Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles
2022; Springer Nature; Volume: 14; Issue: 8 Linguagem: Inglês
10.15252/emmm.202115418
ISSN1757-4684
AutoresPaladd Asavarut, Sajee Waramit, Keittisak Suwan, Gert Marais, Aitthiphon Chongchai, Surachet Benjathummarak, Mariam Al‐Bahrani, Paula Vila-Gómez, Matthew Williams, Prachya Kongtawelert, Teerapong Yata, Amin Hajitou,
Tópico(s)Virus-based gene therapy research
ResumoArticle27 June 2022Open Access Transparent process Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles Paladd Asavarut Paladd Asavarut Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Sajee Waramit Sajee Waramit Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Software, Formal analysis, Validation, Methodology Search for more papers by this author Keittisak Suwan Keittisak Suwan orcid.org/0000-0001-6542-5841 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Resources, Data curation, Software, Formal analysis, Validation, Investigation, Methodology Search for more papers by this author Gert J K Marais Gert J K Marais Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Validation, Methodology Search for more papers by this author Aitthiphon Chongchai Aitthiphon Chongchai orcid.org/0000-0002-1193-1738 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Thailand Excellence Centre for Tissue Engineering and Stem Cells, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Contribution: Data curation, Methodology Search for more papers by this author Surachet Benjathummarak Surachet Benjathummarak orcid.org/0000-0003-2959-6728 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Contribution: Data curation, Methodology Search for more papers by this author Mariam Al-Bahrani Mariam Al-Bahrani Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Methodology Search for more papers by this author Paula Vila-Gomez Paula Vila-Gomez orcid.org/0000-0001-6101-9838 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Methodology Search for more papers by this author Matthew Williams Matthew Williams Department of Surgery and Cancer, Imperial College London, London, UK Contribution: Resources, Funding acquisition Search for more papers by this author Prachya Kongtawelert Prachya Kongtawelert Thailand Excellence Centre for Tissue Engineering and Stem Cells, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Contribution: Resources, Funding acquisition Search for more papers by this author Teerapong Yata Teerapong Yata Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Conceptualization, Methodology Search for more papers by this author Amin Hajitou Corresponding Author Amin Hajitou [email protected] orcid.org/0000-0003-1119-5686 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration Search for more papers by this author Paladd Asavarut Paladd Asavarut Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Sajee Waramit Sajee Waramit Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Software, Formal analysis, Validation, Methodology Search for more papers by this author Keittisak Suwan Keittisak Suwan orcid.org/0000-0001-6542-5841 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Resources, Data curation, Software, Formal analysis, Validation, Investigation, Methodology Search for more papers by this author Gert J K Marais Gert J K Marais Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Validation, Methodology Search for more papers by this author Aitthiphon Chongchai Aitthiphon Chongchai orcid.org/0000-0002-1193-1738 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Thailand Excellence Centre for Tissue Engineering and Stem Cells, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Contribution: Data curation, Methodology Search for more papers by this author Surachet Benjathummarak Surachet Benjathummarak orcid.org/0000-0003-2959-6728 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Contribution: Data curation, Methodology Search for more papers by this author Mariam Al-Bahrani Mariam Al-Bahrani Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Methodology Search for more papers by this author Paula Vila-Gomez Paula Vila-Gomez orcid.org/0000-0001-6101-9838 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Data curation, Methodology Search for more papers by this author Matthew Williams Matthew Williams Department of Surgery and Cancer, Imperial College London, London, UK Contribution: Resources, Funding acquisition Search for more papers by this author Prachya Kongtawelert Prachya Kongtawelert Thailand Excellence Centre for Tissue Engineering and Stem Cells, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Contribution: Resources, Funding acquisition Search for more papers by this author Teerapong Yata Teerapong Yata Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Conceptualization, Methodology Search for more papers by this author Amin Hajitou Corresponding Author Amin Hajitou [email protected] orcid.org/0000-0003-1119-5686 Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration Search for more papers by this author Author Information Paladd Asavarut1,†, Sajee Waramit1,†, Keittisak Suwan1,†, Gert J K Marais1, Aitthiphon Chongchai1,2, Surachet Benjathummarak1,3, Mariam Al-Bahrani1, Paula Vila-Gomez1, Matthew Williams4, Prachya Kongtawelert2, Teerapong Yata1,5 and Amin Hajitou *,1 1Cancer Phagotherapy, Department of Brain Sciences, Imperial College London, London, UK 2Thailand Excellence Centre for Tissue Engineering and Stem Cells, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 3Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 4Department of Surgery and Cancer, Imperial College London, London, UK 5Present address: Department of Physiology, Chulalongkorn University, Bangkok, Thailand † These authors contributed equally to this work as first authors *Corresponding author. Tel: +44 207 594 6546; E-mail: [email protected] EMBO Mol Med (2022)14:e15418https://doi.org/10.15252/emmm.202115418 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNF α) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses. Synopsis This study describes the characterization of a new phage-derived gene delivery particle capable of systemic targeting (TPA), demonstrating its superiority over existing phage systemic viral vectors (AAVP), and its application in targeted cytokine gene therapy of cancer. TPA particles are targeted bacteriophage capsids that carry only an expression cassette flanked by AAV-2 inverted terminal repeat sequences (ITRs), and a phage origin of replication. Following systemic administration in tumor-bearing mice, the tumor-targeted RGD4C.TPA binds to αvβ3 integrin receptor in tumors, resulting in selective expression of IL15, IL12, or TNFα cytokines in the tumor microenvironment. TPA-guided cytokine delivery results in complete tumor eradication in 50% of the treated animals. Targeted expression of IL15 results in proliferation of the immune CD8+ T, natural killer (NK), and T helper 1 (Th1) cells in tumors. Introduction Immunotherapy has the potential to create an enormous impact in cancer treatment if it is able to overcome existing limitations associated with delivery methods and specificity. Because cytokines are pleiotropic molecules produced by immune cells predominantly in the systemic circulation, achieving specific control of their synthesis and release has been an insurmountable challenge (Riley et al, 2019). To address this unmet need, we designed and characterised transmorphic particles that systemically target the delivery of recombinant adeno-associated virus (rAAV) DNA-bearing cytokine genes using the capsid of a tumour-targeted prokaryotic viral capsid. Recruitment of native immunity to target and destroy tumour cells is an effective treatment approach due to the complex array of cellular mechanisms that induce cytotoxicity directly to the pathology (Conforti et al, 2018). Since the 1980s, systemically delivered anti-tumour cytokines and immunoglobulins have been employed in cancer treatment. Today, the five main tactical approaches are the use of cytokines, tumour vaccines, antibodies, immune checkpoint inhibitors and Chimeric Antigen Receptor T-cells (CAR-T; Cao et al, 2020; Kennedy & Salama, 2020). The use of cytokines is most robust, as much of their signalling pathways are known; however, a fundamental problem is control over when and where they are present to exert the biological effects (Berraondo et al, 2018, 2019). The reactivity of cytokines across the diverse cellular and tissue targets is a double-edged sword. Overactivation of the immune system, that is, a “cytokine storm,” is potentially fatal on the host, and remains a primary concern in clinical translation of immunological agents. Delivering cytokines using gene delivery vectors is a solution, but the inherent biology of existing vectors also needs to be addressed if gene delivery limitations are to be overcome (Riley et al, 2019). Gene delivery is a core technology with potentially broad and impactful applications in immunotherapy. The delivery of cytokine genes at the pathology is a lucrative approach that may effectively target malignant cells whilst sparing normal tissues from the non-selective effect of cytokines. Even though mammalian viruses are efficient at gene transfer, a key problem is their inherent tropism and immunogenicity, resulting in off-target transduction, which is of significant concern in the context of cytokine gene delivery (Santiago-Ortiz & Schaffer, 2016). As a result, existing viral vectors do not allow for effective repeated administrations, as well as require localised delivery to avoid non-specific transduction (Riviere et al, 2006). Significant consideration must also be taken on using mammalian viruses for immunotherapy, as the immunogenic effect of cytokines can be compounded by the immune response to the vector itself, leading to further concerns on safety. Since the idea of gene transfer was launched in the 1970s, eukaryotic viral vectors have made their impact in gene therapy and ex vivo gene transfer; however, their approved applications remain narrow in immunotherapy. In contrast, bacteriophages (phages) are another species of viruses which are capable of gene delivery but have not been extensively investigated. Historically, phages have been used for drug discovery and as an antibiotic due to their complete lack of tropism and pathogenicity to human, and their low immunogenicity. These inherent qualities make them a prime candidate for cytokine gene delivery, where high specificity to the target tissue is required and activation of innate immunity should be avoided. We have attempted to address the parallel challenges in targeting both cytokine and gene delivery by developing a novel phage-guided system for the delivery of rAAV DNA encoding cytokine genes for cancer immunotherapy. As a result, we characterised a novel system for highly efficient production of transmorphic Phage/Adeno-associated viral particles (TPA). Mammalian viruses deliver genes by the use of complex infective mechanisms that coevolved with their mammalian hosts (Waehler et al, 2007). Furthermore, they are able to drive stable and long-term gene expression by the use of mechanisms such as chromosomal integration or protective structures that prevent detection and degradation by the host. These evolutionary mechanisms also give rise to unwanted side effects from immunity or delivery to healthy tissues and cells. Currently commercialised gene therapy vectors, such as Luxturna or Zolgensma, are approved by the Food and Drug Administration (FDA) only for rare diseases with a clearly defined spatial and temporal target (Foust et al, 2010; Russell et al, 2017). This is because despite being efficient at gene transfer, viral vectors possess native tropism to a wide range of mammalian tissues, similar to how cytokines are reactive towards multiple tissue targets. Indeed, attempts have been made to reduce or ablate the native tropism of mammalian viral vectors in hope that vectors can be delivered systemically, but significant success permitting targeted delivery has yet to be achieved (Anderson et al, 2000; Grimm et al, 2003; Zincarelli et al, 2008). Furthermore, the production and use of mammalian viral vectors come at a great economic cost, with approved therapies costing over 850,000 to over 1.6 million US dollars per treatment (Darrow, 2019; Dean et al, 2021). Thus, overcoming challenges that mammalian viruses have in their biology is vital for furthering meaningful progress in the field. On another side of virology, prokaryotic viruses such as bacteriophages (phages) are routinely used for the very reasons that limit the clinical application of mammalian viruses. Phages are abundant, simplistic viruses that have no native tropism for mammalian tissues, are not pathogenic, and are economical and efficient to manipulate and produce at GMP standards (Regulski et al, 2021). The use of phages as an antibiotic was widely accepted and used in the pre-antibiotic era, but in modern laboratory research, they play an important role in drug discovery in vitro and in vivo by their ability to tolerate large mutations on their coat proteins with very high binding specificity (Pasqualini & Ruoslahti, 1996; Arap et al, 2002; Kutateladze & Adamia, 2010; Bradbury et al, 2011). As a result, the use of phages in mammalian gene delivery has been explored through the insertion of a mammalian or viral transgene cassettes in its genome, and a receptor-specific mutation on its coat protein genes (Larocca et al, 1998, 1999, 2001, 2002; Burg et al, 2002). An attractive property of the phage in mammalian gene delivery is the target specificity that can be achieved, while at the same time, avoiding significant mammalian immune responses seen in eukaryotic viral vectors. As such, premature vector clearance, as well as harmful and potentially fatal side effects, can be avoided. At present, phage and chimeric phage vectors for mammalian delivery have not yielded sufficient success in eukaryotic gene delivery. The use of phage-derived vectors to circumvent the limitations of mammalian viruses is an attractive strategy; however, prokaryotic vectors also face challenges of their own. A significant impediment is their lack of mechanisms to evade intracellular degradation and efficiently induce gene expression when compared to mammalian viruses. Several groups have tried to develop phage vectors for human gene therapy with limited success due to weak gene expression derived from conventional transgene cassettes (Larocca et al, 1998, 1999, 2001). Hajitou et al, 2006, developed a hybrid vector, the adeno-associated virus/phage (AAVP), which targeted the angiogenic blood vessels of solid tumours and tumour cells by supplying the phage minor coat protein gene (pIII) with a double cyclic CDCRGDCFC (RGD4C) mutation (Hajitou et al, 2006). While enabling applications in targeted therapy and molecular imaging, its efficiency of gene transfer in vitro remains incomparable to mammalian viruses. Current phage and phage-derived vectors continue to possess a fundamental flaw; indeed, the presence of part of the bacteriophage genome or often a full phage genomic sequence dictates the final size of the vector particle. Because filamentous phages have a genome-dependent particle length, an unnecessarily long capsid gives rise to limitations in replication and packaging, cloning capacity and susceptibility to clearance by the reticuloendothelial system. These factors contribute significantly to poor uptake and induction of gene expression observed in bacteriophage vectors. Previous studies on the AAVP have explored a number of strategies to enhance the relatively low transduction efficiency when compared to conventional mammalian viruses (Kia et al, 2013; Przystal et al, 2013; Yata et al, 2014, 2015; Donnelly et al, 2015; Tsafa et al, 2016, 2020; Campbell et al, 2018). Furthermore, alternative studies have explored removing parts of the phage genome, albeit at the cost of packaging efficiency (Chasteen et al, 2006). Together, the challenges present in bacteriophage-guided gene delivery are inherently constrained by its own reproductive biology. In this study, we developed a novel approach to systemically targeted cytokine gene delivery using transmorphic particles based on a filamentous phage capsid and the DNA of rAAV-2 carrying transgene expression cassettes flanked by AAV-2 inverted terminal repeats (ITRs). The rationale behind combining the phenotype and genotype between two viruses of different kingdom classifications is to combine the specificity and less immunogenic properties of the phage capsid with the efficiency of gene expression observed in rAAV vectors. Using a phage capsid to encapsulate rAAV DNA has the potential to eliminate the native tropism and exceed the cloning capacity of rAAV, which are key limitations imposed by the nature of the AAV capsid and its architecture. Most importantly, phage and AAV are both single-stranded DNA (ssDNA) viruses, meaning their genomes are compatible for both manipulation and packaging. To achieve this, we used the phage origin of replication in a rAAV plasmid containing a transgene expression cassette of interest and employed a helper phage to supply a capsid bearing the RGD4C mutation on its pIII coat proteins for tumour targeting. By carefully modifying growth conditions, we were able to generate high-yield TPA particles unseen in any previous study, with low helper phage contamination that can be efficiently removed through ultracentrifugation or fast protein liquid chromatography (FPLC). Unlike previously reported vectors, we completely decoupled the phage genome from the final vector particle, resulting in a compact vector containing only the genetic payload desired for delivery to the target cell. In doing so, we provide evidence of a production system that generates high-yield, high-efficacy particles, without any phage structural genes present in the final vector and significant enhancement of gene delivery compared to a phage vector containing the whole phage genome (Larocca et al, 1999, 2001; Chasteen et al, 2006). While immunotherapy has been extensively evaluated in haematological cancers and melanoma, targeting solid tumours with current approaches has not been successful due to delivery barriers associated with the tumour interstitial pressure, compressed vasculature and dense extracellular matrix (Riley et al, 2019). We performed cancer immunotherapy using three different cytokine genes: interleukin 12 (IL12) and newly designed secreted isoforms of tumour necrosis factor-α (TNFα) and interleukin 15 (IL15), as they have been shown to produce potent, cell-mediated anti-tumour effects (Otani et al, 1999; Johansson et al, 2012; Waldmann et al, 2020). TNFα is a cytokine that achieves tumour killing both through direct induction of apoptosis and recruitment of other immune cells to activate cell-mediated cytotoxicity and disrupt tumour neoangiogenesis (Johansson et al, 2012). Moreover, selective systemic gene delivery of TNFα to cancer by tumour-targeted phage vectors resulted in tumour growth suppression in mice and pet dogs (Paoloni et al, 2009; Tandle et al, 2009; Yuan et al, 2013; Smith et al, 2016). IL12 acts as a bridge between innate and adaptive immunity, activating the Th1 response, resulting in cell-mediated cytotoxicity through the induction of TNFα and interferon gamma (IFN-y; Otani et al, 1999). Similarly, IL15 stimulates cell-mediated immunity through inducing the proliferation of CD8+ cytotoxic T cells and NK cells (Waldmann et al, 2020). Immunotherapy is a potentially safe and powerful tool if met with an equally efficient delivery strategy. Achieving localisation of cytokine expression only at the tumour site will resolve historic concern of side effects when using cytokines for cancer treatment, as well as broadening the application of immunotherapy to a wider range of cancers (Riley et al, 2019). We postulated that using TPA particles can confer the ability to safely and systemically, that is intravenous, target cytokine expression in tumours through repeated administrations, which is not achievable by a eukaryotic viral vector. By demonstrating the ability to perform highly targeted immunotherapy, we hope to show the impact that TPA particles can make in targeted immunological therapeutics and beyond. Results Transmorphic particle design and production Transmorphic phage/AAV particles (TPA) were designed and constructed based on the rAAV serotype-2 DNA, which contains both a pUC high copy-number origin of replication as well as a phage f1 origin of replication, and a transgene expression cassette flanked by AAV-2 ITRs (Figs 1A and EV1A). Moreover, production of TPA particles requires a helper phage (Fig 1B–D). The previously reported AAVP contains a full filamentous phage genomic sequence and an expression cassette flanked by AAV-2 ITRs, thereby significantly increasing its particle size (Figs 1E and EV1B). To generate tumour-targeted TPA particles, we induced an insertion mutation of the double cyclic RGD4C in the pIII gene of the filamentous M13KO7 helper phage (Figs 1B, and EV2A and B), termed RGD4C.M13KO7 (Fig EV2A), which contains a medium copy-number p15A origin of replication. In the presence of RGD4C.M13KO7, the rAAV DNA is packaged in bacteria by the tumour-targeted bacteriophage capsid. Figure 1. TPA construction A–C. Production of TPA particles requires two key elements: (A) a plasmid containing a mammalian transgene cassette flanked by AAV-2 ITRs (TPA plasmid) and (B) tumour-targeted bacteriophage-derived coat proteins containing the RGD4C peptide insertion mutation on the pIII minor coat protein gene of the M13KO7 filamentous phage, RGD4C.M13KO7, whose genome contains other structural genes required for general other protein subunits for phage assembly (C). D. To encapsidate the AAV DNA cassette using the bacteriophage capsid proteins, the AAV plasmid is transformed into F′ competent E. coli hosts and subsequently infected with RGD4C.M13KO7 helper phage. The resulting particle has the external characteristics of a tumour targeted bacteriophage but contains only the AAV DNA transgene cassette encoding a gene of interest. E. The AAVP vector genome contains an inserted transgene cassette from AAV-2, and an insertion of the RGD4C ligand on the pIII minor coat proteins of the phage display vector fUSE5. The genome of AAVP thus contains both phage structural genes and an AAV transgene cassette. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Genetic maps of Transmorphic Phage/AAV, TPA and Adeno-associated Virus/Phage, AAVP A. A schematic diagram of the TPA DNA encoding enhanced eGFP. TPA contains two origins of replication: pUC (high copy-number, in yellow), which enables double-stranded DNA replication in prokaryotic hosts, and f1 ori (phage origin of replication, in red), which enables single-stranded DNA replication and packaging into the phage capsid. B. A schematic diagram of the chimeric genome of AAVP encoding eGFP. AAVP contains the full genomic sequence of filamentous bacteriophage, and a transgene cassette from AAV-2 inserted in to an intergenomic region. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Genetic map of M13KO7 helper phage bearing the RGD4C peptide for tumour targeting A. A schematic diagram of the genome of M13KO7 helper phage used for packaging the TPA DNA (shown in Fig EV1) to produce non-targeted TPA particles. The M13KO7 genome contains a medium copy-number origin of replication (p15A, in yellow). B. A schematic diagram of the genome of RGD4C.M13KO7 helper phage used for packaging the TPA DNA (shown in Fig EV1) to produce the tumour-targeted RGD4C.TPA particles. The RGD4C coding sequence is inserted in-frame in to the M13 gene III, which encodes the pIII minor coat proteins. Download figure Download PowerPoint To identify the most efficient production protocol, superinfection and chemically (calcium chloride) competent cell methods were explored at 18 and 40-h incubation time endpoints with or without the presence of kanamycin (a selection marker for the helper phage). The infective method, done according to the standard reference protocol (Nissim et al, 1994), yielded an average of 7 × 108 bacterial transducing units (TU) per μl of TPA in the presence of kanamycin and 3 × 108 TU/μl of TPA without kanamycin. Despite the high number of particles produced, helper phage contamination was significantly higher than the TPA particle yield. In an attempt to identify whether the order of infection will affect particle yield, we generated calcium chloride competent TG1 Escherichia coli bearing the RGD4C.M13KO7 genome and transformed the cells with the TPA plasmid. Using identified colonies as a seed for particle production, we observed over four orders of magnitude lower TPA yield after incubation at both 18 and 40-h timepoints. The presence of kanamycin enabled TPA particle production, but also resulted in low particle yields as well as helper phage contamination. The standardised infective method, based on a standard phagemid packaging protocol, yielded higher particles compared to the competent cell method (Fig 2A; Larocca et al, 2001; Larocca et al, 1999; Larocca et al, 1998). In addition, a shorter 18-h incubation period is favourable for higher particle yield; however, extending the incubation period for the infective method is not feasible as the bacterial culture begins to die. These results indicate that RGD4C.M13KO7 and kanamycin are both required for TPA production, and that particle production seems to occur transiently after infection rather than constitutively over time. One problem we had to address, however, was to decrease helper phage contamination. This requires suppression of the competition the helper phage has in packaging its genome in the presence of the TPA DNA. Taking into account that the TPA DNA carries a high copy-number origin of replication, and RGD4C.M13KO7 carries a medium copy-number origin, we attempted to generate RGD4C.M13KO7 carrying a low copy-number origin of replication (pSC101) instead of its medium copy-number origin (p15A). While positive clones (RGD4C.M13KO7pSC101) could be generated and verified by sequencing, no TPA particles could be produced and quantified using this particular mutant. Our findings indicate efficient transmorphic particle production is dependent on sufficient transient expression of RGD4C.M13KO7. Figure 2. Optimisation of production and physical characterisation of TPA A. Yield comparison from standardised particle production using either superinfection by RGD4C.M13KO7 of E. coli transformed with TPA plasmid (i), or a calcium chloride competent E. coli carrying the RGD4C.M13KO7 genome (c), with or without kanamycin for selective expression, and at different incubation time endpoints. Data are expressed as mean ± S
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