- Email: [email protected]
190. Generation of Lentivirus Based Melanocortin α, β- and γ-MSH Gene Delivery Vectors
METABOLIC DISEASES I the livers of dead mice and transplanted them into fumarylacetoacetate hydrolase (Fah) deficient mice, a model for the human metabolic liver disease, hereditary tyrosinemia (HT1). These mice represent a stringent in vivo model for hepatic cell transplantation. Surprisingly, complete functional liver repopulation could be achieved with cells isolated up to 27 hours post mortem. Furthermore, competitive repopulation experiments demonstrated that cadaveric cells had a similar repopulation capacity when compared to freshly isolated hepatocytes. This suggests the exciting possibility that a continuous source of viable hepatocytes for transplantation could be acquired from human cadavers.
188. DNA Microarray Analysis of Whole Blood Cells and Insulin-Sensitive Tissues Reveals the Usefulness of Blood RNA Profiling as a Source of Markers for Predicting Type 2 Diabetes
Yasuhiro Hayashi,1 Kazuaki Kajimoto,1 Shinya Iida,1 Yuichiro Sato,1 Noritada Kaji,2,3 Hiroyuki Kamiya,1 Yoshinobu Baba,2,3,4,5 Hideyoshi Harashima.1 1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; 2Graduate School of Engineering, Nagoya University, Nagoya, Japan; 3MEXT Innovative Reasarch Center of Preventive Medical Engineering, Nagoya University, Nagoya, Japan; 4Graduate School of Medicine, Nagoya University, Nagoya, Japan; 5Health Technology Reseach Center, National Institute of Advanced Industrial Science and Technology, Takamatsu, Japan. [Background] It is well known that diabetes is one of the most prevalent diseases worldwide. It is estimated that 246 million people have diabetes, and the number is expected to reach 380 million by 2025. Type 2 diabetes is caused by many factors, such as multigenetic factors, obesity, physical inactivity, and unhealthful diet so that it is difficult to cure effectively after it is progressed. Therefore, development of early detection method that predicts the onset of type 2 diabetes and treatment method at an early stage are greatly needed. [Materials and Methods] Male OLETF rats and non-diabetic control male LETO rats (6 weeks) of age were used as a model of spontaneous type 2 diabetes. After an overnight fast, blood was collected from the tail vein, and the glucose and insulin concentration were quantified. Then, total RNA was isolated from liver, adipose tissue, skeletal muscle, and whole blood cells. DNA microarray analysis was performed to examine the gene expression profiles in four tissues. [Results] Fasting plasma glucose (LETO=5.3±0.8 mmol/L, OLETF=5.1±0.5 mmol/L) and insulin (LETO=0.80±0.11 ng/mL, OLETF=0.92±0.16 ng/mL) concentrations were not significantly different between OLETF and LETO rats (p=0.255, 0.185, respectively). RNA profiling of whole blood cells showed that expression of 300 genes in OLETF rats was at least 1.5-fold greater or less than expression in LETO rats. In particular, 4 genes (PC-1, ectonucleotide pyrophosphatase / phosphodiesterase 1; SIRP, signal-reguratory protein alpha; Grb2, growth factor receptor bound protein 2; Pten, phosphatase and tensin homolog) are involved in the insulin-signaling pathway. In these four genes, gene expression of PC-1 in adipose tissue was 2.1-fold higher in OLETF than in LETO rats. [Conclusions] Results of this study support our hypothesis that gene expression profiling of whole blood cells might be a useful source of markers to predict the onset of type 2 diabetes. And PC-1 in adipose tissue might be a promising target gene to treat type 2 diabetes at an very early stage.
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189. Targeted Transduction of Hepatic Progenitor Cells after Early In Utero Gene Transfer Jessica L. Roybal, Masayuki Endo, David Stitelman, Antoneta Radu, Bryce Spitze, Philip W. Zoltick, Alan W. Flake. Department of Surgery, The Center for Fetal Research, Children’s Hospital of Philadelphia, Philadelphia, PA.
Orthotopic liver transplantation is the only curative treatment for a number of inherited liver-based metabolic disorders. Hepatocyte transplantation has been postulated as a less morbid approach, but inefficient engraftment has limited its application. An ideal gene transfer system would target hepatocytes prior to the onset of parenchymal injury. Our laboratory has found that early intracardiac injection of vectors leads to longterm transduction of a number of cell types. We hypothesized that an opportunity exists early in gestation when hepatic progenitor cells are accessible for gene transfer. Time-dated pregnant Balb/c mice were used for in utero gene transfer. Under ultrasound guidance, 700nl of replication incompetent, selfinactivating third generation HIV-1-based lentiviral vector encoding the green fluorescent protein (GFP) reporter gene was injected into the circulation of murine embryonic day 9 (E9) and E10 fetuses via an intracardiac approach. Vectors driven by a nonspecific and a liver-specific promoter were compared for transduction efficiency. Stereoscopic fluorescence microscopy was used to analyze the GFP expression in vector-injected mice. Tissue specimens were collected for histology and immunohistochemistry. Intracardiac injection of lentiviral vectors driven by the nonspecific promoter MND into E9 and E10 fetuses results in GFP expression in several tissues including the liver, heart, bones, and cartilage. Under stereomicroscopy, postnatal mice injected at E10 showed equal cardiac expression but greater liver expression than those injected at E9. Use of a liver-specific promoter, containing enhancing elements of alpha-1 antitrypsin and apolipoprotein E, resulted in even greater GFP expression in the liver. Immunostaining with antibodies to GFP revealed that within the liver, GFP expression was specific to hepatocytes, with no GFP expression in other tissues. Efficient gene transfer to hepatocytes is possible after early gestational intracardiac injection of lentiviral vectors. Reporter gene expression can be optimized using E10 fetuses and a liver-specific promoter. Early gestational in utero gene transfer may be applicable to a wide range of liver-based genetic disorders, particularly those with early onset and those that shorten hepatocyte longevity.
190. Generation of Lentivirus Based Melanocortin α, β- and γ-MSH Gene Delivery Vectors
Kim Eerola, Eriika Savontaus, Sini Virtanen, Mikko Savontaus. Pharmacology, Drug development and Therapeutics, University of Turku, Turku, Finland; The Centre of Biotechnology, University of Turku, Turku, Finland. Obesity and Type II Diabetes are an increasing health issue in the world and the negative effects of these conditions on health and life expectancy are evident. The pro-opiomelanocortin (POMC) pathway is involved in the metabolic regulation, mediating the effects of leptin in the hypothalamus. The melanocortin stimulating hormones; α-MSH, β-MSH and γ-MSH decrease feeding behavior and stimulate energy expenditure centrally. In addition, MSH peptides have been shown to have anti-inflammatory effects and are involved in the regulation of blood pressure. The three MSH peptides are derived from pro-opiomelanocortin (POMC) and are agonists of melanocortin receptors (MCR1-5). The MC-receptors can be found in different tissues such as melanocytes (MCR1) and in the arcuate and paraventricular nucleus of the hypothalamus (MCR3 and MCR4). In order to study the long term effects of MSH peptides, an accurate delivery and a stable expression system of the target gene is needed. Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
MUSCLE AND CONNECTIVE TISSUE - NON-MUSCLE Therefore, the aim of this study was to generate lentivirus based α-MSH, β-MSH and γ-MSH gene delivery vectors, and test their ability to produce bioactive MSH peptides. First, α-MSH, β-MSH and γ-MSH gene constructs including N’terminal POMC sequence with the signal-sorting element needed for a proper intracellular processing of the prohormones were generated and subcloned to the lentiviral vector constructs. Second generation lentiviruses were produced by cotransfection of packaging, envelope and target gene vector to HEK293T cells. GFP (Green Fluorescent Protein) control virus was used for titering and to determine the infectivity of cells of intrest: B16F10 melanoma cells and 3T3-L1 adipocytes. Stimulation of melanin production by melanocytes was used as an indicator of biological activity of the MSH vectors. B16F10 cells were infected with α-MSH, β-MSH, γ-MSH or GFP lentivirus and melanin concentration of the growth medium was measured spectrophotometrically at 405 nm. B16F10 cells infected with GFP lentivirus MOI 10 showed 4% positive cells compared to 75% in HepG2 and 90% in HEK293T infected similarly. Infections of 3T3-L1 preadipocytes and differentiated adipocytes with MOI10 produced 53% and 38% GFP positive cells, respectively. Despite of the low infectivity, melanocytes infected with α-MSH (MOI 10) virus produced 33 % more melanin compared to cells infected with control GFP virus showing that infection with α-MSH virus results in production of biologically active MSH peptide. MSH peptides have been shown to be involved in several regulatory processes such as regulation of body weight and blood pressure. Investigations of the chronic effects of MSH peptides have proved to be difficult due to their short half-life. We have shown that MSH lentiviral vectors containing the N’terminal POMC signal-sorting sequence followed by the MSH sequence leads to production of bioactive MSH peptide. These vectors give a new tool to study the effects of MSH peptides in vitro and in vivo.
Muscle and Connective Tissue - Non-muscle 191. Examining the Role of Elevated Blood Glucose in the Severity of Arthrofibrosis Using Intra-Articular Gene Delivery of TGF-β1
Rachael S. Watson,1 Padraic P. Levings,1 Marsha L. Bush,1 Jesse D. Kay,1 Steven C. Ghivizzani.1 1 Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL.
Idiopathic adhesive capsulitis (IAC) of the shoulder is a disease of unknown etiology characterized by painful, chronic fibrotic expansion of the synovium and joint capsule, which gradually leads to loss of joint motion, often resolving after a period of years. Although IAC affects approximately 3% of the population and 20% of diabetics, little is known about its pathogenesis or the effects of elevated glucose on arthrofibrosis. While the underlying causes are diverse, it is likely that many of the harmful aspects of fibrosis are mediated by transforming growth factor-β1 (TGF-β1), a pleiotropic cytokine. In an effort to establish an animal model of IAC, examine the role elevated glucose plays and develop an understanding of the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver TGF-β1 cDNA (AdTGF-β1) in the knee joints of streptozotocin-induced diabetic and non-diabetic Wistar rats. Following injection of the vector, animals were sacrificed periodically, and the joint tissues were examined macroscopically and histologically, as well as by PCR-array for alterations in the patterns of expression of extracellular matrix-associated genes. Overexpression of the TGF-b1 transgene stimulated a dramatic fibrotic event, causing a rapid increase in knee diameter and the complete encasement of the joints in dense scar-like tissue, locking the joints at 90o of flexion. Histologically, massive proliferation of resident synovial fibroblasts was seen followed by their differentiation into myofibroblasts. The Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
fibrotic expansion was observed to overrun and displace the normal architecture of the joint capsule and fuse with the articular cartilage. Day 10 expression profiles showed exceptionally high levels of MMP production, as well as, tenascin C, osteopontin and thromobospondins 1 and 2. By day 30 the predominant phenotype of the expanded tissue had changed to that of articular cartilage, indicated by tissue and cellular morphology, matrix composition, and increases in expression of collagen type II and link protein. Over the course of 120 days, the joints appeared to remodel, such that much of the cellular fibrotic tissue resolved. Mild differences were observed in diabetic animals. Histologically, diabetic joints appeared to have a more severe response, with an increased proliferation of fibrotic tissue filling the joint space. Expression profiles in diabetic animals at day 10 showed decreases in MMPs 8-13 and collagen II expression and increases in MMP 7 and platelet, endothelial and lymphocyte selectins. By day 30, the expression profiles showed increases in collagens 1-6, integrins, TIMPs 2 and 3, tenascin C and MMP9 expression. Altogether, these results indicate that TGF-b1 is a potent inducer of arthrofibrosis, and elevated glucose as a result of diabetes enhances the fibrotic induction of TFG-b1 resulting in a slightly more severe fibrotic episode.
192. Transduction of Equine Joint Tissues with Self-Complementary Adeno-Associated Virus
Jesse D. Kay,1 Rachael S. Watson,1 Marsha L. Bush,1 Padraic L. Levings,1 Patrick T. Colahan,2 Steve C. Ghivizzani.1 1 Orthopaedics & Rehabilitation, The University of Florida, Gainesville, FL; 2Large Animal Clinical Sciences, The University of Florida, Gainesville, FL.
The degenerate joint disease osteoarthritis affects humans and many large mammals alike, severely decreasing quality of life. Effective gene delivery approaches to treating arthritic conditions have been met with several challenges, including suitable vector and model systems. Self-complementary AAV (scAAV) vectors provide the safety advantages of AAV, while also having the potential to deliver heightened levels of transgene expression with rapid onset. Gene based delivery of the anti-inflammatory protein interleukin-1 receptor antagonist (IL-1Ra) in concentrations around 1 ng/ml has been shown to decrease inflammatory activity inside arthritic joints in rodent models. Horses provide a unique model system in which to study intra-articular gene transfer for joint disease as their forelimb joints are proportional in size to human knees, and they naturally develop osteoarthritis. In this study we tested the utility of scAAV vectors to deliver therapeutic levels of transgenes in equine carpal and midcarpal joints. cDNAs for IL-1Ra or GFP were inserted into the CMV expression cassette of the pHpa-Trs-SK, self complementary AAV2 vector plasmid. Recombinant scAAV was generated by cotransfection of 293 cells with appropriate helper plasmids to yield serotypes 1, 2, 3, 5, 7, and 8. scAAV-GFP packaged in this battery of serotypes was used to transduce primary equine cells derived from synovium, cartilage, tendon, and ligament at 10³ viral genomes/cell. The results indicate that serotypes 2 and 5 are most effective across all cell types. When measured by flow cytometry three days post infection, over 98% of equine synovial fibroblasts were GFP positive for both type 2 and type 5 infections. When IL-1Ra is used as the transgene, similar results were observed: medium collected from cells infected with serotypes 2 and 5 contained 10-25 ng/ml of IL-1Ra as measured by ELISA, while medium from other serotype infections yielded IL1Ra concentrations in the low pg/ml range. Data from horses was obtained after direct injections of both serotypes of scAAV-IL-1Ra into the carpal and midcarpal joints of thoroughbred race horses. Synovial fluid was collected for ELISA every week for five weeks. Results were positive for the entire length of the study, with synovial fluid from type 5 infected joints averaging 1.5 ng/ml of IL-1Ra, and type 2 joints averaging 0.5 ng/ml. The data from this study indicate scAAV vectors can effectively transduce equine joint tissues to a level of therapeutic relevance. S75
188. DNA Microarray Analysis of Whole Blood Cells and Insulin-Sensitive Tissues Reveals the Usefulness of Blood RNA Profiling as a Source of Markers for Predicting Type 2 Diabetes
Yasuhiro Hayashi,1 Kazuaki Kajimoto,1 Shinya Iida,1 Yuichiro Sato,1 Noritada Kaji,2,3 Hiroyuki Kamiya,1 Yoshinobu Baba,2,3,4,5 Hideyoshi Harashima.1 1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; 2Graduate School of Engineering, Nagoya University, Nagoya, Japan; 3MEXT Innovative Reasarch Center of Preventive Medical Engineering, Nagoya University, Nagoya, Japan; 4Graduate School of Medicine, Nagoya University, Nagoya, Japan; 5Health Technology Reseach Center, National Institute of Advanced Industrial Science and Technology, Takamatsu, Japan. [Background] It is well known that diabetes is one of the most prevalent diseases worldwide. It is estimated that 246 million people have diabetes, and the number is expected to reach 380 million by 2025. Type 2 diabetes is caused by many factors, such as multigenetic factors, obesity, physical inactivity, and unhealthful diet so that it is difficult to cure effectively after it is progressed. Therefore, development of early detection method that predicts the onset of type 2 diabetes and treatment method at an early stage are greatly needed. [Materials and Methods] Male OLETF rats and non-diabetic control male LETO rats (6 weeks) of age were used as a model of spontaneous type 2 diabetes. After an overnight fast, blood was collected from the tail vein, and the glucose and insulin concentration were quantified. Then, total RNA was isolated from liver, adipose tissue, skeletal muscle, and whole blood cells. DNA microarray analysis was performed to examine the gene expression profiles in four tissues. [Results] Fasting plasma glucose (LETO=5.3±0.8 mmol/L, OLETF=5.1±0.5 mmol/L) and insulin (LETO=0.80±0.11 ng/mL, OLETF=0.92±0.16 ng/mL) concentrations were not significantly different between OLETF and LETO rats (p=0.255, 0.185, respectively). RNA profiling of whole blood cells showed that expression of 300 genes in OLETF rats was at least 1.5-fold greater or less than expression in LETO rats. In particular, 4 genes (PC-1, ectonucleotide pyrophosphatase / phosphodiesterase 1; SIRP, signal-reguratory protein alpha; Grb2, growth factor receptor bound protein 2; Pten, phosphatase and tensin homolog) are involved in the insulin-signaling pathway. In these four genes, gene expression of PC-1 in adipose tissue was 2.1-fold higher in OLETF than in LETO rats. [Conclusions] Results of this study support our hypothesis that gene expression profiling of whole blood cells might be a useful source of markers to predict the onset of type 2 diabetes. And PC-1 in adipose tissue might be a promising target gene to treat type 2 diabetes at an very early stage.
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189. Targeted Transduction of Hepatic Progenitor Cells after Early In Utero Gene Transfer Jessica L. Roybal, Masayuki Endo, David Stitelman, Antoneta Radu, Bryce Spitze, Philip W. Zoltick, Alan W. Flake. Department of Surgery, The Center for Fetal Research, Children’s Hospital of Philadelphia, Philadelphia, PA.
Orthotopic liver transplantation is the only curative treatment for a number of inherited liver-based metabolic disorders. Hepatocyte transplantation has been postulated as a less morbid approach, but inefficient engraftment has limited its application. An ideal gene transfer system would target hepatocytes prior to the onset of parenchymal injury. Our laboratory has found that early intracardiac injection of vectors leads to longterm transduction of a number of cell types. We hypothesized that an opportunity exists early in gestation when hepatic progenitor cells are accessible for gene transfer. Time-dated pregnant Balb/c mice were used for in utero gene transfer. Under ultrasound guidance, 700nl of replication incompetent, selfinactivating third generation HIV-1-based lentiviral vector encoding the green fluorescent protein (GFP) reporter gene was injected into the circulation of murine embryonic day 9 (E9) and E10 fetuses via an intracardiac approach. Vectors driven by a nonspecific and a liver-specific promoter were compared for transduction efficiency. Stereoscopic fluorescence microscopy was used to analyze the GFP expression in vector-injected mice. Tissue specimens were collected for histology and immunohistochemistry. Intracardiac injection of lentiviral vectors driven by the nonspecific promoter MND into E9 and E10 fetuses results in GFP expression in several tissues including the liver, heart, bones, and cartilage. Under stereomicroscopy, postnatal mice injected at E10 showed equal cardiac expression but greater liver expression than those injected at E9. Use of a liver-specific promoter, containing enhancing elements of alpha-1 antitrypsin and apolipoprotein E, resulted in even greater GFP expression in the liver. Immunostaining with antibodies to GFP revealed that within the liver, GFP expression was specific to hepatocytes, with no GFP expression in other tissues. Efficient gene transfer to hepatocytes is possible after early gestational intracardiac injection of lentiviral vectors. Reporter gene expression can be optimized using E10 fetuses and a liver-specific promoter. Early gestational in utero gene transfer may be applicable to a wide range of liver-based genetic disorders, particularly those with early onset and those that shorten hepatocyte longevity.
190. Generation of Lentivirus Based Melanocortin α, β- and γ-MSH Gene Delivery Vectors
Kim Eerola, Eriika Savontaus, Sini Virtanen, Mikko Savontaus. Pharmacology, Drug development and Therapeutics, University of Turku, Turku, Finland; The Centre of Biotechnology, University of Turku, Turku, Finland. Obesity and Type II Diabetes are an increasing health issue in the world and the negative effects of these conditions on health and life expectancy are evident. The pro-opiomelanocortin (POMC) pathway is involved in the metabolic regulation, mediating the effects of leptin in the hypothalamus. The melanocortin stimulating hormones; α-MSH, β-MSH and γ-MSH decrease feeding behavior and stimulate energy expenditure centrally. In addition, MSH peptides have been shown to have anti-inflammatory effects and are involved in the regulation of blood pressure. The three MSH peptides are derived from pro-opiomelanocortin (POMC) and are agonists of melanocortin receptors (MCR1-5). The MC-receptors can be found in different tissues such as melanocytes (MCR1) and in the arcuate and paraventricular nucleus of the hypothalamus (MCR3 and MCR4). In order to study the long term effects of MSH peptides, an accurate delivery and a stable expression system of the target gene is needed. Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
MUSCLE AND CONNECTIVE TISSUE - NON-MUSCLE Therefore, the aim of this study was to generate lentivirus based α-MSH, β-MSH and γ-MSH gene delivery vectors, and test their ability to produce bioactive MSH peptides. First, α-MSH, β-MSH and γ-MSH gene constructs including N’terminal POMC sequence with the signal-sorting element needed for a proper intracellular processing of the prohormones were generated and subcloned to the lentiviral vector constructs. Second generation lentiviruses were produced by cotransfection of packaging, envelope and target gene vector to HEK293T cells. GFP (Green Fluorescent Protein) control virus was used for titering and to determine the infectivity of cells of intrest: B16F10 melanoma cells and 3T3-L1 adipocytes. Stimulation of melanin production by melanocytes was used as an indicator of biological activity of the MSH vectors. B16F10 cells were infected with α-MSH, β-MSH, γ-MSH or GFP lentivirus and melanin concentration of the growth medium was measured spectrophotometrically at 405 nm. B16F10 cells infected with GFP lentivirus MOI 10 showed 4% positive cells compared to 75% in HepG2 and 90% in HEK293T infected similarly. Infections of 3T3-L1 preadipocytes and differentiated adipocytes with MOI10 produced 53% and 38% GFP positive cells, respectively. Despite of the low infectivity, melanocytes infected with α-MSH (MOI 10) virus produced 33 % more melanin compared to cells infected with control GFP virus showing that infection with α-MSH virus results in production of biologically active MSH peptide. MSH peptides have been shown to be involved in several regulatory processes such as regulation of body weight and blood pressure. Investigations of the chronic effects of MSH peptides have proved to be difficult due to their short half-life. We have shown that MSH lentiviral vectors containing the N’terminal POMC signal-sorting sequence followed by the MSH sequence leads to production of bioactive MSH peptide. These vectors give a new tool to study the effects of MSH peptides in vitro and in vivo.
Muscle and Connective Tissue - Non-muscle 191. Examining the Role of Elevated Blood Glucose in the Severity of Arthrofibrosis Using Intra-Articular Gene Delivery of TGF-β1
Rachael S. Watson,1 Padraic P. Levings,1 Marsha L. Bush,1 Jesse D. Kay,1 Steven C. Ghivizzani.1 1 Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL.
Idiopathic adhesive capsulitis (IAC) of the shoulder is a disease of unknown etiology characterized by painful, chronic fibrotic expansion of the synovium and joint capsule, which gradually leads to loss of joint motion, often resolving after a period of years. Although IAC affects approximately 3% of the population and 20% of diabetics, little is known about its pathogenesis or the effects of elevated glucose on arthrofibrosis. While the underlying causes are diverse, it is likely that many of the harmful aspects of fibrosis are mediated by transforming growth factor-β1 (TGF-β1), a pleiotropic cytokine. In an effort to establish an animal model of IAC, examine the role elevated glucose plays and develop an understanding of the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver TGF-β1 cDNA (AdTGF-β1) in the knee joints of streptozotocin-induced diabetic and non-diabetic Wistar rats. Following injection of the vector, animals were sacrificed periodically, and the joint tissues were examined macroscopically and histologically, as well as by PCR-array for alterations in the patterns of expression of extracellular matrix-associated genes. Overexpression of the TGF-b1 transgene stimulated a dramatic fibrotic event, causing a rapid increase in knee diameter and the complete encasement of the joints in dense scar-like tissue, locking the joints at 90o of flexion. Histologically, massive proliferation of resident synovial fibroblasts was seen followed by their differentiation into myofibroblasts. The Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
fibrotic expansion was observed to overrun and displace the normal architecture of the joint capsule and fuse with the articular cartilage. Day 10 expression profiles showed exceptionally high levels of MMP production, as well as, tenascin C, osteopontin and thromobospondins 1 and 2. By day 30 the predominant phenotype of the expanded tissue had changed to that of articular cartilage, indicated by tissue and cellular morphology, matrix composition, and increases in expression of collagen type II and link protein. Over the course of 120 days, the joints appeared to remodel, such that much of the cellular fibrotic tissue resolved. Mild differences were observed in diabetic animals. Histologically, diabetic joints appeared to have a more severe response, with an increased proliferation of fibrotic tissue filling the joint space. Expression profiles in diabetic animals at day 10 showed decreases in MMPs 8-13 and collagen II expression and increases in MMP 7 and platelet, endothelial and lymphocyte selectins. By day 30, the expression profiles showed increases in collagens 1-6, integrins, TIMPs 2 and 3, tenascin C and MMP9 expression. Altogether, these results indicate that TGF-b1 is a potent inducer of arthrofibrosis, and elevated glucose as a result of diabetes enhances the fibrotic induction of TFG-b1 resulting in a slightly more severe fibrotic episode.
192. Transduction of Equine Joint Tissues with Self-Complementary Adeno-Associated Virus
Jesse D. Kay,1 Rachael S. Watson,1 Marsha L. Bush,1 Padraic L. Levings,1 Patrick T. Colahan,2 Steve C. Ghivizzani.1 1 Orthopaedics & Rehabilitation, The University of Florida, Gainesville, FL; 2Large Animal Clinical Sciences, The University of Florida, Gainesville, FL.
The degenerate joint disease osteoarthritis affects humans and many large mammals alike, severely decreasing quality of life. Effective gene delivery approaches to treating arthritic conditions have been met with several challenges, including suitable vector and model systems. Self-complementary AAV (scAAV) vectors provide the safety advantages of AAV, while also having the potential to deliver heightened levels of transgene expression with rapid onset. Gene based delivery of the anti-inflammatory protein interleukin-1 receptor antagonist (IL-1Ra) in concentrations around 1 ng/ml has been shown to decrease inflammatory activity inside arthritic joints in rodent models. Horses provide a unique model system in which to study intra-articular gene transfer for joint disease as their forelimb joints are proportional in size to human knees, and they naturally develop osteoarthritis. In this study we tested the utility of scAAV vectors to deliver therapeutic levels of transgenes in equine carpal and midcarpal joints. cDNAs for IL-1Ra or GFP were inserted into the CMV expression cassette of the pHpa-Trs-SK, self complementary AAV2 vector plasmid. Recombinant scAAV was generated by cotransfection of 293 cells with appropriate helper plasmids to yield serotypes 1, 2, 3, 5, 7, and 8. scAAV-GFP packaged in this battery of serotypes was used to transduce primary equine cells derived from synovium, cartilage, tendon, and ligament at 10³ viral genomes/cell. The results indicate that serotypes 2 and 5 are most effective across all cell types. When measured by flow cytometry three days post infection, over 98% of equine synovial fibroblasts were GFP positive for both type 2 and type 5 infections. When IL-1Ra is used as the transgene, similar results were observed: medium collected from cells infected with serotypes 2 and 5 contained 10-25 ng/ml of IL-1Ra as measured by ELISA, while medium from other serotype infections yielded IL1Ra concentrations in the low pg/ml range. Data from horses was obtained after direct injections of both serotypes of scAAV-IL-1Ra into the carpal and midcarpal joints of thoroughbred race horses. Synovial fluid was collected for ELISA every week for five weeks. Results were positive for the entire length of the study, with synovial fluid from type 5 infected joints averaging 1.5 ng/ml of IL-1Ra, and type 2 joints averaging 0.5 ng/ml. The data from this study indicate scAAV vectors can effectively transduce equine joint tissues to a level of therapeutic relevance. S75