Human Nerve Growth Factor Receptor Research Articles

A customized retroviral vector confers marker gene expression in osteoclast lineage cells

Osteoclasts play a seminal role in many skeletal diseases and therefore are candidates for cell-based gene delivery systems to treat disorders of bone. As an initial step toward developing osteoclast-mediated gene delivery systems, we have made and analyzed a customized Molony-Murine leukemia virus (MMLV)-based retroviral vector containing elements of the osteoclast-specific tartrate-resistant acid phosphatase (TRAP) gene. RAW 264.7 cells were transduced with the customized vector (E3) and differentiated along macrophage or osteoclast lineages. E3 contained a truncated form of the human nerve growth factor receptor (NGFR) as a reporter gene. NGFR expression increased with RANK-ligand (RANK-L) treatment but not with macrophage (gamma-IFN/LPS treatment) differentiation. Enhanced NGFR expression peaked 48 h after RANK-L treatment. Electrophoretic mobility shift assays (EMSA) analysis of the TRAP gene regulatory elements in E3 identified a single 27 bp DNA probe, which specifically bound protein from RANK-L-treated cells. DNA sequence revealed AP-1 binding sites, and analysis with mutant probes implied that the sites were functional. EMSA supershift analysis identified Fos protein interacting with the 27 bp probe. In summary, insertion of sequence -962 to -868 from the TRAP gene into the U3 region of the MMLV LTR confers RANK-L induced retroviral gene expression via Fos family protein interaction at AP-1 sites.

Osteoclasts direct bystander killing of cancer cells in vitro

Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells ( P < 0.006) and GFP-4T1 cells ( P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.

Analysis of Distinct Tartrate-resistant Acid Phosphatase Promoter Regions in Transgenic Mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Co-Expression of the UPRT Gene with the Human Nerve Growth Factor Receptor and Cytosine Deaminase Fusion Gene Using a Single Retroviral Vector Augments Cytotoxicity of Transduced Human T-Cells Exposed to 5-Fluorocytosine.

Donor T lymphocytes engineered to express a ‘suicide gene' that facilitates negative selection following exposure to a non-toxic pro-drug provides an alternate strategy for the management of graft versus host disease occurring after allogeneic hematopoietic cell transplantation. The herpes simplex virus thymidine kinase (HSV-tk) gene remains the most widely studied suicide gene, but has limitations in this setting; these include restriction of ganciclovir (GCV) induced cytotoxicity to proliferating HSV tk+ cells, immunogenicity of HSV-tk, an inability to use acyclovir or GCV for viral therapy and acquired resistance to GCV. Cytosine deaminase (CD), an alternate suicide gene, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by exposure to a gene encoding uracil phosphoribosyl transferase (UPRT) which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate (5-FUMP). Using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor for positive selection linked by a [Gly4Ser]2 linker to Sacchromyces CD gene, we examined strategies to achieve optimal CD and UPRT activity on a single retroviral vector. Human T-cells co-expressing NG/CD and UPRT gene achieved through sequential transduction with two independent vectors (NG/CD + U) were compared to 3 construct strategies using a single vector. The vector NG/CD-i-U utilizes an internal ribosomal entry site to express both genes from a single bicistronic message, and this was compared, to 2 viruses using direct fusions of the NG/CD and UPRT genes with or without a [Gly4Ser]2 linker termed NG/CD-L-U and NG/CD-U respectively. Parameters tested included expression based on flow cytometry and UPRT Western analysis, CD and UPRT activity by enzymatic determinations, cytotoxicity and the induction of apoptosis in proliferating and quiescent human T-cells. UPRT was found to enhance cytotoxicity to 5-FC in all constructs compared to the NG/CD alone (LD 90, 38.7 micrograms/ml). Cytoxicity was comparable in the NG/CD+U (double transductant) and NG/CD-i-U with a documented LD 90 of 0.13 micrograms/ml of 5-FC; in addition, the LD 90 in 5-FU was 0.03 and 0.01 micrograms/ml for the doubly transduced and NG/CD-i-U transduced cells respectively. In contrast, cytoxicity was decreased using the tri-chimeric contructs (LD 90, 6.4 micrograms/ml, and 10.9 micrograms/ml 5-FC respectively for NG/CD-L-U and NG/CD-U). Based on enzymatic testing, a decrease in both UPRT and CD is consistent with these differences. Cytotoxicity occurs through apoptosis, with increased annexin-V binding following exposure to 5-FC. Additionally, we demonstrated the ability to induce cytotoxicity and apoptosis in quiescent cell populations. In summary, we have demonstrated that in association with NG/CD, sufficient UPRT activity can be achieved using a single retroviral vector resulting in markedly enhanced cytotoxicity to 5-FC. The 5-FC concentration necessary to eliminate virtually 100% of transduced human T-cells can easily be achieved in human serum. This approach may have advantages over the HSV-tk system in eradicating T cells in a clinical setting.

Spontaneous Memory CD4+ T Cells Preserve Graft-Versus-Leukemia without Causing Graft-Versus-Host Disease.

We have recently shown in a major histocompatibility complex (MHC) compatible, multiple minor histocompatibility antigen mismatched CD4-dependent murine model of allogeneic stem cell transplantation (alloSCT) that donor spontaneous effector memory (EM) CD4 cells depleted of regulatory (Treg) CD25+ cells (CD4+CD44+CD62L−CD25−) do not cause graft-vs.-host disease (GVHD), but engraft and mount recall responses to a model antigen (Anderson et al., JCI, 2003). Thus EM cells might allow immune reconstitution with less GVHD. However, as alloSCT is most frequently used to treat malignant diseases, it is important to determine whether spontaneous EM cells can mediate graft-vs.-leukemia (GVL). Chen et al (Blood, 2004) found that memory T cells from donors immunized to host antigens can mediate GVL in an MHC disparate model; however Tregs were not depleted and whether spontaneous memory T cells can mediate GVL, which would be a likely clinical scenario, was not addressed. Here, we report that donor CD25− EM CD4 cells do not cause GVHD in the MHCII-disparate B6bm12→B6 model, but mediate GVL against a murine model of chronic phase CML (mCP-CML) induced by retroviral insertion into bone marrow (BM) cells of the bcr-abl (p210) fusion cDNA, the defining genetic abnormality in human CML (Matte et al., Blood, 2004). mCP-CML is manifest by a high WBC count and splenomegaly dominated by maturing myeloid cells. The retroviral construct also expresses the human nerve growth factor receptor (NGFR) which allows the tracking of transduced cells by FACS. We first addressed GVHD. Lethally irradiated B6 mice were reconstituted with T cell depleted B6bm12 BM with no T cells or with 5x105 CD4+CD25−CD62L+CD44− naïve (N) or CD4+CD25−CD62L−CD44+ EM B6bm12 T cells. In 2 independent experiments EM cells induced neither clinical nor histologic GVHD. In contrast, N CD4 cells induced GVHD manifest by weight loss (P<0.007; EM vs. N), hunched posture and ruffled fur. GVHD was confirmed histologically in the liver, intestine and skin (P<0.0002 for liver and bowel, P=0.0553 for skin; EM vs. N). To determine whether EM CD4 cells mediate GVL, lethally irradiated B6 mice were reconstituted with p210-infected B6 BM, donor B6bm12 BM, with or without a source of donor T cells (2.5x105 or 5x105 N CD4 cells or 106 EM CD4 cells). 5/5 mice that did not receive donor T cells died from mCP-CML between days 18-21 post BMT. N CD4 recipients (n=5 for both 2.5 and 5x105groups) cleared blood NGFR+ cells by day 22, but suffered from GVHD as expected. Strikingly, EM CD4 cells also mediated GVL as measured by improved survival and a 10-fold reduction in NGFR+ WBCs as compared to mice that received no T cells. Only 1/6 recipients died of mCP-CML; 5/6 recipients were still alive with a low WBC count at day 28 (P<0.0001; BM vs. BM/EM), although some NGFR+ cells were still present. Importantly, none of the EM recipients developed GVHD. EM cells were 99% pure and it is unlikely that less that 104 contaminating N cells were responsible for the GVL we observed. Taken together, our prior published work and this new data suggest that donor EM cells can contribute to both immune reconstitution and GVL and thus selective infusion of EM cells may be a successful strategy in clinical alloSCT.

Generation of Novel, High Titre, Multi-Cistronic Retroviral Vectors Which Simultaneously Express Individual Proteins in Different Subcellular Locations of Hematopoietic Cells.

As gene therapy strategies become ever more sophisticated, there is a need for vectors which efficiently deliver and express more than one gene product to target cells e.g. to enable in vivo selection; to correct multiple genetic loci; to provide a suicide mechanism for safety purposes. Employing alternate codon usage, we have synthesised a novel version of the FMDV 2A self-processing moiety which encodes the same primary amino-acid sequence as the wild type protein (and which possesses equivalent cleavage activity to the wild type protein). By including this synthetic FMDV 2A sequence in retroviral vectors which also contain the wild type sequence, we are able to facilitate the efficient co-translational separation of 3 different proteins encoded by a single transcript. This vector configuration also permits the inclusion of an IRES sequence and thus expression of a fourth protein. Using this technology we have created two tetracistronic retroviral vectors which express eGFP; O6-methylguanine DNA-methyltransferase and truncated human nerve growth factor receptor courtesy of 2A mediated cleavage, and a mitochondrially targeted dsRED via an IRES. Stable amphotropic producer lines containing these vectors generated supernatant which contained a high titre of retroviral particles (>106 IU/ml) which was sufficient for the transduction of K562 human erythroleukemic cells. Analysis of transduced cells confirmed that all four proteins were expressed individually and segragated to their correct subcellular locations. Furthermore, limiting dilution assay revealed that proteins expressed courtesy of the 2A sequences were still generated at high levels, even when cells were infected with a low MOI of virus. We propose that this technology will allow the creation of more sophisticated vectors for use in gene therapy of hematopoietic and other cells.

Susceptibility of cell populations to transduction by retroviral vectors.

Retroviral transduction efficiency is related to the multiplicity of infection and the physiological state of the target cells. It is generally not known what proportion of a cell population is susceptible to transduction. We used coinfection with two retroviral vectors containing the marker genes for green fluorescent protein and the truncated human nerve growth factor receptor. In the CD34+ cell line TF-1 or human primary CD34+ hematopoietic progenitor cells, it was found that cells transduced with one vector had a better than random chance of transduction by the other vector. A probability model was developed to estimate target cell susceptibility; susceptibility was calculated as the product of the proportions of transgene-positive cells divided by the proportion of double-positive cells. By using this relationship, it was found that susceptibility was related to the target cell type and culture conditions but not the retroviral titer or the retroviral packaging envelope protein used in this study. Cotransduction with two vectors is a relatively simple procedure that provides a means to assess the maximum transduction level possible in a given cell population.

Genetic marking with the ΔLNGFR-gene for tracing goat cells in bone tissue engineering

The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (ΔLNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40–60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with ΔLNGFR is applicable to trace goat BMSC's in bone tissue engineering research.

Human nerve growth factor receptor and cytosine deaminase fusion genes.

Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.

Long-term high-level reconstitution of NADPH oxidase activity in murine X-linked chronic granulomatous disease using a bicistronic vector expressing gp91phox and a Delta LNGFR cell surface marker.

A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91(phox) gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91(phox) and a linked gene for Delta LNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91(phox) expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91(phox) and Delta LNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for Delta LNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for Delta LNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection for Delta LNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed.

CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy

The introduction of an inducible suicide gene has been proposed as a strategy to exploit the antitumor reactivity of donor T cells after allogeneic hematopoietic stem cell transplantation but permit control of graft-versus-host disease. However, there are several obstacles to this approach that may impair the ability of T cells to function and survive in vivo. These include the requirement for in vitro activation or long-term culture to introduce the transgene and obtain therapeutic cell numbers, the toxicity of drug selection to enrich transduced cells, and the immunogenicity of the transgene-encoded products. Here we have developed a transduction and selection strategy for generating large numbers of polyclonal T cells transduced with a retroviral vector encoding the human low-affinity nerve growth factor receptor (LNGFR) for selection and a Fas-based suicide construct (LV'VFas). Ligation of CD28 in conjunction with a T-cell receptor signal permitted efficient transduction, substantially promoted T-cell growth, and contributed to the generation of gene-modified T cells that retained clonal diversity, functional properties, and a homing receptor profile similar to untransduced peripheral blood lymphocytes. Microbeads conjugated directly to antibody specific to LNGFR significantly improved the immunomagnetic selection of LV'VFas-modified T cells and assisted in scaling of the selection procedure to therapeutic cell numbers. Thus, these studies identified a strategy that requires only a brief ex vivo culture and does not use drug selection to obtain large numbers of functional gene-modified polyclonal T cells that can be used for adoptive immunotherapy.

Inducible expression of the deltaNGFr/F12Nef fusion protein as a new tool for anti-human immunodeficiency virus type 1 gene therapy.

Expression of the human immunodeficiency virus type 1 (HIV-1) Nef triple mutant F12Nef strongly inhibits HIV-1 replication. We exploited such a unique feature in a novel anti-HIV-1 gene therapy design by constructing an HIV-1 Tat-defective lentivirus vector expressing the product of fusion between the low-affinity human nerve growth factor receptor truncated in its intracytoplasmic domain (deltaNGFr, NH(2) moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (deltaNGFr) and the anti-HIV-1 effector are comprised in the same fusion protein, the expression of which is targetable by HIV-1 infection. Such a vector was proved to transduce human cells efficiently and, on HIV-1 infection, it expressed high levels of the fusion protein. In addition, strong antiviral activity of the deltaNGFr/F12Nef-expressing vector was demonstrated in cell lines as well as in primary cell cultures challenged with T- or M-tropic HIV-1 isolates. Thus, the HIV-1-targetable expression of the deltaNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.

Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor.

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.

Single‐step, multiple retroviral transduction of human T cells

Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells. Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T-cell line (MT-2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double- and triple-transduced cells were isolated by fluorescence cell sorting. Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), beta-galactosidase, and truncated versions of human nerve growth factor receptor (Delta NGFR) and human growth hormone receptor (Delta GHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti-CD3. The transient production of viral particles encoding EGFP, Delta NGFR, and Delta GHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells. This study describes new experimental conditions for efficient single-step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches.

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

Objectives: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. Methods: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38–CD34++lineage– (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (ΔNGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 µg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10–21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. Results: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.

Remyelination of Demyelinated CNS Axons by Transplanted Human Schwann Cells: The Deleterious Effect of Contaminating Fibroblasts.

Areas of demyelination can be remyelinated by transplanting myelin-forming cells. Schwann cells are the naturally remyelinating cells of the peripheral nervous system and have a number of features that may make them attractive for cell implantation therapies in multiple sclerosis, in which spontaneous but limited Schwann cell remyelination has been well documented. Schwann cells can be expanded in vitro, potentially affording the opportunity of autologous transplantation; and they might also be spared the demyelinating process in multiple sclerosis. Although rat, cat, and monkey Schwann cells have been transplanted into rodent demyelinating lesions, the behavior of transplanted human Schwann cells has not been evaluated. In this study we examined the consequences of injecting human Schwann cells into areas of acute demyelination in the spinal cords of adult rats. We found that transplants containing significant fibroblast contamination resulted in deposition of large amounts of collagen and extensive axonal degeneration. However, Schwann cell preparations that had been purified by positive immunoselection using antibodies to human low-affinity nerve growth factor receptor containing less than 10% fibroblasts were associated with remyelination. This result indicates that fibroblast contamination of human Schwann cells represents a greater problem than would have been appreciated from previous studies.

Systematic analysis of clinically applicable conditions leading to a high efficiency of transduction and transgene expression in human T cells.

The transduction of human peripheral blood T cells with retroviral vectors constitutes an attractive approach for the correction of a number of genetic diseases. In this study we have conducted a systematic analysis of the relevance of a large number of parameters currently considered to affect the transduction of, and transgene expression in, human T cells. Retroviral vectors encoding the human nerve growth factor receptor (NGFR) were used for transducing human T cells from normal volunteers. The proportion of T cells that expressed the marker transgene was determined by flow cytometry using anti-NGFR antibodies. Spinoculation and static fibronectin (FN)-assisted infections improved to a similar extent the transduction efficiency of PHA/IL-2 stimulated T cells, when compared with samples subjected to standard static infections. When immobilized anti-CD3 (anti-CD3i) or anti-CD3i/28i-stimulated T cells were considered, static infections in FN-coated plates were reproducibly more efficient than spinoculation infections performed on FN-uncoated plates. Under optimized manipulation conditions (three infection cycles of anti-CD3i/28i-stimulated T cells in FN-coated plates) the total number of NGFR+ T cells harvested after 7 days of incubation represented, on average, twice the total number of T cells seeded at Day 0, and up to 95% of the human T cells efficiently expressed the marker transgene. Similar results were obtained regardless of whether samples were manipulated in medium supplemented with fetal bovine serum or with heat-inactivated autologous serum. Our study offers new experimental conditions for the transduction of human T cells, with obvious implications for the development of gene therapy protocols.

Systematic analysis of clinically applicable conditions leading to a high efficiency of transduction and transgene expression in human T cells

Background The transduction of human peripheral blood T cells with retroviral vectors constitutes an attractive approach for the correction of a number of genetic diseases. In this study we have conducted a systematic analysis of the relevance of a large number of parameters currently considered to affect the transduction of, and transgene expression in, human T cells. Methods Retroviral vectors encoding the human nerve growth factor receptor (NGFR) were used for transducing human T cells from normal volunteers. The proportion of T cells that expressed the marker transgene was determined by flow cytometry using anti-NGFR antibodies. Results Spinoculation and static fibronectin (FN)-assisted infections improved to a similar extent the transduction efficiency of PHA/IL-2 stimulated T cells, when compared with samples subjected to standard static infections. When immobilized anti-CD3 (anti-CD3i) or anti-CD3i/28i-stimulated T cells were considered, static infections in FN-coated plates were reproducibly more efficient than spinoculation infections performed on FN-uncoated plates. Under optimized manipulation conditions (three infection cycles of anti-CD3i/28i-stimulated T cells in FN-coated plates) the total number of NGFR+ T cells harvested after 7 days of incubation represented, on average, twice the total number of T cells seeded at Day 0, and up to 95% of the human T cells efficiently expressed the marker transgene. Similar results were obtained regardless of whether samples were manipulated in medium supplemented with fetal bovine serum or with heat-inactivated autologous serum. Conclusions Our study offers new experimental conditions for the transduction of human T cells, with obvious implications for the development of gene therapy protocols. Copyright © 2000 John Wiley & Sons, Ltd.

Ex-vivo expansion after gene marking of hematopoietic stem cells from cord blood under serum free conditions

Combined transplantation of expanded and non expanded cord blood (CB) hematopoietic stem cells has recently been proposed for transplantation of adult patients. Retroviral gene marking is used to investigate the role of expanded cells in hematopoietic reconstitution. We used a Moloney murine leukemia virus retroviral vector containing the human nerve growth factor receptor (NGFR) cDNA as a marker to transduce CB CD34+ cells purified from 200,000 nucleated cells. Prestimulation with interleukin (IL)-6, IL-11, Flt-3 ligand and thrombopoietin in StemPro serum-free medium was performed for 20 hours for samples A–C and for 68 hours for sample D. Infection was carried out in serum-free medium on retronectine coated plates. Then, samples C and D were expanded for 2 weeks with the above cytokines and medium. Results are shown in table.Between 3.6 and 5% of CD34+ cells show the NGFR marker after 20–68 hours prestimulation and 2 week expansion.

Amphotropic and VSV-G-pseudotyped retroviral vectors transduce human hematopoietic progenitor cells with similar efficiency.

One restriction of retroviral gene transfer into hematopoietic stem cells is the low level of amphotropic virus receptor. In the present study, we examined whether retroviral vectors pseudotyped with the G-protein of vesicular stomatitis virus (VSV) can overcome this restriction. Human progenitor cells purified by magnetic beads and cell sorting were transduced with an amphotropic or VSV-G-pseudotyped retroviral vector containing the truncated human nerve growth factor receptor as a marker gene. Cells were prestimulated with flt-3 ligand, stem cell factor, and interleukin-3 and transduced on fibronectin. Marker gene expression was analyzed by flow cytometry. Transduction efficiencies of amphotropic and VSV-G-pseudotyped virus for CD34+ cells did not differ significantly. Gene transfer into CD34+CD38- cells, which are enriched in more immature progenitors, was not restricted and transfer efficiencies for this subset were also similar for both pseudotypes. The addition of fibronectin improved gene transfer with the amphotropic vector considerably (5- to 19.3-fold, mean 12.6), while the effect on the VSV-G-pseudotype was far less pronounced (1- to 3.9-fold, mean 2.1, P = 0.04). In conclusion, high levels of gene transfer to human hematopoietic progenitors were achieved with an optimized transduction protocol, and transduction efficiencies could not be improved further by the use of VSV-G-pseudotypes.