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Masters Thesis I

Masters Thesis I

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Published by Kumar Appari
construction and analysis of ad5 vectors for prostate cancer gene therapy
construction and analysis of ad5 vectors for prostate cancer gene therapy

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Published by: Kumar Appari on Mar 16, 2010
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1Department of Molecular BiologyUmeå UniversityS-901 87 Umeå, SwedenDegree thesis in Molecular Biology 15 ECTS-creditsSwedish Master’s level2007-03-14
Construction and Analysis of Tissue Specific Ad5 Vectors for Prostate Cancer Gene Therapy
Kumar Swamy Appari
Supervisor:Ya-fang MeiVirology,Department of ClinicalMicrobiologyUmeå University,SE-901 87 Umeå, Sweden.
The Prostate-Specific Antigen (PSA) promoter region (initial 5’ sequence), PSA promoter withenhancer sequence, and the constitutively expressed Phospho Glycerate Kinase (PGK) promoter were cloned into an Ad5 vector system with an intention to compare the efficiencyand specificity of gene expression in Prostate cancer and the control HEK293 cell lines using aGFP reporter gene. The PGK promoter was found to be more efficient compared to the PSA promoter with enhancer region; on the other hand the PSA promoter plus enhancer region wasmore efficient compared to the PSA promoter only. Further studies can be carried out in order to evaluate the specificity and the infectivity of the different types of adenoviral vectors byusing promoters of variable strength.
Prostate cancer is the third most common cause of cancer death in men after lung andcolorectal cancers (1). The conventional strategies for the treatment of metastatic prostatecancers, like chemotherapy, radiation therapy, and hormone therapy can provide onlysymptomatic palliation. On the other hand, gene therapy rather eliminates the cause of thedisease (2). Gene therapy provides the opportunity to selectively introduce genes into cancer cells targeting multiple biological processes including induction of cytotoxic and apoptoticresponses, correction of aberrant cell growth regulation and elicit anti-tumour immuneresponses in the tumour. Despite the initial setbacks due to side effects, the gene therapystrategy has evolved to an efficient approach to cancer treatment by the recent advances indefining the genetic alterations in cancer, in gene regulation, and delivery vectorology (3).The practical advantages and the application potential of the adenoviral vectors have drawn theattention of many scientists in the field of gene therapy. Adenoviral vectors are the potentialcandidates to meet the requirements of the ideal gene delivery vector for cancer treatment for the following reasons: adenoviral vectors are well characterized and thoroughly studied as amodel system for eukaryotic gene regulation, their size (around 36 kilobases) is suitable for development of large-capacity vectors with minimal viral sequence. Adenoviral vectors can beeasily generated and manipulated with good stability and can be produced in high titers (10
– 10
plaque-forming units (PFU)/mL). The adenoviral vectors exhibit a broad host range in
in vivo
with high infectivity in both dividing and quiescent cells, they do not integrateinto the host cell genome and genes thus delivered are expressed episomally with lowgenotoxicity, they exhibit wide variety of routes of administration in different tissue types and,no significant side effects were reported while demonstrating the safety of the adenoviralvectors for gene transfer (4). Here experiments were made with the
most commonly usedadenovirus vector systems, which are based on adenovirus
serotype 5 (Ad5), belonging tospecies C. These have shown promising
oncolytic activity and are being tested in the clinic for antitumor 
efficacy (5).Human adenoviruses belong to the family
. They form alarge group comprising 51 different serotypes. These 51 serotypes of human adenoviruses arefurther divided into six different species (designated from A to F) (6, 7). Ad5 from species Chas been the focus of vector development for more than two decades (8).The Ad5 vector used is replication incompetent due to deletions of the E1 and E3 genes. Thesedeletions render the virus replication defective and provide space for large foreign DNA, up to7.5 kb. The E1 gene is required in the viral assembly and is complemented in the adenovirus packaging cell line HEK293. The E3 gene encodes proteins involved in evading host immunityand is dispensable (9).
3Prostate cancer is diagnosed through the most commonly employed test called PSA testing.PSA or the prostate-specific membrane antigen is expressed exclusively by benign hyperplasticand metastatic, or the malignant prostatic epithelial, cells. The rising levels of PSA in theserum are the indications of the prostate cancer (10). PSA, which is specifically restricted tothe prostate tissue, provides an opportunity for further investigations of PSA-mediated generegulation for the purpose of prostate cancer gene therapy (11). In this experiment, the PSA promoter and the PSA enhancer regulatory DNA sequences were used to control GFPexpression. Wide ranges of possibilities are available with the tissue-specific gene expressionvector system. Tissue-specific promoters represent a powerful tool for decreasing the toxicityof the cancer gene therapy in the normal tissues. These have previously been utilized for specific mutation compensation or delivery of prodrug-converting enzymes (12).However, tissue-specific promoters can also be used for controlling crucial viral replicationregulators and consequently restriction of replication in tumor cells. Here along with the tissue-specific promoters, an investigation of the tissue non-specific promoter-based gene regulationwas attempted. For this purpose the mouse PGK promoter was chosen. The PGK gene promoter confers the highest activity among cellular promoters (13), and PGK-regulated geneexpression is independent of androgen induction. Construction of adenoviral vectors, withdifferent promoters of variable strength regulating the GFP expression, helps to evaluate theinfectivity, specificity and efficiency of the vectors. Better strategies, for cancer gene therapy,can also be developed based on these studies.
Materials and Methods:
Plasmids Used:
 pDRIVE-hPSA (Invivogen), pQBI pgk(Quantum Biotechnologies), pShuttle-CMV-GFP-SV40polyA vector, pAdEasy-Ad5 with E1 and E3 deletions (Stratagene).PCR fragments:PSA upstream promoter region (initial 5’ sequence of the PSA) was amplified from the pDRIVE-hPSA plasmid using the primers, EcoRI-PSApro-5´primer (5´CCG GAA TTC CTAGTA CAT TGT TTG C) and EcoRI-PSApro-3´primer (5´CCG GAA TTC GTG ACA CAGCTC TCC G). PSA promoter with enhancer sequence was also amplified from the pDRIVE-hPSA plasmid using the primers EcoRI-PSA-En-5´primer (5´CCG GAA TTC TGC AGG CCTCTA GAA ATC) and EcoRI-PSApro-3´primer. Further the PGK promoter sequence wasamplified from pQBI pgk plasmid using, KpnI-PGKpro-5´primer (5’CCG GGT ACC GTGCCA CCT GAC GTC G) and KpnI-PGKpro-3´primer (5’CCG GGT ACC GGC TGG ATACGT GTC C).Plasmid Constructs:
PCR-amplified PSA upstream promoter region (initial 5’ sequence of the PSA) was digestedwith EcoRI restriction enzyme to cleave the EcoRI flanks. Further the EcoRI overhangs werefilled in using Klenow enzyme (Roche) to regenerate blunt ends. The pShuttle-CMV-GFP-SV40polyA vector was subjected to KpnI digestion, to remove the CMV promoter (cytomegalovirus promoter) and to open the pShuttle vector for cloning. The opened plasmidswere then treated with Klenow enzyme to generate blunt ends and further treated with phosphatase to prevent self-religation of plasmid vector. T4 DNA Ligase (Invitrogen) was usedto clone the PCR-amplified, EcoRI-digested and Klenow-filled EcoRI-DNA-fragment, into theopened pShuttle vector. In this way, the
 pShuttle-PSApromotor-GFP-SV40polyA plasmid wasconstructed. Similarly the pShuttle-PSAenhancer-GFP-SV40polyA plasmid was constructed

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