Publications by authors named "Johan Skog"

50 Publications

BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles.

Mol Ther Nucleic Acids 2013 Jul 23;2:e109. Epub 2013 Jul 23.

1] Department of Neurology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, Boston, Massachusetts, USA [2] Department of Radiology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, Boston, Massachusetts, USA [3] Program, Harvard-MIT Division of Health, Science, and Technology, Harvard Medical School, Boston, Massachusetts, USA.

Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.Molecular Therapy-Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28; published online 23 July 2013.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mtna.2013.28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732870PMC
July 2013

Current methods for the isolation of extracellular vesicles.

Biol Chem 2013 Oct;394(10):1253-62

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1515/hsz-2013-0141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075462PMC
October 2013

Alternative methods for characterization of extracellular vesicles.

Front Physiol 2012 7;3:354. Epub 2012 Sep 7.

Harvard Catalyst Laboratory for Innovative Translational Technologies, Harvard Medical School Boston, MA, USA.

Extracellular vesicles (ECVs) are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins, and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize ECVs. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some ECVs-specific evidence. Characterization of ECVs has also recently seen many advances with the use of Nanoparticle Tracking Analysis, flow cytometry, cryo-electron microscopy instruments, and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphys.2012.00354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435888PMC
October 2012

Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles.

Front Physiol 2012 28;3:162. Epub 2012 May 28.

Harvard Catalyst Laboratory for Innovative Translational Technologies, Harvard Medical School Boston, MA, USA.

Microvesicles are nano-sized lipid vesicles released by all cells in vivo and in vitro. They are released physiologically under normal conditions but their rate of release is higher under pathological conditions such as tumors. Once released they end up in the systemic circulation and have been found and characterized in all biofluids such as plasma, serum, cerebrospinal fluid, breast milk, ascites, and urine. Microvesicles represent the status of the donor cell they are released from and they are currently under intense investigation as a potential source for disease biomarkers. Currently, the "gold standard" for isolating microvesicles is ultracentrifugation, although alternative techniques such as affinity purification have been explored. Viscosity is the resistance of a fluid to a deforming force by either shear or tensile stress. The different chemical and molecular compositions of biofluids have an effect on its viscosity and this could affect movements of the particles inside the fluid. In this manuscript we addressed the issue of whether viscosity has an effect on sedimentation efficiency of microvesicles using ultracentrifugation. We used different biofluids and spiked them with polystyrene beads and assessed their recovery using the Nanoparticle Tracking Analysis. We demonstrate that MVs recovery inversely correlates with viscosity and as a result, sample dilutions should be considered prior to ultracentrifugation when processing any biofluids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphys.2012.00162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362089PMC
October 2012

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

Nat Med 2012 Jun;18(6):883-91

Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, Cell and Developmental Biology, Weill Cornell Medical College, New York, New York, USA.

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.2753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645291PMC
June 2012

Microvesicle-associated AAV vector as a novel gene delivery system.

Mol Ther 2012 May 7;20(5):960-71. Epub 2012 Feb 7.

Department of Neurology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, Boston, Massachusetts, USA.

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2011.303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345986PMC
May 2012

RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls.

BMC Cancer 2012 Jan 17;12:22. Epub 2012 Jan 17.

Department of Neurology, Neurosurgery and Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA 02114, USA.

Background: RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients.

Methods: Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups).

Results: Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production.

Conclusions: Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-12-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329625PMC
January 2012

Blood platelets contain tumor-derived RNA biomarkers.

Blood 2011 Sep 10;118(13):3680-3. Epub 2011 Aug 10.

Neuro-oncology Research Group, VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands.

Diagnostic platforms providing biomarkers that are highly predictive for diagnosing, monitoring, and stratifying cancer patients are key instruments in the development of personalized medicine. We demonstrate that tumor cells transfer (mutant) RNA into blood platelets in vitro and in vivo, and show that blood platelets isolated from glioma and prostate cancer patients contain the cancer-associated RNA biomarkers EGFRvIII and PCA3, respectively. In addition, gene-expression profiling revealed a distinct RNA signature in platelets from glioma patients compared with normal control subjects. Because platelets are easily accessible and isolated, they may form an attractive platform for the companion diagnostics of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2011-03-344408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224637PMC
September 2011

Brain tumor microvesicles: insights into intercellular communication in the nervous system.

Cell Mol Neurobiol 2011 Aug 8;31(6):949-59. Epub 2011 May 8.

Department of Neurology, Boston, MA, USA.

Brain tumors are heterogeneous tumors composed of differentiated tumor cells that resemble various neural cells and a small number of multipotent cancer stem cells. These tumors modify normal cells in their environment to promote tumor growth, invasion and metastases by various ways. Recent publications show that glioblastoma cells release microvesicles that contain a select subset of cellular proteins and RNAs. These microvesicles are avidly taken up by normal cells in cell culture and can change the translational profile of these cells through delivery of tumor-derived mRNAs, which are translated into functional proteins. In addition to mRNA and proteins, microvesicles have been shown to contain microRNAs, non-coding RNAs and DNA. This commentary explores the recent advances in this novel intercellular communication route and discusses the potential physiological role of microvesicles in brain tumorigenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10571-011-9697-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702172PMC
August 2011

Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences.

Nat Commun 2011 Feb 1;2:180. Epub 2011 Feb 1.

Department of Neurology, Harvard Medical School, Boston, MA 02129, USA.

Tumour cells release an abundance of microvesicles containing a selected set of proteins and RNAs. Here, we show that tumour microvesicles also carry DNA, which reflects the genetic status of the tumour, including amplification of the oncogene c-Myc. We also find amplified c-Myc in serum microvesicles from tumour-bearing mice. Further, we find remarkably high levels of retrotransposon RNA transcripts, especially for some human endogenous retroviruses, such as LINE-1 and Alu retrotransposon elements, in tumour microvesicles and these transposable elements could be transferred to normal cells. These findings expand the nucleic acid content of tumour microvesicles to include: elevated levels of specific coding and non-coding RNA and DNA, mutated and amplified oncogene sequences and transposable elements. Thus, tumour microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms1180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040683PMC
February 2011

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.

Kidney Int 2010 Jul 28;78(2):191-9. Epub 2010 Apr 28.

Division of Nephrology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ki.2010.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451567PMC
July 2010

Microfluidic isolation and transcriptome analysis of serum microvesicles.

Lab Chip 2010 Feb 8;10(4):505-11. Epub 2009 Dec 8.

BioMEMS Resource Center, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/b916199fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136803PMC
February 2010

Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.

Nat Cell Biol 2008 Dec 16;10(12):1470-6. Epub 2008 Nov 16.

Department of Neurology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA.

Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb1800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423894PMC
December 2008

miR-296 regulates growth factor receptor overexpression in angiogenic endothelial cells.

Cancer Cell 2008 Nov;14(5):382-93

Department of Neurology, Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, Boston, MA 02115, USA.

A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here, we demonstrate that a small regulatory microRNA, miR-296, has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRbeta. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccr.2008.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597164PMC
November 2008

Adenoviruses 16 and CV23 efficiently transduce human low-passage brain tumor and cancer stem cells.

Mol Ther 2007 Dec 18;15(12):2140-5. Epub 2007 Sep 18.

Department of Virology, Umeå University, Umeå, Sweden.

Most clinical protocols involving adenovirus (Ad) vectors for gene therapy use a vector based on serotype 5 (Ad5). We believe that this serotype is not suitable for all gene therapy applications and that alternative vectors based on other serotypes should be developed. We have compared the ability of Ad5, Ad11p, Ad16p, and a chimpanzee Ad (CV23) to infect human low-passage brain tumor cells as well as primary glioma cells sorted into a CD133(+) and CD133(-) population. Cancer stem cells have been shown to reside in the CD133(+) population of cells in human glioma tumors and they are of considerable interest in glioma therapy. Ad16p and CV23 infected the low-passage brain tumor cell lines and also the CD133(+) and CD133(-) primary tumor cells most efficiently. Interestingly, as the passage number of the cells increased, the infection capacity of Ad5 increased significantly, whereas this was not seen for CV23. To ensure the therapeutic effect of Ad vectors on brain tumors, the vector must be capable of addressing both the CD133(+) cancer stem cells and the CD133(-) cells of the tumor. In particular, Ad16 and CV23 are meeting this challenge.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.mt.6300315DOI Listing
December 2007

Glioma-specific antigens for immune tumor therapy.

Authors:
Johan Skog

Expert Rev Vaccines 2006 Dec;5(6):793-802

Umea University, Department of Virology, SE-901 85, Umea, Sweden.

This review describes glioma-specific antigens important in immunotherapy of glioma tumors. The structure and function of these antigens and recent immunotherapy data are summarized. Also, some important aspects of tumor formation are outlined. The roles of neuronal precursor cells and tumor stroma cells are discussed. The stroma cells of the tumor may be of interest as a target for tumor therapy, especially since they are less heterogeneous than the tumor cells. To date, the clinical benefit of immunotherapy has been very limited. Immunotherapy is, however, still an extremely promising approach to tumor therapy and it will most likely be implemented as a future treatment option for many types of tumors. The current shortcomings of immunotherapy will probably diminish as we start to understand and are able to modulate tumor-induced immunosuppression. There is also a need for a continued search for new tumor-specific antigens and to optimize protocols for vaccine administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1586/14760584.5.6.793DOI Listing
December 2006

Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells.

J Virol 2007 Jan 1;81(2):954-63. Epub 2006 Nov 1.

Department of Virology, Umeå University, Umeå, Sweden.

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01995-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797453PMC
January 2007

Efficient internalization into low-passage glioma cell lines using adenoviruses other than type 5: an approach for improvement of gene delivery to brain tumours.

J Gen Virol 2004 Sep;85(Pt 9):2627-2638

Department of Virology, Umeå University, SE-901 85 Umeå, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/vir.0.80084-0DOI Listing
September 2004

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.

J Gen Virol 2003 Aug;84(Pt 8):2061-2071

Department of Virology, Umeå University, SE-901 85 Umeå, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/vir.0.19178-0DOI Listing
August 2003

Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin.

J Gen Virol 2002 Jun;83(Pt 6):1299-1309

Department of Virology, Umeå University, SE-901 85 Umeå, Sweden1.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/0022-1317-83-6-1299DOI Listing
June 2002