRACT
Top
 Abstract
Animal Models for HSC...
 Safety Evaluation
 Improved Transduction of Primate...
 New Insights into Primate...
 Conclusion
 References
 
Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation abilities. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. Using these models, clinically relevant levels (around 10% or even more) of gene-modified cells in peripheral blood have recently been achieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction, and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus or feline endogenous retrovirus envelope. Other strategies including the use of lentiviral vectors and in vivo selective expansion of gene-modified cells with the drug resistance gene or selective amplifier gene (also designated the molecular growth switch) are now being tested to further increase the fraction of gene-modified cells using nonhuman primate models. In addition to the high gene transfer efficiency, high-level and long-term expression of transgenes in human HSCs and their progeny is also required for effective HSC gene therapy. For this purpose, other backbones of retroviral vectors such as the murine stem cell virus and cis-DNA elements, such as the ß-globin locus control region and the chromatin insulator, also need to be tested in nonhuman primate models. Nonhuman primate studies will continue to provide an important framework for human HSC gene therapy. Well-designed nonhuman primate studies will also offer unique insights into the HSCs, immune system, and transplantation biology characteristic of large animals.


   ANIMAL MODELS FOR HSC GENE THERAPY
Top
 Abstract
 Animal Models for HSC...
Safety Evaluation
 Improved Transduction of Primate...
 New Insights into Primate...
 Conclusion
 References
 
Hematopoietic stem cells (HSCs) have been pursued as highly desirable targets for gene therapy because of their self-renewal and multilineage differentiation capabilities [1]. Retroviral vectors based on murine leukemia viruses have been most extensively used to transduce HSCs, since these vectors are well characterized and can integrate into the target cell genome, allowing transmission of the vector to daughter cells in vivo [2]. Another class of retroviral vectors based on lentiviral genomes has been developed and is now widely used to transduce HSCs [3]. Although adenovirus and adeno-associated virus (AAV) vectors have been utilized for a number of gene therapy trials, they do not seem appropriate for applications requiring long-term in vivo expression of transgenes in hematopoietic cells since those vectors do not usually integrate into the host genome [4, 5]. Most applications directed at HSCs are dependent on in vivo proliferation and differentiation of the cells post-transplant, and thus nonintegrating, nonreplicating adenovirus and AAV vectors would have limited applicability in this field.

In the initial clinical human studies with retroviral vectors, gene-modified cells were detected at very low levels in peripheral blood (usually less than 1%) after transplantation of transduced HSCs, while the results obtained in murine studies showed a much higher percentage of gene-modified cells (10%-100%) [6, 7]. As a matter of course, very little clinical utility has been achieved in most of the HSC gene therapy trials [8]. The successful HSC gene therapy for X-linked severe combined immunodeficiency (X-SCID) has recently been reported [9]. In this study, two infants who received nonmyeloablative autologous transplantation of CD34+ cells transduced with a retroviral vector expressing the common {gamma} chain that is deficient in the disease, showed restored lymphocytes and improved immunity. The success of the gene therapy was presumably brought by the extremely high selective growth advantage of lymphocytes transduced with the common {gamma} chain gene, but not by the improved gene transfer efficiency into human HSCs [10-12]. The low gene transfer efficiency is still a problem that remains to be solved for more widely applicable HSC gene therapy. There are two major reasons for the low efficiency of gene transfer into human HSCs compared to murine HSCs. First, the nature of human HSCs is far more quiescent than that of murine HSCs. The self-renewal frequency of murine HSCs is presumably high, so that the cells are expected to divide at least once during a few days of a standard retroviral transduction period. However, in larger animals, the frequency of self-renewal of HSCs may be much lower even in the presence of multiple hematopoietic cytokines. It is reported that feline HSCs divide at most once every three weeks [13]. It is therefore unlikely that these cells cycle during a transduction period, even in the presence of hematopoietic cytokines, and instead may undergo differentiation or lose engraftment capabilities. Second, human HSCs seem to have much less density of retroviral receptors on their cell surface than murine HSCs. Most murine experiments have used retroviral vectors packaged in an ecotropic envelope protein that binds to a receptor not found on primate cells [14]. Instead, human trials have utilized vector particles incorporating an amphotropic envelope protein. The most primitive human hematopoietic cells (CD34+/CD38–) have extremely few amphotropic receptors, while primitive murine HSCs appear to have much more ecotropic receptors. The low level of the cell surface retroviral receptor therefore limits the transduction of human HSCs [15-17].

Interestingly, low in vivo transduction levels after gene transfer into HSCs and their autologous transplantation have also been documented in other large animals such as baboons, rhesus monkeys, common marmosets, dogs, and cats [18-23, 57]. The low gene transfer efficiency into HSCs with retroviral vectors may be the common feature not only of humans but also of large animals. This is one of the reasons why large animal models are required for study of the human HSC gene transfer. Among large animals, nonhuman primates such as macaque monkeys and baboons may provide the best animal models because of their close phylogenetic distance to humans [24-26]. Furthermore, there is considerable cross-reactivity in many cytokines and antibodies between humans and these animals [27-29]. However, these animals are usually outbred just as humans are although experimental mice are inbred. The genetic backgrounds of each animal are completely different and there are significant individual differences among animals. Other disadvantages are that there are very few nonhuman primate models for human diseases compared to mice [30, 31] and that research is more costly [32].

Animal transplantation models have proved to be essential for the assay of HSCs since at present, there are no reliable in vitro assays for cells capable of establishing long-term hematopoiesis in vivo [33]. Therefore, to transplant cells into humans might be the best way to assess human HSCs, but it is impossible to design this kind of experiment. Instead, xenograft models of human hematopoiesis have been used for the study of the in vivo engraftment/proliferation potential of human HSCs. There are two xenotropic transplantation models available now. One recipient is the nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) mouse and the other is the fetal sheep [34-36]. These models take advantage of the immunologically naïve state. Since the NOD/SCID mice are severe immunodeficient and the fetal sheep is immunologically immature, human HSCs can engraft and generate their progeny in these animals. However, the behaviors of human HSCs might be very different in xenotropic recipients such as mice and sheep. The relevance of these xenograft models to natural human in vivo hematopoiesis remains unclear. On the other hand, the autologous transplantation models using large animals as discussed above are ideal for evaluating the engraftment, proliferation, and differentiation of HSCs, since the natural hematopoiesis can be studied in these models [25]. We are using the cynomolgus macaque (Macaca fascicularis) as the autologous transplantation model (Fig. 1Go). Figure 2Go shows the overview of the cynomolgus HSC transduction and autologous transplantation conducted in our institute.



View larger version (168K):
[in this window]
[in a new window]
  		Figure 1. The cynomolgus macaque (Macaca fascicularis). It is a macaque monkey and belongs to the same family as the rhesus monkey.

 


View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. The cynomolgus macaque HSC transduction and autologous transplantation. A central venous catheter is placed in each animal to allow administration of fluids, antibiotics and transfusions. CD34+ cells are isolated from their bone marrow or G-CSF/SCF-mobilized peripheral blood [145]. The animals receive myeloablative total body irradiation (500 cGy daily for two days) just prior to transplantation. The animals are transplanted with CD34-enriched autologous cells and are kept in the intensive care units with the high efficiency particulate air-filtered airflow from the day of irradiation until hematopoiesis is recovered. The animals are then examined for the provirus and its expression.

 

   SAFETY EVALUATION
Top
 Abstract
 Animal Models for HSC...
 Safety Evaluation
Improved Transduction of Primate...
 New Insights into Primate...
 Conclusion
 References
 
Even large amounts of mouse retrovirus injected into monkeys intravenously or intraperitoneally cannot replicate in primates in vivo [37, 38]. The immune system of primates seems to recognize and suppress mouse retrovirus replication effectively. However, there is evidence that, in a few cases of immunosuppressive monkeys, retrovirus replicated in vivo. Development of aggressive T-cell lymphoma in rhesus monkeys that received autologous HSCs exposed to replication-competent retrovirus (RCR) provided the evidence of pathogenesis of such vectors in primates when it is contaminated with RCR [39]. The study showed the importance of the use of RCR-negative vector stock. It is not difficult to avoid RCR, given that the absence of RCR in gene therapy products can be verified very effectively [40]. An RCR developed in vivo would be of a completely different nature than RCR originating from recombination in retroviral vector-producing cell lines. Mouse retroviral vectors show too little homology with endogenous human or monkey retroviruses to generate RCR by recombination in vivo. Until now, the in vivo emergence of RCR has not been reported even in many mice subjected to transplantation of bone marrow cells transduced with retroviral vectors. Thus, this may be regarded as an unlikely event. However, in such a case when HIV vectors are used to transduce HSCs or lymphocytes of HIV-infected patients followed by autologous reinfusion of the cells, in vivo generation of RCR might become a concern. Removal of viral sequences as much as possible is required for safety [41-45].


   IMPROVED TRANSDUCTION OF PRIMATE HSCS
Top
 Abstract
 Animal Models for HSC...
 Safety Evaluation
 Improved Transduction of Primate...
New Insights into Primate...
 Conclusion
 References
 
Nonhuman primate models have provided important frameworks for improving gene transfer into human HSCs. The strategies are summarized in Table 1Go.


View this table:
[in this window]
[in a new window]
  		Table 1. Strategies for improvement of gene transduction of primate HSCs
 
Optimization of Gene Transfer Conditions
Previous HSC gene therapy protocols utilized the transduction conditions which had been optimized in mice with murine retroviral vectors. One can re-optimize the transduction conditions for primates using nonhuman primate models. For instance, addition of Flt-3 ligand in the ex vivo culture during transduction resulted in a one-log improvement in terms of in vivo long-term marking levels in peripheral blood mononuclear cells and granulocytes, compared to the transduction without Flt-3 ligand [46]. Cytokine priming with G-CSF and stem cell factor (SCF) may also improve susceptibility of primitive hematopoietic cells to retroviral transduction by allowing collection of increased numbers of primitive cells from both peripheral blood and bone marrow that are cycling [47].

The distance that an average retroviral particle can travel within one half-time (several hours) in medium is estimated to be 480-610 µm, and only those particles close enough to target cells will be captured and deliver the gene [48]. To overcome such physical limitations imposed by Brownian motion on retroviral gene delivery, several methods have been developed. Centrifugation of target cells in vector-supernatant was the first approach with some success in increasing the likelihood of interaction between target cells and retroviral vectors [49-51]. The second approach is flow-through transduction, or transduction in continuous flow culture flasks or vessels resulting in improved transduction efficiency of murine fibroblasts and human bone marrow cells [48, 52]. The use of fibronectin fragment-coated flasks is another approach shown to increase the likelihood of interaction between target cells and retroviral vectors [53]. In particular, a C-terminal fragment of fibronectin containing integrin-binding and heparin-binding domains (CH-296, retronectin) is the most potent in enhancing retroviral transduction [54]. It has been reported that retronectin increases the efficiency of gene transfer into baboon HSCs [55] and it is now used for transduction of human HSCs in clinical protocols [9, 56].

Pseudotyped Retroviral Vectors
Pseudotyping vector particles, or the utilization of alternative retroviral envelopes that target unique receptors on the cell surface, is an effective method to change target cell specificity or the physical properties of the vector particles. Pseudotyping retroviral vectors with the gibbon ape leukemia virus (GALV) envelope improves the transduction efficiency of baboon and canine HSCs and of human SCID-repopulating cells [57-59]. The cell-surface receptors for GALV and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters, but the GALV envelope targets a distinct surface receptor (Glvr-1) that is 60% homologous but not cross-reactive to the amphotropic receptor (Ram-1) [60]. Pseudotyping with the vesicular stomatitis G glycoprotein (VSV-G) has also been tried for gene transfer into human HSCs. Since the cellular receptors for VSV-G including phosphatidylserine, phosphatidylinositol, and GM3 ganglioside appear to be very abundant and ubiquitous membrane components of most mammalian cells [61, 62], VSV-G-enveloped viruses can infect a wide variety of cells and tissues including human HSCs. In addition to the broader range, VSV-G-pseudotyped viruses are physically more stable than naturally occurring retroviruses and can be concentrated by ultracentrifugation [63, 64]. It is reported that the use of high-titer VSV-G-pseudotyped retroviral vectors allows efficient gene transfer without extended ex vivo culture, minimizing the loss of HSCs during culture [65]. Retroviral vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope may also be good candidates to transduce primate HSCs [66]. The cellular receptor for RD114 is a neutral amino acid receptor [67]. The retroviral vector pseudotyped with RD114 could transduce human NOD/SCID-repopulating cells far more efficiently than those pseudotyped with the GALV or amphotropic envelopes [68]. In engrafted NOD/SCID mice, as much as 90% of human blood cells were positive for the green fluorescent protein (GFP) as transgene expression.

Lentiviral Vectors
Unlike murine oncoretroviruses that infect only dividing cells, lentiviruses can also infect nondividing, terminally differentiated cells of specific lineages [69]. Lentiviral vectors have been developed by modifying HIV 1 or HIV 2 [3, 70, 71], feline immunodeficiency virus [72], equine infectious anemia virus [73], and simian immunodeficiency virus [74-76]. Instead of depending on specific viral entry such as HIV 1 infection via CD4 and other coreceptors, the VSV-G envelope has generally been used to pseudotype lentiviral vectors, although the GALV-pseudotyped lentiviral vector has also recently been developed [77]. It is reported that human SCID-repopulating cells can be efficiently transduced (10%-50%) with VSV-G-pseudotyped HIV vectors, even without hematopoietic cytokines in the transduction culture [78, 79]. In rhesus monkeys, in vivo low levels of expression (~1%) with a lentiviral vector encoding GFP were observed in granulocytes, lymphocytes, and monocytes, implying successful HSC transduction with the lentiviral vector [80]. However, the marking levels were comparable to those with a murine retroviral vector and further study of the requirements for optimal use seems to be needed.

Lentiviral vectors clearly have more real and perceived safety concerns than standard retroviral vectors. A series of safety modifications have been made, decreasing further and further the chance of recombination events resulting in RCR. More and more HIV sequences have been removed from the vectors, but the ability to produce high-titer vector that can transduce nondividing cells and integrate efficiently is dependent on the inclusion of at least some residual HIV elements other than the long-terminal repeats (LTRs) [41-43]. Current lentiviral vectors take advantage of the fact that the 3' LTR duplicates and becomes the 5' LTR in vivo after reverse transcription and integration. This allows production of "self-inactivating" or SIN vectors, which lose the transcriptional capacity of the LTR once transferred to target genome, further increasing safety [44, 45].

Transgene Expression
One major limitation to the use of retroviral vectors for gene therapy may be decline of expression of transgenes in vivo [81, 82]. The insertion of retroviral DNA can trigger transcriptional silencing of the inserted sequences, usually via mechanisms that involve methylation of DNA within regulatory regions, thus far demonstrated primarily in the murine model [83, 84]. DNA methylation inhibits transcription largely by the recruitment of repressive chromosomal proteins such as histone deacetylases [85]. In an attempt to improve expression, retroviral vectors derived from the myeloproliferative sarcoma virus (MPSV), murine stem cell virus (MSCV), and spleen focus-forming virus have recently been developed [86-89]. In general, these vectors differ from the Moloney murine leukemia virus (MoMLV) vectors in the LTR promoter-enhancer region and the primer-binding site. These vectors have been shown to result in improved expression of transgenes compared to MoMLV vectors in vivo in mice, and in vitro in human primary hematopoietic cells. These vectors are being examined in vivo in nonhuman primate models.

An alternative approach to enhancing vector expression is to use cis-acting DNA elements such as the locus control region (LCR) and the chromatin insulator. The LCR lies 5' to the human ß-globin-like gene cluster, compromises three DNase I hypersensitive sites (HS4, HS3, and HS2 in order from 5'), and directs high-level and position-independent ß-globin gene expression [90]. This region is 20 kb long, and can be reduced to shorter forms of a few hundred base pairs long [91, 92]. Although the reduced LCR elements are still able to confer high-level position-independent expression to a transgene, it has been difficult to generate stable and high-titer retroviral vectors encoding the LCRs, indicating a significant degree of genetic instability of the LCRs in the retroviral constructs [93-95]. However, it has recently been reported that the LCRs can be stable in lentiviral vectors and bring long-term, high levels of ß-globin expression in mice [96]. On the other hand, the chromatin insulator is located within the chicken ß-globin LCR [97]. It contains the chicken HS4 element and appears to constitute the 5' boundary of the chicken ß-globin locus. Within the region, a 49-bp chicken HS4 core is associated with the ability to protect expression cassettes from position effects [98]. When this fragment (1.2kb) was used to flank a reporter vector, the fraction of transduced cells that expressed the reporter increased in cultures and mice transplanted with transduced marrow [99]. The chromatin insulator may improve the transgene expression by protecting the provirus from chromosomal position effects. These strategies need to be examined in vivo in nonhuman primate models.


   NEW INSIGHTS INTO PRIMATE HSC BIOLOGY
Top
 Abstract
 Animal Models for HSC...
 Safety Evaluation
 Improved Transduction of Primate...
 New Insights into Primate...
Conclusion
 References
 
Selection and Expansion of Primate HSCs
High levels of transduction efficiency are required for applications involving hemoglobinopathies and cancer gene therapy. If close to 100% transduction of a target population is desirable, then selection of transduced cells via a vector-encoded cell surface marker such as CD24, the truncated nerve growth factor receptor or GFP could be utilized. This strategy worked out well in murine transplantation models [100-102]; however, there have been no successful reports in nonhuman primate models thus far. Given the low gene transfer efficiency into primate HSCs, many HSCs will be lost during the selection procedures, possibly resulting in failure of hematopoietic reconstitution after transplantation.

Many papers describe so-called ex vivo expansion of human HSCs, either in suspension culture with cytokines or in bioreactors or other devices containing a stromal support layer [103-107]. Most studies report increases in total cell number, total CD34+ cells, colony-forming units, or SCID-repopulating cells, all probably inadequate surrogates for true long-term repopulating HSCs. Clinical studies using ex vivo-expanded cell populations have not proven maintenance or expansion of either short- or long-term repopulating ability [108]. A nonhuman primate HSC marking study will be helpful to clarify the kinetics of hematopoiesis originating from these ex vivo-expanded populations. Gene marking studies in rhesus monkeys indicate that ex vivo expansion of mobilized peripheral blood cells for 10-14 days with multiple cytokines (interleukin 3 [IL-3], IL-6, SCF, and Flt-3 ligand) and stromal supports results in no increase in initial engraftment and diminished long-term engraftment [46]. It suggests that present "expansion" conditions may damage engrafting cells and that committed progenitors do not contribute to even short-term engraftment.

On the other hand, if in vivo expansion of transduced HSCs is feasible, the low gene transfer efficiency into HSCs can be circumvented [109]. Several investigators have tried to confer HSC drug resistance by introducing the multidrug resistance (MDR)-1 gene into the cells with retroviral vectors, and selecting and expanding the transduced HSCs in vivo after transplantation by administration of an MDR-pumped drug such as taxol [56, 110-113]. However, in vivo selection with the MDR-1 gene may have resulted from selection at the level of relatively mature precursor cells, not at the level of HSCs, since HSCs seem to express the MDR-1 gene [114-116]. In addition, aberrant splicing of the vector-derived MDR-1 transcript in human hematopoietic cells has been shown, which leads to the generation of a nonfunctional, truncated MDR-1 gene product [117]. It has also been reported that mice transplanted with ex vivo-expanded MDR1-transduced HSCs developed a myeloproliferative disorder [118]. A number of alternative drug resistance genes have been studied in vitro and in murine models, including O6-alkylguanine-DNA-alkyltransferase, which confers protection to some alkylating agents [119, 120] and mutant dihydrofolate reductase [121, 122]. Although the strategies using the drug resistance genes worked out well in murine experiments [110, 122], it has been difficult to show evidence of in vivo expansion of the transduced cells in humans or nonhuman primates. There were too few HSCs that were successfully transduced with the drug resistance genes to be expanded to clinically relevant levels [56, 111-113]. Another strategy of in vivo expansion of transduced cells is to confer a direct proliferation advantage on the gene-modified cells relative to their nontransduced counterparts. Some groups developed the selective amplifier gene (or also designated the molecular growth switch), a chimeric gene which expresses a fusion protein of growth factor receptor signaling domain and its specific molecular switch [123-125]. One group reported the successful in vivo expansion of gene-modified hematopoietic cells with the molecular growth switch in a murine model [126]. Future work will focus on adapting these in vivo selection systems for use in nonhuman primates.

Nonmyeloablative HSC Transplantation
The engraftment of significant numbers of genetically modified HSCs and progenitor cells, without the requirement for fully myeloablative conditioning therapy and thus without the high toxicity or potential damage to marrow stroma associated with lethal radiation, is a highly desirable goal for the treatment of many nonmalignant hematological disorders. Using a rhesus macaque model, it has been reported that transduction of cytokine-mobilized peripheral blood CD34+ cells resulted in significant levels of gene marking in vivo (exceeding 12% in peripheral blood mononuclear cells in one animal for 33 weeks) with nonmyeloablative conditioning irradiation (500 cGy) [127]. Rosenzweig et al. have reported that 5%-10% of peripheral blood cells expressed the murine CD24 surface antigen as transgene for at least six months after transplantation of transduced peripheral blood CD34+ cells by conditioning the animals with sublethal radiation (320-400 cGy) [128]. Even with only small numbers of gene-modified cells engrafted after nonmyeloablative transplantation, the cells could be expanded in vivo to reach clinically relevant levels if growth or survival advantage is conferred on the cells, for instance using the drug-resistance genes or the selective amplifier genes as discussed above [129, 130].

Immune Response to Gene-Modified HSCs and Their Progeny
There is evidence that immune responses against transgene products recognized as foreign could be a major obstacle to the long-term persistence of genetically corrected cells in vivo. In vivo immune destruction of cells expressing xenogeneic genes such as peripheral blood lymphocytes, myocytes and fibroblasts has been reported [131-135]. Many retroviral vectors utilized in preclinical animal models and early clinical gene marking and gene therapy trials expressed xenogeneic genes such as the bacterial neomycin phosphotransferase (neo) gene, the ß-galactosidase gene, the jellyfish GFP gene, or the herpes thymidine kinase suicide gene. These foreign proteins are likely to be processed and presented as antigenic peptides to T cells of the recipients. This would elicit cellular immune responses against the foreign gene products, resulting in destruction of the cells expressing the foreign transgenes. Therefore, it is important to consider this issue when designing gene therapy strategies and vectors, and avoid use of xenogeneic or foreign genes in vectors whenever possible. Even vectors that contain only normal human genes designed to replace a deleted or poorly expressed endogenous gene in patients with congenital deficiency disorders such as chronic granulomatous disease or Gaucher's disease, could elicit an immune response to a previously absent gene product. However, long-term in vivo expression of xenogeneic genes such as murine adenosine deaminase (ADA), human ADA, and human glucocerebrosidase without immune rejection has been documented after retroviral gene transfer into rhesus monkey HSCs [136-139]. In addition, it has been reported that introduction of a foreign gene via HSCs compared to mature lymphocytes or other mature cell types can result in specific tolerance against the gene product, thus avoiding a destructive immune response against transduced cells [135]. Therefore, HSCs may be more effective targets, even if the goal is expression of the new gene in differentiated progeny such as T lymphocytes, when there is a risk of an immune response against transduced cells.

Clonal Analyses of Primate HSCs
Although, in mice, the entire hematopoiesis can be reconstituted by a single or few HSCs [140, 141], no data about clonality of hematopoiesis in larger animals have been available. The hematopoietic demand of a mouse is very small. A typical mouse (25 g) makes, in a two-year lifetime, the same amount of red blood cells as does man in one day, raising the possibility that HSC kinetics in large animals are more complex [142]. Kim et al. have described for the first time the tracking of clonal contributions to hematopoiesis in a nonhuman primate model [143]. Relatively high levels of stable retroviral gene transfer (5%-15%) allowed them to use inverse polymerase chain reaction to characterize the specific retrovirally marked clones (by the unique insertion site flanking the provirus) contributing to hematopoiesis over time [144]. In two animals studied intensively, over 45 clones were identified up to 18 months post-transplantation. Some persisted for prolonged periods, and multilineage contributions were documented. This result contrasts sharply with the murine hematopoiesis and shows importance of large animal study to understand human hematopoiesis.


   CONCLUSION
Top
 Abstract
 Animal Models for HSC...
 Safety Evaluation
 Improved Transduction of Primate...
 New Insights into Primate...
 Conclusion
References
 
Nonhuman primate studies have provided an important framework for human clinical studies. In a real sense, nonhuman primates as animal models represent a "step to human." Well-designed nonhuman primate study will continue to offer unique insights into the biology of HSCs, the immune system, transplantation, and many diseases.


   ACKNOWLEDGMENTS
Top
 Abstract
 Animal Models for HSC...
 Safety Evaluation
 Improved Transduction of Primate...
 New Insights into Primate...
 Conclusion
 References
 
The authors thank Dr. Cynthia E. Dunbar (Hematology Branch, NHLBI, NIH, Bethesda, MD) for helpful comments on the manuscript.


   REFERENCES
Top
 Abstract
 Animal Models for HSC...
 Safety Evaluation
 Improved Transduction of Primate...
 New Insights into Primate...
 Conclusion
 References
 

  1. Halene S, Kohn DB. Gene therapy using hematopoietic stem cells: sisyphus approaches the crest. Hum Gene Ther 2000;11:1259-1267.[CrossRef][Medline]

  2. Verma IM, Somia N. Gene therapy—promises, problems and prospects. Nature 1997;389:239-242.[CrossRef][Medline]

  3. Naldini L, Blomer U, Gallay P et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263-267.[Abstract]

  4. Benihoud K, Yeh P, Perricaudet M. Adenovirus vector for gene therapy. Curr Opin Biotechnol 1999;10:440-447.[CrossRef][Medline]

  5. Russell DW, Kay MA. Adeno-associated virus vectors and hematology. Blood 1999;94:864-874.[Free Full Text]

  6. Dunbar CE, Cottler-Fox M, O'Shaughnessy JA et al. Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. Blood 1995;85:3048-3057.[Abstract/Free Full Text]

  7. Hanazono Y, Dunbar CE. Genetic marking. In: Forman SJ, Blume KG, Thomas ED, eds. Hematopoietic Cell Transplantation, 2nd Edition. Malden, MA: Blackwell Science, Inc., 1999:89-96.

  8. Kohn DB, Nolta JA, Crooks GM. Clinical trials of gene therapy using hematopoietic stem cells. In: Forman SJ, Blume KG, Thomas ED, eds. Hematopoietic Cell Transplantation, 2nd Edition. Malden, MA: Blackwell Science, Inc., 1999:97-102.

  9. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288:669-672.[Abstract/Free Full Text]

  10. Stephan V, Wahn V, Le Deist F et al. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells. N Engl J Med 1996;335:1563-1567.[Free Full Text]

  11. Fischer A, Cavazzana-Calvo M, De Saint Basile G et al. Naturally occurring primary deficiencies of the immune system. Annu Rev Immunol 1997;15:93-124.[CrossRef][Medline]

  12. Soudais C, Shiho T, Sharara LI et al. Stable and functional lymphoid reconstitution of common cytokine receptor {gamma} chain deficient mice by retroviral-mediated gene transfer. Blood 2000;95:3071-3077.[Abstract/Free Full Text]

  13. Abkowitz JL, Catlin SN, Guttorp P. Evidence that hematopoiesis may be a stochastic process in vivo. Nat Med 1996;2:190-197.[CrossRef][Medline]

  14. Miller AD. Cell-surface receptors for retroviruses and implications for gene transfer. Proc Natl Acad Sci USA 1996;93:11407-11413.[Abstract/Free Full Text]

  15. Orlic D, Girard LJ, Jordan CT et al. The level of mRNA encoding the amphotropic retrovirus receptor in mice and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc Natl Acad Sci USA 1996;93:11097-11102.[Abstract/Free Full Text]

  16. Kurre P, Kiem HP, Morris J et al. Efficient transduction by an amphotropic retrovirus vector is dependent on the high-level expression of the cell surface virus receptor. J Virol 1999;73:495-500.[Abstract/Free Full Text]

  17. MacDonald C, Walker S, Watts M et al. Effect of changes in expression of the amphotropic retroviral receptor PiT-2 on transduction efficiency and viral titer: implications for gene therapy. Hum Gene Ther 2000;11:587-595.[CrossRef][Medline]

  18. Schuening FG, Kawahara K, Miller AD et al. Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs. Blood 1991;78:2568-2576.[Abstract/Free Full Text]

  19. Kiem HP, Darovsky B, von Kalle C et al. Retrovirus-mediated gene transduction into canine peripheral blood repopulating cells. Blood 1994;83:1467-1473.[Abstract/Free Full Text]

  20. Kiem HP, Darovsky B, Von Kalle C et al. Long-term persistence of canine hematopoietic cells genetically marked by retrovirus vectors. Hum Gene Ther 1996;7:89-96.[Medline]

  21. Lutzko C, Kruth S, Abrams-Ogg AC et al. Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine {alpha}-L-iduronidase deficiency. Blood 1999;93:1895-1905.[Abstract/Free Full Text]

  22. Hibino H, Tani K, Ikebuchi K et al. The common marmoset as a target preclinical primate model for cytokine and gene therapy studies. Blood 1999;93:2839-2848.[Abstract/Free Full Text]

  23. Josephson NC, Sabo KM, Abkowitz JL. Transduction of feline hematopoietic cells by oncoretroviral vectors pseudotyped with the subgroup A feline leukemia virus (FeLV-A). Mol Ther 2000;2:56-62.[CrossRef][Medline]

  24. King FA, Yarbrough CJ, Anderson DC et al. Primates. Science 1988;240:1475-1482.[Abstract/Free Full Text]

  25. Van Beusechem VW, Valerio D. Gene transfer into hematopoietic stem cells of nonhuman primates. Hum Gene Ther 1996;7:1649-1668.[Medline]

  26. VandeBerg JL, Williams-Blangero S. Advantages and limitations of nonhuman primates as animal models in genetic research on complex diseases. J Med Primatol 1997;26:113-119.[Medline]

  27. Jonker M. The importance of non-human primates for preclinical testing of immunosuppressive monoclonal antibodies. Semin Immunol 1990;2:427-436.[Medline]

  28. Reimann KA, Waite BC, Lee-Parritz DE et al. Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys. Cytometry 1994;17:102-108.[CrossRef][Medline]

  29. Ozwara H, Niphuis H, Buijs L et al. Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey. J Med Primatol 1997;26:164-171.[Medline]

  30. McClure HM. Nonhuman primate models for human disease. Adv Vet Sci Comp Med 1984;28:267-304.[Medline]

  31. Walsh GP, Tan EV, dela Cruz EC et al. The Philippine cynomolgus monkey (Macaca fascicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease. Nat Med 1996;2:430-436.[CrossRef][Medline]

  32. Fitzgerald TA. Comparison of research cost: man—primate animal—other animal models. J Med Primatol 1983;12:138-145.[Medline]

  33. Bock TA. Assay systems for hematopoietic stem and progenitor cells. STEM CELLS 1997;15(suppl 1):185-195.

  34. Srour EF, Zanjani ED, Cornetta K et al. Persistence of human multilineage, self-renewing lymphohematopoietic stem cells in chimeric sheep. Blood 1993;82:3333-3342.[Abstract/Free Full Text]

  35. 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 1996;2:1329-1337.[CrossRef][Medline]

  36. Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038-1045.[CrossRef][Medline]

  37. Cornetta K, Moen RC, Culver K et al. Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum Gene Ther 1990;1:15-30.[Medline]

  38. Cornetta K, Morgan RA, Gillio A et al. No retroviremia or pathology in long-term follow-up of monkeys exposed to a murine amphotropic retrovirus. Hum Gene Ther 1991;2:215-219.[Medline]

  39. 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 1992;176:1125-1135.[Abstract/Free Full Text]

  40. Otto E, Jones-Trower A, Vanin EF et al. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum Gene Ther 1994;5:567-575.[Medline]

  41. Zufferey R, Nagy D, Mandel RJ et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997;15:871-875.[CrossRef][Medline]

  42. Kim VN, Mitrophanous K, Kingsman SM et al. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J Virol 1998;72:811-816.[Abstract/Free Full Text]

  43. Dull T, Zufferey R, Kelly M et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998;72:8463-8471.[Abstract/Free Full Text]

  44. Miyoshi H, Blomer U, Takahashi M et al. Development of a self-inactivating lentivirus vector. J Virol 1998;72:8150-8157.[Abstract/Free Full Text]

  45. Zufferey R, Dull T, Mandel RJ et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998;72:9873-9880.[Abstract/Free Full Text]

  46. Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998;92:1131-1141.[Abstract/Free Full Text]

  47. Dunbar CE, Seidel NE, Doren S et al. Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor. Proc Natl Acad Sci USA 1996;93:11871-11876.[Abstract/Free Full Text]

  48. Chuck AS, Palsson BO. Consistent and high rates of gene transfer can be obtained using flow-through transduction over a wide range of retroviral titers. Hum Gene Ther 1996;7:743-750.[Medline]

  49. Kotani H, Newton PB 3rd, Zhang S et al. Improved methods of retroviral vector transduction and production for gene therapy. Hum Gene Ther 1994;5:19-28.[Medline]

  50. Bahnson AB, Dunigan JT, Baysal BE et al. Centrifugal enhancement of retroviral mediated gene transfer. J Virol Methods 1995;54:131-143.[CrossRef][Medline]

  51. Sanyal A, Schuening FG. Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes. Hum Gene Ther 1999;10:2859-2868.[CrossRef][Medline]

  52. Eipers PG, Krauss JC, Palsson BO et al. Retroviral-mediated gene transfer in human bone marrow cells growth in continuous perfusion culture vessels. Blood 1995;86:3754-3762.[Abstract/Free Full Text]

  53. Moritz T, Patel VP, Williams DA. Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors. J Clin Invest 1994;93:1451-1457.

  54. Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 1996;2:876-882.[CrossRef][Medline]

  55. Kiem HP, Andrews RG, Morris J et al. Improved gene transfer into baboon marrow repopulating cells using recombinant fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998;92:1878-1886.[Abstract/Free Full Text]

  56. 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 2000;6:652-658.[CrossRef][Medline]

  57. Kiem HP, Heyward S, Winkler A et al. Gene transfer into marrow repopulating cells: comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons. Blood 1997;90:4638-4645.[Abstract/Free Full Text]

  58. Kiem HP, McSweeney PA, Bruno B et al. Improved gene transfer into canine hematopoietic repopulating cells using CD34-enriched marrow cells in combination with a gibbon ape leukemia virus-pseudotype retroviral vector. Gene Ther 1999;6:966-972.[CrossRef][Medline]

  59. Demaison C, Brouns G, Blundell MP et al. A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector. Hum Gene Ther 2000;11:91-100.[CrossRef][Medline]

  60. Kavanaugh MP, Miller DG, Zhang W et al. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci USA 1994;91:7071-7075.[Abstract/Free Full Text]

  61. Schlegel R, Tralka TS, Willingham MC et al. Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site? Cell 1983;32:639-646.[CrossRef][Medline]

  62. Mastromarino P, Conti C, Goldoni P et al. Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH. J Gen Virol 1987;68:2359-2369.[Abstract/Free Full Text]

  63. Burns JC, Friedmann T, Driever W et al. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 1993;90:8033-8037.[Abstract/Free Full Text]

  64. Friedmann T, Yee JK. Pseudotyped retroviral vectors for studies of human gene therapy. Nat Med 1995;1:275-277.[CrossRef][Medline]

  65. Rebel VI, Tanaka M, Lee JS et al. One-day ex vivo culture allows effective gene transfer into human nonobese diabetic/severe combined immune-deficient repopulating cells using high-titer vesicular stomatitis virus G protein pseudotyped retrovirus. Blood 1999;93:2217-2224.[Abstract/Free Full Text]

  66. Kelly PF, Bonifacino AC, Vandergriff JA et al. Multilineage transduction of non-human primate CD34+ hematopoietic cells using RD-114-pseudotyped oncoretroviruses. Mol Ther 2000;1:S125.[CrossRef]

  67. Rasko JE, Battini JL, Gottschalk RJ et al. The RD114/simian type D retrovirus receptor is a neutral amino acid transporter. Proc Natl Acad Sci USA 1999;96:2129-2134.[Abstract/Free Full Text]

  68. Kelly PF, Vandergriff J, Nathwani A et al. Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. Blood 2000;96:1206-1214.[Abstract/Free Full Text]

  69. Buchschacher GL, Wong-Staal F. Development of lentiviral vectors for gene therapy for human diseases. Blood 2000;95:2499-2504.[Abstract/Free Full Text]

  70. Shimada T, Fujii H, Mitsuya H et al. Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector. J Clin Invest 1991;88:1043-1047.

  71. Poznansky M, Lever A, Bergeron L et al. Gene transfer into human lymphocytes by a defective human immunodeficiency virus type 1 vector. J Virol 1991;65:532-536.[Abstract/Free Full Text]

  72. Poeschla EM, Wong-Staal F, Looney DJ. Efficient transduction of nondividing human cells by feline immunodeficiency lentiviral vectors. Nat Med 1998;4:354-357.[CrossRef][Medline]

  73. Olsen JC. Gene transfer vectors derived from equine infectious anemia virus. Gene Ther 1998;5:1481-1487.[CrossRef][Medline]

  74. White SM, Renda M, Nam NY et al. Lentivirus vectors using human and simian immunodeficiency virus elements. J Virol 1999;73:2832-2840.[Abstract/Free Full Text]

  75. Schnell T, Foley P, Wirth M et al. Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum Gene Ther 2000;11:439-447.[CrossRef][Medline]

  76. Nakajima T, Nakamura K, Ido E et al. Development of novel simian immunodeficiency virus vectors carrying a dual gene expression system. Hum Gene Ther 2000;11:1863-1874.[CrossRef][Medline]

  77. Stitz J, Buchholz CZ, Engelstadter M et al. Lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10A1. Virology 2000;273:16-20.[CrossRef][Medline]

  78. Miyoshi H, Smith KA, Mosier DE et al. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 1999;283:682-686.[Abstract/Free Full Text]

  79. Guenechea G, Gan OI, Inamitsu T et al. Transduction of human CD34+CD38– bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol Ther 2000;1:566-573.[CrossRef][Medline]

  80. An DS, Wersto RP, Agricola BA et al. Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34+ cells. J Virol 2000;74:1286-1295.[Abstract/Free Full Text]

  81. Bestor TH. Gene silencing as a threat to the success of gene therapy. J Clin Invest 2000;105:409-411.[Medline]

  82. Klug CA, Cheshier S, Weissman IL. Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their progeny. Blood 2000;96:894-901.[Abstract/Free Full Text]

  83. Challita PM, Kohn DB. Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc Natl Acad Sci USA 1994;91:2567-2571.[Abstract/Free Full Text]

  84. Gram GJ, Nielsen SD, Hansen JE. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation. J Hematother 1998;7:333-341.[Medline]

  85. Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science 1999;286:481-486.[Abstract/Free Full Text]

  86. Cheng L, Du C, Lavau C et al. Sustained gene expression in retrovirally transduced, engrafting human hematopoietic stem cells and their lympho-myeloid progeny. Blood 1998;92:83-92.[Abstract/Free Full Text]

  87. 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 1999;94:3349-3357.[Abstract/Free Full Text]

  88. Marx JC, Allay JA, Persons DA et al. High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells. Hum Gene Ther 1999;10:1163-1173.[CrossRef][Medline]

  89. Tsuji T, Itoh K, Baum C et al. Retroviral vector-mediated gene expression in human CD34+CD38– cells expanded in vitro: cis elements of FMEV are superior to those of Mo-MuLV. Hum Gene Ther 2000;11:271-284.[CrossRef][Medline]

  90. Grosveld F, van Assendelft GB, Greaves DR et al. Position-independent, high-level expression of the human ß-globin gene in transgenic mice. Cell 1987;51:975-985.[CrossRef][Medline]

  91. Forrester WC, Novak U, Gelinas R et al. Molecular analysis of the human ß-globin locus activation region. Proc Natl Acad Sci USA 1989;86:5439-5443.[Abstract/Free Full Text]

  92. Pasceri P, Pannell D, Wu X et al. Full activity from human ß-globin locus control region trangenes requires 5'HS1, distal ß-globin promoter, and 3' ß-globin sequences. Blood 1998;92:653-663.[Abstract/Free Full Text]

  93. Novak U, Harris EA, Forrester W et al. High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells. Proc Natl Acad Sci USA 1990;87:3386-3390.[Abstract/Free Full Text]

  94. Plavec I, Papayannopoulou T, Maury C et al. A human ß-globin gene fused to the human ß-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells. Blood 1993;81:1384-1392.[Abstract/Free Full Text]

  95. Li Q, Emery DW, Fernandez M et al. Development of viral vectors for gene therapy of ß-chain hemoglobinopathies: optimization of a {gamma}-globin gene expression cassette. Blood 1999;93:2208-2216.[Abstract/Free Full Text]

  96. May C, Rivella S, Callegari J et al. Therapeutic haemoglobin synthesis in ß-thalassaemic mice expressing lentivirus-encoded human ß-globin. Nature 2000;406:82-86.[CrossRef][Medline]

  97. Prioleau MN, Nony P, Simpson M et al. An insulator element and condensed chromatin region separate the chicken ß-globin locus from an independently regulated erythroid-specific folate receptor gene. EMBO J 1999;18:4035-4048.[CrossRef][Medline]

  98. Bell AC, West AG, Felsenfeld G. The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 1999;98:387-396.[CrossRef][Medline]

  99. Emery DW, Yannaki E, Tubb J et al. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA 2000;97:9150-9155.[Abstract/Free Full Text]

  100. Phillips K, Gentry T, McCowage G et al. Cell-surface markers for assessing gene transfer into human hematopoietic cells. Nat Med 1996:2;1154-1156.[CrossRef][Medline]

  101. 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 1997;8:1595-1604.[Medline]

  102. 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 1998;4:1201-1205.[CrossRef][Medline]

  103. Williams DA. Ex vivo expansion of hematopoietic stem and progenitor cells—robbing Peter to pay Paul? Blood 1993;81:3169-3172.[Free Full Text]

  104. Emerson SG. Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics. Blood 1996;87:3082-3088.[Free Full Text]

  105. Srour EF, Abonour R, Cornetta K et al. Ex vivo expansion of hematopoietic stem and progenitor cells: are we there yet? J Hematother 1999;8:93-102.[CrossRef][Medline]

  106. Shih CC, Hu MC, Hu J et al. Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells. Blood 1999;94:1623-1636.[Abstract/Free Full Text]

  107. Dorrell C, Gan OI, Pereira DS et al. Expansion of human cord blood CD34+CD38– cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 2000;95:102-110.[Abstract/Free Full Text]

  108. Holyoake TL, Alcorn MJ, Richmond L et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Tranplant 1997;19:1095-1101.[CrossRef][Medline]

  109. Licht T, Herrmann F, Gottesman MM et al. In vivo drug-selectable genes: a new concept in gene therapy. STEM CELLS 1997;15:104-111.[Abstract/Free Full Text]

  110. Sorrentino BP, Brandt SJ, Bodine D et al. Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1. Science 1992;257:99-103.[Abstract/Free Full Text]

  111. Hanania EG, Giles RE, Kavanagh J et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy. Proc Natl Acad Sci USA 1996;93:15346-15351.[Abstract/Free Full Text]

  112. Hesdorffer C, Ayello J, Ward M et al. Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation. J Clin Oncol 1998;16:165-172.[Abstract/Free Full Text]

  113. Moscow JA, Huang H, Carter C et al. Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy. Blood 1999;94:52-61.[Abstract/Free Full Text]

  114. Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 1991;66:85-94.[CrossRef][Medline]

  115. Drach D, Zhao S, Drach J et al. Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistance phenotype. Blood 1992;80:2729-2734.[Abstract/Free Full Text]

  116. Blau CA, Neff T, Papayannopoulou T. Cytokine prestimulation as a gene therapy strategy: implications for using the MDR1 gene as a dominant selectable marker. Blood 1997;89:146-154.[Abstract/Free Full Text]

  117. Sorrentino BP, McDonagh KT, Woods D et al. Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice. Blood 1995;86:491-501.[Abstract/Free Full Text]

  118. Bunting KD, Galipeau J, Topham D et al. 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 1998;92:2269-2279.[Abstract/Free Full Text]

  119. Allay JA, Dumenco LL, Koc ON et al. Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells. Blood 1995;85:3342-3351.[Abstract/Free Full Text]

  120. Jelinek J, Fairbairn LJ, Dexter TM et al. Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase. Blood 1996;87:1957-1961.[Abstract/Free Full Text]

  121. Spencer HT, Sleep SE, Rehg JE et al. A gene transfer strategy for making bone marrow cells resistant to trimetrexate. Blood 1996;87:2579-2587.[Abstract/Free Full Text]

  122. Allay JA, Persons DA, Galipeau J et al. In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med 1998;4:1136-1143.[CrossRef][Medline]

  123. Blau CA, Peterson KR, Drachman JG et al. A proliferation switch for genetically modified cells. Proc Natl Acad Sci USA 1997;94:3076-3081.[Abstract/Free Full Text]

  124. Ito K, Ueda Y, Kokubun M et al. Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells. Blood 1997;90:3884-3892.[Abstract/Free Full Text]

  125. Richard RE, Wood B, Zeng H et al. Expansion of genetically modified primary human hematopoietic cells using chemical inducers of dimerization. Blood 2000;95:430-436.[Abstract/Free Full Text]

  126. Jin L, Zeng H, Chien S et al. In vivo selection using a cell-growth switch. Nat Genet 2000;26:64-66.[CrossRef][Medline]

  127. Huhn RD, Tisdale JF, Agricola B et al. Retroviral marking and transplantation of rhesus hematopoietic cells by nonmyeloablative conditioning. Hum Gene Ther 1999;10:1783-1790.[CrossRef][Medline]

  128. Rosenzweig M, MacVittie TJ, Harper D et al. Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning. Blood 1999;94:2271-2286.[Abstract/Free Full Text]

  129. James RI, Warlick CA, Diers MD et al. Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity. Blood 2000;96:1334-1341.[Abstract/Free Full Text]

  130. Davis BM, Koc ON, Gerson SL. Limiting numbers of G156A O6-methylguanine-DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection. Blood 2000;95:3078-3084.[Abstract/Free Full Text]

  131. Tripathy SK, Black HB, Goldwasser E et al. Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat Med 1996;2:545-550.[CrossRef][Medline]