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HOST EPIGENETIC REGULATOR BMI1 AND VIRAL FIBER PROTEIN FOR EQUILIBRIUM BETWEEN ADENOVIRUS AND HOST

Na, Manli LU (2013) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2013:9.
Abstract (Swedish)
Popular Abstract in Swedish

Adenovirus från människa (Ad) används ofta vid genterapi mot cancer, vaccinutveckling och genöverföring. Modifierade Ad5 och Ad2 virus har framgångsrikt använts som cancercelldödande (onkolytiska) agens i experimentella studier som dock inte kunnat överföras till kliniskt bruk av flera skäl, inklusive bristande spridning av virusinfektionen inom tumörvävnaden. De onkolytiska virusens kapacitet att döda cancerceller är beroende inte endast av effektiviteten i virusreplikationen utan också av hur effektivt nybildat virus sprider sig och infekterar nya celler i cancervävnaden. I denna avhandling förutsättes både faktorer som rör virus och host värdceller reglera Ad infektionens spridning i... (More)
Popular Abstract in Swedish

Adenovirus från människa (Ad) används ofta vid genterapi mot cancer, vaccinutveckling och genöverföring. Modifierade Ad5 och Ad2 virus har framgångsrikt använts som cancercelldödande (onkolytiska) agens i experimentella studier som dock inte kunnat överföras till kliniskt bruk av flera skäl, inklusive bristande spridning av virusinfektionen inom tumörvävnaden. De onkolytiska virusens kapacitet att döda cancerceller är beroende inte endast av effektiviteten i virusreplikationen utan också av hur effektivt nybildat virus sprider sig och infekterar nya celler i cancervävnaden. I denna avhandling förutsättes både faktorer som rör virus och host värdceller reglera Ad infektionens spridning i cancervävnaden och påverka hur de interagerar. Arbetet syftar till att identifiera sådana faktorer och klargöra deras respektive roll i Ad virusets replikation och spridningen av infektionen.

För att identifiera virala faktorer studerades med användning av flödescytometri och mikroskopisk konfokalanalys cellavdödningens kinetik och virusproduktionen efter infektion med litet antal viruspartiklar per cell. Resultaten visade att Ad infekterade host värdceller frigör fria virusfiber-molekyler innan intakta viruspartiklar frigörs. De fria virusfiber-molekylerna maskerar Ad virusets receptormolekyler på ytan av icke-infekterade celler och hindrar dessa celler från att bli virusinfekterade. Härigenom underlättas Ad virusets samexistens med värdcellerna eftersom det ökar chansen för viruset att föröka sig och fortleva i jämförelse med om viruset snabbt skulle döda alla värdceller. Emellertid är detta fenomen ofördelaktigt för användningen av Ad som ett onkolytiskt agens för terapi i klinik.

För att identifiera väsentliga värdcellsfaktorer har vi undersökt effekten av polycomb-genen BMI1 på cellernas virusproduktion. BMI1 är överuttryckt i de flesta typerna av cancer. Genom retrovirus-medierat överuttryck av BMI1 eller siRNA-medierad nedreglering av endogent BMI1 uttryck, demonstreras en omvänd korrelation mellan ny Ad-produktion och graden av BMI1 uttryck. Denna effect var inte relaterad till cellens läge i cellcykeln eller den receptorberoende Ad infektiviteten för värdceller. Inte heller var den relaterad till Ad genomets replication eller produktionen av strukturella Ad proteiner. Istället skadade överuttryck av BMI1 viruspartiklarnas uppbyggnad, vilket kunde motverkas med hjälp av TSA som hämmar histon-deacetylas (HDAC)-aktiviteten. Fynden indikerar att överuttryck av BMI1 begränsar effektiviteten av cancerterapi med onkolytiskt Ad. Omvänt kan inhibition av BMI1-uttryck eller BMI1-relaterad HDAC-aktivitet förbättra funktionaliteten av onkolytiska Ad vid cancerterapi.

För att etablera ny metoder där Ad kan användas för att och uttrycka överföra dubbla gener till mänskliga hematopoietiska stamceller genererades en Ad vector, Ad5F35-ΔLNGFR-BiDp-GFP, som innehåller den genetiska infomationen för “kinase domain deleted low-affinity NGF receptor” (ΔLNGFR) och grönt fluorescent protein (GFP) som en expressionscassette kontollerad av en syntetisk bi-directional “tvåvägs promoter”. Denna Ad5F35-ΔLNGFR-BiDp vector visas vara högaktiv för att ge dubbla genuttryck i HSC och de flesta testade leukemiska celltyper, men den relativa nivån av dubbla genuttryck är starkt beroende av celltypen. (Less)
Abstract
Human adenoviruses (Ads) are broadly used in cancer gene therapy, vaccine development and gene delivery. The modified Ad5 or Ad2 viruses have been successfully used as oncolytic agents in pre-clinical studies. However they could not be translated into clinic utility due to a number of limitations including inefficient Ad spread among tumor cells. The cancer cell killing capacity of oncolytic Ad is dependent not only on the efficiency of Ad replication, but also on the efficiency of progeny dispersal and propagation of infection within cancer tissue. In this thesis, we believe that both viral and host cell factors regulate Ad propagation and spread process and consequently affect the co-existence interplay between Ad and host cells. Our... (More)
Human adenoviruses (Ads) are broadly used in cancer gene therapy, vaccine development and gene delivery. The modified Ad5 or Ad2 viruses have been successfully used as oncolytic agents in pre-clinical studies. However they could not be translated into clinic utility due to a number of limitations including inefficient Ad spread among tumor cells. The cancer cell killing capacity of oncolytic Ad is dependent not only on the efficiency of Ad replication, but also on the efficiency of progeny dispersal and propagation of infection within cancer tissue. In this thesis, we believe that both viral and host cell factors regulate Ad propagation and spread process and consequently affect the co-existence interplay between Ad and host cells. Our projects aim to identify such factors from Ad as well as host cell side, and to clarify their role in regulating Ad propagation and spread.

To identity viral factors, we investigated the kinetics of cell killing and Ad propagation following Ad infection at low multiplicity. Our results showed that prior to the release of Ad progenies, Ad infected cells secrete free fiber molecules in an excess, which mask Ad receptor molecules on non-infected bystander host cells, thus preventing these cells from efficient Ad infection and thereby promoting Ad and host cell co-existence. This is advantageous to Ad propagation and persistency of infection compared to the killing of all host cells with rapid kinetics. However, this is disadvantageous if Ad is used as oncolytic agents for therapeutic purposes.

To identity host factors, we investigated the effect of polycomb gene BMI1 on Ad propagation. BMI1 is broadly overexpressed in various cancers. By retroviral vector mediated enforced BMI1 overexpression or siRNA mediated down-regulation of endogenous BMI1 expression, we demonstrated an inverse correlation between the Ad progeny production and the levels of BMI1 expression. This effect was not related to the cell cycle status and the receptor dependent Ad infectivity in host cells; nor to the replication of Ad genome and the production of Ad structural proteins. Instead, BMI1 overexpression impaired the morphogenesis of Ad particles, which could be reversed by TSA mediated inhibition of HDAC activity. Our findings indicate overexpression of BMI1 as a limiting factor in cancer therapy based on oncolytic Ad. So inhibition of BMI1 expression or BMI1-related HDAC activity may improve the functionality of oncolytic Ads in cancer therapy.

To explore new approaches in Ad vector mediated dual gene expression in human hematopoietic stem cells (HSC), we generated an Ad vector, Ad5F35- ΔLNGFR-BiDp-GFP, encoding kinase domain deleted low-affinity NGF receptor (ΔLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter. Our data showed that Ad5F35-ΔLNGFR-BiDp vector is highly active in directing dual gene expression in HSCs and most leukemic cell types tested, but the relative levels of dual gene expression by this vector is strongly cell-type dependent. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Kochanek, Stefan, Division of Gene Therapy, Uniklinik Ulm, Ulm, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Adenovirus, BMI1, Dual-gene expression, Epigenetic, Equilibrium, Fiber, HSC, HDAC, TSA
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2013:9
pages
130 pages
publisher
Neurosurgery, Department of Clinical Sciences, Lund
defense location
Segerfalk lecture hall, Sölvegatan 17, Lund
defense date
2013-02-08 09:15
ISSN
1652-8220
ISBN
978-91-87189-77-7
language
English
LU publication?
yes
id
827d997b-cdcc-4562-8da9-52370dde747d (old id 3351468)
date added to LUP
2013-01-16 10:54:29
date last changed
2019-05-22 00:21:17
@phdthesis{827d997b-cdcc-4562-8da9-52370dde747d,
  abstract     = {Human adenoviruses (Ads) are broadly used in cancer gene therapy, vaccine development and gene delivery. The modified Ad5 or Ad2 viruses have been successfully used as oncolytic agents in pre-clinical studies. However they could not be translated into clinic utility due to a number of limitations including inefficient Ad spread among tumor cells. The cancer cell killing capacity of oncolytic Ad is dependent not only on the efficiency of Ad replication, but also on the efficiency of progeny dispersal and propagation of infection within cancer tissue. In this thesis, we believe that both viral and host cell factors regulate Ad propagation and spread process and consequently affect the co-existence interplay between Ad and host cells. Our projects aim to identify such factors from Ad as well as host cell side, and to clarify their role in regulating Ad propagation and spread.<br/><br>
To identity viral factors, we investigated the kinetics of cell killing and Ad propagation following Ad infection at low multiplicity. Our results showed that prior to the release of Ad progenies, Ad infected cells secrete free fiber molecules in an excess, which mask Ad receptor molecules on non-infected bystander host cells, thus preventing these cells from efficient Ad infection and thereby promoting Ad and host cell co-existence. This is advantageous to Ad propagation and persistency of infection compared to the killing of all host cells with rapid kinetics. However, this is disadvantageous if Ad is used as oncolytic agents for therapeutic purposes.<br/><br>
To identity host factors, we investigated the effect of polycomb gene BMI1 on Ad propagation. BMI1 is broadly overexpressed in various cancers. By retroviral vector mediated enforced BMI1 overexpression or siRNA mediated down-regulation of endogenous BMI1 expression, we demonstrated an inverse correlation between the Ad progeny production and the levels of BMI1 expression. This effect was not related to the cell cycle status and the receptor dependent Ad infectivity in host cells; nor to the replication of Ad genome and the production of Ad structural proteins. Instead, BMI1 overexpression impaired the morphogenesis of Ad particles, which could be reversed by TSA mediated inhibition of HDAC activity. Our findings indicate overexpression of BMI1 as a limiting factor in cancer therapy based on oncolytic Ad. So inhibition of BMI1 expression or BMI1-related HDAC activity may improve the functionality of oncolytic Ads in cancer therapy.<br/><br>
To explore new approaches in Ad vector mediated dual gene expression in human hematopoietic stem cells (HSC), we generated an Ad vector, Ad5F35- ΔLNGFR-BiDp-GFP, encoding kinase domain deleted low-affinity NGF receptor (ΔLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter. Our data showed that Ad5F35-ΔLNGFR-BiDp vector is highly active in directing dual gene expression in HSCs and most leukemic cell types tested, but the relative levels of dual gene expression by this vector is strongly cell-type dependent.},
  author       = {Na, Manli},
  isbn         = {978-91-87189-77-7},
  issn         = {1652-8220},
  keyword      = {Adenovirus,BMI1,Dual-gene expression,Epigenetic,Equilibrium,Fiber,HSC,HDAC,TSA},
  language     = {eng},
  pages        = {130},
  publisher    = {Neurosurgery, Department of Clinical Sciences, Lund},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {HOST EPIGENETIC REGULATOR BMI1 AND VIRAL FIBER PROTEIN FOR EQUILIBRIUM BETWEEN ADENOVIRUS AND HOST},
  volume       = {2013:9},
  year         = {2013},
}