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The quest for new improved adenovirus gene therapy vectors against glioma tumours
Umeå University, Faculty of Medicine, Clinical Microbiology, Virology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene therapy has received much attention the last decade as a method to correct a number of disorders arising from a defective gene. Gene therapy can be defined as the introduction of a functional genetic element into a cell for a therapeutic purpose. This is a very broad term and gene therapy can be applied to a wide range of diseases from genetic diseases such as cystic fibrosis to infectious diseases or even acquired genetic diseases such as cancers.

Adenoviruses (Ad) are the second most common vector for gene therapy in clinical trials today, and these vectors are mostly based on serotype 2 or 5 (Ad2 and Ad5). It has been shown that Ad2 and Ad5 use a receptor that is often downregulated in malignant cells and they also suffer from shortcomings because of the high levels of pre-existing immunity against these serotypes in the society. Hence, new and improved vectors serving as alternatives to these serotypes need to be developed to make gene therapy a successful treatment option.

The work presented herein is devoted to analyse what alternative adenovirus vectors could be used for treatment of glioma brain tumours. A number of different adenovirus serotypes were screened for their ability to infect human glioma tumour cells in vitro. Established cell lines as well as low-passage glioma cells from different donors were used. Adenovirus serotype 11p (Ad11p) proved to be a promising vector candidate because of its capacity to efficiently infect the glioma cells and its low prevalence in the society. The complete genome of this serotype was sequenced to further develop this as an alternative adenovirus vector. Furthermore, a number of cell lines were produced to generate E1 deleted Ad11p vectors. Other promising vector candidates were Ad16 and a chimpanzee adenovirus called CV23. Ad16 was the most efficient human serotype to infect human low-passage glioma cells and the prevalence for this serotype is also very low. The overall most efficient virus was surprisingly the non-human CV23 virus. This adenovirus has no prevalence in humans, but efficiently infects human cells in vitro. The first analysis was made on established glioma cell lines and was followed up by using low passage glioma cells from a number of different patients. The glioma cells were analysed when subjected to <20 passages (low passage) and then again at >40 passages (high passage). The cells at a higher passage number were significantly more permissive to Ad5 than the cells analysed at a low passage number. This could in part explain why some of the promising in vitro data for Ad5 have shown a limited success in vivo. In contrast, CV23 infected the low and high passage gliomas equally. This indicates that CV23 uses an internalisation mechanism subjected to less variation than the mechanism used by Ad5.

We further characterised the receptor interaction of CV23 and found that none of the previously known primary receptors for adenoviruses were of importance for binding. We found that bovine serum albumin present in the growth medium was responsible for the high binding capacity to cells. Binding is a criterion for the first step of the infection, but not necessarily a good correlate to the infection capacity. CV23 infected human cells efficiently also in the absence of bovine serum albumin.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi , 2005. , 80 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 994
Keyword [en]
Gene therapy, virology, adenovirus, glioma, glioblastoma
Keyword [sv]
Genterapi, virologi, adenovirus, gliom, glioblastom
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-624ISBN: 91-7305-973-0 (print)OAI: oai:DiVA.org:umu-624DiVA: diva2:144026
Public defence
2005-11-25, 09:00 (English)
Supervisors
Available from: 2005-11-04 Created: 2005-11-04 Last updated: 2009-11-27Bibliographically approved
List of papers
1. Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin
Open this publication in new window or tab >>Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin
2002 (English)In: Journal of General Virology, ISSN 0022-1317, Vol. 83, no 6, 1299-1309 p.Article in journal (Refereed) Published
Abstract [en]

Most currently used adenovirus vectors are based upon adenovirus serotypes 2 and 5 (Ad2 and Ad5), which have limited efficiencies for gene transfer to human neural cells. Both serotypes bind to the known adenovirus receptor, CAR (coxsackievirus and adenovirus receptor), and have restricted cell tropism. The purpose of this study was to find vector candidates that are superior to Ad5 in infecting human neural tumours. Using flow cytometry, the vector candidates Ad4p, Ad11p and Ad17p were compared to the commonly used adenovirus vector Ad5v for their binding capacity to neural cell lines derived from glioblastoma, medulloblastoma and neuroblastoma cell lines. The production of viral structural proteins and the CAR-binding properties of the different serotypes were also assessed in these cells. Computer-based models of the fibre knobs of Ad4p and Ad17 were created based upon the crystallized fibre knob structure of adenoviruses and analysed for putative receptor-interacting regions that differed from the fibre knob of Ad5. The non CAR-binding vector candidate Ad11p showed clearly the best binding capacity to all of the neural cell lines, binding more than 90% of cells of all of the neural cell lines tested, in contrast to 20% or less for the commonly used vector Ad5v. Ad4p and Ad11p were also internalized and produced viral proteins more successfully than Ad5. Ad4p showed a low binding ability but a very efficient capacity for infection in cell culture. Ad17p virions neither bound or efficiently infected any of the neural cell lines studied.

Identifiers
urn:nbn:se:umu:diva-4782 (URN)12029144 (PubMedID)
Available from: 2005-11-04 Created: 2005-11-04Bibliographically approved
2. Efficient internalization into low-passage glioma cell lines using adenoviruses other than type 5: an approach for improvement of gene delivery to brain tumours.
Open this publication in new window or tab >>Efficient internalization into low-passage glioma cell lines using adenoviruses other than type 5: an approach for improvement of gene delivery to brain tumours.
Show others...
2004 (English)In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 85, no Pt 9, 2627-2638 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for improvement of the commonly used adenovirus vectors based on serotype 5. This study was performed on three adenovirus serotypes with a CAR-binding motif (Ad4p, Ad5p and Ad17p) and three non-CAR-binding serotypes (Ad11p, Ad16p and Ad21p). The capacity of these alternative adenovirus vector candidates to deliver DNA into low-passage glioma cell lines from seven different donors was evaluated. The non-CAR-binding serotype Ad16p was the most efficient serotype with regard to import of its DNA, as well as initiation of hexon protein expression. Ad16p established hexon expression in 60-80 % of the cell population in gliomas from all donors tested. The other non-CAR-binding serotypes, Ad11p and Ad21p, showed hexon expression in 25-60 and 40-80 % of cells, respectively. The corresponding figure for the best CAR-binding serotype, Ad5p, was only 25-65 %, indicating greater variability between cells from different donors than serotype Ad16p had. The other CAR-binding serotypes, Ad4p and Ad17p, were refractory to some of the gliomas, giving a maximum of only 45 and 40 % hexon expression, respectively, in the most permissive cells. Interestingly, the transduction capacity of the CAR-binding serotypes was not correlated to the level of CAR expression on the cells.

Identifiers
urn:nbn:se:umu:diva-20661 (URN)10.1099/vir.0.80084-0 (DOI)15302956 (PubMedID)
Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2011-04-04
3. Comparative analysis of the genome organization of human adenovirus 11, a member of the human adenovirus species B, and the commonly used human adenovirus 5 vector, a member of species C
Open this publication in new window or tab >>Comparative analysis of the genome organization of human adenovirus 11, a member of the human adenovirus species B, and the commonly used human adenovirus 5 vector, a member of species C
2003 (English)In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 84, no 8, 2061-2071 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus type 11 (Ad11), a member of the human adenovirus species B (HAdV-B), has a tropism for the urinary tract. The genome of Ad11 was found to comprise 34 794 bp and is 1141 bp shorter than the Ad5 genome of species HAdV-C. The G+C content of the Ad11 genome is 48.9 %, whereas that of Ad5 is 55.2 %. Ad11 and Ad5 share 57 % nucleotide identity and possess the same four early regions, but the E3 region of Ad11 could not be divided into E3A and E3B. The late genes of Ad11 and Ad5 are organized into six and five regions, respectively. Thirty-eight putative ORFs were identified in the Ad11 genome. The ORFs in the late regions, the E2B region and IVa2 show high amino acid identity between Ad11 and Ad5, whereas the ORFs in E1, E2A, E3 and E4, protein IX and the fibre protein show low amino acid identity. The highest and lowest identities were noted in the pre-terminal protein and fibre proteins: 85 % and 24.6 %, respectively. The E3 20.3K and 20.6K ORFs and the L6 agnoprotein were present in the Ad11 genome only, whereas the E3 11.6K cell death protein was identified only in Ad5. All ORFs but the E3 10.3K and L4 pVIII protein vary not only in composition but also in size. Ad11 may have a higher vector capacity than Ad5, since it has a shorter genome and a shorter fibre. Furthermore, in the E3 region, two additional ORFs can be deleted to give extra capacity for foreign DNA.

Identifiers
urn:nbn:se:umu:diva-4784 (URN)10.1099/vir.0.19178-0 (DOI)12867636 (PubMedID)
Available from: 2005-11-04 Created: 2005-11-04 Last updated: 2011-04-04Bibliographically approved
4. Construction of an E1-transcomplementing cell line for adenovirus 11p vectors
Open this publication in new window or tab >>Construction of an E1-transcomplementing cell line for adenovirus 11p vectors
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4785 (URN)
Available from: 2005-11-04 Created: 2005-11-04 Last updated: 2010-01-13Bibliographically approved
5. Analysis and characterisation of a chimpanzee adenovirus that is highly infectious to human low-passage glioma cells
Open this publication in new window or tab >>Analysis and characterisation of a chimpanzee adenovirus that is highly infectious to human low-passage glioma cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4786 (URN)
Available from: 2005-11-04 Created: 2005-11-04 Last updated: 2010-01-13Bibliographically approved

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