Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro

Autologous bone marrow stromal cells engineered to produce 3,4,-dihydroxyphenylalanine (L-DOPA) can potentially be used as donor cells for neural transplantation in Parkinson's disease. Here, we examined the possibility of using several different promoters and either a self-inactivating retrovirus (pSIR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors were constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP reporter gene or a bicistronic sequence containing the genes for human tyrosine hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced with both standard retroviral vectors and pSIR containing the PGK promoter. Transduced rMSCs expressed GFP (90.4--94.4% of cells) or were able to synthesize and secrete L-DOPA (89.0--283 pmols/10(6) cells/h). After transduced rMSCs were plated at low density (3--6 cells/cm(2)), the cells expanded over 1000-fold in 3--4 weeks, and the rMSCs continued to either express GFP or produce L-DOPA. Furthermore, two high-expressing clones were isolated and expanded at low-density from rMSCs transduced with pSIR driven by the PGK promoter (97.0% GFP+ or 1096.0 pmols L-DOPA/10(6) cells/h).     

Long-term phenotypic, functional and genetic stability of cancer-specific T-cell receptor (TCR) alphabeta genes transduced to CD8+ T cells

In adoptive T-cell transfer as an intervention for malignant diseases, retroviral transfer of T-cell receptor (TCR) genes derived from CD8(+) cytotoxic T-lymphocyte (CTL) clones provides an opportunity to generate a large number of T cells with the same antigen specificity. We cloned the TCR-alphabeta genes from a human leukocyte antigen (HLA)-A(*)2402-restricted CTL clone specific for MAGE-A4(143-151). The TCR-alphabeta genes were transduced to 99.2% of non-TCR expressing SupT1, a human T-cell line, and to 12.7-32.6% of polyclonally activated CD8(+) T cells by retroviral transduction. As expected, TCR-alphabeta gene-modified CD8(+) T cells showed cytotoxic activity and interferon-gamma production in response to peptide-loaded T2-A(*)2402 and tumor cell lines expressing both MAGE-A4 and HLA-A(*)2402. A total of 24 clones were established from TCR-alphabeta gene-transduced peripheral blood mononuclear cells and all clones were functional on a transduced TCR-dependent manner. Four clones were kept in culture over 6 months for analyses in detail. The transduced TCR-alphabeta genes were stably maintained phenotypically, functionally and genetically. Our results indicate that TCR-transduced alphabeta T cells by retroviral transduction represent an efficient and promising strategy for adoptive T-cell transfer for long term.     

Myeloablation enhances engraftment of transduced murine hematopoietic cells,but does not influence long-term expression of the transgene

To investigate to what extent myeloablation, graft size, and ex vivo manipulation influence the engraftment and long-term survival of transduced murine hematopoietic cells, groups of C57BL/6J (CD45.2) mice receiving total body irradiation (TBI) (1-9 Gy) or no irradiation were transplanted with either transduced bone marrow (BM) cells, at two cell doses, or with fresh BM cells from B6/SJL (CD45.1) congenic mice. Short (40 days) and long-term (5 months) engraftment and transgene expression were measured by FACS analysis. No donor cells were detected in the hematopoietic tissues of non-myeloablated mice, whereas in the irradiated animals, levels of engraftment correlated well with the dose of TBI administered. Similar percentages of transgene-expressing cells were found in the grafted hematopoietic cells of all groups of mice, regardless of the dose of TBI administered or the level of engraftment achieved. This suggests that the engrafted animals could become tolerant to the transgene product (enhanced green fluorescent protein, EGFP). Our results indicate that TBI facilitates the engraftment of manipulated hematopoietic cells in a dose-dependent manner, that mice engrafted with EGFP(+) hematopoietic cells probably acquire tolerance to EGFP, and that increasing the graft size and reducing the ex vivo manipulation required for retroviral gene transfer of hematopoietic cells also enhances their engrafting potential.     

Efficient engraftment of in utero transplanted mice with retrovirally transduced hematopoietic stem cells

Using an experimental mouse model, we have investigated the kinetics of hematopoietic reconstitution of recipients transplanted during fetal development with fresh and transduced hematopoietic stem cells (HSCs). Total bone marrow (BM) and purified Lin(-)Sca-1(+) cells, either fresh or transduced ex vivo with enhanced green fluorescent protein (EGFP)-encoding retroviral vectors, were in utero transplanted (IUT) into fetal mice. Data obtained 2 months after transplantation showed a similar proportion of engrafted animals, regardless of the fact that samples were purified or not on HSCs, and subjected or not to ex vivo transduction with retroviral vectors. The transplantation of grafts enriched in HSCs, either fresh or transduced, always improved the levels of donor chimerism of IUT mice in comparison with results obtained in mice transplanted with unpurified BM grafts (6.8 and 7.3% versus 1.15% median values, respectively). Significantly, engrafted recipients that were transplanted with the transduced graft always contained transduced EGFP(+) cells in peripheral blood (around 5% of donor cells were EGFP(+) at 2 months post-transplantation). This proportion was essentially maintained at longer times post-transplantation, as well as in secondary recipients transplanted with the BM of IUT mice. Our study describes for the first time a significant and stable engraftment of unconditioned mice subjected to IUT with HSCs transduced with retroviral vectors.     

双耐药基因导人脐血干/祖细胞降低联合化疗骨髓毒性作用的临床前研究

目的探讨转染醛脱氢酶基因(ALDH3)和多药耐药基因(mdr-1)的人脐血CD     

In vivo methotrexate selection of murine hemopoietic cells transduced with a retroviral vector for Gaucher disease.

The studies described were performed to investigate whether in vivo selection of retrovirus-transduced hemopoietic cells is feasible starting from a low percentage of transduced hemopoietic stem cells (PHSCs). The vector used is an amphotropic bicistronic retroviral vector carrying a cDNA for human lysosomal glucocerebrosidase (hGC) for treatment of Gaucher disease and a methotrexate (MTX) resistant mutant cDNA encoding human dihydrofolate reductase (DHFR). We tested the effect of MTX selection in mice that were either myeloablated or not before infusion of transduced cells. In addition, we determined whether repeated administration of transduced bone marrow cells has an additional effect on the percentage of hGC expressing cells. The results obtained have shown that, in myeloablated mice transplanted once with transduced bone marrow and treated twice weekly with 10 mg/kg of MTX for a total of 6 months, a two- to three-fold increased numbers of hGC expressing cells could be detected in both peripheral blood and bone marrow as compared with non-MTX treated mice. In mice transplanted with transduced bone marrow once every 2 weeks for a total of four times, percentages of hGC expressing cells were not significantly increased as compared with mice transplanted once. In non-ablated mice neither MTX selection nor multiple infusions of transduced bone marrow resulted in detection of hGC expressing cells 6 months after transplantation, indicating that the success of in vivo selection using MTX is highly dependent on the ratio of transduced hemopoietic stem cells transplanted versus residing and untransduced stem cells.     

Gene therapy of murine solid tumors with T cells transduced with a retroviral vascular endothelial growth factor--immunotoxin target gene

Solid tumor growth can be inhibited by targeting its neovasculature with vascular endothelial growth factor (VEGF)-toxin fusion proteins (FPs), but these agents have been limited by their inability to localize at the tumor site. In this study, we devised a gene therapy approach intended to deliver VEGF-toxin directly to tumor. Antigen-specific cytotoxic T lymphocytes (CTLs) served as vehicles to deliver a retroviral VEGF-toxin fusion protein to its specific leukemia cell target in vivo. A retroviral vector was constructed for gene therapy with VEGF positioned downstream of its 27-amino acid leader sequence, which promoted secretion of a catalytic immunotoxin containing either truncated diphtheria toxin or Pseudomonas exotoxin A. VEGF was chosen on the basis of the expression of VEGF receptor on endothelial cells in the tumor neovasculature. The VEGF FP was first expressed and secreted by mammalian NIH 3T3 cells. Intracellular expression of both VEGF and toxin was verified by immunofluorescence. In vitro, supernatants collected from transfected cells specifically inhibited the growth of VEGF receptor-expressing human umbilical vein endothelial cells (HUVECs), but not a control cell line. In vivo findings correlated with in vitro findings. A retroviral vector containing the target gene and a nerve growth factor receptor (NGFR) reporter gene was used to transiently transduce T15, a CD8(+) CTL line that specifically recognizes C1498, a lethal C57BL/6 myeloid tumor. Transduced T15 cells injected intravenously significantly inhibited the growth of subcutaneous tumor, whereas nontransduced controls did not. Together, these data indicate that gene therapy of T cells with retrovirus containing a VEGF-immunotoxin target gene may be a valid means of inhibiting a broad range of solid tumors dependent on angiogenesis.     

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.     

Genetic modification of human T cells with CD20: a strategy to purify and lyse transduced cells with anti-CD20 antibodies

A retroviral vector has been constructed that contains the human CD20 cDNA under the control of the Moloney murine leukemia virus (Mo-MuLV) LTR. Freshly isolated mononuclear cells are infected for three consecutive days in the presence of PHA and hrlL-2, and a mean 15.9% of the cells (range, 6.5 to 31.7%) acquire a CD3+CD20+ phenotype. Transduced T lymphocytes grow and expand in vitro for up to 3 weeks like mock-infected cells and, as observed for the T lymphoblastoid CEM cell line, CD20 expression is maintained for several months with no change in the growth curve of the cells. CD20-expressing CEM and fresh T lymphocytes can be positively immunoselected on columns using different anti-CD20 antibodies. Exposure to monoclonal chimeric anti-CD20 IgG1(kappa) Rituximab antibody (Roche), in the presence of complement, results in effective and rapid killing of the transduced CD3+CD20+ human T cells in vitro. This approach represents a new and alternative method to gene manipulation with "suicide" genes for the production of drug-responsive T cell populations, a crucial step for the future management of graft-versus-host disease in bone marrow transplant patients.     

Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.     

Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC).

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.     

Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation

Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cells from chemotherapeutic damage. Electroporation is a non-chemical, nonviral, highly reproducible means to introduce foreign genes into mammalian cells that has been used primarily for rapidly dividing cells. CD34(+) cells isolated from mobilized peripheral blood of patients were cultured for 48 h in serum-free culture medium supplemented with Flt-3 ligand, stem cell factor and thrombopoietin. Cell cycle analysis showed an increase in % S-phase from 2% on day 0 to 28% on day 2 without significant loss of mean fluorescence intensity (MFI). Optimal electroporation conditions for CD34(+) cells were 550 V/cm, 38 ms, 30 microg DNA/500 microl at cell densities between 0.2 x 10(6) and 10 x 10(6) cells/ml resulting in transient EGFP gene expression in 21% (+/- 1%) of CD34(+) precursor cells, as determined by flow cytometry 48 h after electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjugated monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) and 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The reporter gene was found to be integrated into the LTC-IC genomic DNA as determined by inverse PCR and DNA sequencing. These results suggest that electroporation has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells.     

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.     

AML1a在小鼠造血细胞增殖分化异常中的作用

本研究旨在探讨AML1a在小鼠造血细胞增殖分化异常中的作用及其作用机制。将构建的pMSCV-FLAG-AML1a-IRES-YFP和pMSCV-IRES-YFP分别与辅助质粒pV Pack-Eco（含Env）、pV Pack-GP（含gal-pol）组合,通过磷酸钙沉淀法转染293T细胞,制备逆转录病毒。通过逆转录病毒将AML1a及YFP转导至C57 BL/6J雄性小鼠的骨髓单个核细胞（BMMNC）,接种于M3434甲基纤维素完全培养液进行集落形成实验,并置于含mSCF、mIL-3、mIL-6的M5300液体培养液中进行长期培养,观察BMMNC形态变化。结果显示,转导了AML1a的BMMNC集落形成能力增强,集落数量和体积均明显大于对照组,集落以CFU-E和CFU-GEMM为主。长期培养实验中,转导AML1a组细胞形态显示分化阻滞,而对照组细胞则处于更成熟的阶段。结论：AML1a增加了造血干/祖细胞的增殖能力,并将小鼠造血干/祖细胞阻滞于较早的发育阶段。     

Expression of retroviral transduced human CD18 in murine cells: an in vitro model of gene therapy for leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by a defective CD18 gene. The cell-surface glycoprotein encoded by this gene CD18 is normally expressed in cells of the hematopoietic system. An in vitro murine model of CD18 gene replacement therapy was developed to investigate the feasibility of an in vivo murine hematopoietic stem cell gene therapy model. Human CD18-transducing retroviruses were used to transfer a functional human CD18 gene into a variety of cells including (i) murine lymphoblasts (which express murine CD11a and murine CD18), (ii) murine fibroblasts (which have no endogenous murine CD11a/CD18 expression), and (iii) murine fibroblasts, which have been stably transfected with a human CD11a gene. In murine lymphoblasts, human CD18 was expressed on the cell surface as a heterodimer with murine CD11a. Cell-surface expression of human CD18 had no apparent effect on the level of endogenous murine CD11a/CD18 expression. Immunoprecipitation of cell-surface labeled proteins in murine lymphoblasts with a human CD18 specific antibody co-precipitated murine CD11a. Human CD18 can be detected by immunochemistry in the cytoplasm of fibroblasts infected with CD18 encoding retrovirus, but coexpression with CD11a is required for cell-surface expression of either subunit in fibroblasts. These studies suggest that human CD18 will form a heterodimer with murine CD11a and that human CD18 is not expressed on the cell surface of cells not expressing CD11. This provides the basis for the development of a murine hematopoietic stem cell gene replacement therapy model for the treatment of LAD.     

Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF-induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34(+) cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34(+) cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.     

人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

背景:研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化.目的:以人主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞,并比较不同移植途径对造血干细胞体内造血重建能力的影响.方法:将小鼠E14 胚胎干细胞诱导为拟胚体,采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6 d,接种NOD-SCID小鼠检测体内致瘤性.再将诱导后的拟胚体细胞移植经致死量60Co γ射线辐照的BALB/C雌鼠,受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组.结果与结论:拟胚体细胞经人AGM区基质细胞诱导后Sca-1+c-Kit+细胞占(13.12±1.30)%.NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤,经骨髓腔接种未见肿瘤形成.静脉移植组动物全部死亡,骨髓腔移植组生存率为55.6%,移植后21 d外周血象基本恢复,存活受鼠检测到供体来源Sry基因.提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力.