Gene transfer using retroviral transduction offers the advantage of long‐term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential.
Methods
Immature DCs were generated from murine bone marrow (BM) using either GM‐CSF alone or GM‐CSF plus IL‐4. The cells were transduced directly with retroviral supernatants or by co‐culture with the GP + E‐86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed.
Results
Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL‐4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM‐CSF plus IL‐4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down‐regulated transgene expression in the co‐cultured packaging cell line in a promoter‐dependent manner.
Gene therapy has recently been advanced by the development of HIV‐based vectors that are able to transduce some non‐dividing cells. The manipulation of most non‐dividing cells remains, however, scarcely efficient. One of the biological mechanisms postulated to prevent powerful transduction of quiescent cells by lentiviral vectors is the paucity of deoxynucleotides (dNTPs). In this study, a novel delivery strategy is developed to improve significantly the efficiency of HIV‐based vectors in transducing non‐dividing cells. This approach is based on increasing the intracellular availability of dNTPs by incubating target cells with the dNTP precursors, deoxynucleosides (dNSs).
Methods
Mature human monocyte‐derived macrophages (14–21 days old) were transduced at a low multiplicity of infection (MOI) of HIV vectors carrying a reporter gene. dNSs were added to the medium during transduction (5 mM dNS) and immediately before post‐transduction culture (2.5 mM dNS). Macrophages were harvested 2–7 days after transduction and assayed for transgene expression by cytofluorimetry.
Results
The addition of dNS to the medium significantly enhanced the efficiency of transduction of human macrophages by HIV‐based vectors. The percentage of cells expressing the transgene rose up to 50% in the presence of dNS, increasing the basal transduction levels up to 35‐fold (average=10.8‐fold). Furthermore, treatment with dNTP precursors compensated for the wide inter‐donor variability, allowing the highest enhancement effects in donors with the lowest basal transduction efficiencies.
The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34+/CD38low and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes.
Methods
CB cells were transduced on Retronectin using an MSCV‐based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13‐MGIN (GALV), 293GPG‐MGIN (VSV‐G) or AM12‐MGIN (amphotropic).
Results
Sorted CD34+/CD38low cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP‐positive cells was 61.8±6.6% (PG13‐MGIN), 26.9±3.5% (293GPG‐MGIN), and 39.3±4.8% (AM12‐MGIN). For transplantation experiments, CD34+ cells were pre‐stimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5×105 cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum‐free medium and transduction of CD34+ cells using VSV‐G‐pseudotyped vectors under serum‐free conditions was very inefficient. In contrast, transduction with PG13‐MGIN under serum‐free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3±6.6%), and efficient gene transfer to SRCs (46.2±4.8%).
Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron‐like cell lines over‐expressing the mitochondrial GSH transporter, the 2‐oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up‐regulation of the anti‐apoptotic protein, B cell lymphoma 2 (Bcl‐2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl‐2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co‐immunoprecipitated with Bcl‐2 from rat brain lysates in a GSH‐dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl‐2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels.
CD22 is a cell surface glycoprotein restricted to normal and malignant B‐cells and is the target of several anti‐CD22 antibody‐based cancer therapies. For therapeutic antibody‐payload conjugates, it is important to understand the subcellular trafficking of anti‐CD22 antibodies to optimize antibody and/or linker–drug properties to maximize antitumor efficacy. It is agreed that anti‐CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell‐type dependent. No studies examined anti‐CD22 trafficking to either pathway in B‐cells over time by dual immunofluorescence microscopy, likely partly because multiple samples of suspension cells are tedious to stain. We overcame this by using DropArray?, a novel wall‐less 96‐well plate technology allowing rapid simultaneous staining of suspension or adherent cells in small (10–20 μL) volumes. We examined the time‐course of trafficking of five different anti‐CD22 antibodies in eight B‐cell lines representing four B‐cell cancer types and show that in all cases antibodies internalize within 5 min and recycle, with only small amounts eventually trafficking to lysosomes. CD22 also localizes to recycling endosomes at steady state in the absence of antibody. Our data may help explain the differential efficacies of anti‐CD22 antibodies conjugated to different therapeutic payloads. 相似文献
A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA−) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA− severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.Retroviral vectors have been the most common gene transfer vehicles in clinical gene therapy trials (15). These vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells (20). The first generation of retroviral vectors have been useful in demonstrating the feasibility of gene therapy approaches, but vectors capable of higher levels of gene transfer and transgene expression would be beneficial. For example, gene transfer levels achieved by first-generation retroviral vectors in large mammals (28) and in human gene therapy trials (7, 13) have been disappointing. There are at least two avenues for improving retroviral vectors. First, molecular changes can be made in the retroviral vector sequence. Second, different packaging cell lines could be tested to modify the host range, increase transduction in a given cell type, and/or render the virions resistant to inactivation by human complement.A clinically useful model for improving retroviral vector design is the vector LASN packaged in the amphotropic line PA317. PA317/LASN was the first therapeutic vector used in a gene therapy clinical trial (1). This vector has yielded gene transfer levels of generally less than 10% in peripheral blood T cells of adenosine deaminase-deficient (ADA−) severe combined immunodeficiency (SCID) patients. Two possibilities to improve this vector include eliminating the dominant selectable marker gene and changing the long terminal repeat (LTR) promoter to optimize expression. LASN, like many of the retroviral vectors used in clinical trials to date, contains two genes: the therapeutic gene (the ADA gene) and a dominant selectable marker gene (the bacterial neomycin phosphotransferase II gene; neo). Dominant selectable marker genes have historically been included to facilitate the generation, isolation, and titration of retroviral producer cell clones and to permit the evaluation and selection of successfully targeted cells. neo is the most commonly used selectable marker gene, although other genes have been used, including a mutant dihydrofolate reductase gene (dhfr) (19), the multidrug resistance gene (mdr) (10), and genes for cell surface markers such as cd24 (24) and the human nerve growth factor receptor (2). Vectors carrying dominant selectable marker genes, particularly those of nonhuman origin, have two theoretical disadvantages. First, careful analysis of some patients has revealed an immune response directed against the dominant selectable marker protein expressed from the retroviral integrant (20a, 25). Second, the more complex retroviral genomes required to express two separate genes may result in lower titers or suboptimal expression of the therapeutic gene product due to promoter interference (8, 29). On the other hand, cloning and determining the titers of useful retroviral vectors without selectable markers have been laborious. Using a recently developed rapid-screening procedure, we have been able to identify a number of “simple” ADA retroviral vectors which lack dominant selectable markers (23).Different packaging cell lines may also improve gene transfer of retroviral vectors into specific target cells. Retroviral vectors are limited by the host range specified by the envelope protein on the surface of the retrovirus. Most gene therapy trials have used retroviruses with a murine amphotropic (4070A) host range. However, packaging cell lines with the gibbon ape leukemia virus (GALV) envelope (PG13 cells) (18) and the cat endogenous virus RD114 envelope (FLYRD18 cells) (5) have become available; these may improve transduction frequencies into various target cell populations. For example, there is evidence that GALV-pseudotyped retroviral vectors may facilitate gene transfer into human peripheral blood T cells with greater efficiency than vectors with an amphotropic envelope (3). Packaging cell lines derived from murine cells have the additional disadvantage that they produce retroviruses which are inactivated by complement in human sera. Packaging cell lines of human origin (FLYA13 and FLYRD18) (5) produce vectors which are complement resistant. Testing both new simple retroviral vector designs and new packaging cells may therefore improve retrovirus-mediated gene transfer.We report the construction and characterization of three simplified ADA vectors by using either the Moloney murine leukemia virus (MLV) LTR, the myeloproliferative sarcoma virus (MPSV) LTR, or the SL3-3 LTR. We tested these vectors to determine which LTR provided the highest level of ADA expression in our target cells of interest: human ADA− lymphohematopoietic cells. The ADA retroviral vector with the highest level of transduction/expression was then evaluated in different packaging cell lines including PG13, FLYA13, and FLYRD18. 相似文献
Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. 相似文献
Recombinant retroviral vectors are still the most common gene delivery vehicles for gene therapy purposes, especially for construction of genetically modified tumor vaccines (GMTV). However, these vehicles are characterized by relatively low titre and in the case of many tumor cell lines, low transduction efficiency. We constructed bicistronic retroviral vector pseudotypes of amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV), encoding enhanced green fluorescent protein (EGFP) as a rapid and easily detectable reporter gene. Transduction of five different human melanoma and four renal carcinoma cell lines by these two virus pseudotypes revealed differences in transduction efficiency, which wase markedly lower for the renal carcinoma cell lines. Stimulation of retroviral receptor expression (PiT1 and PiT2) by phosphate depletion induced a limited increase of receptor mRNA levels, but did not improve the gene transfer efficiency. In contrast, simultaneous transduction with both vector pseudotypes markedly increased the transduction efficiency, compared to GaLV or A-MuLV alone. The same effect could be achieved by several repeated exposures of target cells to fresh vector preparation. Overexpression of GaLV receptor (PiT1) in target cells significantly increased the transduction rate and enabled retrovirus mediated gene transfer into the cells which normally are not transducible by GaLV pseudotypes. We demonstrated that, using different transduction strategies, the relatively inefficient, widely used retroviral vector systems could be significantly improved. 相似文献
Lentiviral vectors allow gene transfer into non‐dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing.
Methods
To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon‐optimized gag‐pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV‐G) under the control of an ponasterone‐inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized.
Results
The RT activity and vector titers of cell clones stably transfected with the inducible gag‐pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone‐inducible VSV‐G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone‐induced producer clones vector titers of more than 1×105 transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production.
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.
Methods
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.
Results
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), β‐galactosidase, and truncated versions of human nerve growth factor receptor (ΔNGFR) and human growth hormone receptor (ΔGHR). 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, ΔNGFR, and ΔGHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells.
In a strategy termed “Protease Targeting”, retroviral vectors carrying an EGF infectivity‐blocking domain fused to the N‐terminus of the envelope SU via a MMP (matrix metalloproteinase)‐cleavable linker were successfully used to target gene delivery to EGF receptor‐(EGF‐R‐)positive tumour cells over‐expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune‐stimulatory properties of GALV via local, MMP‐mediated release human granulocyte‐macrophage colony stimulating factor (GM‐CSF).
Methods
We generated GALV envelope expression constructs displaying EGF or GM‐CSF blocking ligands at the N‐terminus of GALV envelope SU via a non‐cleavable, Factor Xa protease or MMP‐cleavable linker and investigated their cytotoxicity on MMP‐positive and negative cell lines.
Results
The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non‐cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease‐cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the ‘blocking domain’ were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.
Adhesion receptors expressed on the surfaces of tumor-activated endothelial cells provide an advantageous locus for targeting gene therapy vectors to angiogenic tissues and/or tumor vasculature. In this study, we engineered a series of Asn-Gly-Arg (NGR)-containing congeners of the presumptive cell binding motif contained within the ninth type III repeat of fibronectin and displayed these tumor vasculature targeting motifs (TVTMs) within the context of Moloney murine leukemia envelope "escort" proteins. Comparative studies of envelope incorporation into viral particles and evaluation of the cell binding properties of the targeted vectors revealed critical structural features, thus identifying a subset of optimal TVTMs. Utilizing a modified ELISA to evaluate viral binding to target cells, we observed a significant down-regulation of TVTM-virion binding to human endothelial cells following sustained (48-h) exposure to VEGF. Normalized for equivalent titers (10(6) CFU/ml), as assayed on NIH 3T3 cells, vectors displaying TVTM escort proteins significantly enhanced the transduction efficiency from 12.2 to 37.4% in human KSY-1 endothelial cell cultures (P < 0.001) and from 0.4 to 4.1% in human umbilical vein endothelial cell (HUVEC) cultures (P < 0.001). In summary, these studies utilized an engineering approach to identify a subset of TVTMs that are stably incorporated as envelope "escort" proteins into retroviral vectors and that, by functioning to improve the binding efficiency and transduction of both HUVEC and KSY1 endothelial cells, may have therapeutic potential for targeting gene delivery to the tumor-associated vasculature. 相似文献
The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited.
Principal Findings
We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments.
Conclusions/Significance
Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays. 相似文献
Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR‐192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR‐192/215 family in the pathogenesis of HSCR. Quantitative real‐time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual‐luciferase reporter assays, and the functional effects of miR‐192/215 on human 293T and SH‐SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR‐192/215 was significantly down‐regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH‐SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR‐192/215 by dual‐luciferase reporter gene assay and its expression was inversely correlated with that of miR‐192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR‐192/215 inhibitor. The down‐regulation of miR‐192/215 may contribute to HSCR development by targeting NID1.
An efficient method has been developed to improve preparation of phage particles by ammonium sulfate precipitation and to yield high quality DNA. The method, that has been used to screen plant DNA libraries constructed in vectors, is inexpensive, does not require purification of phage particles, and can be used from either plate stocks or liquid lysates. Up to 1100 g DNA was produced from 5 ml lysate obtained from agar plates. 相似文献
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