Transient depletion of specific immune cell populations to improve adenovirus-mediated transgene expression in the liver.

BACKGROUND & AIMS: Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. METHODS: First-generation Ad vectors encoding reporter genes (luciferase or ß-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4(+) , CD8(+) T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. RESULTS: The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100-1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. CONCLUSIONS: Transient CD4(+) T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.

Trauma surgical simulation: discussing the replacement of live animals used as human patient simulators.

BACKGROUND: Despite advances in simulator technology, live anaesthetised animals continue to be used as human patient simulators for medical professionals to practice techniques in the management of surgical trauma. This article describes the process of convening a working group of individuals with a professional interest in simulation to discuss the use of live animals and consider if and how they can be replaced in the future. MAIN BODY: A working group was formed of voluntary attendees to a workshop held at the SESAM 2023 conference. Iterative discussions reflecting on the topic were used to produce statements summarising the working group's opinions. The working group determined that live animals are used as human patient simulators due to the presence of accurate and responsive physiology in the presence of bleeding, realistic tissue tactility and an emotional response experienced by the learner due to interaction with the animal. They were unable to reach a consensus on replacement, determining that there is currently no single model which is able to provide all the learning aspects which a live animal model can provide. Several suggestions were made regarding development of technologies and pedagogical change. CONCLUSION: Replacement of live animals in surgical simulation is not straightforward but should be an aspiration, if possible. For the ongoing development of trauma surgical simulation models, it is important to combine the knowledge, skills and perspectives of medical stakeholders and educators, academic researchers and industry experts in producing alternative options to the use of live animal simulators.

Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice.

BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 µg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine. CONCLUSIONS: Long-term controlled expression of IL-12 using an HC-Ad vector in combination with oxaliplatin is effective and clinically applicable against hepatic colon cancer metastases.

Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy.

BACKGROUND: Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies. RESULTS: A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival. CONCLUSION: We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

Lethality in an anti-angiogenic tumor gene therapy model upon constitutive but not inducible expression of the soluble vascular endothelial growth factor receptor 1.

BACKGROUND: Neoangiogenesis is essential for tumor growth. The present study aimed to test the hypothesis that vector-mediated expression of sflt1 at high levels would result in the blockade of vascular endothelial growth factor (VEGF) function and therefore the inhibition of tumor growth. METHODS: To sequester VEGF, we tested, in a subcutaneous LLC tumor model, 'gutless' high-capacity adenovirus vectors expressing the soluble VEGF receptor 1 (sflt1) in a liver-specific manner, either in a constitutive or in a RU486 induced manner. RESULTS: High serum levels of sflt1 were observed upon in vivo injection of both vectors. Despite the differences in expression kinetics, both modes of sflt1 expression resulted in significant though transient suppression of tumor growth. Unexpectedly, constitutive but not intermittent sflt1 expression resulted in ascites and death of all animals. Morphological analyses by light and electron microscopy indicated that the animals had died from a nephropathy, which apparently was due to the blockade of VEGF function. CONCLUSIONS: Although confirming earlier results of toxic effects of prolonged VEGF sequestration, the present study suggests that therapeutic anti-tumor effects can be achieved without side-effects with intermittent VEGF blockade or the use of drugs with short half-lives, as shown by the use of an inducible gene expression system.

Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector.

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy.

Human adenovirus replicates in immunocompetent models of pancreatic cancer in Syrian hamsters.

The preclinical evaluation of toxicity and antitumor effect of conditionally replicative (oncolytic) adenoviruses is hampered by the inability of human adenoviruses to replicate efficiently in murine cells. The Syrian golden hamster (Mesocricetus auratus) has been suggested as a permissive animal for adenoviral replication, and cancer cell lines derived from various hamster tumors are available. We provide evidence that wild-type adenovirus type 5 is able to infect and replicate in the pancreatic cancer cell lines HaP-T1 and H2T both in vitro and in vivo. Determination of cytopathic effect, viral spread, progeny production, and the expression of late viral proteins indicates that the complete viral cycle of adenovirus takes place, albeit less efficiently than in highly permissive human cancer cell lines A549 and HuH7. Intrahepatic inoculation of HaP-T1 and H2T cells gave rise to tumors in the liver of hamsters that resemble metastases of pancreatic cancer. The growth of HaP-T1-induced nodules was faster compared with those derived from H2T, but both caused progressive liver infiltration and peritoneal dissemination. When adenovirus was inoculated in these lesions, productive replication took place and newly formed infective virions could be recovered 4 days after administration. In conclusion, the Syrian hamster models described here offer the opportunity to evaluate the effect of oncolytic adenoviruses in an immunocompetent animal and may be a valuable tool in the preclinical evaluation of these agents.

Target-mediated disposition model describing the dynamics of IL12 and IFNγ after administration of a mifepristone-inducible adenoviral vector for IL-12 expression in mice.

Interleukin-12 (IL12) is a cytokine with potential applications in the treatment of cancer given the potent immune response that it triggers, in part due to its ability to stimulate expression of interferon-γ (IFNγ). To avoid the toxicity associated with systemic exposure to IL12, a high-capacity adenoviral vector carrying a liver-specific, mifepristone-inducible IL12 expression system (HC-Ad/RUmIL12) has been developed. However, the maintenance of IL12 expression at therapeutic levels is compromised by the inhibitory effect of IFNγ on inducible systems. The aim of this work is to develop a semi-mechanistic model to characterize the relationship between IL12 and IFNγ in wild-type and knock-out mice for the IFNγ receptor treated with HC-Ad/RUmIL12 under different dosing regimens in order to better understand the key mechanisms controlling the system. Rapid binding was considered to account for target-mediated disposition exhibited by both cytokines (equilibrium dissociation constant were 18 and 2.28 pM for IL12 and IFNγ, respectively). The final model included: (1) IFNγ receptor turnover, (2) irreversible free cytokine elimination from the serum compartment, (3) internalization of the IL12 receptor complex, (4) IL12 expression upregulated by the co-administration of the adenoviral vector and mifepristone and downregulated by the IFNγ receptor, and (5) synthesis of IFNγ controlled by the relative increments in the bound IL12. In conclusion, a model simultaneously describing the kinetics of IL12 and IFNγ in the context of gene therapy was developed and validated with additional data. The model was applied to design an experimental dosing protocol intended to maintain sustained therapeutic IL12 levels.

Protection against liver damage by cardiotrophin-1: a hepatocyte survival factor up-regulated in the regenerating liver in rats.

BACKGROUND & AIMS: Cardiotrophin-1 (CT-1) is a member of the interleukin 6 (IL-6) family of cytokines, which protect cardiac myocytes against thermal and ischemic insults. In this study, we investigated the expression of CT-1 by liver cells and its possible hepatoprotective properties. METHODS: We analyzed the production, signaling, and antiapoptotic properties of CT-1 in hepatocytes and the expression of this cytokine during liver regeneration. We also investigated whether CT-1 might exert protective effects in animal models of liver damage. RESULTS: We found that CT-1 is up-regulated during liver regeneration and exerts potent antiapoptotic effects on hepatocytic cells. Hepatocytes cultured under serum starvation or stimulated with the pro-apoptotic cytokine transforming growth factor beta (TGF-beta) produce CT-1, which behaves as an autocrine/paracrine survival factor. Treatment with an adenovirus encoding CT-1 efficiently protects rats against fulminant liver failure after subtotal hepatectomy, an intervention that causes 91% mortality in control animals whereas 54% of those receiving CT-1 gene therapy were long-term survivors. This protective effect was associated with reduced caspase-3 activity and activation of the antiapoptotic signaling cascades signal transducer and activator of transcription (Stat-3), extracellular regulated kinases (Erk) 1/2, and Akt in the remnant liver. Gene transfer of CT-1 to the liver also abrogated Concanavalin A (Con-A) liver injury and activated antiapoptotic pathways in the hepatic tissue. Similar protection was obtained by treating the animals with 5 microg of recombinant CT-1 given intravenously before Con-A administration. CONCLUSIONS: We show that CT-1 is a hepatocyte survival factor that efficiently reduces hepatocellular damage in animal models of acute liver injury. Our data point to CT-1 as a new promising hepatoprotective therapy.