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A D Miller

Publications and source records attributed to A D Miller.

At least 37 records · Page 2Linked to original sources
PubMed2001

Many nonmammalian cells exhibit postentry blocks to transduction by gammaretroviruses pseudotyped with various viral envelopes, including vesicular stomatitis virus G glycoprotein.

Previous studies have suggested that Moloney murine leukemia virus (MoMLV)-based vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) have extensive ability to transduce nonmammalian cells. However, we have identified multiple cell lines from fish (FHM), mosquitoes (Mos-55), moths (Sf9 and High-5), flies (S2), and frogs (XPK2) that are not efficiently transduced by MoMLV-based vectors pseudotyped with many different viral envelope proteins, including VSV-G, while the same vectors are functional in these cells following transfection. A comparison of MoMLV-based vector transduction in mammalian and nonmammalian cells shows that the nonmammalian cells exhibit blocks at either entry, reverse transcription, or integration. Additionally, VSV-G-pseudotyped MoMLV-based vector transduction is attenuated in the zebrafish cell line ZF4 at entry and/or reverse transcription, whereas other transduction processes are unaffected. We show that the variation of transduction by MoMLV-based vectors in mammalian and nonmammalian cells is not due to differences in culture conditions or cell division rate but is likely the result of divergence in cellular factors required for retroviral transduction.

Animals↗
PubMed2001

Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors.

Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis.

Administration, Intranasal↗
PubMed2001

Jaagsiekte sheep retrovirus Env protein stabilizes retrovirus vectors against inactivation by lung surfactant, centrifugation, and freeze-thaw cycling.

Jaagsiekte sheep retrovirus (JSRV) replicates in the lungs of sheep and causes the secretion of copious lung fluid containing the virus. Adaptation of JSRV to infection and replication in the lung and its apparent resistance to the denaturing activity of lung fluid suggest that vectors based on JSRV would be useful for gene therapy targeted to the lung. We show here that a retrovirus vector bearing the JSRV Env is stable during treatment with lung surfactant while an otherwise identical vector bearing an amphotropic Env is inactivated. Furthermore, the JSRV vector was stable during centrifugation, allowing facile vector concentration, and showed no loss of activity after six freeze-thaw cycles. However, the JSRV vector was inactivated by standard disinfectants, indicating that JSRV vectors pose no unusual safety risk related to their improved stability under other conditions.

Animals↗
PubMed2001

Receptors and entry cofactors for retroviruses include single and multiple transmembrane-spanning proteins as well as newly described glycophosphatidylinositol-anchored and secreted proteins.

In the past few years, many retrovirus receptors, coreceptors, and cofactors have been identified. These molecules are important for some aspects of viral entry, although in some cases it remains to be determined whether they are required for binding or postbinding stages in entry, such as fusion. There are certain common features to the molecules that many retroviruses use to gain entry into the cell. For example, the receptors for most mammalian oncoretroviruses are multiple membrane-spanning transport proteins. However, avian retroviruses use single-pass membrane proteins, and a sheep retrovirus uses a glycosylphosphatidylinositol-anchored molecule as its receptor. For some retroviruses, particularly the lentiviruses, two cell surface molecules are required for efficient entry. More recently, a soluble protein that is required for viral entry has been identified for a feline oncoretrovirus. In this review, we will focus on the various strategies used by mammalian retroviruses to gain entry into the cell. The choice of receptors will also be discussed in light of pressures that drive viral evolution and persistence.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Precise gene localization by phenotypic assay of radiation hybrid cells.

A high resolution map of the human genome previously has been constructed by using the G3 panel of human/hamster radiation hybrid cell lines and >15,000 unique human genetic markers. By determining whether human DNA sequences are present or absent in each of the hybrids, localization of single genes may routinely be achieved at approximately 250-kb resolution. In this paper we have tested whether similarly precise localization might be achieved by phenotypic screening of the hybrids to facilitate positional cloning of unknown genes. We assayed the susceptibility of each of the hybrid cell lines to transduction by retroviral vectors bearing different retroviral envelope proteins that recognize receptors present on human but not on hamster cells. The results for each of the retroviral vectors were informative and allowed precise localization of the receptor genes for the RD114 cat endogenous retrovirus, xenotropic murine leukemia virus, and type C feline leukemia virus. After cloning of the receptors for these retroviruses, we found that standard genotypic mapping by PCR gave results that were nearly identical to those from phenotypic mapping. These experiments show that precise gene localization by phenotypic assay of radiation hybrids is practical and was not appreciably impacted by the known instability of such hybrid cells. This technique should be applicable to many other human genes having discernible phenotypes in hamster cells and, with completion of the human genome project, will allow rapid identification of unknown genes on the basis of phenotype.

Animals↗
PubMed2000

Systemic production of human granulocyte colony-stimulating factor in nonhuman primates by transplantation of genetically modified myoblasts.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Animals↗
PubMed2000

Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo.

Adeno-associated virus (AAV) vectors might offer solutions for restenosis and angiogenesis by transducing nondividing cells and providing long-term gene expression. We investigated the feasibility of vascular cell transduction by AAV vectors in an in vivo rabbit carotid artery model. Time course of gene expression, inflammatory reaction to the vector, and effects of varying viral titer, exposure time, and intraluminal pressures on gene expression were examined. Recombinant AAV vectors with an Rous sarcoma virus promoter and alkaline phosphatase reporter gene were injected intraluminally into transiently isolated carotid segments. Following transduction, gene expression increased significantly over 14 days and then remained stable to 28 days, the last time point examined. Medial vascular smooth muscle cells were the main cell type transduced even with an intact endothelial layer. Increasing the viral titer and intraluminal pressure both enhanced transduction efficiency to achieve a mean of 34 +/- 7% of the subintimal layer of smooth muscle cells expressing gene product. A mild inflammatory reaction, composed of T cells with only rare macrophages, with minimal intimal thickening was demonstrated in 40% of transduced vessels; inflammatory cells were not detected in sham-operated control arteries. These findings demonstrate that AAV is a promising vector for intravascular applications in coronary and peripheral vascular diseases.

Alkaline Phosphatase↗
PubMed2000

Synthesis of high-mannose type neoglycolipids: active targeting of liposomes to macrophages in gene therapy.

The concise synthesis of five biantennary oligomannose neoglycolipids is presented. Employing a strategy based on the principles of reactivity tuning and orthogonal activation, the oligomannose moieties, isolated from the glycoprotein 63 of the parasite Leishmania mexicana amazonensis, were rapidly assembled taking advantage of common structural motifs found in these N-glycans. Deprotection of all structures was achieved in high yield by hydrogenolysis. The deprotected glycoconjugates were subsequently coupled to a cholesteroldiamine derivative using diethylsquarate as a linker. The resulting neoglycolipids will be used as additives to cationic liposome formulations in the active targeting of liposomes to macrophages.

Carbohydrate Sequence↗
PubMed2000

The affinity of the GroEL/GroES complex for peptides under conditions of protein folding.

The affinity of four short peptides for the Escherichia coli molecular chaperone GroEL was studied in the presence of the co-chaperone GroES and nucleotides. Our data show that binding of GroES to one ring enhances the interaction of the peptides with the opposite GroEL ring, a finding that was related to the structural readjustments in GroEL following GroES binding. We further report that the GroEL/GroES complex has a high affinity for peptides during ATP hydrolysis when protein substrates would undergo repeated cycles of assisted folding. Although we could not determine at which step(s) during the cycle our peptides interacted with GroEL, we propose that successive state changes in GroEL during ATP hydrolysis may create high affinity complexes and ensure maximum efficiency of the chaperone machinery under conditions of protein folding.

Adenosine Diphosphate↗
PubMed2000

Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6.

Recent advances in adeno-associated virus (AAV) vector production have eliminated the need for adenovirus infection by transfection of plasmids encoding the adenovirus E2A, E4orf6, and VA RNA transcription units. We report here the generation of significantly higher AAV vector titers with transfection of the single adenovirus gene, E4orf6, when used in conjunction with the split AAV packaging plasmids MTrep and CMVcap. Transduction in a murine lung model with these higher titer vector stocks was greater than that observed with traditional preparation methods. The generation of higher titer AAV vector stocks with fewer adenovirus gene products and free of replication-competent AAV will enhance the potential for AAV in clinical applications.

Adenoviridae↗
PubMed2000

Superior transduction of mouse hematopoietic stem cells with 10A1 and VSV-G pseudotyped retrovirus vectors.

The inefficient transduction of human hematopoietic stem cells (HSC) with amphotropic retroviral vectors has been an obstacle to gene therapy for hematopoietic diseases. We have previously reported low levels of amphotropic retrovirus receptor (Pit-2) mRNA and higher levels of gibbon ape leukemia virus (GALV) or 10A1 retrovirus receptor (Pit-1) mRNA in mouse and human HSC. The vesicular stomatitis virus (VSV-G) uses an abundant membrane phospholipid as a receptor. We hypothesized that transduction of HSC requires relatively high levels of retrovirus receptor molecules. Because mouse HSC can be efficiently transduced by ecotropic virus through the abundant ecotropic receptor, the mouse is an ideal model to compare receptor levels and transduction. We have developed a cotransduction assay where ecotropic retrovirus transduction is a positive internal control for downstream steps in retrovirus transduction. A comparison of mouse HSC transduction with amphotropic, 10A1, and VSV-G envelopes showed that the level of amphotropic and 10A1 receptor mRNA in HSC correlated with the frequency of transduction. Transduction with VSV-G vectors was similar to that with 10A1 vectors. We conclude that the level of retrovirus receptor on HSC is critical for HSC transduction and that GALV or VSV-G vectors would be better for human HSC transduction.

Animals↗
PubMed2000

Perfluorochemical liquid enhances adeno-associated virus-mediated transgene expression in lungs.

Use of adeno-associated virus (AAV) vectors for lung gene therapy is limited, in part, by low levels of AAV-mediated transgene expression in lungs. Generally, less than 1% of total airway and alveolar epithelial cells express transgene activity following vector administration. A means of improving AAV vector delivery could potentially enhance AAV-mediated gene expression in lungs. We have previously demonstrated that use of perfluorochemical (PFC) liquids improved overall levels of adenovirus vector-mediated gene expression as well as distribution of expression in lungs of spontaneously breathing rodents. To evaluate whether use of PFC liquids might similarly enhance AAV-mediated expression, spontaneously breathing rodents received intratracheal instillation of the AAV vectors CWRAP and ARAP4 (2-5 x 10(8) FFU/animal) with or without 10 cc/kg body wt PFC liquid (FC-75, ACROS). Animals were sacrificed 4 weeks later and lungs assessed for overall and in situ alkaline phosphatase (AP) expression. Animals receiving vector alone exhibited scattered sparse in situ activity, predominantly in alveolar epithelium. In contrast, animals receiving vector with FC-75 exhibited increased and more widespread AP expression as well as up to a 26-fold increase in AP activity. These results demonstrate that use of the PFC liquid FC-75 improves overall and in situ AAV-mediated gene expression in rodent lungs.

Animals↗
PubMed2000

Pregnancy, labour and delivery in a Jehovah's Witness with esophageal varices and thrombocytopenia.

PURPOSE: An increasing number of women with cirrhosis are conceiving and carrying their pregnancies to term. However, the maternal mortality rate remains high (10-61%). This case report describes the management of a parturient with esophageal varices and thrombocytopenia. She was also a Jehovah's Witness. CLINICAL FEATURES: A 25-yr-old Jehovah's Witness parturient with portal hypertension and esophageal varices secondary to cryptogenic cirrhosis was referred to our obstetrical unit at eight weeks gestation. In addition she was thrombocytopenic with platelet counts ranging from 42,000-67,000 x microl(-1). Her esophageal varices were banded prophylactically on three occasions during her pregnancy. Magnetic resonance imaging at 32 wk gestation showed extensive caput medusa and dominant midline varix. Therefore, the planned mode of delivery was changed from Cesarean section which could result in massive hemorrhage, to elective induction of labour with an assisted second stage. The patient refused any blood product transfusion except acute hemodilution and cell saving if necessary during labour and delivery. Despite elaborate preparations for a planned vaginal delivery, she underwent an unanticipated rapid labour. Spinal analgesia was provided to facilitate smooth assisted vacuum delivery. CONCLUSION: Multidisciplinary care is the key for a successful outcome in parturients with cirrhosis. Periodic examination and banding of esophageal varices is recommended during pregnancy. Active consideration should be given to availing of the benefits of regional anesthesia.

Adult↗
PubMed2000

Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells.

The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some correlated with an increase in the rate of transfer of the pDNA to lysosomes, a decrease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes-lysosomes was beneficial for expression. After 30 min both the peptide and pDNA localized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellular mechanisms by which vectors transfer to and traffic in cells should help design improved vectors.

Cation Exchange Resins↗
PubMed2000

Amelioration of established collagen induced arthritis by systemic IL-10 gene delivery.

A novel formulation of cationic liposomes containing the novel cytofectin ACHx was used for delivery of an anti-inflammatory cytokine gene, IL-10, to mice with established collagen induced arthritis. A single intraperitoneal injection of human IL-10 expression plasmid complexed with liposomes 2 to 4 days after the onset of arthritis was sufficient to give significant and prolonged amelioration of arthritis for 30 days. Preliminary experiments suggested that the therapeutic effect was IL-10 dose-dependent. The distribution of the human IL-10 DNA after injection was widespread, including the inflamed paws. Human IL-10 mRNA was also detected in the paws 24 h after injection. IL-10 protein was below the level of detection in paws and serum but was detected in some tissues up to 10 days after injection. The target cell of transfection was demonstrated to be the macrophage. These results suggest that systemic therapy with plasmid DNA complexed with cationic liposomes merits further development as an alternative method for anti-inflammatory treatment of arthritis.

Animals↗
PubMed2000

Retrovirus vectors bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3.

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus associated with a contagious lung tumor of sheep, ovine pulmonary carcinoma. Other than sheep, JSRV is known to infect goats, but there is no evidence of human infection. Until now it has not been possible to study the host range for JSRV because of the inability to grow this virus in culture. Here we show that the JSRV envelope protein (Env) can be used to pseudotype Moloney murine leukemia virus (MoMLV)-based retrovirus vectors and that such vectors can transduce human cells in culture. We constructed hybrid retrovirus packaging cells that express the JSRV Env and the MoMLV Gag-Pol proteins and can produce JSRV-pseudotype vectors at titers of up to 10(6) alkaline phosphatase-positive focus-forming units/ml. Using this high-titer virus, we have studied the host range for JSRV, which includes sheep, human, monkey, bovine, dog, and rabbit cells but not mouse, rat, or hamster cells. Considering the inability of the JSRV-pseudotype vector to transduce hamster cells, we used the hamster cell line-based Stanford G3 panel of whole human genome radiation hybrids to phenotypically map the JSRV receptor (JVR) gene within the p21.3 region of human chromosome 3. JVR is likely a new retrovirus receptor, as none of the previously identified retrovirus receptors localizes to the same position. Several chemokine receptors that have been shown to serve as coreceptors for lentivirus infection are clustered in the same region of chromosome 3; however, careful examination shows that the JSRV receptor does not colocalize with any of these genes.

Animals↗
PubMed2000

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes.

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.

Alkaline Phosphatase↗