Title:
Quantitative analysis of lentivirus incorporation of heterologous viral and non-viral proteins for lung gene therapy

Simple item page
dc.contributor.advisor Le Doux, Joseph M.
dc.contributor.author Jung, Cindy en_US
dc.contributor.committeeMember García, Andrés J.
dc.contributor.committeeMember Cheng Zhu
dc.contributor.committeeMember Nael McCarty
dc.contributor.committeeMember Richard Compans
dc.contributor.department Biomedical Engineering en_US
dc.date.accessioned 2009-01-22T15:53:43Z
dc.date.available 2009-01-22T15:53:43Z
dc.date.issued 2007-11-12 en_US
dc.description.abstract 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. en_US
dc.description.degree Ph.D. en_US
dc.identifier.uri http://hdl.handle.net/1853/26648
dc.publisher Georgia Institute of Technology en_US
dc.subject Retrovirus en_US
dc.subject Lung gene therapy en_US
dc.subject Pseudotyping en_US
dc.subject Lipid rafts en_US
dc.subject Polarized en_US
dc.subject.lcsh Gene therapy
dc.subject.lcsh Lungs Diseases
dc.subject.lcsh Lentiviruses
dc.title Quantitative analysis of lentivirus incorporation of heterologous viral and non-viral proteins for lung gene therapy en_US
dc.type Text
dc.type.genre Dissertation
dspace.entity.type Publication
local.contributor.advisor Le Doux, Joseph M.
local.contributor.corporatename Wallace H. Coulter Department of Biomedical Engineering
local.contributor.corporatename College of Engineering
relation.isAdvisorOfPublication c6e9e0de-b6b5-455e-b7ad-7b27f6ef0e31
relation.isOrgUnitOfPublication da59be3c-3d0a-41da-91b9-ebe2ecc83b66
relation.isOrgUnitOfPublication 7c022d60-21d5-497c-b552-95e489a06569
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
jung_cindy_200712_phd.pdf
Size:
2.99 MB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Theses and Dissertations