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The year 2023 marks the 20th anniversary of the British Society for Gene and Cell Therapy (BSGCT). In these 20 years, the field of gene and cell therapy has gone from promising strategy to clinical reality. This report describes the history, objectives, organization, and activities of BSGCT to advance research and practice of gene and cell therapy in the United Kingdom.
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Terapia Genética , Sociedades Médicas , Sociedades Médicas/historia , Reino Unido , Aniversarios y Eventos Especiales , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
It is now well accepted that many tumors undergo a process of clonal selection which means that tumor antigens arising at various stages of tumor progression are likely to be represented in just a subset of tumor cells. This process is thought to be driven by constant immunosurveillance which applies selective pressure by eliminating tumor cells expressing antigens that are recognized by T cells. It is becoming increasingly clear that the same selective pressure may also select for tumor cells that evade immune detection by acquiring deficiencies in their human leucocyte antigen (HLA) presentation pathways, allowing important tumor antigens to persist within cells undetected by the immune system. Deficiencies in antigen presentation pathway can arise by a variety of mechanisms, including genetic and epigenetic changes, and functional antigen presentation is a hard phenomenon to assess using our standard analytical techniques. Nevertheless, it is likely to have profound clinical significance and could well define whether an individual patient will respond to a particular type of therapy or not. In this review we consider the mechanisms by which HLA function may be lost in clinical disease, we assess the implications for current immunotherapy approaches using checkpoint inhibitors and examine the prognostic impact of HLA loss demonstrated in clinical trials so far. Finally, we propose strategies that might be explored for possible patient stratification.
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Antígenos HLA/inmunología , Inmunoterapia/métodos , Neoplasias/inmunología , Linfocitos T/inmunología , Animales , Humanos , RatonesRESUMEN
Vaccinia virus (VV) is a powerful tool for cancer treatment with the potential for tumor tropism, efficient cell-to-cell spread, rapid replication in cancer cells, and stimulation of anti-tumor immunity. It has a well-defined safety profile and is being assessed in late-stage clinical trials. However, VV clinical utility is limited by rapid bloodstream neutralization and poor penetration into tumors. These factors have often restricted its route of delivery to intratumoral or intrahepatic artery injection and may impede repeat dosing. Chemical stealthing improves the pharmacokinetics of non-enveloped viruses, but it has not yet been applied to enveloped viruses such as VV. In the present study, amphiphilic polymer was used to coat VV, leading to reduced binding of a neutralizing anti-VV antibody (81.8% of polymer-coated VV [PCVV] staining positive versus 97.1% of VV [p = 0.0038]). Attachment of anti-mucin-1 (aMUC1) targeting antibody, to give aMUC1-PCVV, enabled binding of the construct to MUC1. In high MUC1 expressing CAPAN-2 cells, infection with PCVV was reduced compared to VV, while infection was restored with aMUC1-PCVV. Pharmacokinetics of aMUC1-PCVV, PCVV, and VV were evaluated. After intravenous (i.v.) injection of 1 × 108 viral genomes (VG) or 5 × 108 VG, circulation time for PCVV and aMUC1-PCVV was increased, with ~5-fold higher circulating dose at 5 min versus VV.
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Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.
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Anticuerpos Monoclonales/aislamiento & purificación , Molécula de Adhesión Celular Epitelial/inmunología , Proteínas de la Membrana/inmunología , Receptores Quiméricos de Antígenos/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Cricetinae , Cricetulus , Biblioteca de Genes , Humanos , Células Jurkat , Unión ProteicaRESUMEN
Oncolytic viruses infect, replicate in, and kill cancer cells selectively without harming normal cells. The rapidly expanding clinical development of oncolytic virotherapy is an exciting interdisciplinary field that provides insights into virology, oncology, and immunotherapy. Recent years have seen greater focus on rational design of cancer-selective viruses together with strategies to exploit their immunostimulatory capabilities, ultimately to develop powerful oncolytic cancer vaccines. However, despite great interest in the field, many important experiments are still conducted under optimum conditions in vitro, with many nutrients present in excess and with cellular stress kept to a minimum. Whilst this provides a convenient platform for cell culture, it bears little relation to the typical conditions found within a tumour in vivo, where cells are often subject to a range of metabolic and environmental stresses. Viral infection and cancer will both lead to production of metabolites that are also not present in media in vitro. Understanding how oncolytic viruses interact with cells exposed to more representative metabolic conditions in vitro represents an under-explored area of study that could provide valuable insight into the intelligent design of superior oncolytic viruses and help bridge the gap between bench and bedside. This review summarises the major metabolic pathways altered in cancer cells, during viral infection and highlights possible targets for future studies.
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Vacunas contra el Cáncer , Inmunoterapia , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , Neoplasias/terapia , Virus Oncolíticos/inmunologíaRESUMEN
Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.
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The interplay between oncolytic virus infection and tumour hypoxia is particularly unexplored in vivo, although hypoxia is present in virtually all solid carcinomas. In this study, oncolytic adenovirus infection foci were found within pimonidazole-reactive, oxygen-poor areas in a colorectal xenograft tumour, where the expression of VEGF, a target gene of the hypoxia-inducible factor (HIF), was attenuated. We hypothesised that adenovirus infection interferes with the HIF-signalling axis in the hypoxic tumour niche, possibly modifying the local vascular supply. In vitro, enadenotucirev (EnAd), adenovirus 11p and adenovirus 5 decreased the protein expression of HIF-1α only during the late phase of the viral life cycle by transcriptional down-regulation and not post-translational regulation. The decreasing HIF levels resulted in the down-regulation of angiogenic factors such as VEGF, coinciding with reduced endothelial tube formation but also increased T-cell activation in conditioned media transfer experiments. Using intravital microscopy, a decreased perfused vessel volume was observed in infected tumour nodules upon systemic delivery of EnAd, encoding the oxygen-independent fluorescent reporter UnaG to a tumour xenograft grown under an abdominal window chamber. We conclude that the attenuation of the HIF pathway upon adenoviral infection may contribute to anti-vascular and immunostimulatory effects in the periphery of established infection foci in vivo.
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The translational success of oncolytic virotherapies would benefit from the widespread use of clinically relevant ex vivo models. Malignant ascites, an accumulation of fluid in the peritoneum due to disseminated cancer, recapitulates many features of the tumor microenvironment, making it a valuable model for studying oncolytic virus activity. Here, we describe a method for the separation and storage of cellular and acellular components of malignant ascites, followed by flow cytometric characterization of the cellular fraction. We then outline a simple experiment using whole ascites to assess the activity of a bispecific T cell engager (BiTE)-expressing oncolytic adenovirus.
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Ascitis , Líquido Ascítico , Vectores Genéticos , Biopsia Líquida , Viroterapia Oncolítica , Virus Oncolíticos , Adenoviridae/genética , Biomarcadores , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Vectores Genéticos/genética , Humanos , Inmunofenotipificación , Biopsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genéticaRESUMEN
BACKGROUND: Tumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid "on-target off-tumour" toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials. METHOD: A panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor ß (FRß) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers. RESULTS: T cells activated by the CD206- and FRß-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding - the first of its kind - demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state. CONCLUSIONS: This study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.
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Linfocitos Infiltrantes de Tumor/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Subgrupos de Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Adenoviridae/genética , Biomarcadores , Comunicación Celular/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Expresión Génica , Humanos , Inmunofenotipificación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias/metabolismo , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Unión Proteica , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , TransgenesRESUMEN
The 11th International Oncolytic Virus Conference (IOVC) was held from April 9-12, 2018 in Oxford, UK. This is part of the high-profile academic-led series of meetings that was started back in 2002 by Steve Russell and John Bell, with most of the previous meetings being held in North America (often in Banff). The conference brought together many of the major players in oncolytic virotherapy from all over the world, addressing all stages of research and development-from aspects of basic science and cellular immunology all the way through to early- and late-phase clinical trials. The meeting welcomed 352 delegates from 24 countries. The top seven delegate countries, namely, the UK, US, Canada, The Netherlands, Germany, Japan and South Korea, contributed 291 delegates while smaller numbers coming from Australia, Austria, Bulgaria, China, Finland, France, Iraq, Ireland, Israel, Italy, Latvia, Malaysia, Poland, Slovenia, Spain, Sweden and Switzerland. Academics comprised about half of the attendees, industry 30% and students 20%. The next IOVC is scheduled to be held on Vancouver Island in autumn 2019. Here we share brief summaries of the oral presentations from invited speakers and proffered papers in the different subtopics presented at IOVC 2018.
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Congresos como Asunto , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Ensayos Clínicos como Asunto , Terapia Combinada/métodos , Terapia Combinada/tendencias , Humanos , Neoplasias/inmunología , Viroterapia Oncolítica/tendencias , Resultado del TratamientoRESUMEN
Oncolytic viruses represent an emerging approach to cancer therapy. However, better understanding of their interaction with the host cancer cell and approaches to enhance their efficacy are needed. Here, we investigate the effect of chemically induced endoplasmic reticulum (ER) stress on the activity of the chimeric group B adenovirus Enadenotucirev, its closely related parental virus Ad11p, and the archetypal group C oncolytic adenovirus Ad5. We show that treatment of colorectal and ovarian cancer cell lines with thapsigargin or ionomycin caused an influx of Ca2+, leading to an upregulation in E1A transcript and protein levels. Increased E1A protein levels, in turn, increased levels of expression of the E2B viral DNA polymerase, genome replication, late viral protein expression, infectious virus particle production, and cell killing during Enadenotucirev and Ad11p, but not Ad5, infection. This effect was not due to the induction of ER stress, but rather the influx of extracellular Ca2+ and consequent increase in protein kinase C activity. These results underscore the importance of Ca2+ homeostasis during adenoviral infection, indicate a signaling pathway between protein kinase C and E1A, and raise the possibility of using Ca2+ flux-modulating agents in the manufacture and potentiation of oncolytic virotherapies.
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BACKGROUND: Oncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses. METHODS: Transgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines. RESULTS: Our results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication. CONCLUSION: Together, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.
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Adenoviridae/patogenicidad , Proteína Cofactora de Membrana/metabolismo , Viroterapia Oncolítica/métodos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones TransgénicosRESUMEN
BACKGROUND: Enadenotucirev (formerly ColoAd1) is a tumor-selective chimeric adenovirus with demonstrated preclinical activity. This phase 1 Mechanism of Action study assessed intravenous (IV) delivery of enadenotucirev in patients with resectable colorectal cancer (CRC), non-small-cell lung cancer (NSCLC), urothelial cell cancer (UCC), and renal cell cancer (RCC) with a comparator intratumoral (IT) dosed CRC patient cohort. METHODS: Seventeen patients scheduled for primary tumor resection were enrolled. IT injection of enadenotucirev (CRC only) was administered as a single dose (≤ 3 × 1011 viral particles [vp]) on day 1, followed by resection during days 8-15. IV infusion of enadenotucirev was administered by three separate doses (1 × 1012 vp) on days 1, 3, and 5, followed by resection during days 8-15 (CRC) or days 10-25 (NSCLC, UCC, and RCC). Enadenotucirev activity was measured using immunohistochemical staining of nuclear viral hexon and quantitative polymerase chain reaction for viral genomic DNA. RESULTS: Delivery of enadenotucirev was observed in most tumor samples following IV infusion, with little or no demonstrable activity in normal tissue. This virus delivery (by both IV and IT dosing) was accompanied by high local CD8+ cell infiltration in 80% of tested tumor samples, suggesting a potential enadenotucirev-driven immune response. Both methods of enadenotucirev delivery were well tolerated, with no treatment-associated serious adverse events. CONCLUSIONS: This study provides key delivery and feasibility data to support the use of IV infusion of enadenotucirev, or therapeutic transgene-bearing derivatives of it, in clinical trials across a range of epithelial tumors, including the ongoing combination study of enadenotucirev with the checkpoint inhibitor nivolumab. It also provides insights into the potential immune-stimulating properties of enadenotucirev. TRIAL REGISTRATION: This MOA study was a phase 1, multicenter, non-randomized, open-label study to investigate the administration of enadenotucirev in a preoperative setting (ClinicalTrials.gov: NCT02053220).
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Adenovirus Humanos/fisiología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Carcinoma de Células Renales/terapia , Carcinoma de Células Transicionales/terapia , Neoplasias Colorrectales/terapia , Neoplasias Pulmonares/terapia , Adenovirus Humanos/genética , Administración Intravenosa , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Transicionales/inmunología , Neoplasias Colorrectales/inmunología , Terapia Combinada , ADN Viral/genética , Procedimientos Quirúrgicos del Sistema Digestivo , Humanos , Neoplasias Pulmonares/inmunología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Procedimientos Quirúrgicos Pulmonares , Resultado del Tratamiento , Procedimientos Quirúrgicos UrológicosRESUMEN
Oncolytic viruses (OVs) are quickly moving toward the forefront of modern medicines. The reward for the decades of research invested into developing viral platforms that selectively replicate in and lyse tumor cells while sparking anticancer adaptive immunity is presenting in the form of durable therapeutic responses. While this has certainly been a concerted global effort, in this review for the 25th anniversary of the European Society of Gene and Cell Therapy, we focus on the contributions made by European researchers. Research centers across Europe have held central roles in advancing OVs, from the earliest reports of coincidental viral infections leading to antitumor efficacy, to advanced mechanistic studies, and now through Phase I-III trials to imminent regulatory approvals. While challenges still remain, with limitations in preclinical animal models, antiviral immune clearance, and manufacture restrictions enforced by poor viral yields in certain cases, the field has come a very long way in recent years. Thoughtful mechanistic integration of OVs with standard of care strategies and other newly approved therapies should provide potent novel approaches. Combination with immunotherapeutic regimes holds significant promise, and the ability to arm the viral platform with therapeutic proteins for localized expression at the tumor site provides an opportunity for creating highly effective synergistic treatments and brings a new age of targeted cancer therapeutics.
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Inmunidad Adaptativa/genética , Neoplasias/terapia , Viroterapia Oncolítica/tendencias , Virus Oncolíticos/genética , Europa (Continente) , Humanos , Neoplasias/genética , Virus Oncolíticos/inmunologíaRESUMEN
Enadenotucirev is an oncolytic group B adenovirus identified by a process of bio-selection for the ability to selectively propagate in and rapidly kill carcinoma cells. It is resistant to inactivation by human blood components, potentially enabling intravenous dosing in patients with metastatic cancer. However, there are no known permissive animal models described for group B adenoviruses that could facilitate a conventional approach to preclinical safety studies. In this manuscript, we describe our tailored preclinical strategy designed to evaluate the key biological properties of enadenotucirev. As enadenotucirev does not replicate in animal cells, a panel of primary human cells was used to evaluate enadenotucirev replication selectivity in vitro, demonstrating that virus genome levels were >100-fold lower in normal cells relative to tumor cells. Acute intravenous tolerability in mice was used to assess virus particle-mediated toxicology and effects on innate immunity. These studies showed that particle toxicity could be ameliorated by dose fractionation, using an initial dose of virus to condition the host such that cytokine responses to subsequent doses were significantly attenuated. This, in turn, supported the initiation of a phase I intravenous clinical trial with a starting dose of 1 × 1010 virus particles given on days 1, 3, and 5.
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Effective therapeutics for ovarian cancer continue to be urgently needed, particularly for chemotherapy-resistant cases. Here we present both a 3D-Matrigel culture-based expansion of our directed evolution method for generation of oncolytic virotherapies and two promising ovarian-cancer targeted oncolytic viruses, OvAd1 and OvAd2. OvAd1 was developed using Matrigel cell cultures, whereas OvAd2 was developed in parallel using traditional monolayer tissue culture methods. Both viruses are potent against a panel of platinum-resistant ovarian cancer cell lines and are attenuated on normal cells in vitro, resulting in therapeutic windows of â¼200-fold. We observed two benefits of the use of Matrigel-based cultures for directed evolution of these oncolytics: (1) use of Matrigel generated a bioselected pool that was more strongly attenuated on normal cells while retaining its potency against ovarian cancer cells, and (2) in an ovarian carcinomatosis model, the Matrigel-derived virus OvAd1 suppressed all tumor growth while the non-Matrigel-derived virus was 50% effective. Neither virus stimulated formation of peritoneal adhesions as seen for Ad5-based therapies. Consequently, these viruses are novel candidates for development as new effective treatments for aggressive ovarian cancer.
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New classes of biologically active materials, such as viruses, siRNA, antibodies and a wide range of engineered nanoparticles have emerged as potent agents for diagnosing and treating diseases, yet many of these agents fail because there is no effective route of delivery to their intended targets. Focused ultrasound and its ability to drive microbubble-seeded cavitation have been shown to facilitate drug delivery. However, cavitation is difficult to control temporally and spatially, making prediction of therapeutic outcomes deep in the body difficult. Here, we utilized passive acoustic mapping in vivo to understand how ultrasound parameters influence cavitation dynamics and to correlate spatial maps of cavitation to drug delivery. Focused ultrasound (center frequency: 0.5 MHz, peak-rarefactional pressure: 1.2 MPa, pulse length: 25 cycles or 50,000 cycles, pulse repetition interval: 0.02, 0.2, 1 or 3 s, number of pulses: 80 pulses) was applied to murine xenograft-model tumors in vivo during systemic injection of microbubbles with and without cavitation-sensitive liposomes or type 5 adenoviruses. Analysis of in vivo cavitation dynamics through several pulses revealed that cavitation was more efficiently produced at a lower pulse repetition frequency of 1 Hz than at 50 Hz. Within a pulse, inertial cavitation activity was shown to persist but reduced to 50% and 25% of its initial magnitude in 4.3 and 29.3 ms, respectively. Both through several pulses and within a pulse, the spatial distribution of cavitation was shown to change in time due to variations in microbubble distribution present in tumors. Finally, we demonstrated that the centroid of the mapped cavitation activity was within 1.33 ± 0.6 mm and 0.36 mm from the centroid location of drug release from liposomes and expression of the reporter gene encoded by the adenovirus, respectively. Thus passive acoustic mapping not only unraveled key mechanisms whereby a successful outcome is achieved, but also a predicted drug delivery outcome.
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Algoritmos , Liposomas/farmacocinética , Ondas Ultrasónicas , Ultrasonografía/métodos , Animales , Células Hep G2 , Humanos , Liposomas/efectos de la radiación , RatonesRESUMEN
BACKGROUND: Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with this disease. METHODS: In a multicentre clinical trial, six male patients (aged 35-63 years) with choroideremia were administered AAV.REP1 (0·6-1·0×10(10) genome particles, subfoveal injection). Visual function tests included best corrected visual acuity, microperimetry, and retinal sensitivity tests for comparison of baseline values with 6 months after surgery. This study is registered with ClinicalTrials.gov, number NCT01461213. FINDINGS: Despite undergoing retinal detachment, which normally reduces vision, two patients with advanced choroideremia who had low baseline best corrected visual acuity gained 21 letters and 11 letters (more than two and four lines of vision). Four other patients with near normal best corrected visual acuity at baseline recovered to within one to three letters. Mean gain in visual acuity overall was 3·8 letters (SE 4·1). Maximal sensitivity measured with dark-adapted microperimetry increased in the treated eyes from 23·0 dB (SE 1·1) at baseline to 25·3 dB (1·3) after treatment (increase 2·3 dB [95% CI 0·8-3·8]). In all patients, over the 6 months, the increase in retinal sensitivity in the treated eyes (mean 1·7 [SE 1·0]) was correlated with the vector dose administered per mm(2) of surviving retina (r=0·82, p=0·04). By contrast, small non-significant reductions (p>0·05) were noted in the control eyes in both maximal sensitivity (-0·8 dB [1·5]) and mean sensitivity (-1·6 dB [0·9]). One patient in whom the vector was not administered to the fovea re-established variable eccentric fixation that included the ectopic island of surviving retinal pigment epithelium that had been exposed to vector. INTERPRETATION: The initial results of this retinal gene therapy trial are consistent with improved rod and cone function that overcome any negative effects of retinal detachment. These findings lend support to further assessment of gene therapy in the treatment of choroideremia and other diseases, such as age-related macular degeneration, for which intervention should ideally be applied before the onset of retinal thinning. FUNDING: UK Department of Health and Wellcome Trust.
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Proteínas Adaptadoras Transductoras de Señales/administración & dosificación , Coroideremia/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Proteínas Adaptadoras Transductoras de Señales/genética , Adenoviridae/genética , Adulto , Anciano , Coroideremia/fisiopatología , Fluorescencia , Técnicas de Transferencia de Gen , Humanos , Inyecciones Intraoculares , Masculino , Persona de Mediana Edad , Desprendimiento de Retina/fisiopatología , Desprendimiento de Retina/terapia , Transgenes/genética , Agudeza Visual/fisiologíaRESUMEN
The encapsulation of cytotoxic drugs within liposomes enhances pharmacokinetics and allows passive accumulation within tumors. However, liposomes designed to achieve good stability during the delivery phase often have compromised activity at the target site. This problem of inefficient and unpredictable drug release is compounded by the present lack of low-cost, non-invasive methods to measure such release. Here we show that focused ultrasound, used at pressures similar to those applied during diagnostic ultrasound scanning, can be utilised to both trigger and monitor release of payload from liposomes. Notably, drug release was influenced by liposome composition and the presence of SonoVue® microbubbles, which provided the nuclei for the initiation of an event known as inertial cavitation. In vitro studies demonstrated that liposomes formulated with a high proportion of 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) released up to 30% of payload following ultrasound exposure in the presence of SonoVue®, provided that the exposure created sufficient inertial cavitation events, as characterised by violent bubble collapse and the generation of broadband acoustic emissions. In contrast a 'Doxil'-like liposome formulation gave no such triggered release. In pre-clinical studies, ultrasound was used as a non-invasive, targeted stimulus to trigger a 16-fold increase in the level of payload release within tumors following intravenous delivery. The inertial cavitation events driving this release could be measured remotely in real-time and were a reliable predictor of drug release.