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Master’s level 2007-03-14
Construction and Analysis of Tissue Specific Ad5 Vectors for Prostate Cancer Gene Therapy
Kumar Swamy Appari
Supervisor: Ya-fang Mei Virology, Department of Clinical Microbiology Umeå University, SE-901 87 Umeå, Sweden.
The Prostate-Specific Antigen (PSA) promoter region (initial 5’ sequence), PSA promoter with enhancer sequence, and the constitutively expressed Phospho Glycerate Kinase (PGK) promoter were cloned into an Ad5 vector system with an intention to compare the efficiency and specificity of gene expression in Prostate cancer and the control HEK293 cell lines using a GFP 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 was more 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 by using promoters of variable strength.
Prostate cancer is the third most common cause of cancer death in men after lung and colorectal cancers (1). The conventional strategies for the treatment of metastatic prostate cancers, like chemotherapy, radiation therapy, and hormone therapy can provide only symptomatic palliation. On the other hand, gene therapy rather eliminates the cause of the disease (2). Gene therapy provides the opportunity to selectively introduce genes into cancer cells targeting multiple biological processes including induction of cytotoxic and apoptotic responses, correction of aberrant cell growth regulation and elicit anti-tumour immune responses in the tumour. Despite the initial setbacks due to side effects, the gene therapy strategy has evolved to an efficient approach to cancer treatment by the recent advances in defining 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 the attention of many scientists in the field of gene therapy. Adenoviral vectors are the potential candidates 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 a model 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 be easily generated and manipulated with good stability and can be produced in high titers (1011 – 1012 plaque-forming units (PFU)/mL). The adenoviral vectors exhibit a broad host range in in vitro and in vivo with high infectivity in both dividing and quiescent cells, they do not integrate into the host cell genome and genes thus delivered are expressed episomally with low genotoxicity, 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 adenoviral vectors for gene transfer (4). Here experiments were made with the most commonly used adenovirus vector systems, which are based on adenovirus serotype 5 (Ad5), belonging to species C. These have shown promising oncolytic activity and are being tested in the clinic for antitumor efficacy (5).Human adenoviruses belong to the family Adenoviridae. They form a large group comprising 51 different serotypes. These 51 serotypes of human adenoviruses are further divided into six different species (designated from A to F) (6, 7). Ad5 from species C has 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. These deletions render the virus replication defective and provide space for large foreign DNA, up to 7.5 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 immunity and is dispensable (9).
Prostate cancer is diagnosed through the most commonly employed test called PSA testing. PSA or the prostate-specific membrane antigen is expressed exclusively by benign hyperplastic and metastatic, or the malignant prostatic epithelial, cells. The rising levels of PSA in the serum are the indications of the prostate cancer (10). PSA, which is specifically restricted to the prostate tissue, provides an opportunity for further investigations of PSA-mediated gene regulation 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 GFP expression. Wide ranges of possibilities are available with the tissue-specific gene expression vector system. Tissue-specific promoters represent a powerful tool for decreasing the toxicity of 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 replication regulators and consequently restriction of replication in tumor cells. Here along with the tissuespecific promoters, an investigation of the tissue non-specific promoter-based gene regulation was 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 gene expression is independent of androgen induction. Construction of adenoviral vectors, with different promoters of variable strength regulating the GFP expression, helps to evaluate the infectivity, 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-GFPSV40polyA 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 CTA GTA CAT TGT TTG C) and EcoRI-PSApro-3´primer (5´CCG GAA TTC GTG ACA CAG CTC TCC G). PSA promoter with enhancer sequence was also amplified from the pDRIVEhPSA plasmid using the primers EcoRI-PSA-En-5´primer (5´CCG GAA TTC TGC AGG CCT CTA GAA ATC) and EcoRI-PSApro-3´primer. Further the PGK promoter sequence was amplified from pQBI pgk plasmid using, KpnI-PGKpro-5´primer (5’CCG GGT ACC GTG CCA CCT GAC GTC G) and KpnI-PGKpro-3´primer (5’CCG GGT ACC GGC TGG ATA CGT GTC C). Plasmid Constructs: PCR-amplified PSA upstream promoter region (initial 5’ sequence of the PSA) was digested with EcoRI restriction enzyme to cleave the EcoRI flanks. Further the EcoRI overhangs were filled in using Klenow enzyme (Roche) to regenerate blunt ends. The pShuttle-CMV-GFPSV40polyA vector was subjected to KpnI digestion, to remove the CMV promoter (cytomegalovirus promoter) and to open the pShuttle vector for cloning. The opened plasmids were 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 used to clone the PCR-amplified, EcoRI-digested and Klenow-filled EcoRI-DNA-fragment, into the opened pShuttle vector. In this way, the pShuttle-PSApromotor-GFP-SV40polyA plasmid was constructed. Similarly the pShuttle-PSAenhancer-GFP-SV40polyA plasmid was constructed
by cloning the amplified PSA promoter with enhancer sequence into the pShuttle-CMV-GFPSV40polyA vector.
CMV promoter GFP SV40polyA
Fig 1: The pShuttle plasmid constructs with the CMV promoter, PSA promoter, PSA enhancer, PGK promoter respectively for driving the expression of the GFP reporter gene.
The pShuttle-PGKpromoter-GFP-SV40polyA plasmid was constructed by sticky end ligation of the amplified KpnI-flanked PGK promoter sequence into the KpnI-opened pShuttle-CMVGFP-SV40polyA vector, replacing the CMV promoter.
Fig 2: pShuttle plasmid Constructs: (a) pShuttle-CMV-GFP-SV40ployA. (b) pShuttle-PSApromoter-GFPSV40polyA. (c) pShuttle-PSAenhancer-GFP-SV40polyA. (d) pShuttle-PGK-GFP-SV40polyA.
Recombinant Adenoviral plasmid construction: Through the efficient homologous recombination machinery in Escherichia coli (E. coli), a recombinant adenoviral plasmid is produced by a double recombination event between co-transformed adenoviral backbone plasmid vector, pAdEasy, and a shuttle vector carrying 4
the gene of interest (14). Once the pshuttle vectors were constructed, they were linearized with PmeI and co-transformed into BJ5183 E. coli, along with pAdEasy vector by electroporation at 200 Ω, 2.5 kV, and 25 µF. Such transformed E. coli cells were selected for kanamycin resistance. The recombinant adenoviral plasmids were isolated from the transformed E. coli, using Quiagen plasmid purification kit and further identified by restriction digestion. The positive recombinants were amplified in E. coli DH5α and produced in bulk through the CsClEthidium Bromide gradient purification method. CsCl-Ethidium Bromide gradient purification procedure: 1 gm of CsCl was added to each ml of DNA solution and 0.8 ml of EtBr solution was added to 10 ml of DNA/CsCl solution after the complete dissolution of CsCl in the DNA solution (Concentration of ethidium bromide should be approximately, 740µg/ml). Final density of the solution was around 1.55 g/ml. A Beckman’s quick seal ultracentrifuge tube was filled with the prepared solution and centrifuged at 50,000 rpm for 18 hours in Beckman VTi65 rotor. Two bands of DNA were found in the centre of the tube. The upper band contained nicked circular plasmid DNA and the lower band consisted of closed circular plasmid. The red pellet at the bottom of the tube consisted of ethidium bromide and RNA complexes. The lower band, containing the closed circular plasmid DNA, was collected from the tube using an 18-gauge hypodermic needle. CsCl was removed from the solution by diluting with 3 volumes of water and precipitating DNA with 2 volumes of ethanol and then centrifuging this solution at 10,000g for 15 minutes at 4oC. Finally, the precipitated DNA was dissolved in approximately 1 ml of TE buffer of pH 8.0. Production of Virus Particles: For the production of the Virus particles, the purified recombinant Ad plasmid DNA was linearized with PacI (Fig. 3) and then transfected into HEK293 cells, where the deleted viral assembly genes were complemented.
Fig 3: Pac I Linearized Recombinant Adenoviral DNA with the promoter of choice and the GFP and SV40polyA tail.
Cell culture: Prostate Cancer cell line, LNCaP was grown in RPMI 1640 medium. This medium was supplemented with 10% FCS, 2% HEPES, 100 Units/ml penicillin and 100 µg/ml streptomycin. HEK293 cell line was maintained in DMEM, supplemented with 10% FCS, 2% HEPES, 100 Units/ml penicillin, and 100 µg/ml streptomycin. R1881 synthetic steroid hormone, which binds with high affinity to the androgen receptors, was used at 10-9 M as a final concentration to maintain the LNCaP cells. Transfection of HEK293 cells and LNCap cells: Lipofectamine 2000 (Invitrogen) was used in transfection of HEK293 and LNCaP cells. On an average 800 ng of DNA, from each of the construct, was used to transfect the cell lines in 24 well plates. Antibiotic-free medium was used during transfection and after 12 hrs, the medium
was replaced with medium containing antibiotics (100 units/ml penicillin, and 100 µg/ml streptomycin).
Results and Discussion:
One in ten cases of cancer in men is accounted to Prostate Cancer. The conventional treatments provide only short time relief from the disease, while gene therapy continues to emerge as a promising adjuvant to treatment of Prostate cancer. Several viral vectors are designed to make use of the qualities of viruses as delivery agents, where the virally encoded genes are removed to introduce therapeutic genes (15). Adenoviral vectors have been investigated extensively for gene therapy. Adenovirus type 5 is the most commonly used vector in clinical trials on gene therapy for prostate cancer. Adenoviral vectors have been modified by deletion of the E1 and other genes, including E2 and E3. These deletions were made not only to increase the capacity for large foreign DNA but also to decrease the immunogenicity (16, 17). Tissue-specific gene expression using tissue-specific promoters is a very useful way to restrict the gene expression to the target tissue. Gene therapy, using tissue-specific promoters, provides the opportunity to selectively target the therapeutic genes to malignant cells in the specific target tissue and thus avoiding toxic side effects in the non-malignant cells (18). The prostatespecific promoters, PSA enhancer and the initial 5’ sequence of the PSA promoter, are specific to the prostate organ. Adenoviral vectors, being specific to the malignant and dividing cells, can help target the malignant prostate cancer cells, for efficient gene transfer and expression. The PSA expression is induced primarily by androgens at the transcriptional level via the androgen receptor (AR). The AR interacts with its consensus DNA binding site, GGTACAnnnTGTT/CCT, called the androgen response element (ARE) and thus regulates transcription (19). The PSA promoter region without enhancer sequence has low activity and specificity but the addition of enhancer sequence that contains ARE, increases expression (1). Three pShuttle plasmids were constructed, with initial 5’ sequence of the PSA promoter, PSA promoter with enhancer sequence and PGK promoter, with the GFP reporter gene under their control. The GFP expression system allows comparison between the gene expression levels from the different promoters; tissue-specific PSA promoter (with initial 5’ sequence), PSA promoter with enhancer sequence, and the tissue non-specific and constitutively expressed PGK promoter. Thus the relative efficiency and specificity of the different promoters can be evaluated easily. The pShuttle plasmid constructs were subjected to restriction digestion analysis and gene sequencing for confirmation of successful cloning. Restriction digestion of pShuttle-CMV-GFP-SV40polyA construct with EcoRI (Fig 4A: lane C20) shows the CMV promoter band of around 0.44 kb. EcoRI digestion of pShuttlePSApromoter-GFP-SV40polyA (Fig 4A: lane PSAp) demonstrates the PSA promoter band of approximately 0.7 kb. Similarly the EcoRI digestion of pShuttle-PSApromoter-GFPSV40polyA (Fig 4A: lane PSAe) resulted in a 1.5 kb band corresponding to the size of the PSA enhancer sequence. KpnI digestion of pShuttle-PGK-GFP-SV40polyA (Fig 4C: lane PGK) resulted in an approximately 0.5 kb band, which confirms the successful cloning.
23-kb 9.4-kb 1,3-kb 1-kb 0.87-kb 0.6kb C)
Fig 4: Restriction digestion analysis of the pShuttle plasmid constructs and the recombinant adenoviral plasmid with PSA enhancer. A)EcoRI digestion of the constructs: pShuttle-CMV-GFP-SV40polyA (C20), pShuttle-CMVde-GFP-SV40 polyA (CMVde: CMV Promoter deleted), pShuttle-PGK-GFP-SV40polyA (PGK), pShuttle-PSApromoterGFP-SV40polyA (PSAp), pShuttle-PSAenhancer-GFP-SV40polyA (PSAe). B) HindIII digestion for detection of the orientation (forward) of the promoters. C) KpnI digestion for further confirmation of the pShuttle-PGK-GFP-SV40polyA construct (PGK). D) Restriction analysis for the confirmation of the Ad5 recombinant Ad5-PSAenhancer-GFP-SV40.
Further HindIII restriction digestion was done on all the constructs to detect the orientation of the promoters cloned upstream to the GFP reporter gene (Fig 4B). HindIII digestion of the pShuttle contructs with CMV promoter, PGK promoter, PSA promoter and PSA promoter with enchancer resulted in bands of approximately 1.7 kb, 1.8 kb, 1.3 kb, and 2.6 kb, respectively (Fig 4B lanes: C20, PGK, PSAp, PSAe). Sequencing of all the pShuttle constructs further confirmed the successful cloning of the promoters in right orientation upstream of the GFP reporter gene. After successful cloning of the promoters into the pShuttle vector upstream of the GFP reporter gene, GFP expression studies were carried out in both the HEK293 and LNCaP prostate cancer cell lines. GFP expression studies in two different cell lines help in evaluating the tissue-specific nature of the constructs. GFP expression of the construct pShuttle-CMVGFP-SV40polyA in HEK293 cells (Fig 5:1a) was used as a control for the experiment. The pShuttle-PGK-GFP-SV40polyA construct demonstrated an efficient and strong GFP expression in HEK293 cells (Fig 5:1b). pShuttle-PSApromoter-GFP-SV40polyA, pShuttlePSAenhancer-GFP-SV40polyA constructs did not show any GFP expression in the HEK293 cells confirming their tissue-specific nature.
HEK 293 cells 1a 1b
Fig 5: GFP expression studies. 1a) GFP expression from the CMV promoter in HEK 293 cells can be observed. 1b) Strong GFP expression from the mouse PGK promoter can be seen in the HEK 293 cells. 2a), 2b) and 2c) represent the LNCap cells transfected with CMV promoter, PGK promoter and PSA enhancer constructs, respectively.
A similar experiment was performed in the LNCaP prostate cancer cell lines, where the GFP expression of pShuttle-CMV-GFP-SV40polyA and pShuttle-PGK-GFP-SV40polyA was found to be similar to that in the HEK293 cells, but very few cells were expressing GFP protein as the transfection was not as efficient as in HEK293 cells (Fig 5 2a, 2b). Further, the GFP expression of the pShuttle-PSAenhancer-GFP-SV40polyA construct in LNCaP cells was found to be lower than the GFP expression of pShuttle-PGK-GFP-SV40polyA (Fig 5:2c). The GFP expression of pShuttle-PSApromoter-GFP-SV40polyA was much lower than the pShuttlePSAenhancer-GFP-SV40polyA (data not shown).The GFP expression studies demonstrated that the constitutively expressed PGK promoter is more efficient than the PSA promoter with enhancer sequence and the PSA promoter with enhancer sequence is more efficient than the PSA promoter without enhancer sequence. The PSA promoter and PSA promoter with enhancer sequence did not show GFP expression in the HEK293 cells, while GFP expression can be found in the LNCaP prostate cancer cell lines (Fig. 5). Thus, the tissue-specific nature of both the PSA promoter and the PSA promoter with enhancer was clearly established. The tissue-specific promoters can be further improved through construction of chimeric promoters. Chimeric constructs of PSA enhancer through multimerization of the key enhancer and the promoter elements augment the prostate-specific expression (20). Combining the PSA enhancer and rat probasin or PGK promoter for strong specificity and high expression of transgenes strengthens tissue-specific therapeutic transgene expression in prostate cancer cells and tissues (21). However the tissue-specific promoters based on PSA are androgen-dependent and cannot be of much use in androgen-independent metastatic prostate cancer or recurrent hormone refractory prostate cancer (HRPC). Such a limiting factor of androgen requirement can be overcome. This was made possible by using long (5837-bp) PSA promoter for cancerspecific gene toxic gene therapy of PSA-positive disease without the requirement of androgen. This long PSA promoter could be used to target both the androgen-dependent and androgen-
independent prostate cancers (22). A new highly augmented prostate-specific two-step transcriptional amplification (TSTA) was developed by Sato M. et.al. Though the TSTA activity is AR-dependent, it recapitulates the functional status of endogenous AR, where the AR function is activated in HRPC despite the castrated levels of androgen (23). After successful cloning of the fragments of interest into the pShuttle vector in front of the GFP reporter gene and successful GFP expression in both the HEK 293 and LNCaP cell lines, recombinations were performed in BJ5183 E.coli bacteria to introduce the promoters with the GFP-SV40polyA expression system into the Ad5 adenoviral plasmids (AdEasy). The recombinants were screened for kanamycin resistance and then subjected to restriction digestion analysis to check the integrity of the adenoviral genome in the recombinant adenoviral plasmids. Recombinant pAdEasy-PSApromoter-GFP-SV40polyA (Pr7) and recombinant pAdEasy-PSAenhancer-GFP-SV40polyA (Er5 and Er8) were digested with PacI and SpeI restriction enzymes (Fig: 6, lane 1, 2 and 3 respectively). The double digestion of recombinants named Pr7, Er5 and Er8 with PacI and SpeI resulted in larger bands of sizes around 27.2 kb and 6.2 kb corresponding to the adenoviral genome as expected.
27.2 kb 6.2 kb 9.3 & 7.6 kb 4.5 kb 3.8 kb 2.6 kb 2.2 kb 2.0 kb
Fig 6: Restriction enzyme digestion of recombinant Ad5 vector constructs. Recombinant Ad plasmids with PSApromoter (Pr7) and PSAenhancer (Er5 and Er8) genes were digested with PacI and SPeI (lanes: 1, 2, and 3, respectively). Recombinant Ad plasmids with PGK promoter fragment insertions (Kr7 and Kr8) were digested with PacI and EcoRV (lanes: 4, 5). All the expected bands from the Ad plasmids were found in the restriction digestion analysis.
Recombinant pAdEasy-PGK-GFP-SV40polyA (Kr7 and Kr8) were identified by PacI and EcoRV restriction digestion (Fig: 6B, lane 4 and 5). The restriction digestion of Kr7 and Kr8 recombinants produced bands of sizes around 9.3 kb, 7.6 kb, 4.5 kb, 3.8 kb, 2.6 kb, 2.2 kb, 2.0 kb, and 1.2 kb according to the PacI and EcoRV sites in the adenoviral genome. A few extra bands were also found corresponding to the insertions from the shuttle vector. Thus, the adenoviral genome in the potential recombinants was established to be intact.
The recombinant plasmids can be easily identified by PacI restriction endonuclease digestion. After PacI digestion a fragment of more than 30 kb and a smaller fragment of 3 kb or 4.5 kb are produced. A smaller 3 kb fragment is produced if the recombination took place between the left arms of the pShuttle vector and the pAdEasy adenoviral plasmid. A larger 4.5 kb fragment is produced if the recombination took place between the origins of replication; such recombinants are as useful as those generated by recombination of the left arm sequences (14).
30 kb or more 4.5 kb 3.0 kb
Fig 7: Identification of recombinant Ad5 vector constructs by using PacI restriction endonuclease digestion. Recombinant Ad plasmids with PSA promoter (Pr7) and PSA enhancer (Er5) fragments and recombinant Ad plasmids with PGK promoter fragment insertion (Kr7) were digested with PacI (lanes: 1, 2, and 3, respectively).
PacI digestion of pAdEasy-PSAenhancer-GFP-SV40polyA (Er5) produced a larger fragment of more than 30 kb and a smaller fragment of 4.5 kb, indicating that the recombination took place at origins of replication (Fig 7, lane 1). PacI digestion of pAdEasy-PSApromoter-GFPSV40polyA (Pr7) and pAdEasy-PGK-GFP-SV40polyA (Kr7) yielded a larger fragment of more than 30 kb and a smaller fragment of 3 kb, indicating that the recombination took place between left arm sequences (Fig 7, lane 2 and 3). The cloning of the PSA Promoter and the PSA promoter with enhancer sequence was confirmed through sequencing of the recombinant adenoviral plasmid constructs Pr7 and Er5. However, sequencing of the recombinant adenoviral plasmid Kr7, for the presence of PGK promoter, failed. Further, the recombinant adenoviral plasmids are to be transfected into the LNCaP and HEK293 cell lines for GFP expression studies. Recombinant adenoviral particles can be produced in the HEK293 cell lines. The recombinant adenoviral plasmid constructs are linearized by PacI digestion and transfected into HEK293 cells for the production of the virus particles. The prostate cancer can be transcriptionally targeted by making the adenoviral vectors prostate-specific, using prostate-specific gene promoters such as PSA promoter and PSA promoter with enhancer sequence. Given that the PSA promoter/enhancer are transcriptionally regulated by AR and the previous studies indicating the detection of AR and
PSA expression in all stages of prostate cancer, the PSA-based expression strategy in designing the adenoviral vectors might evolve as a potential approach in prostate cancer gene therapy. Further studies can be carried out using a similar promoter strategy, to evaluate the efficiency, specificity and infectivity of Novel adenoviral vectors such as, Adenovirus type 11. Ad11 a novel plasmid based adenovirus vector, made replication-incompetent by deletion of the E1 gene. Ad11 is a newly characterized subspecies B:2 member that could be a vector candidate for human gene therapy and vaccination since it has lower seroprevalance in the human population and because of its affinity for a non-CAR receptor which is expressed in all human cells (24). Ad11 has markedly higher binding affinity and infectivity than Ad5 for the endothelial cell lines (HMEC) and all carcinoma cell lines studied (cell lines of hepatoma (HepG2), breast cancer (CAMA and MG7), prostatic cancer (DU145 and LNCaP) and laryngeal cancer (Hep2) (25). Compared to Ad5, Ad11 seroprevalence is significantly low and Ad11 has higher efficiency to transduce different cell types of the human body. Thus an Ad11based vector is a novel candidate as a vehicle of gene transfer for gene therapy and vaccination (26). Comparative studies to evaluate the adenoviral vectors based on both tissue-specific and tissue non-specific promoters will provide better options in choice of the efficient vector for gene therapy. Such studies can help improve the gene therapy strategy for prostate cancer.
I sincerely thank Prof. Göran Wadell for providing me an opportunity to do my thesis in his lab and for his valuable advice. I thank Ya-fang Mei my supervisor for guiding me throughout the project with valuable suggestions. I also thank lab assistant Kristina Lindman for her technical support. Finally I thank the people at Virology department for their kind cooperation.
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