Four in one-Combination therapy using live Lactococcus lactis expressing three therapeutic proteins for the treatment of chronic non-healing wounds.

Diabetes mellitus is one of the major concerns for health care systems, affecting 382 million people worldwide. Among the different complications of diabetes, lower limbs chronic ulceration is a common, severe and costly cause of morbidity. Diabetic foot ulcers are a leading cause of hospitalization in diabetic patients and its rate exceed the ones of congestive heart failure, depression or renal disease. Diabetic non-healing ulcers account for more than 60% of all non-traumatic lower limb amputations and the five-year mortality after amputation is higher than 50%, being equal to several types of advanced cancer. The primary management goals for an existing diabetic foot ulcer are to achieve primary healing as expeditiously as possible and to achieve a reduction of the amputation rate in the patients. Unfortunately, approximately a quarter of patients do not partially or fully respond to the standard of care. Advanced therapies for chronic wounds are existing, however, recent guidelines including the latest reviews and meta-analyses of the scientific and clinical evidence available from current treatment strategies and new therapeutic agents revealed that there is a lack of clinical data and persistent gap of evidence for many of the advanced therapeutic approaches. In addition, no pharmacological wound healing product has gained authority approval for more than 10 years in both US and EU, constituting a highly unmet medical need. In this publication we present data from a live biopharmaceutical product AUP1602-C designed as a single pharmaceutical entity based on the non-pathogenic, food-grade lactic acid bacterium Lactococcus lactis subsp. cremoris that has been genetically engineered to produce human fibroblast growth factor 2,interleukin4 and colony stimulating factor 1. Designed to address different aspects of wound healing (i.e. fibroblast proliferation, angiogenesis and immune cell activation) and currently in phase I clinical study, we show how the combination of the individual components on the wound micro-environment initiates and improves the wound healing in chronic wounds.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

Preclinical Proof-of-Concept, Analytical Development, and Commercial Scale Production of Lentiviral Vector in Adherent Cells.

The therapeutic efficacy of a lentiviral vector (LV) expressing the herpes simplex virus thymidine kinase (HSV-TK) was studied in an immunocompetent rat glioblastoma model. Intraperitoneal ganciclovir injections (50 mg/kg/day) were administered for 14 consecutive days, resulting in reduced tumor volumes as monitored by MRI. Survival analyses revealed a significant improvement among the LV-expressing HSV-TK (LV-TK)/ganciclovir-treated animals when compared to non-treated control rats. However, a limiting factor in the use of LV has been the suboptimal small-scale production in flasks. Our aim during the translation phase, prior to entering the final pre-clinical and early clinical phases, was to develop a scalable, robust, and disposable manufacturing process for LV-TKs. We also aimed to minimize future process changes and enable production upscaling to make the process suitable for larger patient populations. The upstream process relies on fixed-bed iCELLis technology and transient plasmid transfection. This is the first time iCELLis 500 commercial-scale bioreactor was used for LV production. A testing strategy to determine the pharmacological activity of LV-TK drug product by measuring cell viability was developed, and the specificity of the potency assay was also proven. In this paper we focus on upstream process development while showing analytical development and the proof-of-concept of LV-TK functionality.

Brain pharmacokinetics of ganciclovir in rats with orthotopic BT4C glioma.

Ganciclovir (GCV) is an essential part of the Herpes simplex virus thymidine kinase (HSV-tk) gene therapy of malignant gliomas. The purpose of this study was to investigate the brain pharmacokinetics and tumor uptake of GCV in the BT4C rat glioma model. GCV's brain and tumor uptakes were investigated by in vivo microdialysis in rats with orthotopic BT4C glioma. In addition, the ability of GCV to cross the blood-brain barrier and tumor vasculature was assessed with in situ rat brain perfusion. Finally, the extent to which GCV could permeate across the BT4C glioma cell membrane was assessed in vitro. The areas under the concentration curve of unbound GCV in blood, brain extracellular fluid (ECF), and tumor ECF were 6157, 1658, and 4834 µM⋅min, respectively. The apparent maximum unbound concentrations achieved within 60 minutes were 46.9, 11.8, and 25.8 µM in blood, brain, and tumor, respectively. The unbound GCV concentrations in brain and tumor after in situ rat brain perfusion were 0.41 and 1.39 nmol/g, respectively. The highly polar GCV likely crosses the fenestrated tumor vasculature by paracellular diffusion. Thus, GCV is able to reach the extracellular space around the tumor at higher concentrations than that in healthy brain. However, GCV uptake into BT4C cells at 100 µM was only 2.1 pmol/mg of protein, and no active transporter-mediated disposition of GCV could be detected in vitro. In conclusion, the limited efficacy of HSV-tk/GCV gene therapy may be due to the poor cellular uptake and rapid elimination of GCV.

Soluble vascular endothelial growth factor receptor-1 improves therapeutic efficacy of suicide gene therapy in an angiogenesis-independent manner.

Abstract Malignant gliomas (MGs) are highly vascularized, aggressive brain cancers carrying a dismal prognosis. Because of their high vascularity, anti-angiogenic therapy is a potential treatment option. Indeed, the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has demonstrated promising results in clinical trials. Similarly, adenovirus-medicated Herpes simplex virus thymidine kinase and ganciclovir (AdHSV-tk/GCV) suicide gene therapy has established itself in clinical trials as a potential novel therapeutic strategy for MGs. In this study, we demonstrate the feasibility of combining adenovirus-mediated soluble VEGF receptor-1 anti-angiogenic gene therapy with AdHSV-tk/GCV suicide gene therapy to treat experimental MGs. Our results reveal that, apart from inhibiting angiogenesis, other anti-tumor mechanisms, such as reduction of infiltration by tumor-associated macrophages/microglia, may contribute to the improved therapeutic benefit of combination therapy.

Lymphatic vessel insufficiency in hypercholesterolemic mice alters lipoprotein levels and promotes atherogenesis.

OBJECTIVE: Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. APPROACH AND RESULTS: Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)/ApoB(100/100)) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had higher plasma cholesterol levels compared with LDLR(-/-)/ApoB(100/100) control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR(-/-)/ApoB(100/100) mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). CONCLUSIONS: We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.

Oxidative stress-regulated lentiviral TK/GCV gene therapy for lung cancer treatment.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.

Efficient gene therapy based targeting system for the treatment of inoperable tumors.

BACKGROUND: A considerable percentage of tumors are not amenable to surgery. We have designed a simple and powerful targeting system that offers an alternative option for the multi-component pre-targeting strategies used clinically. This targeting system can be used for any type of solid tumors independent of the tumor type, thereby omitting the need to engineer unique antibodies for each specific application or tumour type. In the present study, we show the expression of a chimeric fusion protein, which contains the low-density lipoprotein receptor transmembrane domains and avidin, after local gene transfer and its ability to bind biotinylated compounds in vivo. METHODS: Semliki Forest virus and lentivirus vectors were used to express the fusion protein with a high affinity binding site for biotinylated compounds in the tumor. Three different animal models and imaging modalities were used for the demonstration of the functionality and efficacy of the targeting system in vitro and in vivo. RESULTS: We demonstrate targeting of biotinylated compounds after local gene transfer in vivo using two different gene transfer vectors. The findings were confirmed by immunohistochemistry, single-photon emission computed tomography and magnetic resonance imaging. The therapeutic efficacy was tested in a syngeneic rat glioma model by injecting biotinylated-(90) Yttrium into the tail vein of glioma bearing rats. The study demonstrates that animals, which were treated by using the gene therapy based targeting system, lived significantly longer than control animals. CONCLUSIONS: Our gene therapy based targeting system is a promising tool for the treatment of inoperable tumors and other disease conditions, as well as diagnostic imaging.

Specific inhibition of SRC kinase impairs malignant glioma growth in vitro and in vivo.

Malignant glioma is a severe cancer with a poor prognosis. Local occurrence and rare metastases of malignant glioma make it a suitable target for gene therapy. Several studies have demonstrated the importance of Src kinase in different cancers. However, these studies have focused mainly on Src-deficient mice or pharmacological inhibitors of Src. In this study we have used Src small hairpin RNAs (shRNAs) in a lentiviral backbone to mimic a long-term stable treatment and determined the role of Src in tumor tissues. Efficacy of Src shRNAs was confirmed in vitro demonstrating up to 90% target gene inhibition. In a mouse malignant glioma model, Src shRNA tumors were almost 50-fold smaller in comparison to control tumors and had significantly reduced vascularity. In a syngenic rat intracranial glioma model, Src shRNA-transduced tumors were smaller and these rats had a survival benefit over the control rats. In vivo treatment was enhanced by chemotherapy and histone deacetylase inhibition. Our results emphasise the importance of Src in tumorigenesis and demonstrate that it can be efficiently inhibited in vitro and in vivo in two independent malignant glioma models. In conclusion, Src is a potential target for RNA interference-mediated treatment of malignant glioma.

Future prospects and challenges of antiangiogenic cancer gene therapy.

In 1971 Judah Folkman proposed the concept of antiangiogenesis as a therapeutic target for cancer. More than 30 years later, concept became reality with the approval of the antivascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab as a first-line treatment for metastatic colorectal cancer. Monoclonal antibodies and small molecular drugs are the most widely applied methods for inhibition of angiogenesis. The efficacy of these antiangiogenic modalities has been proven, in both preclinical and clinical settings. Although angiogenesis plays a major role in wound healing, hypoxia, and in the female reproductive cycle, inhibition of angiogenesis seems to be a relatively safe therapeutic option against cancers, and has therefore become a logical arena for a wide range of experimentation. The twentieth century has shown the boom of gene therapy and thus it has been applied also in the antiangiogenic setting. This review summarizes methods to induce antiangiogenic responses with gene therapy and discusses the obstacles and future prospects of antiangiogenic cancer gene therapy.

Clinical trials for glioblastoma multiforme using adenoviral vectors.

Gliomas are among the most lethal malignancies, and constitute more than 70% of all brain tumors. Standard therapy includes surgical resection followed by adjuvant radiotherapy and/or chemotherapy. Malignant gliomas are considered to be non-curable, and the overall prognosis of treatment success is poor with a mean survival of 14.6 months after diagnosis and a 5-year survival rate of 9.8%. The diffusely infiltrating property of the tumors makes total surgical excision often impossible, leading to eventual tumor recurrence. The maximum radiation dose that can be administered to the brain is limited to approximately 60 Gy, which is usually not sufficient to completely eradicate the tumor given that some brain tumors are resistant to radiotherapy. The limitations and short-comings of the available treatment options have provided the impetus to test novel therapy modalities to improve quality of life and increase survival of patients with gliomas.

Adenovirus mediated herpes simplex virus-thymidine kinase/ganciclovir gene therapy for resectable malignant glioma.

With the introduction of sophisticated tools of molecular biology, prodrug activating gene therapies have evolved as a novel therapeutic option for high-grade malignant gliomas. Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) is an extensively studied form of cytotoxic gene therapies. It is especially applicable for localized cancers, such as malignant glioma because of its restricted anatomical location and absence of metastasis. The early successes in the treatment of experimental malignant gliomas in the 1990s, gave impetus to further test this approach in this devastating disease. In malignant glioma, the recurrence after conventional therapy is inevitable, due to the residual cells in the tumor bed. The fascinating feature of adenoviral HSV-tk is that it attacks the residual dividing tumor cells without affecting the non-dividing neurons and furthermore, exploits them to destroy the malignant cells via so-called bystander-effect. Clinical Phase I and II studies have shown significant survival advantage and excellent safety profile when compared to conventional treatments. Thus, the adenoviral mediated HSV-tk gene therapy is a promising new adjuvant treatment for patients with operable high-grade glioma.

Avidin fusion protein-expressing lentiviral vector for targeted drug delivery.

One of the main objectives of cancer therapy is to enhance the effectiveness of the drug by concentrating it at the target site and to minimize the undesired side effects to nontarget cells. We have previously constructed a fusion protein, Lodavin, consisting of avidin and the endocytotic part of the low-density lipoprotein receptor, and demonstrated its applicability to transient drug targeting in vivo. In this study we produced a lentiviral vector expressing this fusion protein and evaluated its safety and efficacy. The results showed that lentivirus-mediated gene transfer led to long-term avidin fusion protein expression on glioma cells and that the receptor was able to bind biotinylated compounds. Repeated administration was proven feasible and the optimal time frame(s) for administration of biotinylated therapeutic and/or imaging compounds was elucidated. Intravenous or intracranial injection of the virus into BDIX rats led to the production of antibodies against transgene (avidin), but repeated administration of the vector was unable to boost this effect. Neutralizing antibodies against the lentivirus were also detected. Furthermore, we showed that the anti-avidin antibodies did not significantly affect the ligand-binding capacity of the avidin fusion protein. The therapeutic efficacy of avidin fusion protein in tumor treatment was tested in vitro with biotinylated and nonbiotinylated nanoparticles loaded with paclitaxel. In vivo applicability of lentivirus was studied in the BDIX rat glioma model, in which high receptor expression was detected in the tumor area. The lentivirus-mediated delivery of the avidin fusion protein thus represents a potential approach for the repeated targeting of cytotoxic compounds to cancer cells.

Challenges in monoclonal antibody-based therapies.

Therapeutic monoclonal antibodies (mAbs) are the fastest growing class of new therapeutic molecules. They hold great promises for the treatment of a variety of diseases, including chronic inflammatory diseases and cancer. However, the current manufacturing and purification processes cause limitations in the production capacity of therapeutic antibodies, leading to an increase in cost. Genetic delivery of therapeutic monoclonal antibodies by in vivo production offers a new potential solution to these problems. Firstly, therapeutic efficacy can be improved by maintaining stable therapeutic, non-toxic levels within the blood circulation over a long period of time. Repeated high-dose bolus injections could be avoided, thereby reducing the possibility of side-effects. Secondly, the high cost of manufacturing and purification of the therapeutic antibodies could be reduced, making an in vivo/ex vivo mAb gene transfer an economically viable and attractive option. In general, three approaches can be used for the stable long-term expression and secretion of therapeutic antibodies in vivo: 1) direct in vivo administration of integrating vectors carrying a mAb gene, 2) grafting of ex vivo genetically modified autologous cells, and 3) implantation of an encapsulated antibody producing heterologous or autologous cells. This paper describes the key factors and problems associated with the current antibody-based immunotherapies and reviews prospects for genetic in vivo delivery of therapeutic antibodies.