ABSTRACT
We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.
Subject(s)
Gene Expression Regulation , Gene Transfer Techniques , Genetic Vectors/genetics , Muscles/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Adenoviridae/genetics , Cell Differentiation , Cell Fusion , Cells, Cultured , Desmin/genetics , Enhancer Elements, Genetic/genetics , Fibroblasts , Flow Cytometry , Genes, Reporter/genetics , Histocytochemistry , Humans , Infant , Male , Muscles/cytology , Organ Specificity , Promoter Regions, Genetic/genetics , Transduction, Genetic/geneticsABSTRACT
Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation. Gene Therapy (2000) 7, 1193-1199.
Subject(s)
Antigens, CD/genetics , Hematopoietic Stem Cells/cytology , Luminescent Proteins/genetics , Membrane Glycoproteins , Retroviridae/genetics , Animals , Antigens, CD/metabolism , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , CD24 Antigen , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Mice , Transduction, Genetic/geneticsABSTRACT
Recombinant adenovirus vectors have provided a major advance in gene delivery systems for post-mitotic neurons. However, the use of these first generation vectors has been limited due to the onset of virally mediated effects on cellular function and viability. In the present study we have used primary cultures of cerebellar granule neurons to examine the efficacy and cytotoxic effects of a helper-dependent adenovirus vector (hdAd) in comparison with a first generation vector. Our results demonstrate that the hdAd system provides equally efficient infectivity with significantly reduced toxicity in comparison to first generation vectors. Neurons transduced with a high titre of a first generation vector exhibited a time-dependent shut down in global protein synthesis and impaired physiological function as demonstrated by a loss of glutamate receptor responsiveness. This was followed by an increase in the fraction of TUNEL-positive cells and a loss of neuronal survival. In contrast, hdAds could be used at titres that transduce >85% of neurons with little cytotoxic effect: cellular glutamate receptor responses and rates of protein synthesis were indistinguishable from uninfected controls. Furthermore, cell viability was not significantly affected for at least 7 days after infection. At excessive viral titres, however, infection with hdAd did cause moderate but significant changes in cell function and viability in primary neuronal cultures. Thus, while these vectors are remarkably improved over first generation vectors, these also have limitations with respect to viral effects on cellular function and viability. Gene Therapy (2000) 7, 1200-1209.
Subject(s)
Adenoviridae/genetics , Genetic Vectors/therapeutic use , Neurons , Animals , Cell Survival , Cells, Cultured , Genetic Vectors/adverse effects , Mice , N-Methylaspartate/metabolism , Neurons/metabolism , Neurons/virology , Protein Biosynthesis , Transduction, Genetic/geneticsABSTRACT
Glycoconjugates bearing the epitope 3-fucosyl-N-acetyllactosamine (CD15) are believed to be involved in cell-cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells. In dissociated cerebellar cell cultures, few glial cells normally express CD15. However, transduction of these cells with an adenoviral vector (AdGFPCMVFucT) that expressed both green fluorescent protein (GFP) and FLAG-tagged rat alpha 1, 3-fucosyltransferase IV (rFuc-TIV) resulted in high CD15 expression on the surface of all transduced glial cells. Likewise, infection of cerebellar slice cultures caused the appearance of CD15-positive transduced cells of glial cell morphology in the internal granule cell layer. Thus, enhancement of Fuc-T activity caused robust CD15 expression in cerebellar glial cells that normally show little expression of CD15, suggesting a role for Fuc-T levels in regulating CD15 expression in this cell type. The manipulation of levels of glycosyltransferases using adenoviral vectors may prove a useful tool to investigate questions of glycoconjugate regulation in glial cells in the developing rodent cerebellum.
Subject(s)
Adenoviridae/genetics , Astrocytes/metabolism , Cerebellum/metabolism , Fucosyltransferases/genetics , Genetic Vectors/pharmacology , Lewis X Antigen/genetics , Transduction, Genetic/genetics , Age Factors , Animals , Animals, Newborn , Astrocytes/cytology , Astrocytes/immunology , Cells, Cultured , Cerebellum/cytology , Cerebellum/growth & development , Chlorocebus aethiops , Gene Expression Regulation, Developmental/physiology , Genetic Vectors/physiology , Lewis X Antigen/immunology , Lewis X Antigen/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Temperate phage PS166 infection of Vibrio eltor MAK757 resulted in complete changes in all biotype-specific determinants. About 10% of the PS166 lysogens of MAK757 lost their eltor-specific determinants, namely, the ability to produce soluble hemolysin, cell-associated hemagglutinin for chicken erythrocytes, and resistance to polymyxin B, as well as resistance to Mukherjee's group IV phage and sensitivity to eltor phage e4. These lysogens were found to have acquired the properties of classical strains, most significantly becoming sensitive to group IV phage but resistant to eltor-specific e4. The remainder of these lysogens, however, retained their parental biotype and serotype but acquired auxotrophy for glycine and histidine. The differential behavior of the two types of lysogen was due to the integration of the phage PS166 genome at different locations in the host chromosome. A 800-bp BglII fragment was found to contain the attP site. Phage PS166 has a polyhedral head (95 nm in diameter) and a contractile tail (98 nm in length). The phage chromosome is a linear double-stranded DNA of 110 kb and a G + C content of 58.7%.
Subject(s)
Bacteriophages/genetics , Bacteriophages/physiology , Lysogeny/genetics , Vibrio cholerae/classification , Vibrio/classification , Vibrio/virology , Absorption , Animals , Attachment Sites, Microbiological/genetics , Bacterial Outer Membrane Proteins/analysis , Bacteriophages/chemistry , Bacteriophages/ultrastructure , Chickens , Chromosomes, Bacterial/genetics , DNA, Viral/genetics , Drug Resistance, Microbial , Erythrocytes/microbiology , Glycine/metabolism , Hemagglutination Tests , Hemagglutinins/biosynthesis , Hemolysin Proteins/metabolism , Histidine/metabolism , Microscopy, Electron , Phenotype , Polymyxin B/pharmacology , Serotyping , Transduction, Genetic/genetics , Vibrio/genetics , Vibrio/metabolism , Vibrio cholerae/genetics , Vibrio cholerae/virology , Viral Proteins/analysisABSTRACT
Gene-transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells. The HIV-1 proteins Matrix, Vpr and Integrase have been implicated in the nuclear import of the viral genome in non-dividing cells. Here we show that a sequence within pol is also required in cis. It contains structural elements previously associated with the progress of reverse transcription in target cells. We restored these elements in cis within late-generation lentiviral vectors. The new vector transduced to a much higher efficiency several types of human primary cells, when both growing and growth-arrested, including haematopoietic stem cells assayed by long-term repopulation of NOD/SCID mice. On in vivo administration into SCID mice, the vector induced higher plasma levels of human clotting factor IX (F.IX) than non-modified vector. Our results indicate that nuclear translocation of the genome is a rate-limiting step in lentiviral infection of both dividing and non-dividing cells, and that it depends on protein and nucleic acid sequence determinants. Full rescue of this step in lentivirus-based vectors improves performance for gene-therapy applications.
Subject(s)
Cell Nucleus/genetics , Gene Products, pol/genetics , Gene Transfer Techniques , Genetic Vectors/genetics , HIV-1/genetics , Transduction, Genetic/genetics , Animals , Base Sequence , Cell Division , Cell Nucleus/metabolism , Cell Nucleus/virology , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Factor IX/analysis , Factor IX/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Products, pol/physiology , Genes, Viral/genetics , Genes, Viral/physiology , HIV-1/physiology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Lymphocytes/cytology , Lymphocytes/metabolism , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, SCID , Molecular Sequence Data , RNA, Viral/genetics , RNA, Viral/metabolism , Virus IntegrationABSTRACT
Retroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer.
Subject(s)
Gene Expression Regulation, Viral/genetics , Genetic Vectors/genetics , Leukemia Virus, Murine/genetics , Promoter Regions, Genetic/genetics , Terminal Repeat Sequences/genetics , Animals , Base Sequence , Cell Line , Doxorubicin/pharmacology , Enhancer Elements, Genetic/genetics , Flow Cytometry , Gene Expression Regulation, Viral/drug effects , Gene Silencing/drug effects , Genes, Reporter/genetics , Green Fluorescent Proteins , Humans , Kinetics , Leukemia Virus, Murine/physiology , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Molecular Sequence Data , Mutagenesis, Insertional/genetics , Response Elements/genetics , Tetracycline/pharmacology , Transduction, Genetic/genetics , Tumor Cells, Cultured , Virus AssemblyABSTRACT
Intravenous administration of an adenovirus human heme oxygenase (HO)-1 gene construct to rats resulted in functional expression of human HO-1 in brain, heart, lung, liver, and kidney. Because accurate assessment of human HO-1 mRNA in various tissues by Northern analysis is not sufficiently sensitive, we developed a method for quantifying human HO-1 mRNA copies with quantitative reverse transcription- polymerase chain reaction techniques; this allowed us to use the same primers for both the sample and internal standard. Administration of the adenovirus human HO-1 gene resulted in the detection of human HO-1 mRNA in various tissues with the highest levels seen in the kidney followed, in order, by lung > liver > brain > heart. Human HO-1 was detectable for up to 4 weeks in all tissues studied. Administration of adenovirus human HO-1 resulted in maximal increase of HO activity after 1 to 2 weeks in rats. The increase in HO activity due to gene transfer also was associated with a parallel decrease (approximately 25%) in cytochrome P-450 (CYP) content and in CYP-dependent arachidonic acid metabolism. In addition, we investigated the possibility that the human HO-1 gene altered the expression of the endogenous rat enzyme after administration of cobalt chloride s.c. Cobalt chloride administration resulted in increased HO activity in all tissues examined in rats transduced with the human HO-1 gene to the same degree as in nontransduced rats. The metal was a more potent inducer of renal HO activity than was the adenoviral-mediated human HO-1 vector. The increase in HO activity after adenoviral-mediated human HO-1 transfer was associated with a decrease in microsomal heme-CYP and CYP activity. The increase in HO-1 activity after adenovirus-mediated human HO-1 gene transfer may prove useful as a means of selectively increasing enzyme activity in a specific organ and regulating homeostasis by modulation of vasoactive molecules such as carbon monoxide and bilirubin and, in addition, providing a means of delivering the human HO-1 gene for experimental purposes.
Subject(s)
Adenoviridae/genetics , Cytochrome P-450 Enzyme System/biosynthesis , Gene Transfer Techniques , Heme Oxygenase (Decyclizing)/genetics , Heme/physiology , Kidney/enzymology , Oxygenases/biosynthesis , Animals , Cobalt/pharmacology , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Genetic Vectors , Humans , Liver/enzymology , Male , Oxygenases/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic/geneticsABSTRACT
OBJECT: The P19 embryonal carcinoma-derived cell line consists of undifferentiated multipotential cells, which irreversibly differentiate into mature neurons after exposure to retinoic acid (RA). In the present study, the authors genetically engineered P19 cells to produce glial cell line-derived neurotrophic factor (GDNF), and grafted the cells in a rat model that had been rendered parkinsonian. METHODS: Undifferentiated P19 cells were grown in vitro and transduced with GDNF complementary DNA. The level of GDNF released from the transduced cells was measured using an enzyme-linked immunosorbent assay, and its neurotrophic activities were assessed by testing the effects on rat embryonic dopamine (DA) neurons in culture. After having been exposed to RA for 48 hours and allowed to differentiate into postmitotic neurons, the GDNF gene-transduced cells were implanted into the midbrain of immunosuppressed rats. A unilateral nigrostriatal lesion was then induced by intrastriatal infusions of 6-hydroxydopamine. Immunohistochemical analyses performed 4 weeks postgrafting revealed that the GDNF-producing cells expressed several neuronal markers without evidence of overgrowth. The grafts expressed GDNF protein and prevented the death of nigral DA neurons. Furthermore, the GDNF-producing cells implanted 4 weeks after nigrostriatal lesions restored the expression of tyrosine hydroxylase in injured DA neurons and induced their dendritic sprouting. CONCLUSIONS: The results indicate that the P19 cell line transduced with the GDNF gene can stably secrete functional levels of GDNF, even after being converted to postmitotic neurons. Because it is has been established that GDNF exerts trophic effects on DA neurons, the means currently used to deliver GDNF into the brain could be a viable strategy to prevent the death of nigral DA neurons in cases of Parkinson's disease.
Subject(s)
Carcinoma, Embryonal/pathology , Dopamine/metabolism , Genetic Engineering , Nerve Growth Factors/metabolism , Neuroglia/metabolism , Neurons/pathology , Parkinson Disease/surgery , Substantia Nigra/pathology , Analysis of Variance , Animals , Carcinoma, Embryonal/genetics , Cell Death , Cell Differentiation/drug effects , Cell Survival , Cells, Cultured , Dendrites/ultrastructure , Disease Models, Animal , Follow-Up Studies , Gene Expression Regulation, Enzymologic , Male , Nerve Growth Factors/genetics , Neurons/transplantation , Rats , Rats, Sprague-Dawley , Regeneration , Stem Cells/drug effects , Stem Cells/pathology , Transduction, Genetic/genetics , Tretinoin/pharmacology , Tumor Cells, Cultured , Tyrosine 3-Monooxygenase/geneticsABSTRACT
The maturation of retrovirus particles involves proteolytic cleavage of the envelope glycoprotein transmembrane component, resulting in conversion of the virus particle to a fusogenic or infectious state. Susceptible murine cells exposed to virus-containing supernatants from ecotropic retroviral helper cells occasionally fused to neighboring cells, resulting in syncytia (giant cells with multiple nuclei). Polycationic molecules dramatically enhanced the effect, leading to widespread cell death. The degree of cell fusion was dependent upon the retroviral envelope subtype (ecotropic-->amphotropic, gibbon ape leukemia virus was negative) as well as on the polycationic reagent used (G9 dendrimer-->Lipofectamine-->polybrene). Cell fusion effects were not mediated by the retroviral vector backbone, because virus-containing supernatants from helper cells (without vector) and vector producer cells had a similar effect. Human target cells were not fused by any type of murine retrovirus; in addition, amphotropic virus from human helper cells was not fusogenic toward murine cells. Thus, fusogenic effects were important during the propagation of vectors using murine helper cells but were not a significant factor during the transduction of human cells.
Subject(s)
Cations/pharmacology , Retroviridae/pathogenicity , 3T3 Cells , Animals , Blood Proteins/pharmacology , Cell Fusion , Cells, Cultured , Cross Infection/physiopathology , Dose-Response Relationship, Drug , Genetic Therapy/methods , Giant Cells/virology , Humans , Hydrogen-Ion Concentration , Mice , Retroviridae/genetics , Transduction, Genetic/genetics , Virion/drug effects , Virion/pathogenicity , Virus AssemblyABSTRACT
Metachromatic leukodystrophy (MLD) is an autosomal recessive, inherited, lysosomal storage disease caused by a deficiency in arylsulfatase A (ASA). This disease is characterized by progressive demyelination leading to severe neurological symptoms. Allogenic bone marrow transplantation at an early stage of clinical course is only effective treatment currently available. Accordingly the corrective transfer of the ASA gene into hematopoietic stem cells is thought to be an important option for curative treatment for MLD. We have recently developed a selectable vector system based on ex vivo sorting of transduced cells (Migita et al. 1995). In this study, we applied this selectable system for development of MLD gene therapy. A bicistronic retroviral vector containing ASA cDNA and CD24 cDNA as a selectable marker gene was constructed. This vector was successfully transduced on fibroblasts from MLD patients, ASA activity was increased 7-fold compared to normal untransduced cells. PCR Southern analysis of hematopoietic colonies showed that transduction efficiency of CD34+ cells was 11-22%. However, after fluorescence-activated cell sorting using anti-CD24 antibody, 75-100% of colonies became vector positive. The sorting raised the ASA activity several fold compared to untransduced CD34+ progenitors. These results suggest that a bicistronic ASA vector containing a CD24 selectable marker could be a useful component of gene therapy for MLD.
Subject(s)
Antigens, CD34/analysis , Antigens, CD/genetics , Cerebroside-Sulfatase/genetics , Fibroblasts/metabolism , Genetic Vectors/therapeutic use , Hematopoietic Stem Cells/immunology , Membrane Glycoproteins , Biomarkers , CD24 Antigen , Cerebroside-Sulfatase/deficiency , DNA, Complementary/genetics , Genetic Therapy , Hematopoietic Stem Cells/metabolism , Humans , Leukodystrophy, Metachromatic/enzymology , Leukodystrophy, Metachromatic/genetics , Leukodystrophy, Metachromatic/therapy , Retroviridae/genetics , Transduction, Genetic/geneticsABSTRACT
Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 +/- 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor alpha (51 +/- 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38- subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 +/- 7% of CD34+ progenitors and 21 +/- 6% of CD34+, CD38- progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient. Gene Therapy (2000) 7, 183-195.
Subject(s)
Adenoviridae/genetics , Antigens, CD34/genetics , Antigens, CD , Antigens, Differentiation/genetics , Genetic Vectors/genetics , Hematopoietic Stem Cells/physiology , NAD+ Nucleosidase/genetics , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Cells, Cultured , Fetal Blood , Gene Transfer Techniques , Helper Viruses/genetics , Humans , Membrane Glycoproteins , Mice , Transduction, Genetic/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
A major problem of tumor gene therapy is the low transduction efficiency of the currently available vectors. One way to circumvent this problem is the delivery of therapeutic genes encoding intracellular enzymes for the conversion of a prodrug to a cytotoxic drug which can then spread to neighboring non-transduced cells (bystander effect). One possibility to improve the bystander effect could be the extracellular conversion of a hydrophilic prodrug to a lipophilic, cell-permeable cytotoxic drug. Toward this end, we have used a secreted form of the normally lysosomal human beta-glucuronidase (s-betaGluc) to establish an extracellular cytotoxic effector system that converts an inactivated glucuronidated derivative of doxorubicin (HMR 1826) to the cytotoxic drug. We demonstrate that s-betaGluc-transduced tumor cells convert HMR 1826 to doxorubicin which is taken up by both transduced and non-transduced cells. s-betaGluc in combination with HMR 1826 efficiently induces tumor cell killing both in cell culture and in vivo. This effect is mediated through a pronounced bystander effect of the generated cytotoxic drug. Most notably, this gene therapeutic strategy is shown to be clearly superior to conventional chemotherapy with doxorubicin. Gene Therapy (2000) 7, 224-231.
Subject(s)
Choriocarcinoma/therapy , Doxorubicin/analogs & derivatives , Glucuronates/therapeutic use , Glucuronidase/genetics , Prodrugs/therapeutic use , Choriocarcinoma/enzymology , Dose-Response Relationship, Drug , Doxorubicin/therapeutic use , Gene Transfer Techniques , Glucuronates/genetics , Glucuronidase/metabolism , Humans , Immunohistochemistry , Transduction, Genetic/genetics , Tumor Cells, CulturedABSTRACT
We have developed a culture method for the foreign serum-free generation of highly immunostimulatory, CD83+ human dendritic cells (DC). In this study, we evaluated the feasibility and consequences of endogenously expressing antigens in mature DC using adenoviral vectors. Transduction of DC with Ad-EGFP demonstrated endogenous fluorescence in 50-85% of CD83+ DC. Ad-transduced DC stimulated the proliferation of allogeneic CD8+ and CD4+ T cells at low DC: T cell ratios. However, at high DC: T cell ratios the stimulatory capacity of Ad-transduced DC was suppressed. This immunosuppressive effect was confirmed by demonstrating that the stimulatory function of untreated DC could be suppressed in a dose-dependent manner by addition of Ad-transduced DC. Furthermore, transwell experiments suggested that direct cell contact was required. Taken together, our results demonstrate the feasibility of efficiently expressing antigens in CD83+ DC using adenoviruses. However, immunosuppressive effects must be considered and carefully studied before Ad-transduced DC are employed for clinical trials. Gene Therapy (2000) 7, 249-254.
Subject(s)
Adenoviridae/genetics , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/metabolism , Transduction, Genetic/genetics , CD4-CD8 Ratio , Flow Cytometry , Humans , Immunity, Active , PhenotypeABSTRACT
Among potential genetic targets for intervention in the HIV-1 life cycle, the tat gene product is a key target. We investigated the ability of an antitat gene to inhibit HIV-1 activation and replication in chronically infected promonocyte (U1) and T cell (ACH-2) lines in vitro. U1 and ACH-2 cells were transduced with an antitat gene expressing RNA with dual (polymeric Tat activation response element and antisense-tat) function that interferes with HIV-1 replication. Tumor necrosis factor-alpha (TNF-alpha) plus phorbol 12- myristate 13-acetate (PMA)-induced HIV-1 expression, as determined by reverse transcribed PCR and reverse transcriptase (RT) assays, was significantly inhibited in U1 and ACH-2 cells transduced with the antitat gene, compared with the cells transduced with control vector and untransduced cells. This resistance to TNF-alpha plus PMA-induced HIV-1 expression was demonstrated in antitat gene-transduced U1 and ACH-2 cells maintained in G418-free media for 5 months, suggesting that functional antitat gene may persist for many months in transduced cells and their progeny. Most importantly, we demonstrate that the antitat gene, when introduced into peripheral blood mononuclear cells (PBMC) isolated from patients with HIV-1 infection, inhibited TNF-alpha plus PMA-induced viral replication as determined by RT-PCR and RT activity. In addition, the antitat gene enhanced the survival of CD4+ T lymphocytes from such patients. These data suggest the feasibility of utilizing antitat gene therapy to block activation and replication of HIV-1 in latently infected monocytes and T- lymphocytes in vivo. Gene Therapy (2000) 7, 321-328.
Subject(s)
Genes, tat/genetics , HIV-1/genetics , Virus Replication/genetics , Cell Line , Flow Cytometry , Gene Expression , Genetic Therapy/methods , HIV Infections/therapy , Humans , Leukocytes, Mononuclear/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Transduction, Genetic/genetics , Virus ActivationABSTRACT
The MDR1 (multidrug resistance) gene, transferred to hematopoietic cells, is expected to protect them from anticancer chemotherapy and may serve as a selectable marker, restoring gene expression in vivo. Appropriate selection strategies, however, need to be established. To investigate whether preselection ex vivo affects chemoresistance, murine bone marrow cells were retrovirally transduced with high-titer or, as a model for suboptimal gene expression, low-titer retroviruses and exposed to daunomycin or colchicine for 48-96 h. Selection significantly increased chemoresistance of clonogenic progenitor cells. In tissue culture, the entire target population was rendered highly drug resistant after MDR1 transfer with high-titer viruses. If transduction was performed under suboptimal conditions, drug selection increased the frequency of chemoresistant colonies up to 40% over the number of unselected cells. Colchicine and daunomycin were equally efficient in increasing drug resistance ex vivo, but colchicine-preselected cells rescued lethally irradiated mice under conditions where daunomycin-selected bone marrow cells failed to do so. Hence, while hematopoietic cells can be protected by MDR1, the selection strategy is critical for repopulation of bone marrow with transduced cells. Preselection in culture before transplantation significantly increased P-gp expression and chemoresistance in vivo in mice reconstituted with transduced bone marrow cells. This study may help to facilitate the use of MDR1 as a selectable marker in gene therapy of the hematopoietic system. Gene Therapy (2000) 7, 348-358.
Subject(s)
Genes, MDR/genetics , Hematopoietic Stem Cells/physiology , Transduction, Genetic/genetics , Transgenes/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Bone Marrow Transplantation , Drug Resistance, Multiple/genetics , Genetic Vectors/genetics , Mice , Mice, Inbred C57BLABSTRACT
Nonhuman primates are increasingly being used as models for pre-clinical assessment of retrovirus vector expression and function following stem and progenitor cell transduction. We compared the relative susceptibility of CD34+ marrow progenitors from four nonhuman primate species and humans to transduction with amphotropic pseudotyped retrovirus vectors containing the Neo gene. The rate of functional gene transfer was measured by colony formation under G418 selection. Marrow progenitors from pigtail macaques (Macaca nemestrina) were transduced at about twice the rate (19.1 +/- 4.3%) as those from rhesus (11.2 +/- 3.7%) and cynomolgus (7.6 +/- 1.9%) macaques, baboons (7.8 +/- 1.8%), and humans (9.6 +/- 1.7%). Semiquantitative RT/PCR analysis suggests this difference may be due to elevated expression of the amphotropic receptor Pit2 in pigtailed macaque CD34+ cells. Further, transduction rates increased an average 1.6 +/- 0.4-fold when the culture temperature was lowered to 33 degrees C, and 2.1 +/- 0.3-fold when the culture dishes were coated with the fibronectin fragment CH-296. The data presented here point to important differences among nonhuman primate models as well as transduction culture conditions, and suggest that pigtailed macaques may be particularly useful for assessing expression and function of therapeutic retrovirus vectors. Gene Therapy (2000) 7, 359-367.
Subject(s)
Hematopoietic Stem Cells/physiology , Retroviridae/genetics , Transduction, Genetic/genetics , Animals , Antigens, CD34/metabolism , Gene Transfer Techniques , Genetic Vectors/genetics , Macaca , Papio , Receptors, Virus/metabolism , TemperatureABSTRACT
Cochlear gene transfer is a promising new approach for inner ear therapy. Previous studies have demonstrated hair cell protection with cochlear gene transfer not only in the inoculated, but also in the uninoculated ear. To characterize the kinetics of viral spread, we investigated the extent of transgene expression in the contralateral (uninoculated) cochlea after unilateral adenoviral cochlear gene transfer. We used a lacZ reporter gene vector, and demonstrated spread of the adenovirus into the cerebrospinal fluid (CSF) after cochlear inoculation of 25 microl viral vector. Direct virus application into the CSF resulted in transduction of both cochleae, whereas virus inoculation into the bloodstream did not. The cochlear aqueduct was identified as the most likely route of virus spread to the contralateral cochlea. These data enhance our understanding of the kinetics of virus-mediated transgene expression in the inner ear, and assist in the development of clinical applications for inner ear gene therapy. Our results showed a functional communication between the CSF and the perilymphatic space of the inner ear, that is not only of importance for otological gene transfer, but also for CNS gene transfer. Gene Therapy (2000) 7, 377-383.
Subject(s)
Adenoviridae/genetics , Cochlea/physiology , Gene Transfer Techniques , Animals , Cerebrospinal Fluid/physiology , Cochlear Aqueduct/physiology , Ear, Middle/physiology , Genetic Vectors/genetics , Guinea Pigs , Lac Operon/genetics , Transduction, Genetic/geneticsABSTRACT
To develop a cellular vehicle able to reach systemically disseminated areas of angiogenesis, we sought to exploit the natural tropism of circulating endothelial progenitor cells (EPCs). Primate CD34+ EPCs were genetically modified with high efficiency and minimal toxicity using a non-replicative herpes virus vector. These EPCs localized in a skin autograft model of angiogenesis in rhesus monkeys, and sustained the expression of a reporter gene for several weeks while circulating in the blood. In animals infused with autologous CD34+ EPCs transduced with a thymidine kinase-encoding herpes virus, skin autografts and subcutaneous Matrigel pellets impregnated with vascular growth factors underwent necrosis or accelerated regression after administration of ganciclovir. Importantly, the whole intervention was perfectly well tolerated. The accessibility, easy manipulation, lack of immunogenicity of the autologous CD34+ cell vehicles, and tropism for areas of angiogenesis render autologous CD34+ circulating endothelial progenitors as ideal candidates for exploration of their use as cellular vehicles when systemic gene delivery to those areas is required. Gene Therapy (2000) 7, 43-52.
Subject(s)
Antigens, CD34/genetics , Leukocytes, Mononuclear/physiology , Transduction, Genetic/genetics , Angiogenesis Inhibitors/genetics , Animals , Cells, Cultured , Flow Cytometry , Gene Transfer Techniques , Humans , Macaca mulatta , Neovascularization, Pathologic , Simplexvirus/genetics , Skin/blood supply , Stem Cells/physiology , beta-Galactosidase/metabolismABSTRACT
The neuronal apoptosis inhibitory protein (NAIP) is known to have anti-apoptotic functions, and its gene is often mutated in severe cases of spinal muscular atrophy (SMA), a disease characterized by motor neuron degeneration. In this study, we examined the distribution of the endogenous NAIP protein in normal human spinal cord and brain tissue by using a polyclonal antibody against NAIP. Immunohistochemical staining demonstrated that NAIP is strongly expressed in anterior horn and motor cortex neurons of normal brains, and it is not altered in the remaining motor neurons of patients with amyotrophic lateral sclerosis (ALS). NAIP is also located in human fetal neurons and in adult choroid plexus cells. These results suggest that the anti apoptotic molecule NAIP may be important in motor neurons, but it specifically does not appear to be altered in ALS.