High serum creatinine concentration is associated with metabolic perturbations in dogs.

BACKGROUND: The kidneys have many essential metabolic functions, and metabolic disturbances during decreased renal function have not been studied extensively. OBJECTIVES: To identify metabolic changes in blood samples with increased serum creatinine concentration, indicating decreased glomerular filtration. ANIMALS: Clinical samples analyzed using a nuclear magnetic resonance (NMR) based metabolomics platform. The case group consisted of 23 samples with serum creatinine concentration >125 µmol/L, and the control group of 873 samples with serum creatinine concentration within the reference interval. METHODS: Biomarker association with increased serum creatinine concentration was evaluated utilizing 3 statistical approaches: Wilcoxon rank-sum test, logistic regression analysis (false discovery rate (FDR)-corrected P-values), and random forest classification. Medians of the biomarkers were compared to reference intervals. A heatmap and box plots were used to represent the differences. RESULTS: All 3 statistical approaches identified similar analytes associated with increased serum creatinine concentrations. The percentages of citrate, tyrosine, branched-chain amino acids, valine, leucine, albumin, linoleic acid and the ratio of phenylalanine to tyrosine differed significantly using all statistical approaches, acetate differed using the Wilcoxon test and random forest, docosapentaenoic acid percentage only using logistic regression (P < .05), and alanine only using random forest. CONCLUSIONS AND CLINICAL IMPORTANCE: We identified several metabolic changes associated with increased serum creatinine concentrations, including prospective diagnostic markers and therapeutic targets. Further research is needed to verify the association of these changes with the clinical state of the dog. The NMR metabolomics test is a promising tool for improving diagnostic testing and management of renal diseases in dogs.

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.

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.

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.

Targeted delivery via avidin fusion protein: intracellular fate of biotinylated doxorubicin derivative and cellular uptake kinetics and biodistribution of biotinylated liposomes.

In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.

Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs. METHODOLOGY/PRINCIPAL FINDINGS: We studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections. CONCLUSIONS/SIGNIFICANCE: Intraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.

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.

Requirements for baculoviruses for clinical gene therapy applications.

Baculoviruses have proven capacity for the production of recombinant proteins including virus-like particles and as viral vectors. Recent progress in preclinical studies suggest that baculoviruses have potential as new vectors for gene therapy but so far no clinical trials have been performed. To date, no specific guidelines for the use of baculoviruses as human gene therapy vectors exist but researchers can utilize existing guidelines made for other biological products. Because of the long history of research on baculoviruses, a lot of knowledge has been obtained that forms a good basis for the gene therapy development process. This article gives an overview of the current status of the application of baculovirus vectors in gene therapy and summarizes some of the challenges to overcome before the first clinical trials with baculoviruses can be accomplished.

A laboratory-scale device for the straightforward production of uniform, small sized cell microcapsules with long-term cell viability.

Microencapsulated and genetically engineered cells may be used for prolonged delivery of therapeutically active proteins. The objective of this study was to develop a simple, inexpensive and flexible laboratory-scale device for the production of cell microcapsules, especially capsules of small diameter (<300 µm). Many microencapsulation devices are expensive, difficult to assemble and to use, and often more suitable for large-scale experiments. However, the simplicity and low price of the encapsulation system should not limit the quality of capsules and reproducibility of the process: for successful in vitro and in vivo experiments it is important to be able to produce uniform, spherical microcapsules without deformities with high reproducibility. In addition, an advantage of the present procedure compared to other similar, co-axial laminar gas flow systems is the possibility to produce also small microcapsules, less than 200 µm in diameter, with narrow size distribution. First, design, optimization and reproducibility testing of this custom-built device were carried out. Second, microencapsulated retinal pigment epithelial cells (ARPE-19) capable of secreting soluble vascular endothelial growth factor receptor 1 (sVEGFR1) were engineered. The cells remained viable in alginate-poly-L-lysine-alginate microcapsules and secreted sVEGFR1 for prolonged periods.

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.

Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma.

Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Delta24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP-treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system.

Replication competent Semliki Forest virus prolongs survival in experimental lung cancer.

We evaluated the therapeutic potential of the replication competent vector VA7-EGFP, which is based on the avirulent Semliki Forest virus (SFV) strain A7 (74) carrying the EGFP marker gene in an orthotopic lung cancer tumor model in nude mice. We have previously shown that this oncolytic vector destroys tumor cells efficiently in vitro and in vivo (in subcutaneous tumor model). Tumor growth in animals with orthotopically implanted adenocarcinoma cells (A549) were monitored during the study with small animal CT. We show that locally administered virotherapy with VA7-EGFP increased survival rate in experimental lung cancer significantly (p < 0.001) comparable to results obtained with the second generation conditionally replicating adenoviral vector Ad5-Delta24TK-GFP, used for comparison. The limited efficacy in systemically administered oncolytic viruses is the essential problem in oncolytic virotherapy and also in this study we were not able to elicit significant response with systemic administration route. Despite the fact that tumor microenvironment in orthotopic lung cancer is more optimal, viruses failed to home to the tumors and were unable to initiate efficient intratumoral replication. Clearly, the efficacy of virotherapy is influenced by many factors such as the route of virus administration, immunological and physiological barriers and cancer cell-specific features (IFN-responsiveness).

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models.

Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication-competent SFV vector VA7-EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7-EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection). This was at least partially due to a strong type I interferon (IFN) response in the tumors. The animals did not display any signs of abnormal behavior or encephalitis, even though SFV-positive foci were detected in the brain after the initial blood viremia. Intracranial rat tumors were injected directly with SFV A7(74) virus and monitored with magnetic resonance imaging. Tumor growth was significantly reduced (p < 0.05) with one virus injection, but the tumor size continued to increase after a lag period and none of the treated animals survived. Three virus injections or T-cell suppression with dexamethasone did not significantly improve treatment efficacy. It appeared that the local virotherapy induced extensive production of neutralizing anti-SFV antibodies that most likely contributed to the insufficient treatment efficacy. In conclusion, we show here that SFV A7(74) is a potential oncolytic agent for cancer virotherapy, but major immunological hurdles may need to be overcome before the virus can be clinically tested.

Transcriptional targeting of virus-mediated gene transfer by the human hexokinase II promoter.

The efficacy of the most commonly used form of suicide gene therapy, the HSV-TK/GCV method, utilizing herpes simplex virus thymidine kinase (HSV-TK) and antiviral drug ganciclovir (GCV) has been demonstrated in clinical trials. However, safer delivery of the therapeutic gene and more controlled regulation of the transgene expression, the essential prerequisites for successful therapeutic use, are still needed. We describe improved suicide gene therapy against cancer through transcripitional targeting by a strong and selective tumor-specific human hexokinase II promoter (hHKII). We examined the targeting properties of the human hHKII promoter in different human non-small cell lung cancer (NSCLC) and other human cancer cell lines using self-inactivating, VSV-G pseudotyped lentiviral vector. To confirm accurate transcriptional targeting of the hHKII promoter, the lack of transgene expression was verified in human primary bronchial epithelial and bronchial fibroblast cells. Furthermore, tissue-specific expression of the promoter was confirmed using transgenic mouse lines carrying the hHKII promoter driven luciferase reporter gene. We also tested the efficacy of the HSV-TK/GCV suicide gene therapy with the hHKII targeted lentiviral vector to NSCLC cells. Our results show that the hHKII promoter is strongly expressed in cancer cells. The targeted vector with the shortest hHKII promoter fragment (352 bp) appeared to have the best targeting properties because it efficiently governed the expression of the therapeutic gene in cancer cell lines, especially in certain non-small cell lung cancer cell lines, the transgene expression in human primary cells was virtually undetectable, and expression of the proximal hHKII promoter in transgenic mice was very low in most tissues. Also, the anti-cancer efficacy of HSV-TK/GCV therapy with the hHKII-targeted vector was comparable to that obtained with the control vector that utilized a commonly used constitutive promoter from the human elongation factor 1 alpha (hEF1alpha) gene. In conclusion, the transcriptionally targeted lentivirus vector with hHKII promoter can successfully direct HSV-TK/GCV suicide gene therapy to non-small cell lung cancer and other tumor cell types. These results warrant further studies with orthotopic animal tumor models and primary human cancer material.

Osteosarcoma and chondrosarcoma as targets for virus vectors and herpes simplex virus thymidine kinase/ganciclovir gene therapy.

Osteosarcoma and chondrosarcoma, the most prevalent primary malignant tumors of the bone, have been demonstrated to be potential target diseases for herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) suicide gene therapy. However, the utility of this gene therapy form for bone tumor cells has not been studied systematically. In this report we show, with the aid of three osteosarcoma cell lines (Saos-2, U-2-OS and MG-63) and one chondrosarcoma cell line (SW1353) that: i) these tumor cells were permissive for adenovirus- or lentivirus-mediated gene delivery; ii) the cell lines appeared to be good or excellent targets for HSV-TK/GCV gene therapy; and iii) the extent of HSV-TK/GCV cytotoxic effect correlated with the presence of the 'bystander effect' in these cells. Our results also suggest that lentiviruses are potential vectors for bone cancer gene therapy. They transduced all four cell lines with high efficiency and provided HSV-TK expression level that was sufficient for cytotoxicity and bystander effect comparable to that obtained with adenovirus vectors.

Non-small cell lung cancer as a target disease for herpes simplex type 1 thymidine kinase-ganciclovir gene therapy.

Lung cancer is a group of diseases that are difficult to cure and new treatment modalities, like gene therapy are actively tested to find alternatives for currently used strategies. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) method is one of the most frequently utilized forms of gene therapy and it has been tested on lung cancer, but no systematic study with comparison of different lung cancer types has been published. In this study, we examined in vitro and in vivo how good targets non-small cell lung cancer (NSCLC) cell lines representing adenocarcinoma, squamous cell lung cancer and large cell lung cancer are for adenovirus-mediated HSV-TK/GCV gene therapy. By using an adenovirus vector carrying a fusion gene of HSV-TK and green fluorescent protein (GFP), we found that: a) adenoviruses were efficient gene transfer vehicles for all types of NSCLCs; b) all adenocarcinoma and large cell lung cancer cells were good targets for HSV-TK/GCV therapy, whereas one of the squamous cell carcinoma cell lines was not responsive to the treatment; c) bystander effect played a major role in the success of this gene therapy form; d) subcutaneous tumors representing all three NSCLC types were efficiently treated with adenovirus-mediated HSV-TK/GCV gene therapy. In summary, this form of gene therapy appeared to be efficient treatment for human NSCLC and these results warrant further studies with primary lung cancer cells and orthotopic lung tumor models.