Unfulfilled Promise of Endostatin in a Gene Therapy-Xenotransplant Model of Human Acute Lymphocytic Leukemia
Authors: Wolfgang Eisterer, Xiaoyan Jiang, Thomas Bachelot, Robert Pawliuk, Carolina Abramovich, Philippe Leboulch, Donna Hogge, Connie Eaves
Journal: Molecular Therapy (2002)

Retroviral transduction of hematopoietic stem cells (HSCs) offers an attractive strategy for treating malignancies that home to the marrow. This approach should therefore be of interest for evaluating the therapeutic activity of anti-angiogenic agents on hematopoietic malignancies whose growth has been associated with enhanced angiogenesis. A variety of studies have indicated endostatin to be a potent anti-angiogenic agent both in vitro and in vivo, and a human malignancy that might be sensitive to endostatin is human B-lineage acute lymphoblastic leukemia (B-ALL). The demonstrated ability of human B-ALL cells to engraft the marrow of immunodeficient mice suggested the potential of this system for testing an endostatin delivery strategy using co-transplanted non-obese diabetic–scid/scid (NOD/SCID) HSCs engineered to express endostatin. Here we show that, in spite of their mutant scid gene, NOD/SCID HSCs can be transduced with an endostatin-encoding retrovirus at efficiencies that result in a several-fold increase in endostatin serum levels in transplanted recipients. However, this did not alter the regrowth of co-transplanted human B-ALL blasts. These findings validate this gene transfer approach for investigating effects of novel therapeutics on primary human malignant cells that engraft NOD/SCID mice and question the utility of native endostatin for controlling human B-ALL in vivo. Retroviral transduction of hematopoietic stem cells (HSCs) offers an attractive strategy for treating malignancies that home to the marrow. This approach should therefore be of interest for evaluating the therapeutic activity of anti-angiogenic agents on hematopoietic malignancies whose growth has been associated with enhanced angiogenesis. A variety of studies have indicated endostatin to be a potent anti-angiogenic agent both in vitro and in vivo, and a human malignancy that might be sensitive to endostatin is human B-lineage acute lymphoblastic leukemia (B-ALL). The demonstrated ability of human B-ALL cells to engraft the marrow of immunodeficient mice suggested the potential of this system for testing an endostatin delivery strategy using co-transplanted non-obese diabetic–scid/scid (NOD/SCID) HSCs engineered to express endostatin. Here we show that, in spite of their mutant scid gene, NOD/SCID HSCs can be transduced with an endostatin-encoding retrovirus at efficiencies that result in a several-fold increase in endostatin serum levels in transplanted recipients. However, this did not alter the regrowth of co-transplanted human B-ALL blasts. These findings validate this gene transfer approach for investigating effects of novel therapeutics on primary human malignant cells that engraft NOD/SCID mice and question the utility of native endostatin for controlling human B-ALL in vivo. IntroductionExpanding populations of malignant cells depend on the continuous outgrowth of new blood vessels, a process referred to as angiogenesis [1Hanahan D. Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6021) Google Scholar]. Previous studies have demonstrated increased angiogenesis in several malignancies, including those of the hematopoietic system [2Perez-Atayde A.R. Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia.Am. J. Pathol. 1997; 150: 815-821PubMed Google Scholar, 3Fiedler W. 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These investigators also found increased levels of a potent angiogenic inducer, basic fibroblast growth factor (bFGF), in the serum and urine of these patients at diagnosis. These findings support the hypothesis that angiogenesis may be an important therapeutic target in several human cancers, including some of hematologic origin. Several reports of potent antitumor effects of endostatin (a recently identified peptide inhibitor of angiogenesis) in experimental animal models [6O'Reilly M.S. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4212) Google Scholar, 7Boehm T. Folkman J. Browder T. O'Reilly M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature. 1997; 390: 404-407Crossref PubMed Scopus (1605) Google Scholar, 8Bergers G. Javaherian K. Lo K.M. Folkman J. Hanahan D. 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Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth.Cancer Res. 2000; 60: 2190-2196PubMed Google Scholar] have created much excitement in the potential clinical application of this agent and clinical trials have been initiated [13Ryan D.P. Penson R.T. Ahmed S. Chabner B.A. Lynch Jr., T.J. Reality testing in cancer treatment: the phase I trial of endostatin.Oncologist. 1999; 4: 501-508PubMed Google Scholar].To improve delivery of endostatin in vivo, a variety of gene transfer approaches have been evaluated. These include in vivo injections into a variety of sites of the endostatin gene itself [14Blezinger P. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene.Nat. Biotechnol. 1999; 17: 343-348Crossref PubMed Scopus (297) Google Scholar, 15Chen Q.-R. Kumar D. Stass S.A. Mixson A.J. 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Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin.Hum. Gene Ther. 2000; 11: 1983-1996Crossref PubMed Scopus (86) Google Scholar, 20Regulier E. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.Cancer Gene Ther. 2001; 8: 45-54Crossref PubMed Scopus (51) Google Scholar, 21Kuo C.J. Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer.Proc. Natl. Acad. Sci. USA. 2001; 98: 4605-4610Crossref PubMed Scopus (272) Google Scholar, 22Wen X.-Y. Bai Y. Stewart A.K. Adenovirus-mediated human endostatin gene delivery demonstrates strain-specific antitumor activity and acute dose-dependent toxicity in mice.Hum. Gene Ther. 2001; 12: 347-358Crossref PubMed Scopus (44) Google Scholar], or cells previously engineered ex vivo to produce endostatin for sustained periods; either directly into the experimental tumor or intravenously into the host animals to convert the vasculature into producers of endostatin [23Yoon S.S. Mouse endostatin inhibits the formation of lung and liver metastases.Cancer Res. 1999; 59: 6251-6256PubMed Google Scholar, 24Joki T. Continuous release of endostatin from microencapsulated engineered cells for tumor therapy.Nat. Biotechnol. 2001; 19: 35-39Crossref PubMed Scopus (341) Google Scholar, 25Read T.-A. Local endostatin treatment of gliomas administered by microencapsulated producer cells.Nat. Biotechnol. 2001; 19: 29-34Crossref PubMed Scopus (267) Google Scholar, 26Scappaticci F.A. Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice.Mol. Ther. 2001; 3: 186-196Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar]. Although significant anti-tumor effects have been reported in many of these studies, in others a positive result has been either partial or not detectable, even when very high levels of endostatin have been produced [20Regulier E. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.Cancer Gene Ther. 2001; 8: 45-54Crossref PubMed Scopus (51) Google Scholar, 21Kuo C.J. Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer.Proc. Natl. Acad. Sci. USA. 2001; 98: 4605-4610Crossref PubMed Scopus (272) Google Scholar, 27Jouanneau E. Lack of antitumor activity of recombinant endostatin in a human neuroblastoma xenograft model.J. Neurooncol. 2001; 51: 11-18Crossref PubMed Scopus (42) Google Scholar].These findings emphasize the need to carefully define the spectrum of primary human tumor types that may be usefully treated by endostatin and under what conditions. For hematopoietic malignancies, a genetic approach using transplantable hematopoietic stem cells (HSCs) transduced with a retroviral vector encoding endostatin offers an alternative strategy for increasing local delivery, as this should ensure sustained endostatin production directly at the site of malignant cell growth, that is, within the extravascular BM space. To test this concept, we first defined conditions for achieving adequate levels of retrovirus-mediated gene transfer and expression in the progeny of transplantable HSCs from nonobese diabetic-scid/scid (NOD/SCID) mice using a murine stem cell virus (MSCV)-based IRES retroviral vector described in detail in the accompanying study [28Pawliuk P. Continuous intravascular secretion of endostatin in mice from transduced hematopoietic stem cells.Mol. Ther. 2002; 79: 345-351Abstract Full Text Full Text PDF Scopus (59) Google Scholar]. We then co-transplanted sublethally irradiated NOD/SCID mice simultaneously with NOD/SCID HSCs engineered to express endostatin together with primary human ALL cells, having shown in parallel experiments that primary human ALL cells engraft the BM of NOD/SCID mice at high efficiency, a finding that was recently confirmed independently [29Nijmeijer B.A. Monitoring of engraftment and progression of acute lymphoblastic leukemia in individual NOD/SCID mice.Exp. Hematol. 2001; 29: 322-329Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar]. Using this approach, we found that the growth of human ALL cells in NOD/SCID mice is unaffected by co-transplanted BM cells able to achieve elevated levels of circulating human endostatin for at least 3 months post-transplant.ResultsRetrovirus-Mediated Gene Transfer to NOD/SCID HSCsIt has been reported that the scid mutation causes defective retroviral integration resulting from associated low levels of DNA-dependent protein kinase [30Daniel R. Katz R.A. Skalka A.M. A role for DNA-PK in retroviral DNA integration.Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar]. This suggested that standard protocols for transducing murine HSCs with retroviral vectors might not be effective on NOD/SCID targets. Moreover, as we planned to use sublethally irradiated NOD/SCID mice as hosts, it was additionally important to determine whether useful levels of engraftment with transduced cells could be achieved under these competitive conditions. Recent studies have defined growth factors that optimize normal mouse HSC self-renewal divisions in vitro (FLT3 ligand (FL), Steel factor (SF), and interleukin (IL)-11) and the period required for such divisions to be initiated (4 days) [31Oostendorp R.A.J. Audet J. Eaves C.J. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo.Blood. 2000; 95: 855-862PubMed Google Scholar, 32Audet J. Miller C.L. Rose-John S. Piret J. Eaves C.J. Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 1757-1762Crossref PubMed Scopus (113) Google Scholar]. This made it possible to design HSC gene transfer protocols that use untreated NOD/SCID mice as BM cell donors rather than mice injected with 5-FU 4 days before, a treatment that reduces the absolute HSC content of the femur by approximately threefold [33Lemieux M.E. Rebel V.I. Lansdorp P.M. Eaves C.J. Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lympho-myeloid differentiation in long-term marrow “switch” cultures.Blood. 1995; 86: 1339-1347PubMed Google Scholar]. Table 1 summarizes the pooled results of several experiments in which the same GFP virus (SM10) was used in the different gene transfer protocols compared. These protocols used variously enriched HSC populations obtained from untreated normal adult NOD/SCID BM cells that were maintained in vitro for a total of 5 days with FL, SF, and IL-11, as well as BM cells from NOD/SCID mice treated 4 days previously with 5-FU and then cultured also for a total of 5 days, but with SF, IL-3, and IL-6 to allow comparison with a procedure that has been more widely used by other groups. It can be seen that all of the procedures evaluated yielded transplantable NOD/SCID HSCs capable of producing detectable levels of GFP+ myeloid progeny for at least 4 months in sublethally irradiated syngeneic NOD/SCID recipients. In all groups it can be estimated from limiting dilution analysis principles that there was approximately one transduced HSC in each transplant dose tested because the proportion of positive mice was generally < 30%. Consistent with this estimation was the observation that, on average, ~ 8% of the circulating WBCs in each of the mice that regenerated any detectable GFP+ cells were GFP+, regardless of the method of transduction used. We have previously shown that each adult BM HSC will produce, on average, 8% of the regenerated blood cells in recipients transplanted with less than 10 HSCs [32Audet J. Miller C.L. Rose-John S. Piret J. Eaves C.J. Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 1757-1762Crossref PubMed Scopus (113) Google Scholar]. Unfortunately, cell losses inherent in obtaining the HSC-enriched suspensions from untreated NOD/SCID mouse BM proved to be much higher than those resulting from the 5-FU treatment (data not shown). Therefore, for practical reasons, we used BM cells from 5-FU-treated donors in subsequent experiments. In addition, given the modest gene transfer efficiencies obtained using the supernatant infection protocol, subsequent experiments also made use of a co-cultivation protocol. As can be seen below, we then obtained slightly higher levels of GFP+ NOD/SCID blood cells in the transplanted recipients.TABLE 1Frequency of transduced NOD/SCID BM stem cells obtained from variously enriched starting populations and levels of GFP+ blood cells 4 months after injection of unselected cellsTarget cells3Cytokines3Exp. No.% GFP+ cells post-transductionbThis value refers to the percent of cells recovered in vitroat the end of the 5 day prestimulation-transduction protocol that were GFP+.Starting equivalent cells injected per mousecEach mouse was injected with the progeny produced during the 5 day prestimulation-transduction protocol from the number and type of original target cells indicated.GFP+ mice/total miceFrequency of transduced stem cells in the transplantdThis value refers to the calculated frequency of transduced repopulating stem cells among the cells actually transplanted based on the proportion of recipient mice found to contain some detectable GFP+ cells in the blood (> 5/1,000 events analyzed). This calculation was performed using the L-calc software program (StemCell).Levels of GFP+ cells in positive miceDay 4 5-FUS,3,614.53.2 × 1062/75.8 × 10−55, 1123.75.2 × 1063/70.7,3,733.96.3 × 1064/90.7,2,21,24F,S,1143.83.4 × 1062/74.8 × 10−58,1055.13.6 × 1062/87,1264.23.8 × 1064/127,11,13,1973.75.1 × 1062/64,585.65.5 × 1064/116,6,13,1594.27.1 × 1063/64,5,11103.61.2 × 1074/69,9,10,14Lin-F,S,1111212.3 × 1042/73.9 × 10−47,812135.3 × 1041/61113226.5 × 1042/101,13Sca-1+Lin−SPaDay 4 5-FU marrow cells or FACS purified subsets of normal marrow cells from NOD/SCID mice were prestimulated with the cytokines indicated for 2 days and exposed to VCM for 3 days in the presence of the same cytokines (as described in the Methods) prior to transplantation into sublethally irradiated syngeneic hosts.F,S,1114141.6 × 1030/52.6 × 10−3−15174.0 × 1031/51116164.5 × 1032/61,10a Day 4 5-FU marrow cells or FACS purified subsets of normal marrow cells from NOD/SCID mice were prestimulated with the cytokines indicated for 2 days and exposed to VCM for 3 days in the presence of the same cytokines (as described in the Methods) prior to transplantation into sublethally irradiated syngeneic hosts.b This value refers to the percent of cells recovered in vitroat the end of the 5 day prestimulation-transduction protocol that were GFP+.c Each mouse was injected with the progeny produced during the 5 day prestimulation-transduction protocol from the number and type of original target cells indicated.d This value refers to the calculated frequency of transduced repopulating stem cells among the cells actually transplanted based on the proportion of recipient mice found to contain some detectable GFP+ cells in the blood (> 5/1,000 events analyzed). This calculation was performed using the L-calc software program (StemCell). Open table in a new tab Engraftment of NOD/SCID Mice with Human ALL CellsWe assessed samples from seven patients with ALL (five with B lineage disease, two with T lineage disease) for their ability to grow in sublethally irradiated (350 cGy) NOD/SCID mice after intravenous injection of 105, 106, and 107 cells (three to six mice per cell dose per sample). None of the recipients of the T-ALL cells showed any evidence of human cells for up to 24 weeks post-transplant, at which time the experiments were terminated. Although T-ALL engraftment has been noted [34Steele J.P. Growth of human T-cell lineage acute leukemia in severe combined immunodeficiency (SCID) mice and non-obese diabetic SCID mice.Blood. 1997; 90: 2015-2019PubMed Google Scholar], 10-fold higher doses of cells were injected. In contrast to our findings with T-ALL, cells from all five B-ALL samples tested did engraft the BM of NOD/SCID mice. The engrafted B-ALL cells were already apparent by 3 weeks post-transplant and at this time they already constituted between 5% and 92% of all the cells present in the BM of the mice. Thereafter, the human B-ALL cells increased in a time- and transplant dose-dependent fashion that was specific for each original B-ALL sample. Figure 1 illustrates the type of patient-specific profile of B-ALL antigen expression that was seen with all five samples at all times post-transplant. From the cell dose response results, frequencies of NOD/SCID repopulating human B-ALL cells in each of the five original B-ALL samples could be determined. These were 0.04 (0.07–0.02), 0.14 (0.28–0.07), 1.4 (2.8–0.7), 95 (1200–1), and > 10 × 10−4 (values shown in brackets indicating the range defined by ± SE). This study thus indicates the relative ease with which human B-ALL cells, even from patients with low-risk disease (absence of cytogenetic abnormalities, n = 2), can engraft irradiated NOD/SCID mice as compared with other immunodeficient strains [35Dick J.E. Normal and leukemic human stem cells assayed in SCID mice.Semin. Immunol. 1996; 8: 197-206Crossref PubMed Scopus (134) Google Scholar, 36Gunther R. Biotherapy of xenografted human central nervous system leukemia in mice with severe combined immunodeficiency using B43 (anti-CD19)-pokeweed anti-viral protein immunotoxin.Blood. 1995; 85: 2537-2545PubMed Google Scholar], as also reported by others [29Nijmeijer B.A. Monitoring of engraftment and progression of acute lymphoblastic leukemia in individual NOD/SCID mice.Exp. Hematol. 2001; 29: 322-329Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar].Unperturbed Engraftment of Human B-ALL Cells in NOD/SCID Mice Co-transplanted with Endostatin-Transduced NOD/SCID BMTo evaluate the ability of endostatin to modulate human B-ALL growth in a vascularized tissue environment, we transplanted irradiated NOD/SCID mice simultaneously with 105 cells from four of the five patients’ samples with B-ALL together with 2 × 105 transduced, FACS-sorted GFP+NOD/SCID BM cells. The latter we obtained by co-cultivating cells from 5-FU-treated donors with GP+E86 cells, producing an endostatin IRES-GFP retrovirus (KA153) or a control GFP virus (SM10). These viruses are shown schematically in Fig. 2A. Figure 2B shows the presence of intact provirus in the producer cells and that these producers also generated immunoreactive endostatin protein. Figure 2D shows the results of experiments with human umbilical vein endothelial cells (HUVEC), whose vascular endothelial growth factor (VEGF)-stimulated proliferation could be specifically inhibited by the addition of supernatant from the endostatin vector-producer cells.FIG. 2Structure and characterization of vectors. (A) Structure of the retroviral vectors used in this study. The SM10 MSCV vector incorporates a 515-bp EMCV-IRES fragment, a 700-bp fragment containing the EGFP coding sequence, and a 565-bp fragment containing the constitutive transport element of the hepatitis B virus. The KA153 vector is a derivative of the SM10 vector and also contains the coding sequence of endostatin that was PCR-derived from the C-terminal sequence of murine collagen XVIII. Expected full-length transcripts are shown. MSCV LTR, murine stem cell virus long terminal repeat; ψ, extended packaging signal; EMCV-IRES, internal ribosome entry site of the encephalomyocarditis virus; CTE, constitutive transport element of the hepatitis virus. (B) Detection of intact provirus in FACS-selected (GFP+) transduced NOD/SCID BM cells (lanes 2 and 11) and NIH3T3 cells (lanes 3 and 10), and in the BM of representative NOD/SCID mice transplanted 12 weeks previously with 105 GFP+ syngeneic BM cells transduced with either KA153 (lanes 4–6) or SM10 (lanes 7–9). DNA (10 μg) from each tissue sample was digested with XbaI, an enzyme that cuts once within each proviral LTR. Shown are the results of a blot probed with a 32P-labeled GFP probe. The positive control represents 50 ng DNA obtained from the original plasmids of KA153 (lane 1) or SM10 (lane 12). Right and left margins, molecular size markers. (C) Detection of endostatin protein in KA153 VCM by immunoprecipitation and western blot. Supernatant of subconfluent KA153 cells (lane 2) and parental GP+E86 control cells (lane 1) was incubated overnight with anti-FLAG M2 beads. Right and left margins, protein size markers. (D) Inhibition of [3H]thymidine-incorporation by HUVEC in culture following the addition of KA153 VCM. HUVEC cells were first plated in medium containing 15% FCS plus 60 μg/mL endothelial cell growth supplement (ECGS) and then for an additional 24 hours in medium containing only 5% FCS (no EGCS). VEGF (5 ng/mL) alone (Ctrl) or with KA153 VCM (KA153) or SM10 VCM (SM10) was then added. After another 24 hours, 1 μα [3H]thymidine was added and the cultures incubated for a final 24 hours. Starved HUVEC to which VEGF was added served as internal negative controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although the pace of human B-ALL cell generation in the BM of the injected mice was again variable according to the particular patient's sample, in none of the four experiments was there any difference between recipients of NOD/SCID BM cells transduced with the endostatin versus the control vector. A representative experiment is shown in Fig. 3. In all groups of transplanted mice, at least 40% of the circulating murine WBCs were GFP+ within 2 weeks post-transplant. However, the numbers of GFP+ WBCs subsequently declined to ~ 20% in each experiment over the ensuing 10 weeks (Fig. 3B). In the serum of the mice that had been co-transplanted with HSCs transduced with the endostatin vector, we observed a consistent, significant (P < 0.05), and sustained two- to threefold elevation in endostatin levels (mean ± SE = 180 ± 7 ng/mL, n = 58, versus 63 ± 4 ng/mL in recipients of control (SM10)-transduced cells, n = 10, and 61 ± 4 ng/mL in untreated NOD/SCID mice, n = 7; Fig. 3C). In addition we obtained evidence of endostatin transcripts in the circulating progeny of the transduced cells from RT-PCR analyses of these cells (data not shown).FIG. 3Kinetics of engraftment of NOD/SCID mice with ALL cells and transduced NOD/SCID BM cells. (A) ALL cell engraftment kinetics in the BM of mice co-transplanted with NOD/SCID BM cells transduced with either the KA153 (endostatin-IRES-GFP) or SM10 (GFP only) vector (eight animals per group). (B) Kinetics of engraftment of the transduced (GFP+) NOD/SCID BM cells in the same mice as in (A) as indicated by their levels of circulating GFP+ WBCs. (C) Endostatin serum levels in the same mice (filled circles show results for recipients of KA153-transduced BM cells, open circles for recipients of SM10-transduced BM cells). Data shown are from mice transplanted with cells from one of four ALL patients.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThese studies describe the development and application of a unique model for assessing gene therapy approaches to human disease where sustained delivery of a therapeutic product by cells of the blood-forming system would offer advantages in terms of tissue penetration or localization. The use of immunodeficient mice as hosts for the engraftment of human hematopoietic cells, both normal and malignant, is now well established and the particular sensitivity of the irradiated NOD/SCID mouse for this purpose has been widely recognized [37Cashman J.D. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.Blood. 1997; 89: 4307-4316PubMed Google Scholar, 38Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-736Crossref PubMed Scopus (5398) Google Scholar]. Here, we have successfully exploited this model to enable a transplant of transduced syngeneic NOD/SCID marrow cells to maximize the in vivo production of an anti-angiogenic agent at the site of an expanding population of human B-ALL cells in the absence of any immune effect, thus simulating what might be expected of a transduced autologous transplant in a clinical setting.The anticipated barrier to this experimental model, posed by the presence of the scid mutation in the NOD/SCID BM cells [30Daniel R. Katz R.A. Skalka A.M. A role for DNA-PK in retroviral DNA integration.Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar], did not prevent the achievement of useful levels of HSC transduction, although the yield of transduced HSCs was reduced several-fold by comparison with our experience with normal mice. In part, our success in transducing NOD/SCID HSCs may be due to the use of a cytokine cocktail (FL, SF, and IL-II) with more potent HSC-stimulating activity than has historically been used in gene transfer protocols with retroviral vectors. Indeed, we have shown here that this enables useful transduction efficiencies to be achieved with partially purified, but otherwise untreated, normal adult NOD/SCID murine BM HSCs.Durably elevated circulating levels of endostatin were thus obtained in recipients of HSCs transduced with an endostatin vector and it is prob