Critical resources for gene therapy in Heart, Lung, and Blood Diseases Working Group.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Savio L C Woo
Publications and source records attributed to Savio L C Woo.
Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA.
We explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine phenylalanine hydroxylase cDNA in the livers of phenylketonuric (PKU) mice. The phiBT1 phage integrase is an enzyme that catalyses the efficient recombination between unique sequences in the phage and bacterial genomes, leading to the site-specific integration of the former into the latter in a unidirectional manner. Here we showed that this phage integrase functions efficiently in mouse cells, and several naturally occurring pseudo-attP sites located in the intergenic regions of the mouse genome have been identified and molecularly characterized. We further demonstrated that the addition of nuclear localization signal sequences to the C terminus of the phage integrase enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system, we delivered mouse phenylalanine hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperphenylalaninemic phenotype in the treated mice decreased significantly. After three applications, serum phenylalanine levels in all treated PKU mice were reduced to the normal range and remained stable thereafter. Their fur color also changed from gray to black, indicating the reconstitution of melanin biosynthesis as a result of available tyrosine derived from reconstituted phenylalanine hydroxylation in the liver. Thus, the phiBT1 bacteriophage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in DNA-mediated gene therapy for a multitude of genetic disorders.
Animals↗
Treatment of multi-focal colorectal carcinoma metastatic to the liver of immune-competent and syngeneic rats by hepatic artery infusion of oncolytic vesicular stomatitis virus.
Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. We report an orthotopic model of multi-focal colorectal cancer (CRC) metastases in the livers of syngeneic and immune-competent rats, which permitted rigorous testing of oncolytic virus vectors as novel therapeutic agents through hepatic arterial infusion for efficacy and safety. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to attenuated anti-viral responses in many tumors. After administration at the maximum tolerated dose, the recombinant VSV vector gained access to multi-focal hepatic CRC lesions that led to tumor-selective viral replication and oncolysis. No relevant vector-associated toxicities were noted and in particular, no damage to the hepatic parenchyma was seen. Moreover, the survival rate of vector-treated rats was significantly improved over that of animals in the control treatment group (p = 0.015). Our results demonstrate that hepatic arterial administration of oncolytic VSV is both effective and safe in an immune-competent and syngeneic rat model of multi-focal CRC liver metastasis, suggesting that it can be developed into an effective therapeutic modality in patients in the future.
Animals↗
Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV.
Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus with intrinsic oncolytic specificity due to attenuated antiviral responses in many tumors. We report that repeated hepatic arterial infusion of recombinant syncytia-forming VSV vector in advanced multifocal hepatocellular carcinoma (HCC)-bearing rats at a 10-fold reduced vector dose resulted in sustained tumor-selective virus replication until the onset of high-titer neutralizing antibodies in blood. No significant elevations in serum transaminases and liver pathology were noted, indicating a lack of hepatotoxicity. Substantially improved tumor response was achieved with completely necrotic tumor nodules surrounded by mononuclear phagocytic cells, followed by fibrosis and calcification of the lesions, angiogenesis, and regeneration of normal hepatic parenchyma. Survival of tumor-bearing rats treated with repeated vector infusions was not only significantly improved over that of animals after a single injection at 10 times the vector dose (P = .001), but 18% of animals in the former treatment group also achieved long-term and tumor-free survival compared with 0% of animals in the latter treatment group. In conclusion, this treatment regimen will be very useful in the future development of VSV-mediated virotherapy as a novel therapeutic modality for patients with advanced HCC.
Animals↗
Systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immune-competent mice.
In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Oncolytic vesicular stomatitis virus (VSV) is a promising novel therapeutic agent for the treatment of cancer. The aim of this study was to evaluate the potential of recombinant VSV containing the M51R mutation in the matrix (M) protein gene administered intravenously as an effective and safe therapeutic agent for treating mice with experimental breast cancer metastases. Recombinant VSV(M51R)-LacZ was generated and characterized in vitro on human and murine breast cancer cells. Breast cancer metastases were established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells. The vector was infused into the tumor-bearing animals via the tail vein, and productive infection of pulmonary breast cancer lesions was assessed by X-gal stainings of frozen lung sections. To evaluate potential systemic toxicity, histology of major organs and serum chemistries were analyzed. To assess effectiveness, buffer- or vector-treated tumor-bearing mice were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. We found that VSV(M51R)-LacZ efficiently replicated and lysed human breast cancer cells but was partially attenuated in 4T1 cells in vitro. We also demonstrated that its maximum tolerated dose after intravenous infusion in normal Balb/c mice was elevated by at least 100-fold over that of the parental VSV vector containing the wild-type M gene. When VSV(M51R)-LacZ was repeatedly injected intravenously into mice bearing syngeneic 4T1 tumors, the virus was able to infect multiple breast cancer lesions in the lungs without apparent toxicities, which led to significant prolongation of animal survival (P=.003). In conclusion, systemic administration of M mutant VSV is both effective and safe in the treatment of experimental breast cancer metastases in immune-competent mice, suggesting that further development of this approach may have potential for clinical application in patients.
Animals↗
Prophylactic alpha interferon treatment increases the therapeutic index of oncolytic vesicular stomatitis virus virotherapy for advanced hepatocellular carcinoma in immune-competent rats.
Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta) in normal cells, we tested if prophylactic treatment with rat IFN-alpha would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-alpha in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-alpha at 66 IU/g body weight (BW), equivalent to a human IFN-alpha dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-alpha, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-alpha were equivalent. The results suggest that prophylactic rat IFN-alpha treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-alpha is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.
Animals↗
Syncytia induction enhances the oncolytic potential of vesicular stomatitis virus in virotherapy for cancer.
Vesicular stomatitis virus (VSV) selectively replicates in tumor but not in normal cells and is being developed as an oncolytic agent for cancer therapy. Here we report the construction of a recombinant VSV capable of inducing syncytia formation between tumor cells through membrane fusion at neutral pH, which led to enhanced oncolytic properties against multifocal hepatocellular carcinoma (HCC) in the livers of immunocompetent rats. Recombinant VSV vectors were constructed by insertion into their genome a transcription unit expressing a control or fusion protein derived from Newcastle disease virus. In vitro characterization of the recombinant fusogenic VSV vector on human and rat HCC cells showed extensive syncytia formation and significantly enhanced cytotoxic effects. In vivo, administration of fusogenic VSV into the hepatic artery of Buffalo rats bearing syngeneic multifocal HCC lesions in their livers resulted in syncytia formation exclusively within the tumors, and there was no collateral damage to the neighboring hepatic parenchyma. The fusogenic VSV also conferred a significant survival advantage over a nonfusogenic control virus in the treated animals (P = 0.0078, log-rank test). The results suggest that fusogenic VSV can be developed into an effective and safe therapeutic agent for cancer treatment in patients, including those with multifocal HCC in the liver.
Animals↗
Oncolysis of multifocal hepatocellular carcinoma in the rat liver by hepatic artery infusion of vesicular stomatitis virus.
Hepatocellular carcinoma (HCC) is a lethal malignancy with poor prognosis and few effective treatments, as well as ever-increasing frequencies in the Western world. Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to significantly attenuated antiviral responses in many tumor cells. The aim of this study was to evaluate the potential of VSV, administered via the hepatic artery, as an effective and safe therapeutic agent for treating "multifocal" HCC in the rat liver. Recombinant VSV vector expressing beta-galactosidase (rVSV-beta-gal) was generated by reverse genetics and infused into the hepatic artery of Buffalo rats bearing orthotopically implanted multifocal HCC. Access by the virus to multifocal HCC lesions in the liver, as well as the kinetic profiles of intratumoral viral replication and spread, was established by X-gal staining of liver and tumor sections. Plaque assays were also performed to determine the infectious viral yields in tumor and normal liver tissues. Pharmacotoxicology studies, including serum chemistries and proinflammatory cytokine production, as well as organ histopathology, were performed. Buffer- or vector-treated tumor-bearing rats were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. Hepatic arterial infusion of rVSV-beta-gal at the maximum tolerated dose in tumor-bearing rats resulted in efficient viral transduction of multifocal HCC lesions in their livers, tumor-selective viral replication, and extensive oncolysis. Importantly, no significant vector-associated toxicities were noted and, in particular, no damage to the hepatic parenchyma was seen. Finally, survival of vector-treated rats was substantially prolonged over that of animals in the control treatment group (p < 0.028). Thus, hepatic arterial administration of VSV is both effective and safe in an orthotopic animal model of multifocal HCC. The results suggest that oncolytic VSV can be developed into an effective and safe therapeutic modality for patients with multifocal HCC in the future.
Animals↗
Oncolytic vesicular stomatitis virus for treatment of orthotopic hepatocellular carcinoma in immune-competent rats.
Tumor-targeted replicating viruses are being developed as a novel class of oncolytic agents. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with inherent specificity for replication in tumor cells due to their attenuated antiviral responses. VSV as an oncolytic virus is particularly appealing for its exceptionally rapid replication rate in tumor cells, such that the oncolytic effects could be maximally manifested before the onset of potentially neutralizing antiviral immune responses in the host. To easily monitor VSV replication, we have rescued a recombinant VSV (rVSV) vector expressing the green fluorescent protein (GFP) gene (rVSV-GFP). Using this GFP-expressing virus, we have demonstrated the oncolytic potential of VSV against human and rat hepatocellular carcinoma (HCC). We found that rVSV-GFP replicated efficiently in cultured human and rat HCC cells, whereas normal human and rat hepatocytes were refractory. When a single dose of the vector was injected intratumorally into large orthotopically implanted HCC in immune-competent rats, rVSV-GFP effectively and selectively replicated throughout the solid tumor mass without apparent hepatotoxicity, caused tumor destruction, and inhibited tumor growth, which led to significant prolongation of animal survival. Our results show that VSV is an effective oncolytic agent against HCC in immune-competent hosts and warrants further development for future therapy in patients with HCC.
Animals↗
Oncolysis of hepatic metastasis of colorectal cancer by recombinant vesicular stomatitis virus in immune-competent mice.
With currently available treatments, patients with metastatic colorectal cancer (CRC) have a median survival of 14.8 months and a 5-year survival rate of less than 10%. In recent years, tumor-targeted replicating viruses have rapidly emerged as potential novel oncolytic agents for cancer treatment. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with inherent selectivity for replication in tumor cells due to their attenuated antiviral response. VSV is particularly appealing as an oncolytic agent for its exceptionally rapid replication cycle in tumor cells, whereby it is capable of manifesting its maximal oncolytic effects before the onset of neutralizing antiviral immune responses in the host. In this study, we used a recombinant VSV vector expressing the green fluorescent protein gene (rVSV-GFP) to monitor VSV replication easily in CRC cells. Using this GFP-expressing virus, we found that rVSV-GFP efficiently replicated and lysed murine and human CRC cell lines in vitro. We also evaluated the potential of rVSV-GFP to treat MCA26 CRC metastases implanted orthotopically into the livers of syngeneic BALB/c mice. We provide conclusive evidence that rVSV-GFP is able to replicate extensively in the tumors, but not in normal liver cells, in tumor-bearing mice. A single intratumoral injection also caused extensive tumor necrosis, which led to a significant prolongation of animal survival. Our results indicate that VSV can be an effective and safe oncolytic agent against hepatic CRC metastasis in immune-competent mice and may be developed for the treatment of cancer patients in the future.
Animals↗
Adenovirus-mediated expression of glucokinase in the liver as an adjuvant treatment for type 1 diabetes.
Glucokinase (GK) plays a crucial role in hepatic glucose disposal. Its activity is decreased in patients with maturity-onset diabetes of the young and in some animal models of diabetes. We investigated the feasibility of manipulating GK expression as an adjuvant treatment for type 1 diabetes, using an E1/E3-deleted adenoviral vector (Ad.EF1(alpha)GK) delivered to the liver of streptozotocin-induced type 1 diabetic rats. First, we studied the metabolic impact of constitutive glucokinase expression in the absence of insulin. Normal blood glucose levels were observed after gene transfer, and glucose tolerance was substantially enhanced compared with diabetic control animals, suggesting that hepatic GK expression is a feasible mechanism to enhance glucose disposal. In a second study we administered Ad.EF1(alpha)GK together with subcutaneous insulin injections to determine whether the combined action of insulin plus GK activity would provide better glucose homeostasis than insulin treatment alone. This combination approach resulted in constant, near-normal glucose values under fed conditions. Furthermore, the animals stayed in the normoglycemic range after an overnight fast, indicating that the risk to develop hypoglycemia is not increased by expression of GK. Alterations of other metabolic routes were observed, suggesting that insulin-regulated expression of GK may be necessary to use the strategy as a treatment of type 1 diabetes.
Adenoviridae↗
Intratumoral delivery of adenovirus-mediated interleukin-12 gene in mice with metastatic cancer in the liver.
Clinical trials of recombinant human interleukin-12 (rhIL-12) delivered by intravenous administration have shown dose-limiting toxicities with limited tumor responses at the doses tested. We have previously reported that intratumoral injection of an adenovirus vector expressing murine interleukin-12 (Adv.RSV-mIL-12) was effective in inducing antitumor immune responses, tumor regression, and long-term survival in mice with established metastatic cancer in the liver. We now report additional studies in the same murine tumor model to assess the safety of intratumoral Adv.RSV-mIL-12 injection. At vector doses that were previously shown to be therapeutically effective, no inflammation in the liver or lungs, and no significant elevations in serum creatinine and aminotransferases were seen after vector injection. Serum elevations of IL-12 and interferon-gamma (IFN-gamma) were 17- and 19-fold lower than peak levels after intravenous recombinant IL-12 at the maximal tolerated dose in clinical trials. No elevations in serum proinflammatory cytokines (interleukin-6, tumor necrosis factor-alpha) were noted up to 2 weeks after vector injection. No systemic dissemination of the vector was detected on polymerase chain reaction (PCR) assays at therapeutically effective vector doses. At higher supratherapeutic vector doses of Adv.RSV-mIL-12, however, inflammation in the liver and lungs with elevation in serum aminotransferases were seen, but not in controls injected with the equivalent particle number of an empty adenoviral vector. These results support the cautious testing in patients with hepatic metastases of adenovirus mediated IL-12 gene delivery by intratumoral injection.
Adenoviridae↗
Functional genomic analysis of type II IL-1beta decoy receptor: potential for gene therapy in human arthritis and inflammation.
Gene expression arrays show that human epithelial cells and human arthritis-affected cartilage lack detectable amounts of mRNA for IL-1 antagonizing molecules: IL-1Ra and IL-1RII, but constitutively express IL-1. Functional genomic analysis was performed by reconstituting human IL-1RII expression in various IL-1RII-deficient cell types to examine its antagonist role using gene therapy approaches. Adenovirus-expressing IL-1RII when transduced into human and bovine chondrocytes, human and rabbit synovial cells, human epithelial cells, and rodent fibroblasts expressed membrane IL-1RII and spontaneously released functional soluble IL-1RII. The IL-1RII(+) (but not IL-1RII(-)) cells were resistant to IL-1beta-induced, NO, PGE(2), IL-6, and IL-8 production or decreased proteoglycan synthesis. IL-1RII inhibited the function of IL-1 in chondrocytes and IL-1- and TNF-alpha-induced inflammatory mediators in human synovial and epithelial cells. IL-1RII(+) chondrocytes were more resistant to induction of NO and PGE(2) by IL-1beta compared with IL-1RII(-) cells incubated with a 10-fold (weight) excess of soluble type II IL-1R (sIL-1RII) protein. In cocultures, IL-1RII(+) synovial cells released sIL-1RII, which in a paracrine fashion protected chondrocytes from the effects of IL-1beta. Furthermore, IL-1RII(+) (but not IL-1RII(-)) chondrocytes when transplanted onto human osteoarthritis-affected cartilage in vitro, which showed spontaneous release of sIL-1RII for 20 days, inhibited the spontaneous production of NO and PGE(2) in cartilage in ex vivo. In summary, reconstitution of IL-1RII in IL-1RII(-) cells using gene therapy approaches significantly protects cells against the autocrine and paracrine effects of IL-1 at the signaling and transcriptional levels.
Adenoviridae↗
Gene transfer in tissue regeneration and reparative medicine.
Explore the source record for details and available documents.
Gene Transfer Techniques↗
Genetic approaches to tissue engineering: breakout session summary.
Explore the source record for details and available documents.
Animals↗
An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis.
Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50-100 of ATF2 (ATF2(50-100)) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF2(50-100) to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.
Activating Transcription Factor 2↗
Basal insulin gene expression significantly improves conventional insulin therapy in type 1 diabetic rats.
Although a conventional insulin regimen for type 1 diabetes with twice-daily insulin injections is effective in preventing postprandial blood glucose excursions, this treatment is limited by its inadequate control of fasting hyperglycemia. Alternatively, sustained basal hepatic insulin gene expression has been shown to result in fasting normoglycemia in type 1 diabetic rats, although the treated animals still exhibited moderate postprandial hyperglycemia. To test the hypothesis that basal hepatic insulin production can be used as an auxiliary treatment to conventional insulin therapy for achieving better glycemic control, streptozotocin-induced diabetic rats were treated with twice-daily insulin injections, basal hepatic insulin production, or both in combination. Diabetic rats treated by conventional insulin therapy still suffered from fasting hyperglycemia, but when complemented with basal hepatic insulin production, near-normoglycemia under both fed and fasting conditions was achieved without fasting hypoglycemia. In addition, the combination-treated animals showed significantly enhanced glucose tolerance and markedly improved profiles in lipid metabolism. Furthermore, the combination treatment reduced the elevated fructosamine, glycated hemoglobin, and advanced glycation end products concentrations to normal. These results provide a proof of concept for basal hepatic insulin production as an adjuvant treatment to conventional insulin therapy in type 1 diabetes.
Animals↗