"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.

"16th September 2002." / Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). / Bibliography: leaves xxix-li. / xxvii, 213, li leaves : ill., plates (some col.) ; 30 cm. + 1 CD-ROM (4 3/4 in.) / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

"16th September 2002." / Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). / Bibliography: leaves xxix-li. / xxvii, 213, li leaves : ill., plates (some col.) ; 30 cm. + 1 CD-ROM (4 3/4 in.) / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Ophthalmology. / Typescript bound. Includes bibliographical references: (leaves 258-281) Also available online.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2005. / Niren Murthy, Committee Member ; Andrš J. Garca̕, Committee Member ; Joseph M. Le Doux, Committee Chair ; Mark R. Prausnitz, Committee Member ; H. Trent Spencer, Committee Member. Includes bibliographical references.

Les IFN-I (interférons de type I), principalement IFN et , constituent un mécanisme de défense primordial de l’hôte contre les pathogènes. Pourtant, dans le cas du VIH-1 (virus de l’immunodéficience humaine), la relation entre les IFN-I et la réplication virale apparaît plus complexe. En effet, si les IFN-I inhibent la réplication du VIH-1 ex vivo, un état d’hyperactivation permanent de la réponse IFN-I a été récemment associé à la progression vers le SIDA ainsi qu’à une forte virémie chez les patients infectés par le VIH-1. De même, la dérégulation de la réponse IFN-I est un critère déterminant dans l’issue pathogénique de certains modèles d’infection virale chez le singe. Si l’hypothèse du rôle pathogénique des IFN-I s’avère correcte, le VIH-1 pourrait avoir évolué afin de se répliquer même en présence d’une telle réponse, qui semble être au final, plus délétère pour l’hôte que pour le virus. L’objectif de ce travail a été d’évaluer la résistance du VIH-1 aux IFN. Dans ce contexte, le VIH-1 a été comparé au VIH-2 et au SIVmac (virus de l’immunodéficience simienne), virus phylogénétiquement proches mais peu ou pas pathogènes pour l’homme, lors de l’infection de plusieurs types cellulaires tels que des lymphocytes, des macrophages et des cellules dendritiques. En accord avec l’hypothèse initiale de travail, les expériences réalisées ont montré que le VIH-1 est capable de se répliquer dans les cellules primaires prétraitées avec des doses d’IFN comparables à celles mesurées in vivo, alors que la réplication des virus VIH-2/SIVmac est complètement bloquée, même à des concentrations très faibles d’IFN. Ce travail a permis de démontrer que le blocage induit par l’IFN s’exerce au niveau des phases précoces de l’infection et plus précisément à l’étape de la transcription inverse. En effet, les données obtenues suggèrent que l’IFN induit l’expression d’un effecteur cellulaire qui affecte différentiellement la stabilité des complexes viraux, ce qui se traduit par un défaut d’accumulation de l’ADN viral plus important pour le VIH-2 et le SIVmac, que pour le VIH-1. La différence de susceptibilité des lentivirus de primates aux IFN-I pourrait ainsi expliquer en partie, les différents niveaux de réplication de ces virus, associés à leurs degrés de pathogénicité in vivo. / Type I Interferons (IFN-α/β, herein IFNs) provide an important mechanism of defense against pathogens and regulate in a paracrine and autocrine manner both intrinsic and adaptive immune responses. In the case of HIV-1 however, the relationship between IFNs and viral replication appears more complex. Indeed, if IFNs have been described to interfere with HIV-1 at basically all phases of its life cycle ex vivo, an IFN-induced state is linked to AIDS progression and to high viral loads in HIV-1 infected individuals. Similarly, a deregulated and prolonged IFN production/state seems one of the main distinguishing features between pathogenic and non-pathogenic SIV infection in primate animal models, suggesting that a deregulated IFN-state may be more detrimental to the host than to the virus itself in vivo.If this hypothesis is correct and if HIV-1 plays an active role in the perpetration of this antiviral state, it is possible that HIV-1 may have overall evolved to cope with this environment, remaining able to replicate despite it.To determine whether HIV-1 was better armed to replicate in the presence of an IFN-state environment than other primate lentiviruses, we compared HIV-1 to SIVmac and more importantly to HIV-2 that albeit capable of inducing AIDS in humans does so in a much less aggressive manner. In agreement with the initial hypothesis, our results indicate that HIV-1 is better fit to replicate in primary cells in the presence of amounts of IFN comparable to the ones measured in vivo, while the replication of HIV-2/SIVmac viruses is completely blocked even in the presence of low levels of IFN. By decorticating the effects of IFNs on the early and late phases of the viral life cycle in primary macrophages, we show here that the main target of the differential action of IFNs are the early phases of infection. More specifically, with time kinetics that we determine herein, IFNs induce cellular factor/s that differentially affect the stability of pre-reverse transcription complexes of HIV-2, but not of HIV-1. Our results could underlie a different evolutionary adaptation of primate lentiviruses to interferons that might be responsible for their different pathogenicity in vivo.

Gene therapy is the delivery of genetic material to cells for a therapeutic effect. Retroviruses are one of the most common viral vectors used for gene therapy, especially lung gene therapy. However the lung has many physical and immunological barriers to gene transfer vectors, and currently, too few cells are genetically modified for the effective treatment of lung diseases such as cystic fibrosis. One of the main reasons for low cell transduction is the lack of commonly-used receptors for gene therapy vectors on the apical surface of polarized epithelial cells. The objective of this project was to determine how to incorporate proteins into the lentiviral lipid bilayer in order to develop a recombinant retrovirus that can efficiently deliver genes to polarized epithelial cells via their apical membranes. We analyzed the process of incorporating heterologous viral and non-viral proteins into lentiviruses and determined key factors that allowed for successful protein incorporation into the lentiviral lipid bilayer. We found that lipid rafts segregated raft proteins, and for a protein to be incorporated into virus particles, it must be colocalized with lentivirus-associated rafts. When cells were treated with the cholesterol-extracting compound, methyl-beta-cyclodextrin, previously sequestered viral and non-viral raft proteins were then colocalized and non-viral proteins were incorporated into lentiviruses. We also created a lentivirus pseudotyped with envelope proteins from human parainfluenza type 3 (HPIV3), which naturally targets polarized epithelial cells of the lung. Lentiviruses were able to incorporate HPIV3 glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), and were able to transduce polarized cells in a manner consistent with lentiviral-mediated transduction via sialated receptors for HPIV3, however titers were too low for clinical use. We increased protein expression of HN and found that while expression, envelope incorporation, and titer increased, lentiviruses still incorporated too few envelope proteins for efficient transduction. We determined low envelope incorporation rates were due to lack of interactions with Gag, and increasing active and passive interactions with Gag enhanced HN and F incorporation into lentiviruses. Overall, this research is significant because it provides insight into viral assembly and protein incorporation for the generation of pseudotyped lentiviruses for human gene transfer.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 139-161).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 92-115).

Gene therapy is the introduction or alteration of genetic material with the intention to treat disease. To support this aim, viruses have been modified, with elements linked to viral pathogenicity removed from their genome and replaced by the genetic material to be delivered. Gene therapy vectors based on lentiviruses have many advantages, such as the ability to transduce non-dividing cells and to target specific cell types via pseudotyping. They have been successfully used in ex vivo clinical trials for several haematopoietic stem cell disorders. Lentiviral vectors, however, suffer from substantially lower titres than the more popular adeno-associated virus (AAV)-based vectors and therefore have limited applicability for in vivo gene therapy which requires much greater quantities of virus. The main aim of this thesis was to investigate strategies to improve lentiviral vector productivity during manufacture, in order to increase the likelihood of lentiviruses being adopted for disease treatment. Initial experiments were based on the lentiviral vector manufacturing process currently being developed by the United Kingdom Cystic Fibrosis Gene Therapy Consortium for the generation of highly concentrated, purified lentivirus for clinical use. Supplementation of FreeStyle 293 Expression Medium used during upstream processing was attempted, but none of the assessed supplements led to significant increases in lentiviral vector production. Investigation into intrinsic immunity to viral infection indicated that over-expression of the protein kinase RNA-activated (PKR) led to lower production titres, but over-expression of its inhibitors was not successful at increasing titres. The focus then shifted to reducing, or 'knocking-down', inhibitory factors present in the host cells, which could adversely affect viral titres. Investigation of the published HIV-1 literature revealed a possible 152 candidate inhibitory factors described as having a negative impact on HIV-1 replication in the late stages of the life cycle of the virus. A novel siRNA screen was developed to assess the effect of ‘knock-down' of inhibitory factors on lentiviral vector titre. Application of the screen to 89 candidate inhibitory factors identified nine genes which, when knocked-down, resulted in increased lentiviral vector production by more than 40%. Further work will be necessary to understand the role of the inhibitory factors in lentiviral vector production, but novel cell lines in which genes encoding these factors have been permanently deleted from producer cells could lead to higher titres, reducing costs in the manufacture of lentiviral vectors and making in vivo gene therapy more feasible from a health economics perspective.