ABSTRACT
We previously reported the efficiency of gene transfer in fetal monkeys using retroviral vectors and an intraperitoneal (IP) approach. Here, we explored intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1 x 10(7) infectious particles/fetus), using the enhanced green fluorescent protein (EGFP) as a reporter, was directly injected into fetal lung with ultrasound guidance (n=4; 55 or 70 days gestation; term 165+/-10 days). Fetuses were monitored sonographically, fetal/maternal blood samples collected during gestation, and four of four healthy newborns were delivered at term. All lung lobes were positive for the transgene (< or = 1%) when assessed by PCR, and transgene expression was observed by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys using an intrapulmonary approach; (2) less transduction of non-pulmonary tissues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-positive by immunohistochemistry; and (4) no evidence of transplacental transport of vector sequences or antibody responses in the dams. The results of these investigations indicate the efficiency of fetal gene transfer by intrapulmonary delivery, and emphasize the importance of the fetal monkey as a preclinical model system for exploring in utero genetic treatment strategies for pulmonary disorders.
Subject(s)
HIV-1/genetics , Lung/embryology , Lung/metabolism , Macaca mulatta/embryology , Animals , DNA Primers/chemistry , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Green Fluorescent Proteins , Hematopoietic Stem Cells/physiology , Humans , Immunoenzyme Techniques , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Macaca mulatta/genetics , Polymerase Chain Reaction , Promoter Regions, GeneticABSTRACT
Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human. Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days). Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer). PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth. Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.
Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Macaca mulatta/genetics , Membrane Glycoproteins , Retroviridae/genetics , Animals , Azacitidine/pharmacology , Cytomegalovirus/genetics , Dose-Response Relationship, Drug , Female , Flow Cytometry , Genes, Reporter , Green Fluorescent Proteins , HIV-1/genetics , Humans , Lentivirus/genetics , Leukocytes, Mononuclear/metabolism , Luminescent Proteins/genetics , Macaca mulatta/embryology , Male , Models, Genetic , Moloney murine leukemia virus/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tissue Distribution , Viral Envelope Proteins/geneticsABSTRACT
Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34(+) cells on fibronectin-coated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells.
Subject(s)
Antigens, CD34/genetics , Genetic Vectors , Hematopoietic Stem Cells/metabolism , Lentivirus/genetics , Membrane Glycoproteins , Transduction, Genetic , Cell Line , Cell Separation , Cytomegalovirus/genetics , Fetal Blood/cytology , Fibronectins/metabolism , Flow Cytometry , Gene Transfer Techniques , Green Fluorescent Proteins , HIV-1/genetics , Heparin Antagonists/pharmacology , Humans , Luminescent Proteins/metabolism , Plasmids/metabolism , Promoter Regions, Genetic , Protamines/pharmacology , Time Factors , Transfection , Viral Envelope Proteins/geneticsABSTRACT
A large nuclear protein complex, termed gammaPE (for gamma-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human y-globin gene. Two proteins, SATB1 (special A-T-rich binding protein 1) and HOXB2, can bind to yPE binding sites. SATB1 binds to nuclear matrix-attachment sites, and HOXB2 is a homeodomain protein important in neural development that is also expressed during erythropoiesis. The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire gammaPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two proteins can bind to the gammaPE binding site simultaneously. When SATB1 or HOXB2 was expressed in vitro, they could bind independently to gammaPE binding sites in EMSA. Interestingly, the proteins expressed in vitro competed effectively with each other for the gammaPE binding site, suggesting that this may occur under certain conditions in vivo. Transient cotransfections of a HOXB2 cDNA and a y-globin-luciferase reporter gene construct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription. Taking into account their potentially opposing effects and binding activities, SATB1 and HOXB2 may modulate the amount of gamma-globin mRNA expressed during development and differentiation.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Globins/genetics , Homeodomain Proteins/metabolism , Matrix Attachment Region Binding Proteins , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Transcription Factors/metabolism , Animals , Binding, Competitive , COS Cells , Cell Extracts , Chlorocebus aethiops , DNA-Binding Proteins/genetics , Electrophoresis, Polyacrylamide Gel/methods , Gene Expression Regulation , Genes, Reporter , Homeodomain Proteins/genetics , Humans , Immune Sera , K562 Cells , Luciferases/genetics , Nuclear Proteins/genetics , Protein Binding , Rabbits , Recombinant Fusion Proteins/biosynthesis , Reticulocytes/metabolism , Transcription Factors/geneticsABSTRACT
We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.
Subject(s)
Antigens, CD , Genetic Therapy , Genetic Vectors , HIV-1 , Hematopoietic Stem Cells/physiology , Transduction, Genetic , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Antigens, CD34 , Antigens, Differentiation , Humans , Lentivirus , Leukemia Virus, Murine , Membrane Glycoproteins , NAD+ NucleosidaseABSTRACT
Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1-based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.
Subject(s)
Gene Transfer Techniques , Genetic Vectors/administration & dosage , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Cation Exchange Resins/pharmacology , Cell Separation , Coculture Techniques , DNA, Recombinant/genetics , Drug Carriers/pharmacology , Electroporation , Flow Cytometry , Genes, Reporter , Genetic Therapy , Genetic Vectors/genetics , Green Fluorescent Proteins , HIV-1/genetics , Humans , Leukemia Virus, Gibbon Ape/genetics , Lipids/pharmacology , Liposomes , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Moloney murine leukemia virus/genetics , Phosphatidylethanolamines/pharmacology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Protamines , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transfection , Tumor Cells, Cultured , Vesicular stomatitis Indiana virus/geneticsABSTRACT
A novel nuclear factor involved in human gamma-globin gene regulation has been identified. Co-migrating and cross-competing complexes were formed with five individual fragments from the 5'- and 3'-flanking regions of the gene in DNA-protein binding assays. This indicates that a nuclear factor, termed gamma PE, has multiple binding sites near the gamma-globin gene. This characteristic is shared by other important factors in globin gene regulation, such as GATA-1. The five gamma PE binding sites can be placed in two categories based on DNA-protein binding affinity and DNA sequence composition. The consensus sequence for the two higher affinity binding sites is ATTANNNGGAANNCT(N)TNNNTAATGG and for the three lower affinity sites is AAAAN(A/T)A(A/T)TT. Both the ATTA and the TAAT motifs of a high affinity binding site are required for efficient DNA-protein binding. The tissue distribution of gamma PE binding activity is broad, including both erythroid and non-erythroid cell types. Transcription of either a gamma-globin or heterologous promoter is increased in the presence of nearby gamma PE binding sites. Therefore, gamma PE may be involved in activating the gamma-globin gene in fetal erythroid cells. UV cross-linking analysis indicates that the major protein interacting with a high affinity gamma PE binding site has a molecular mass of 108 kDa.