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Adenovirus for Cancer Therapy: With a Focus on its Surface Modification
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Magnus Essand)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 914
Keyword [en]
Adenovirus, cancer, therapy, neuroblastoma, neuroendocrine, modification, Tat, PTD, cell penetrating peptide, Helicobacter pylori, NAP
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Cancer and Oncology Microbiology in the medical area
Research subject
Biology with specialization in Molecular Biology; Clinical Virology; Medical Virology; Molecular Medicine; Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-203662ISBN: 978-91-554-8700-3 (print)OAI: oai:DiVA.org:uu-203662DiVA: diva2:637259
Public defence
2013-09-06, Rudbecksalen, The Rudbeck Laboratory C11, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-08-16 Created: 2013-07-17 Last updated: 2014-01-07
List of papers
1. Adenovirus with Hexon Tat-Protein Transduction Domain Modification Exhibits Increased Therapeutic Effect in Experimental Neuroblastoma and Neuroendocrine Tumors
Open this publication in new window or tab >>Adenovirus with Hexon Tat-Protein Transduction Domain Modification Exhibits Increased Therapeutic Effect in Experimental Neuroblastoma and Neuroendocrine Tumors
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2011 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, no 24, 13114-13123 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surfaces of tumor cells. Furthermore, infected cells overproduce adenovirus fiber proteins, which are released prior to cell lysis. The released fibers block CAR on noninfected neighboring cells, thereby preventing progeny virus entry. Our aim was to add a CAR-independent infection route to Ad5 to increase the infectivity of tumor cells with low CAR expression and prevent the fiber-masking problem. We constructed Ad5 viruses that encode the protein transduction domain (PTD) of the HIV-1 Tat protein (Tat-PTD) in hypervariable region 5 (HVR5) of the hexon protein. Tat-PTD functions as a cell-penetrating peptide, and Tat-PTD-modified Ad5 showed a dramatic increased transduction of CAR-negative cell lines compared to unmodified vector. Moreover, while tumor cell infectivity was severely reduced for Ad5 in the presence of fiber proteins, it was only marginally reduced for Tat-PTD-modified Ad5. Furthermore, because of the sequence alteration in the hexon HVR, coagulation factor X-mediated virus uptake was significantly reduced. Mice harboring human neuroblastoma and neuroendocrine tumors show suppressed tumor growths and prolonged survival when treated with Tat-PTD-modified oncolytic viruses. Our data suggest that modification of Ad5 with Tat-PTD in HVR5 expands its utility as an oncolytic agent.

Keyword
adenovirus, cell penetrating peptide, Tat-PTD, neuroblastoma, neuroendocrine
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-165614 (URN)10.1128/JVI.05759-11 (DOI)000297642000029 ()
Funder
Swedish Research Council, K2008-68X-15270-04-3
Available from: 2012-01-16 Created: 2012-01-09 Last updated: 2017-12-08Bibliographically approved
2. Tat‐PTD‐modified Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy
Open this publication in new window or tab >>Tat‐PTD‐modified Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy
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2013 (English)In: Human Gene Therapy, ISSN 1043-0342, E-ISSN 1557-7422, Vol. 24, no 8, 766-775 p.Article in journal (Refereed) Published
Abstract [en]

Secretogranin III (SGC3) belongs to the granin family and is highly expressed in endocrine and neural tissues. The human SCG3 promoterhas not yet been characterized. We identified that a 0.5 kb DNA fragment upstream of the SCG3 gene can selectively drivetransgene expression in neuroblastoma cell lines. The strength of transgene expression was further increased and specificity maintained,by addition of the human achaete‐scute complex homolog 1 (ASH1) enhancer. We developed an oncolytic serotype 5‐basedadenovirus, where the SCG3 promoter and ASH1 enhancer drive E1A gene expression. The virus was further modified with a cellpenetratingpeptide (Tat‐PTD) in the virus capsid, which we have previously shown results in increased adenovirus transductionefficiency of many neuroblastoma cell lines. The virus, Ad5PTD(ASH1‐SCG3‐E1A), shows selective and efficient killing of neuroblastomacell lines in vitro, including cisplatin‐, etoposide‐ and doxorubicin‐insensitive neuroblastoma cells. Furthermore, it delays tumorgrowth and thereby prolonged survival for nude mice harboring subcutaneous human neuroblastoma xenograft. In conclusion, wereport a novel oncolytic adenovirus with potential use for neuroblastoma therapy.

Keyword
Tat-PTD, neuroblastoma, cancer therapy, adenovirus
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Clinical Virology; Medical Virology; Molecular Biotechnology; Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-203651 (URN)10.1089/hum.2012.132 (DOI)000323181200007 ()
Funder
Swedish Cancer Society, 10‐0105Swedish Cancer Society, 10‐0552Swedish Research Council, K2013‐55X‐22191‐01‐3
Note

De två (2) första författarna delar förstaförfattarskapet.

Other funds:

TheSwedish Cancer Society (10‐0105 and 10‐0552), the Swedish ChildrenCancer Foundation (PROJ10/027, NBCNSPDHEL10/013,JIN C. ET AL. 20138PROJ11/062), Gunnar Nilsson’s Cancer Foundation, the SwedishResearch Council (K2013‐55X‐22191‐01‐3) and the Marcus andMarianne Wallenberg’s Foundation.

Available from: 2013-07-16 Created: 2013-07-16 Last updated: 2017-12-06Bibliographically approved
3. Adenovirus Serotype 5 Vectors with Tat-PTD Modified Hexon and Serotype 35 Fiber Show Greatly Enhanced Transduction Capacity of Primary Cell Cultures
Open this publication in new window or tab >>Adenovirus Serotype 5 Vectors with Tat-PTD Modified Hexon and Serotype 35 Fiber Show Greatly Enhanced Transduction Capacity of Primary Cell Cultures
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1, e54952- p.Article in journal (Refereed) Published
Abstract [en]

Recombinant adenovirus serotype 5 (Ad5) vectors represent one of the most efficient gene delivery vectors in life sciences. However, Ad5 is dependent on expression of the coxsackievirus-adenovirus- receptor (CAR) on the surface of target cell for efficient transduction, which limits it's utility for certain cell types. Herein we present a new vector, Ad5PTDf35, which is an Ad5 vector having serotype 35 fiber-specificity and Tat-PTD hexon-modification. This vector shows dramatically increased transduction capacity of primary human cell cultures including T cells, monocytes, macrophages, dendritic cells, pancreatic islets and exocrine cells, mesenchymal stem cells and tumor initiating cells. Biodistribution in mice following systemic administration (tail-vein injection) show significantly reduced uptake in the liver and spleen of Ad5PTDf35 compared to unmodified Ad5. Therefore, replication-competent viruses with these modifications may be further developed as oncolytic agents for cancer therapy. User-friendly backbone plasmids containing these modifications were developed for compatibility to the AdEasy-system to facilitate the development of surface-modified adenoviruses for gene delivery to difficult-to-transduce cells in basic, pre-clinical and clinical research.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-197480 (URN)10.1371/journal.pone.0054952 (DOI)000315210400045 ()
Available from: 2013-03-26 Created: 2013-03-26 Last updated: 2017-12-06Bibliographically approved
4. An infection-enhanced oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors
Open this publication in new window or tab >>An infection-enhanced oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors
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2013 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 21, no 11, 2008-2018 p.Article in journal (Refereed) Published
Abstract [en]

Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is a major virulence factor involved in H. pylori infection. HP-NAP can mediate anti-tumor effects by recruiting neutrophils and inducing Th1-type differentiation in the tumor microenvironment. It therefore holds strong potential as a therapeutic gene. Here, we armed a replication-selective, infection-enhanced adenovirus with secretory HP-NAP, Ad5PTDf35-[Δ24-sNAP], and evaluated its therapeutic efficacy against neuroendocrine tumors. We observed that it could specifically infect and eradicate a wide range of tumor cells lines from different origin in vitro. Insertion of secretory HP-NAP did not affect the stability or replicative capacity of the virus and infected tumor cells could efficiently secrete HP-NAP. Intratumoral administration of the virus in nude mice xenografted with neuroendocrine tumors improved median survival. Evidence of biological HP-NAP activity was observed 24 hours after treatment with neutrophil infiltration in tumors and an increase of proinflammatory cytokines such as TNF-α and MIP2-α in the systemic circulation. Furthermore, evidence of Th1-type immune polarization was observed as a result of increase in IL-12/23 p40 cytokine concentrations 72 hours post-virus administration. Our observations suggest that HP-NAP can serve as a potent immunomodulator in promoting anti-tumor immune response in the tumor microenvironment and enhance the therapeutic effect of oncolytic adenovirus.

Keyword
Helicobacter pylori, Neutrophil Activating Protein, adenovirus, cancer therapy
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine; Clinical Virology; Medical Virology; Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-203649 (URN)10.1038/mt.2013.153 (DOI)000326937000007 ()23817216 (PubMedID)
Funder
Swedish Cancer Society, 10‐0105Swedish Cancer Society, 10‐0552Swedish Research Council, K2013‐55X‐22191‐01‐3
Note

De två (2) sista författarna delar sistaförfattarskapet.

Open access under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License (CC BY-NC-ND). 2013.

Supported funds:

The Swedish Children Cancer Foundation(PROJ10/027), Gunnar Nilsson’s Cancer Foundation, Marcus and Marianne Wallenberg’sFoundation

Available from: 2013-07-16 Created: 2013-07-16 Last updated: 2017-12-06Bibliographically approved

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