GAMER-Ad: a novel and rapid method for generating recombinant adenoviruses
2021; Cell Press; Volume: 20; Linguagem: Inglês
10.1016/j.omtm.2021.01.014
ISSN2329-0501
AutoresFiras Hamdan, Beatriz Martins, Michaela Feodoroff, Yvonne Giannoula, Sara Feola, Manlio Fusciello, Jacopo Chiaro, Gabriella Antignani, Mikaela Grönholm, Erkko Ylösmäki, Vincenzo Cerullo,
Tópico(s)CAR-T cell therapy research
ResumoOncolytic adenoviruses have become ideal agents in the path toward treating cancer. Such viruses have been engineered to conditionally replicate in malignant cells in which certain signaling pathways have been disrupted. Other than such oncolytic properties, the viruses need to activate the immune system in order to sustain a long-term response. Therefore, oncolytic adenoviruses have been genetically modified to express various immune-stimulatory agents to achieve this. However, genetically modifying adenoviruses is very time consuming and labor intensive with the current available methods. In this paper, we describe a novel method we have called GAMER-Ad to genetically modify adenovirus genomes within 2 days. Our method entails the replacement of the gp19k gene in the E3 region with any given gene of interest (GOI) using Gibson Assembly avoiding the homologous recombination between the shuttle and the parental plasmid. In this manuscript as proof of concept we constructed and characterized three oncolytic adenoviruses expressing CXCL9, CXCL10, and interleukin-15 (IL-15). We demonstrate that our novel method is fast, reliable, and simple compared to other methods. We anticipate that our method will be used in the future to genetically engineer oncolytic but also other adenoviruses used for gene therapy as well. Oncolytic adenoviruses have become ideal agents in the path toward treating cancer. Such viruses have been engineered to conditionally replicate in malignant cells in which certain signaling pathways have been disrupted. Other than such oncolytic properties, the viruses need to activate the immune system in order to sustain a long-term response. Therefore, oncolytic adenoviruses have been genetically modified to express various immune-stimulatory agents to achieve this. However, genetically modifying adenoviruses is very time consuming and labor intensive with the current available methods. In this paper, we describe a novel method we have called GAMER-Ad to genetically modify adenovirus genomes within 2 days. Our method entails the replacement of the gp19k gene in the E3 region with any given gene of interest (GOI) using Gibson Assembly avoiding the homologous recombination between the shuttle and the parental plasmid. In this manuscript as proof of concept we constructed and characterized three oncolytic adenoviruses expressing CXCL9, CXCL10, and interleukin-15 (IL-15). We demonstrate that our novel method is fast, reliable, and simple compared to other methods. We anticipate that our method will be used in the future to genetically engineer oncolytic but also other adenoviruses used for gene therapy as well. IntroductionIn the clinic, anti-cancer agents target rapidly dividing cells or single genetic mutations. Advancements in genome sequencing have led us to understand that cancer is not monogenic but rather a complex and heterogeneous disease.1Hyman D.M. Taylor B.S. Baselga J. Implementing Genome-Driven Oncology.Cell. 2017; 168: 584-599Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar This explains why the use of single genetic mutation agents over the past decades have not yielded significant full response rates or cures as once expected. Therefore, scientists have stopped hunting for individual tumor suppressor genes or oncogenes and started investigating methods in disrupting whole tumorigenic biological pathways.2Ylösmäki E. Cerullo V. Design and application of oncolytic viruses for cancer immunotherapy.Curr. Opin. Biotechnol. 2020; 65: 25-36Crossref PubMed Scopus (45) Google Scholar Oncolytic viruses are the ideal agents in achieving this, because such viruses are able to thrive in tumor cells, where such malignant pathways have been activated or disrupted, and exploit metabolic pathways that characterize tumorigenesis.2Ylösmäki E. Cerullo V. Design and application of oncolytic viruses for cancer immunotherapy.Curr. Opin. Biotechnol. 2020; 65: 25-36Crossref PubMed Scopus (45) Google Scholar Also, oncolytic viruses have extensively been shown to stimulate systemic host immune responses. The tumor microenvironment is immunosuppressive and boosting the immune system has been observed to have significant anti-tumor effects.2Ylösmäki E. Cerullo V. Design and application of oncolytic viruses for cancer immunotherapy.Curr. Opin. Biotechnol. 2020; 65: 25-36Crossref PubMed Scopus (45) Google Scholar Hence, the dual mechanism oncolytic viruses possess makes them interesting therapy agents.To date, only one oncolytic virus has been granted US Food and Drug Administration (FDA) approval for treatment despite years of extensive investigation.3Pitt J.M. Marabelle A. Eggermont A. Soria J.C. Kroemer G. Zitvogel L. Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy.Ann. Oncol. 2016; 27: 1482-1492Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar One of the reasons is that oncolytic viruses have been generally seen as direct tools for killing cancer due to their tumor-specific tropism. A growing body of evidence has shown that the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects.4Andtbacka R.H.I. Kaufman H.L. Collichio F. Amatruda T. Senzer N. Chesney J. Delman K.A. Spitler L.E. Puzanov I. Agarwala S.S. et al.Talimogene laherparepvec improves durable response rate in patients with advanced melanoma.J. Clin. Oncol. 2015; 33: 2780-2788Crossref PubMed Scopus (1518) Google Scholar Therefore, to make more significant advances with such therapies there has been a shift in focus from viewing oncolytic viruses not solely as direct oncolytic tools but also as immunotherapies. Scientists have equipped oncolytic viruses with multiple immune-stimulatory molecules that have enhanced anti-tumor effects. For example, oncolytic viruses have been engineered to express molecules like interleukins (IL-2, IL-12, IL-15, and IL-18), chemokines (CCL5, CCL19, CCL20, and CCL21), immune-activating ligands (CD40L), bi-specific T cell engager molecules, and much more (all reviewed in Ylösmäki et al.2Ylösmäki E. Cerullo V. Design and application of oncolytic viruses for cancer immunotherapy.Curr. Opin. Biotechnol. 2020; 65: 25-36Crossref PubMed Scopus (45) Google Scholar).Such oncolytic viruses range from adenoviruses, herpes simplex viruses, vaccinia, Newcastle disease virus, and much more. However, adenoviruses have drawn much of the attention due to their numerous advantages. First, the adenoviral genome is highly stable, which leads to easier large-scale production.5Marelli G. Howells A. Lemoine N.R. Wang Y. Oncolytic viral therapy and the immune system: A double-edged sword against cancer.Front. Immunol. 2018; 9: 866Crossref PubMed Scopus (138) Google Scholar Also, they are able to infect most human cells due to the high expression of the viral coxsackievirus and adenovirus receptor (CAR) throughout the human body. This high infectivity subsequently leads to higher expression levels and sustainability of transgenes. Moreover, anti-adeno immunity can be circumvented by attenuating adenovirus vectors. In addition, because of the extensive research done on adenoviruses, the safety dosing and routes of administrations have been established.6Kennedy M.A. Parks R.J. Adenovirus virion stability and the viral genome: size matters.Mol. Ther. 2009; 17: 1664-1666Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Finally, the tropism of the virus can be easily manipulated by modifying its viral capsid.7Wold W.S. Toth K. Adenovirus vectors for gene therapy, vaccination and cancer gene therapy.Curr. Gene Ther. 2013; 13: 421-433Crossref PubMed Scopus (313) Google Scholar For example, Zsolt and colleagues8Yoon A.R. Hong J. Kim S.W. Yun C.O. Redirecting adenovirus tropism by genetic, chemical, and mechanical modification of the adenovirus surface for cancer gene therapy.Expert Opin. Drug Deliv. 2016; 13: 843-858Crossref PubMed Scopus (35) Google Scholar, 11Sebestyen Z. de Vrij J. Magnusson M. Debets R. Willemsen R. An oncolytic adenovirus redirected with a tumor-specific T-cell receptor.Cancer Res. 2007; 67: 11309-11316Crossref PubMed Scopus (20) Google Scholar were able to redirect adenoviruses to expand solely in tumor cells by replacing the adenoviral fiber knob (responsible for binding to CAR on target cells) with a T cell receptor specific to a unique class of tumor antigens.Despite all the advantages, starting from a cDNA of interest to modifying an adenovirus able to express such genes involves many steps and significant time to be invested. The most common method used is homologous recombination using a shuttle plasmid and the full adenovirus genome. However, this methodology is very time consuming, inefficient, and labor intensive. Here we present GAMER-Ad (Gibson Assembly Mediated Recombination), a novel method for engineering recombinant viruses that is simple, highly efficient, and takes around 4 days to obtain an adeno full genome with the gene of interest (GOI) inserted (Figure 1). Our method is based on directly substituting the GOI into the E3-gp19k region via the well-known molecular cloning method Gibson Assembly (GA).9Gibson D.G. Young L. Chuang R.Y. Venter J.C. Hutchison 3rd, C.A. Smith H.O. Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat. Methods. 2009; 6: 343-345Crossref PubMed Scopus (5232) Google Scholar,10Lee C.S. Bishop E.S. Zhang R. Yu X. Farina E.M. Yan S. Zhao C. Zheng Z. Shu Y. Wu X. et al.Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine.Genes Dis. 2017; 4: 43-63Crossref PubMed Scopus (296) Google Scholar Gibson Assembly is a molecular cloning method allowing the assembly of multiple DNA fragments in one isothermal reaction containing three enzymes: exonuclease, DNA polymerase, and DNA ligase. The method requires a base overlap of 20–40 nucleotides among the fragments to be assembled. This overlap will allow the exonucleases to chew the 5′ ends, allowing the seamless joining of adjacent fragments. In this study, we added overlapping regions of the adenovector backbone into the 3′ and 5′ ends of the GOI, and, using the already commercially available GA Master Mix, we were able to replace the gp19k gene in the E3 region of the adenovector backbone with our GOI. In this paper, we describe the use of this method for the creation of three genetically modified adenoviruses to express CXCL9, CXCL10, and IL-15.ResultsExcising the E3 region and constructing the GOIThe genetic modification of the described viruses was possible by using a vector containing the Ad5/3-Δ24-E3+ genomic DNA. Our novel methodology consists of replacing the gp19k region of the E3 gene with our GOI using the GA method. First, the E3 region from the adenovirus was removed by taking advantage of the convenient and inherent restriction enzymes flanking the gene, BarI and SrfI (Figure 3A). After digestion, products were loaded on an agarose gel, and a clear band between 3 kb and 4 kb can be observed, indicating that the E3 gene (3,398 bp) was excised successfully (Figure 3B).The GOI to replace the gp19k region consisted of a Citomegalovirus (CMV), coding sequence of IL-15, and a poly(A). The CMV and poly(A) entailed 40 overlapping nucleotides to the 5′ and 3′ ends of the excised Ad-5/3-Δ24-E3+, respectively, to ensure a correct assembly. Additionally, the poly(A) fragment also contained all the coding sequences of the E3 region except for the gp19k gene. As shown in Figure 2, all three components were amplified via PCR. Moreover, using our primers, 40 overlapping nucleotides were added to the 5′ and 3′ ends of the IL-15 fragment allowing assembly with poly(A) and CMV fragments (Figure 3C). Using the GA Master Mix, all three components were assembled together. A PCR using a forward primer flanking the 5′ end of the CMV and reverse primer flanking the 3′ of poly(A) demonstrated that assembly was successful and in the correct orientation (Figure 3D).Figure 2Cloning strategy of GAMER-AdShow full captionThe GOI was constructed made up of three components: a CMV starting sequencing, followed by the coding sequencing of the chemokines, and finally ending in a poly(A) tail. The chemokine coding sequences contained 40 nucleotides in their 5′ and 3′, which are homologous to the ends of the CMV and poly(A) fragment, respectively. The CMV and poly(A) tail also contain all the genes required for E3 except the gp19k gene. All three fragments were then assembled together using the GA method. The Ad-5/3 genome was excised with BarI and SrfI to liberate the E3 region. The GOI was then inserted into the excised genome, again using the GA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Releasing the E3 region and constructing the GOIShow full caption(A) A schematic representation of the linearization of the Ad5/3 genome using BarI and SrfI. (B) Ad5/3 was linearized using the mentioned restriction enzymes and loaded onto an agarose gel in lane 2. Lane 1 represents a 1 kb gene ruler from Thermo Fisher. (C) The CMV, poly(A), and chemokine fragments were amplified and loaded onto an agarose gel in lanes 2, 3, and 5, respectively. Lanes 1 and 4 were loaded with 1 kb gene ruler from Thermo Fisher. (D) Schematic representation of the assembly of the GOI. (E) After assembling all three fragments of the GOI using the GA, the final fragment containing CMV-cytokine-poly(A) was amplified using PCR and loaded on a gel. Lane 2 represents the amplified GOI, while lane 1 represents a 1 kb gene ruler from Thermo Fisher.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Assembling the GOI into the adenovirus genomeFollowing adenovector digestion and GOI construction, the two components were assembled together using again the GA Master Mix. The GA products were transformed in E. coli cells, and eight colonies were taken for colony PCR. Using primers flanking the CMV and poly(A) tail (only found in the GOI), we saw that from eight colonies, six of them had the GOI (Figure 4A). Three positive colonies (based on colony PCR) were selected, grown in Lysogeni Broth (LB) medium, and the DNA was isolated. To further check whether replacement of E3 with the GOI in the adenovector was successful, constructs underwent restriction enzyme analysis and Sanger sequencing. Isolated DNA from the three colonies and unmodified adenovector were digested using EcoRI. In case of success in replacing gp19k with the GOI, a band should be observed at 2,075 bp, compared to a 2,718 bp band when gp19k remains (Figure 4B). Digestion with EcoRI demonstrated that gp19k was replaced in all three selected colonies (Figure 4C). Sanger sequencing was then performed and confirmed that all the three selected clones had the gp19k gene replaced by the GOI (Figure 4D). To further illustrate the rapidness and simplicity of our method, we engineered two new Ad5/3 viruses expressing CXCL9 and CXCL10. The same process was used, and each virus took an average of 2 days to be cloned.Figure 4Screening for positive colonies for the IL-5-expressing virusShow full caption(A) After transformation of GA products, eight colonies were chosen (lanes 2–9) and a colony PCR was conducted. Primers flanking the CMV and IL-15 coding region were used. As a positive control, the GA of CMV-IL-5-poly(A) (lane 10) was used. Lane 1 represents a 1 kb gene ruler from Thermo Fisher. (B) Simulation of expected lanes where unmodified adenovector is cut with EcoRI (lane 3) compared to positive adenovirus clones successfully having the gp19k replaced with the GOI (lane 2). Lane1 represents a 1 kb gene ruler from Thermo Fisher. (B) Actual representation of three colonies transformed with the assembled adenovirus containing the GOI (lanes 2, 3, and 4). Lane 5 represents wild-type adenovirus cut with EcoRI, while lane 1 represents a 1 kb gene ruler from Thermo Fisher. (C) Three samples of Sanger sequencing from one of the positive sequences. Original sequence represents the reference sequence of IL-15.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Viral production and Ad5/3-Δ24 contamination checkAfter successfully constructing adenovectors expressing CXCL9, CXCL19, and IL-15, we linearized the genomes with PacI to release the vector sequence and expose the viral ITRs required for the initiation of the viral DNA replication in the host cell. The digestion product was transfected into A549. Viral plaques appeared on the cell layer 7 days post transfection, suggesting successful rescue of recombinant viruses (Figure 5A). Packaging cells were lysed to release the virus, and the viral crude was utilized for further amplification of the virus. Viruses were successfully purified using CsCl, and the viral particle titer and infectious unit titer were determined.Figure 5Viral production with lack of Ad5/3-Δ24 contaminationShow full caption(A) Representative images of A549 cells transfected with IL-15-expressing adenovirus and wild-type virus. Images were taken 9 days post-transfection at two different magnification, 10× and 40×. (B) Ad5/3-Δ24 contamination was checked by amplifying the gp19k region (Ad5/3-Δ24) or the CMV and poly(A) region (GOI). A simulation of what should be expected is presented and type of samples are annotated. Lane MW represents a 1 kb gene ruler from Thermo Fisher. Lanes 4 and 9 represent Ad5/3-Δ24 virus. Lanes 1 and 6 represent CXCL9-expressing adenovirus. Lanes 2 and 7 represent CXCL10-expressing adenovirus. Lanes 3 and 8 represent IL-15-expressing adenovirus. (C) The actual representation of the wild-type contamination PCR assay. Lane 1 represents a 1 kb gene ruler from Thermo Fisher. Lanes 5 and 9 represent the unmodified Ad5/3-Δ24 virus. Lanes 2 and 6 represent CXCL9-expressing adenovirus. Lanes 3 and 7 represent CXCL10-expressing adenovirus. Lanes 4 and 8 represent IL-15-expressing adenovirus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)After, we sought to determine whether there was an Ad5/3-Δ24 contamination in the purified preparations of the cloned chemokine expressing Ad-5/3. We designed two different PCR protocols, with the first protocol having primers flanking the gp19k gene and the second having primers binding to CMV and poly(A) (only found in the cloned Ad5/3-Δ24) (Figure 5B). Purified virus samples from the cytokine-expressing viruses and unmodified Ad5/3-Δ24 were boiled to release the DNA from the viral capsid and were used as template for the PCRs. When primers flanking the gp19k gene were used, a band was only seen with the Ad5/3-Δ24 virus. As expected, when primers binding to the CMV and poly(A) were added, corresponding bands were seen in all cloned Ad-5/3 viruses, but no band was observed with the Ad-5/3-Δ24 virus (Figure 5C). Hence, this demonstrates that our method does lead to Ad5/3-Δ24 virus contamination.Viral replication and fitness of cloned virusesFollowing the cloning and purification of Ad-5/3 viruses expressing CXCL9, CXL10, or IL-15, we wanted to check that method and modification used did not affect the oncolytic fitness or replication of the viruses. All viruses have a well-known 24-bp deletion in the E1A region conditioning such viruses to replicate only in Rb-deficient cells. As a result, two tumor models, lung carcinoma cell (A549) and triple-negative breast cancer cells (MDA-MB-436), were infected with our cloned viruses and unmodified Ad5/3-Δ24 virus. Oncolysis was observed at day 3 post infection in a dose-dependent fashion in both cell lines (Figures 6A and 6B ). Oncolysis levels of the cloned viruses resembled those of the unmodified virus, indicating that our cloning method did not affect oncolytic potency or virus replication. To further corroborate this, two murine cell lines, B16F10 and 4T1, were also studied. Human adenoviruses serotype 5 can infect murine cell lines but are unable to replicate. As expected, no cell death was observed with either murine cell line when infected with the unmodified and cytokine-expressing Ad-5/3 viruses (Figures 6C and 6D). These data illustrate that our novel cloning methodology does not affect oncolytic fitness or replication.Figure 6Oncolytic fitnessShow full caption(A–D) Cell viability assay of (A) A549, (B) MDA-MB-436, (C) B16F10, and (D) B16F1 cell lines. Cell lines were infected with CXCL9 (blue), CXCL10 (red), and IL-15 (green)-expressing adenoviruses along with wild-type virus (purple). Cell viability was checked after 3 days using an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. The data are presented as mean ± SD (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Chemokine expression and migration induction from cloned virusesThe next step of the cloned viruses' in vitro characterization was to investigate the levels of expression of the cytokine upon virus infection. MDA-MB-436 cells were infected with the unarmed and the cytokine viruses, separately. The cell's supernatant was collected at day 2 post infection. All cloned viruses were able to induce expression and secretion of the designated chemokine or cytokine at levels ranging from 0.9–37 ng (Figure 7A).Figure 7Functional activity of chemokine-expressing virusesShow full caption(A) Chemokines from CXCL9, CXCL10, and IL-15-expressing viruses were measured using an ELISA. A549 cells were infected with 100 MOI of the viruses indicated, and at day 2 post-infection the supernatant was collected and tested. (B) Migration was assessed using a Transwell system. Chemokine-expressing viruses were added to MDA-MB-436 cells at MOI 100 at the lower chamber. To the top chamber, calcein green-labeled PBMCs were added, and at day 2 the number of cells at the bottom (migrated cells) were counted manually. (C) Visual representation of migrated green labeled PBMCs. Scale bar, 400 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CXCL9, CXCL10, and IL-15 have a common role in T cell migration. To test whether the secreted chemokines/cytokines from our viruses were functional, we infected MDA-MB-436 cells with the cytokine-encoding viruses and tested peripheral blood mononuclear cell (PBMC) migration using a Transwell system. Calcein green-labeled PBMCs were placed on top of Transwells and migrated green PBMCs were then counted on the lower chamber. When MDA-MB-436 cells were infected with our cloned viruses, an increase in migratory PBMCs was observed compared to uninfected cells or cells infected with the unarmed virus (Figures 7B and 7C). The highest increase in PBMC migration was observed with Ad5/3-CXCL9, followed by Ad5/3-CXCL10. In conclusion, our cloned viruses are able to express functional chemokines/cytokines that induce lymphocyte migration.DiscussionOncolytic adenoviruses have become ideal agents to treat cancer due to their specific tumor tropism. To achieve a long-term response, such viruses need to induce an anti-tumor response.4Andtbacka R.H.I. Kaufman H.L. Collichio F. Amatruda T. Senzer N. Chesney J. Delman K.A. Spitler L.E. Puzanov I. Agarwala S.S. et al.Talimogene laherparepvec improves durable response rate in patients with advanced melanoma.J. Clin. Oncol. 2015; 33: 2780-2788Crossref PubMed Scopus (1518) Google Scholar In doing so, scientist have armed oncolytic adenoviruses with various immune-stimulating agents. Yet, the current methods to genetically modify the adenoviruses are rather complex, time consuming, and expensive. In spite of this, we adapted a novel method to genetically modify oncolytic adenoviruses using GA, which is faster, cheaper, and more convenient than other methods.GA has been previously used, to a certain extent, to construct adenovirus genomes. However, the novelty of GAMER-Ad is that it can be done with routinely used Ad-5, Ad-5/3, and Ad-3 viruses. Moreover, the method entails replacing the gp19k gene from E3 region with a GOI, which has not been done before by using GA. Usually, genetic manipulations are done in limited regions that are not essential for viral production, such as E1, E2A, E3, and E4.5Marelli G. Howells A. Lemoine N.R. Wang Y. Oncolytic viral therapy and the immune system: A double-edged sword against cancer.Front. Immunol. 2018; 9: 866Crossref PubMed Scopus (138) Google Scholar,11Sebestyen Z. de Vrij J. Magnusson M. Debets R. Willemsen R. An oncolytic adenovirus redirected with a tumor-specific T-cell receptor.Cancer Res. 2007; 67: 11309-11316Crossref PubMed Scopus (20) Google Scholar,12Flint S.J. Wewerka-Lutz Y. Levine A.S. Sambrook J. Sharp P.A. Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells.J. Virol. 1975; 16: 662-673Crossref PubMed Google Scholar Yet, much attention has been drawn to the E3 region for genetic manipulation. The E3 region is a complex region, with multiple-gene transcription units expressing around seven different E3 proteins.13Kelly Jr., T.J. Lewis Jr., A.M. Use of nondefective adenovirus-simian virus 40 hybrids for mapping the simian virus 40 genome.J. Virol. 1973; 12: 643-652Crossref PubMed Google Scholar This complexity causes such proteins to be expressed at varying times and levels during the course of infection. If these genes were to be substituted by a therapeutic GOI, this would provide a flexible system in which multiple transgenes could be expressed in varying and predictable levels.14Berkner K.L. Sharp P.A. Generation of adenovirus by transfection of plasmids.Nucleic Acids Res. 1983; 11: 6003-6020Crossref PubMed Scopus (88) Google Scholar Additionally, the E3 proteins have well-known roles in immunomodulation; hence, removing such genes creates an opportunity to direct the immune responses to synergize with the therapeutic GOI.15Wold W.S.M. Tollefson A.E. Hermiston T.W. E3 transcription unit of adenovirus.Curr. Top. Microbiol. Immunol. 1995; 199: 237-274PubMed Google ScholarCurrently, many labs replace the adenoviral genes with GOI via homologous recombination using a shuttle plasmid and full-length adenoviral backbones.16Hawkins L.K. Johnson L. Bauzon M. Nye J.A. Castro D. Kitzes G.A. Young M.D. Holt J.K. Trown P. Hermiston T.W. Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region.Gene Ther. 2001; 8: 1123-1131Crossref PubMed Scopus (81) Google Scholar However, this process has a low efficiency, has wild-type (WT) contamination, and is time and labor intensive. Moreover, recombination takes place in a specialized E. coli strain, BJ5183, containing a Rec-A deficiency.17Wold W.S.M. Doronin K. Toth K. Kuppuswamy M. Lichtenstein D.L. Tollefson A.E. Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic.Curr. Opin. Immunol. 1999; 11: 380-386Crossref PubMed Scopus (162) Google Scholar,18He T.C. Zhou S. Da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. A simplified system for generating recombinant adenoviruses.Proc. Natl. Acad. Sci. USA. 1998; 95: 2509-2514Crossref PubMed Scopus (3230) Google Scholar Even though higher yields of homologous recombination are achieved, there is also a higher chance of secondary recombination events giving rise to unwanted repeat regions or secondary structures to the adenoviral genome. Hence, scientists have directed their efforts in developing easier and more efficient methods for generating recombinant adenoviruses. These methods include directly ligating cDNA into E1-deleted adenoviral genomes using Cre-loxP shuttles19West S.C. The processing of recombination intermediates: mechanistic insights from studies of bacterial proteins.Cell. 1994; 76: 9-15Abstract Full Text PDF PubMed Scopus (89) Google Scholar and E. coli-recombinant systems.20Camerini-Otero R.D. Hsieh P. Homologous recombination proteins in prokaryotes and eukaryotes.Annu. Rev. Genet. 1995; 29: 509-552Crossref PubMed Google Scholar These methods have simplified this process and fixed wild-type viral DNA input problems. Nevertheless, these methods are still time consuming and not very efficient and require several steps in planning.We have shown that our method is robust, with minimal false positives during the screening stage. An average of 75% of the colonies from the transformed GA products of Ad5/3-Il-15 tested had IL-15 assembled to the excised adenovirus genome. Usually, the official GA method entails that the overlapping ends have around 15–20 bp.21Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. Construction of adenovirus vectors through Cre-lox recombination.J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar In our method, 40 overlapping nucleotides seem to be very efficient in assembling products onto the adenovirus genome. Whether this could be further optimized by increasing or decreasing the overlapping
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