Development of improved expression vectors and their applications in cancer gene therapy

Persistent Link:
http://hdl.handle.net/10150/280431
Title:
Development of improved expression vectors and their applications in cancer gene therapy
Author:
Luo, Phoebe Lihong
Issue Date:
2003
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Recombinant DNA vectors are fundamental tools in gene therapy research. A novel cloning system, pLinus, was made to facilitate vector construction by providing 32 unique restriction sites to adapt DNA fragments in a single step. To compensate the low delivery efficiency of the non-viral vector systems, we have constructed two high expression plasmid vectors, pHi1/2, by incorporating a transcriptional amplifier strategy into a single construct. In both pHi1/2 vectors, the amplifier expression cassettes contained two independent transcriptional units. One transcriptional unit contained a transcriptional factor, the tat gene, driven by a strong constitutive CMV promoter. The second transcriptional unit contained either an HIV1 LTR or HIV2 LTR driving the gene of interest. Using the human IL-2 cytokine as a reporter and therapeutic gene, the pHi1/2 amplifier vectors could achieve significantly higher IL-2 expression levels than that observed when using the CMV promoter alone. In vivo injection of the stable pHi2-IL-2 gene modified Lewis Lung (LL/2) tumor clones resulted in slower tumor growth and longer survival as compared to those mice injected with either CMV-driven IL-2 transfected clones or the parental tumor cells. To solve the safety concern, we constructed a novel plasmid vector, pHi-Hot, by combining inducible and amplifier strategies in a single vector. In pHi-Hot, the first transcriptional unit contained an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. Using the human IL-2 cytokine gene as a reporter gene, we demonstrated that, heat shock at 42°C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were significantly higher than achieved by using the hsp promoter or CMV promoter directly. And repeated heat shock at 42°C for 30 min of mice injected with a pHi-Hot-IL-2 gene modified LL/2 clone led to tumor regression. In this study, three major approaches towards facilitating vector construction and improving vector expression cassette design are described.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Health Sciences, Immunology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Microbiology and Immunology
Degree Grantor:
University of Arizona
Advisor:
Harris, David T.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleDevelopment of improved expression vectors and their applications in cancer gene therapyen_US
dc.creatorLuo, Phoebe Lihongen_US
dc.contributor.authorLuo, Phoebe Lihongen_US
dc.date.issued2003en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractRecombinant DNA vectors are fundamental tools in gene therapy research. A novel cloning system, pLinus, was made to facilitate vector construction by providing 32 unique restriction sites to adapt DNA fragments in a single step. To compensate the low delivery efficiency of the non-viral vector systems, we have constructed two high expression plasmid vectors, pHi1/2, by incorporating a transcriptional amplifier strategy into a single construct. In both pHi1/2 vectors, the amplifier expression cassettes contained two independent transcriptional units. One transcriptional unit contained a transcriptional factor, the tat gene, driven by a strong constitutive CMV promoter. The second transcriptional unit contained either an HIV1 LTR or HIV2 LTR driving the gene of interest. Using the human IL-2 cytokine as a reporter and therapeutic gene, the pHi1/2 amplifier vectors could achieve significantly higher IL-2 expression levels than that observed when using the CMV promoter alone. In vivo injection of the stable pHi2-IL-2 gene modified Lewis Lung (LL/2) tumor clones resulted in slower tumor growth and longer survival as compared to those mice injected with either CMV-driven IL-2 transfected clones or the parental tumor cells. To solve the safety concern, we constructed a novel plasmid vector, pHi-Hot, by combining inducible and amplifier strategies in a single vector. In pHi-Hot, the first transcriptional unit contained an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. Using the human IL-2 cytokine gene as a reporter gene, we demonstrated that, heat shock at 42°C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were significantly higher than achieved by using the hsp promoter or CMV promoter directly. And repeated heat shock at 42°C for 30 min of mice injected with a pHi-Hot-IL-2 gene modified LL/2 clone led to tumor regression. In this study, three major approaches towards facilitating vector construction and improving vector expression cassette design are described.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectHealth Sciences, Immunology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHarris, David T.en_US
dc.identifier.proquest3108926en_US
dc.identifier.bibrecord.b44829371en_US
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