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766. Hairpin Ribozymes Encoded in Adenoviral Vectors Reduce Drinking in Alcohol Dependent Rats
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY III 764. Microporous Polymer Scaffolds for Islet Transplantation
Romie F. Gibly,1 Lonnie D. Shea.1 Chemical and Biological Engineering, Northwestern University, Evanston, IL.
1
Background: Type-1 diabetes is a lifelong disease managed with insulin therapy yet typically results in signicant morbidity and early mortality. Islet transplantation as a cell therapy to replace the damaged insulin-producing ß-cells offers the potential of a real cure. Despite a decade of research since the rst transplants, a reliable and long-term solution has been elusive. Currently, islets are infused into the recipient’s hepatic vasculature; a site that may negatively impact the islets, and a process limited by islet availability and graft failure over time. Here we present an extrahepatic approach based on localized control of the islet microenvironment to maximize islet survival, function and reintegration with the host. Integration is accomplished by delivering islets on a porous, biodegradable polymer scaffold, in contrast with the isolating encapsulation strategies of most biomaterials. Methods: Poly(lactide-co-glycolide) microspheres were used to fabricate biocompatible, highly porous and degradable scaffolds for islet transplant in mice with chemically-induced diabetes. Scaffolds were modied to architecturally dene the transplant environment, support the islets, present ECM molecules, and locally deliver IGF to enhance islet survival. Diabetes reversal, as measured by blood glucose, weight gain, and histology of graft explants were used to assay the effectiveness of scaffolds. Pilot studies investigating allotransplants on these scaffolds at two sites in a porcine model were also performed. Results: Scaffolds with varying architecture were investigated for the impact on islet function in mice at a peritoneal fat transplant site. Scaffolds formed from 6% PLG microspheres reversed diabetes with only 75 islets, representing approximately 1/3 of the normal pancreatic mass of islets. Using 2% PLG microspheres required 125 islets under the same conditions. Histological analysis demonstrated complete host cell inltration, islet revascularization, and general biocompatibility based on the lack of an inammatory capsule. In vitro, scaffolds with IGF released bioactive protein for at least 2 wks. We subsequently investigated the transplant of islets on scaffolds in a porcine model; requiring a signicant increase in islet number due to the mass of the recipient. Despite this increase, histological analysis demonstrated similar engraftment. Scaffolds supported signicant islet survival with revascularization, host tissue inltration and no brotic capsule formation at 2 wks post-transplant in both the gastric submucosa and omental fat of pigs. Conclusions: Utilizing PLG scaffolds and an extrahepatic site, syngeneic and allogeneic islets can be delivered as a cell-based therapy for insulin-dependent diabetes. In mice, scaffolds in the epididymal fat pad support successful transplant of what is considered a minimal mass of islets at traditional liver or kidney capsule sites. This platform enables localized control of the islet distribution, and the transplant microenvironment by presenting extracellular matrix molecules to mimic the native islet environment, and locally delivering trophic factors to enhance survival, engraftment and revascularization in a robust, modular package; potentially decreasing the required number of islets signicantly.
765. AAV2- and AAV9 -Mediated Gene Transfer of LDL Receptor to Hypercholesterolemic WHHL Rabbits
Elisa Vähäkangas,1 Anniina Laurema,1 Atsushi Miyanohara,2 Seppo Ylä-Herttuala.1 1 Department of Biotechnology and Molecular medicine, A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; 2Department of Medicine, University of California San Diego, La Jolla, CA. Liver-directed gene therapy is a potential new treatment for a variety of inherited liver disorders. Familial hypercholesterolemia (FH) is a monogenic disease of cholesterol metabolism caused by a defect in the receptor for low density lipoprotein (LDLR), which leads to increased circulating low-density lipoprotein (LDL) levels and premature cardiovascular disease. Adeno-Associated viruses (AAVs) are non-pathogenic parvoviruses that can replicate only in the presence of a helper virus. Vectors based on AAV are promising tools for gene therapy due to their ability to mediate long term gene expression, necessary for inherited disorder such as FH, without signicant immune responses or toxicity. To determine the efciency of AAV-mediated LDLR gene transfer in the treatment of FH, a serotype 2 (AAV2) or 9 (AAV9) adenoassociated virus (AAV) based vector encoding rabbit LDL recptor was injected into the portal vein of WHHL (Watanabe Heritable Hypercholesterolemic) rabbits. Control animals received either AAV2- or AAV9-LacZ. Blood samples were drawn regularly to monitor lipid levels and other clinical chemistry parameters. Liver biopsies were obtained 4 weeks and animals sacriced 12 months after gene transfer. Presence of transgene in the liver was veried by PCR 12 months after gene transfer from AAV2-LacZ and AAV2-LDLR groups and with immunohistochemistry from AAV2-LacZ group. Gene transfer of AAV9-LDLR led to a decrease in serum total cholesterol levels 5 months after gene transfer but the decrease was not statistically signicant (-20.4% ± 27.6% for AAV9-LDLR and 2.3% ± 16% for AAV9-LacZ, mean ± SD ). The effect was attenuated at later time points. AAV-2 mediated gene transfer of LDLR did not lead to a decrease in total cholesterol levels compared to controls but in turn increased 12 months after gene transfer in comparison to LacZ controls. Measeurment of liver enzymes showed no major changes in liver function. Histology of the liver revealed some inammation in animals from the control groups. Also in two animals from the AAV2-LDLR group bile duct proliferation was seen, the signicance of which remains to be studied. In conclusion, although AAV mediated gene transfer of LDLR resulted in long lasting and sustained expression of the transgene in WHHL-rabbit liver, the achieved transgene expression did not result in sustained correction of the FH phenotype with either AAV serotype.
766. Hairpin Ribozymes Encoded in Adenoviral Vectors Reduce Drinking in Alcohol Dependent Rats
Amalia Sapag,1 Thergiory Irrazábal,1 María Elena Quintanilla,2 Lutske Tampier.2 1 Gene Pharmacotherapy Laboratory, Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile, Santiago, Chile; 2Program of Molecular and Clinical Pharmacology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile.
Alcoholism is a multifactorial disease in which genetic factors play a major role (60%). Ethanol is metabolized in the liver to acetaldehyde by alcohol dehydrogenases and then to acetate by S298
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY III mitochondrial aldehyde dehydrogenase (ALDH2). Natural genetic protection from alcoholism is provided by an inactivating mutation in ALDH2 which results in increased blood acetaldehyde that leads to unpleasant physical effects and aversion to ethanol. The drug disulram mimics this protection by covalent inactivation of ALDH2 but its efcacy in the treatment of alcoholics is offset by toxicity, individual variability and the requirement of daily intake. Thus, alternative drugs based on silencing the ALDH2 mRNA are worth exploring. A hairpin ribozyme (RzCG20) targeted to the rat ALDH2 mRNA has been shown to reduce ALDH2 activity in a rat hepatoma cell line by both cleavage and blockade, as seen by comparison to a control ribozyme (RzCG20c) designed to bind but not cleave the target. The aim of this work was to test RzCG20 and RzCG20c in vivo for their capacity to reduce drinking in alcohol dependent rats. Wistar rats bred as heavy alcohol drinkers (UChB rats) were allowed free access to both 10% ethanol and water for 30 days (ethanol intake ∼7 g/kg/day). Animals were injected in the tail vein with a ∆(E1,E3) adenoviral vector (2x1012 vp/kg) encoding either RzCG20, RzCG20c, or a control RNA, or with vehicle; gene constructs had a CMV promoter and an SV40 polyadenylation signal. After four days of ethanol withdrawal, animals were allowed to choose between 10% ethanol and water for only one hour each day; water was available ad libitum. A single administration of the adenoviral vectors encoding RzCG20 or RzCG20c (n=5) yielded a 50% decrease in alcohol consumption for 34 days while the control adenovirus and the vehicle did not decrease consumption (p<0.001). Arterial blood acetaldehyde was measured at 39 days postinjection after alcohol disintoxication (one day withdrawal) and administration of a standard dose of ethanol (1 g/kg i.p.). In rats receiving either ribozyme gene, acetaldehyde increased to more than twice that of animals receiving the control adenovirus or the vehicle (p<0.001). The ALDH2 activity measured in liver homogenates showed a 24% reduction (p<0.02) while the activity of other ALDHs was not affected. In conclusion, both RzCG20 and RzCG20c are effective in vivo, being capable of silencing the ALDH2 mRNA in rats. The mechanistic differences seen between these ribozymes in vitro were not reected in their biochemical effects in vivo after a month, suggesting that binding of RzCG20c to 16 nucleotides in the target mRNA is sufcient to reduce the liver ALDH2 activity to an extent resulting in increased blood acetaldehyde and reduced alcohol consumption. Overall, hairpin ribozymes that reduce the expression of the ALDH2 mRNA are promising candidates for further development of gene therapy for alcoholism. Support: FONDECYT 1040555, VOLO 2008-02.
767. Sustained Therapeutic Levels of FVIII Activity from Lentivirally-Engineered Blood Outgrowth Endothelial Cells Requires Cell Stability and Augmented Angiogenesis
Hideto Matsui,1,2 Margareth Ozelo,1 Andrea Labelle,1 Carol Hegadorn,1 Patrick Thompson,1 Erin Burnnet,1 Christine Hough,1 David Lillicrap.1 1 Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada; 2Regulatory Medicine for Thrombosis, Nara Medical University, Kashihara, Nara, Japan. An ex vivo gene transfer strategy using gene modied blood outgrowth endothelial cells (BOECs) for hemophilia A has been proposed as a therapeutic alternative to systemic administration of viral vectors. We have previously reported that FVII gene-modied BOECs are sequestered in subcutaneous implants and cells are viable and capable of synthesizing therapeutic levels of plasma FVIII over a prolonged period of time. However, one of the major challenges in establishing this as a new treatment modality is the loss of BOEC function and viability after engraftment. This may in part be due to a less than optimal environment within the extracellular matrix. The aim of the present study was to assess the inuence of incorporating Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Fibroblast Growth Factor-2 (FGF) and brinogen (Fbg) within the implanted matrix in enhancing BOEC viability and function. BOECs were isolated from mouse peripheral blood and transduced with a lentiviral vector encoding the canine FVIII transgene under the transcriptional control of the CMV promoter. Two million transduced BOECs were resuspended in Matrigel, supplemented with either FGF, Fbg or both FGF and Fbg, and implanted subcutanenously into NOD/SCID mice. Canine FVIII antigen levels were assayed at weekly intervals and compared to control mice that received Matrigel implants consisting of the same numbers of cells without FGF and Fbg. FVIII antigen was assayed with the Asserachrom VIII:Ag ELISA that detects canine FVIII against a background of normal murine FVIII. After 4 weeks, the mean FVIII antigen in mice that received implants incorporating FGF, Fbg and FGF+Fbg was 36.9 mU/mL, 24.0 mU/ml and 52.3 mU/mL, respectively compared to 25.0 mU/ mL, for Matrigel alone. These therapeutic levels were sustained for up to 12 weeks as expected for cells engineered with a gene under the control of a viral promoter. To evaluate if the observed elevation of FVIII expression resulted from enhanced cellular viability resulting from inclusion of FGF or Fbg, mice were sacriced after 20 weeks and grafts were excised and processed for histological examination. Immuno-staining shows that the numbers of BOECs within Matrigel were similar between the 4 different groups of mice. However, the extent of the capillary architecture observed in the various retrieved implants was signicantly higher in those that contained FGF and FGF+Fbg compared to implants with Fbg and Matrigel without supplements. In conclusion, these results indicate that incorporation of adhesive proteins and vascular growth factors into the transplanted scaffolds enhances FVIII expression at least in part due to increased neo-vascularization.
768. Effects of Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanlippo Syndrome Type B Mice
Coy D. Heldermon,1 Elizabeth Qin,2 Kevin K. Ohlemiller,3 Erik Herzog,4 Carole Vogler,6 David F. Wozniak,5 Mark S. Sands.2 1 Department of Medicine, University of Florida, Gainesville, FL; 2 Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; 3Department of Otolaryngology, Washington University School of Medicine, St. Louis, MO; 4 Department of Biology, Washington University, St. Louis, MO; 5 Department of Psychiatry, Washington University School of Medicine, St. Louis, MO; 6Department of Pathology, St. Louis University School of Medicine, St. Louis, MO. Sanfilippo Syndrome type B (MPS IIIB) is a lysosomal storage disease (LSD) resulting from the deciency of N-acetyl glucosaminidase (NAGLU). We previously showed that intracranial AAV2/5-based gene therapy improves several aspects of the disease such as lifespan. However, there is growing evidence that LSDs will be most effectively treated by using a combination of approaches that target different aspects of disease. Therefore, we initially combined bone marrow transplantation (BMT) with intracranial AAV. Unfortunately, BMT provided little or no additional therapeutic benet. In fact, BMT actually interfered with the benet provided by AAV with respect to motor function and life span. In an attempt to discover a more effective combination of approaches to treat the disease in the mouse model of MPS IIIB, affected mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (AAV), intravenous lentiviralNAGLU (MND) or both (AAV/MND). Compared to untreated MPS IIIB animals, preliminary results indicate that all treatments resulted in some improvement in the clinical phenotype. AAV, MND and AAV/MND treatment results in improved motor function compared to untreated controls using a rocking rotarod paradigm. Early results from Auditory-evoked Brainstem Response (ABR) measurements of hearing also demonstrate improvement in all treatment groups. S299
Romie F. Gibly,1 Lonnie D. Shea.1 Chemical and Biological Engineering, Northwestern University, Evanston, IL.
1
Background: Type-1 diabetes is a lifelong disease managed with insulin therapy yet typically results in signicant morbidity and early mortality. Islet transplantation as a cell therapy to replace the damaged insulin-producing ß-cells offers the potential of a real cure. Despite a decade of research since the rst transplants, a reliable and long-term solution has been elusive. Currently, islets are infused into the recipient’s hepatic vasculature; a site that may negatively impact the islets, and a process limited by islet availability and graft failure over time. Here we present an extrahepatic approach based on localized control of the islet microenvironment to maximize islet survival, function and reintegration with the host. Integration is accomplished by delivering islets on a porous, biodegradable polymer scaffold, in contrast with the isolating encapsulation strategies of most biomaterials. Methods: Poly(lactide-co-glycolide) microspheres were used to fabricate biocompatible, highly porous and degradable scaffolds for islet transplant in mice with chemically-induced diabetes. Scaffolds were modied to architecturally dene the transplant environment, support the islets, present ECM molecules, and locally deliver IGF to enhance islet survival. Diabetes reversal, as measured by blood glucose, weight gain, and histology of graft explants were used to assay the effectiveness of scaffolds. Pilot studies investigating allotransplants on these scaffolds at two sites in a porcine model were also performed. Results: Scaffolds with varying architecture were investigated for the impact on islet function in mice at a peritoneal fat transplant site. Scaffolds formed from 6% PLG microspheres reversed diabetes with only 75 islets, representing approximately 1/3 of the normal pancreatic mass of islets. Using 2% PLG microspheres required 125 islets under the same conditions. Histological analysis demonstrated complete host cell inltration, islet revascularization, and general biocompatibility based on the lack of an inammatory capsule. In vitro, scaffolds with IGF released bioactive protein for at least 2 wks. We subsequently investigated the transplant of islets on scaffolds in a porcine model; requiring a signicant increase in islet number due to the mass of the recipient. Despite this increase, histological analysis demonstrated similar engraftment. Scaffolds supported signicant islet survival with revascularization, host tissue inltration and no brotic capsule formation at 2 wks post-transplant in both the gastric submucosa and omental fat of pigs. Conclusions: Utilizing PLG scaffolds and an extrahepatic site, syngeneic and allogeneic islets can be delivered as a cell-based therapy for insulin-dependent diabetes. In mice, scaffolds in the epididymal fat pad support successful transplant of what is considered a minimal mass of islets at traditional liver or kidney capsule sites. This platform enables localized control of the islet distribution, and the transplant microenvironment by presenting extracellular matrix molecules to mimic the native islet environment, and locally delivering trophic factors to enhance survival, engraftment and revascularization in a robust, modular package; potentially decreasing the required number of islets signicantly.
765. AAV2- and AAV9 -Mediated Gene Transfer of LDL Receptor to Hypercholesterolemic WHHL Rabbits
Elisa Vähäkangas,1 Anniina Laurema,1 Atsushi Miyanohara,2 Seppo Ylä-Herttuala.1 1 Department of Biotechnology and Molecular medicine, A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; 2Department of Medicine, University of California San Diego, La Jolla, CA. Liver-directed gene therapy is a potential new treatment for a variety of inherited liver disorders. Familial hypercholesterolemia (FH) is a monogenic disease of cholesterol metabolism caused by a defect in the receptor for low density lipoprotein (LDLR), which leads to increased circulating low-density lipoprotein (LDL) levels and premature cardiovascular disease. Adeno-Associated viruses (AAVs) are non-pathogenic parvoviruses that can replicate only in the presence of a helper virus. Vectors based on AAV are promising tools for gene therapy due to their ability to mediate long term gene expression, necessary for inherited disorder such as FH, without signicant immune responses or toxicity. To determine the efciency of AAV-mediated LDLR gene transfer in the treatment of FH, a serotype 2 (AAV2) or 9 (AAV9) adenoassociated virus (AAV) based vector encoding rabbit LDL recptor was injected into the portal vein of WHHL (Watanabe Heritable Hypercholesterolemic) rabbits. Control animals received either AAV2- or AAV9-LacZ. Blood samples were drawn regularly to monitor lipid levels and other clinical chemistry parameters. Liver biopsies were obtained 4 weeks and animals sacriced 12 months after gene transfer. Presence of transgene in the liver was veried by PCR 12 months after gene transfer from AAV2-LacZ and AAV2-LDLR groups and with immunohistochemistry from AAV2-LacZ group. Gene transfer of AAV9-LDLR led to a decrease in serum total cholesterol levels 5 months after gene transfer but the decrease was not statistically signicant (-20.4% ± 27.6% for AAV9-LDLR and 2.3% ± 16% for AAV9-LacZ, mean ± SD ). The effect was attenuated at later time points. AAV-2 mediated gene transfer of LDLR did not lead to a decrease in total cholesterol levels compared to controls but in turn increased 12 months after gene transfer in comparison to LacZ controls. Measeurment of liver enzymes showed no major changes in liver function. Histology of the liver revealed some inammation in animals from the control groups. Also in two animals from the AAV2-LDLR group bile duct proliferation was seen, the signicance of which remains to be studied. In conclusion, although AAV mediated gene transfer of LDLR resulted in long lasting and sustained expression of the transgene in WHHL-rabbit liver, the achieved transgene expression did not result in sustained correction of the FH phenotype with either AAV serotype.
766. Hairpin Ribozymes Encoded in Adenoviral Vectors Reduce Drinking in Alcohol Dependent Rats
Amalia Sapag,1 Thergiory Irrazábal,1 María Elena Quintanilla,2 Lutske Tampier.2 1 Gene Pharmacotherapy Laboratory, Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile, Santiago, Chile; 2Program of Molecular and Clinical Pharmacology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile.
Alcoholism is a multifactorial disease in which genetic factors play a major role (60%). Ethanol is metabolized in the liver to acetaldehyde by alcohol dehydrogenases and then to acetate by S298
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY III mitochondrial aldehyde dehydrogenase (ALDH2). Natural genetic protection from alcoholism is provided by an inactivating mutation in ALDH2 which results in increased blood acetaldehyde that leads to unpleasant physical effects and aversion to ethanol. The drug disulram mimics this protection by covalent inactivation of ALDH2 but its efcacy in the treatment of alcoholics is offset by toxicity, individual variability and the requirement of daily intake. Thus, alternative drugs based on silencing the ALDH2 mRNA are worth exploring. A hairpin ribozyme (RzCG20) targeted to the rat ALDH2 mRNA has been shown to reduce ALDH2 activity in a rat hepatoma cell line by both cleavage and blockade, as seen by comparison to a control ribozyme (RzCG20c) designed to bind but not cleave the target. The aim of this work was to test RzCG20 and RzCG20c in vivo for their capacity to reduce drinking in alcohol dependent rats. Wistar rats bred as heavy alcohol drinkers (UChB rats) were allowed free access to both 10% ethanol and water for 30 days (ethanol intake ∼7 g/kg/day). Animals were injected in the tail vein with a ∆(E1,E3) adenoviral vector (2x1012 vp/kg) encoding either RzCG20, RzCG20c, or a control RNA, or with vehicle; gene constructs had a CMV promoter and an SV40 polyadenylation signal. After four days of ethanol withdrawal, animals were allowed to choose between 10% ethanol and water for only one hour each day; water was available ad libitum. A single administration of the adenoviral vectors encoding RzCG20 or RzCG20c (n=5) yielded a 50% decrease in alcohol consumption for 34 days while the control adenovirus and the vehicle did not decrease consumption (p<0.001). Arterial blood acetaldehyde was measured at 39 days postinjection after alcohol disintoxication (one day withdrawal) and administration of a standard dose of ethanol (1 g/kg i.p.). In rats receiving either ribozyme gene, acetaldehyde increased to more than twice that of animals receiving the control adenovirus or the vehicle (p<0.001). The ALDH2 activity measured in liver homogenates showed a 24% reduction (p<0.02) while the activity of other ALDHs was not affected. In conclusion, both RzCG20 and RzCG20c are effective in vivo, being capable of silencing the ALDH2 mRNA in rats. The mechanistic differences seen between these ribozymes in vitro were not reected in their biochemical effects in vivo after a month, suggesting that binding of RzCG20c to 16 nucleotides in the target mRNA is sufcient to reduce the liver ALDH2 activity to an extent resulting in increased blood acetaldehyde and reduced alcohol consumption. Overall, hairpin ribozymes that reduce the expression of the ALDH2 mRNA are promising candidates for further development of gene therapy for alcoholism. Support: FONDECYT 1040555, VOLO 2008-02.
767. Sustained Therapeutic Levels of FVIII Activity from Lentivirally-Engineered Blood Outgrowth Endothelial Cells Requires Cell Stability and Augmented Angiogenesis
Hideto Matsui,1,2 Margareth Ozelo,1 Andrea Labelle,1 Carol Hegadorn,1 Patrick Thompson,1 Erin Burnnet,1 Christine Hough,1 David Lillicrap.1 1 Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada; 2Regulatory Medicine for Thrombosis, Nara Medical University, Kashihara, Nara, Japan. An ex vivo gene transfer strategy using gene modied blood outgrowth endothelial cells (BOECs) for hemophilia A has been proposed as a therapeutic alternative to systemic administration of viral vectors. We have previously reported that FVII gene-modied BOECs are sequestered in subcutaneous implants and cells are viable and capable of synthesizing therapeutic levels of plasma FVIII over a prolonged period of time. However, one of the major challenges in establishing this as a new treatment modality is the loss of BOEC function and viability after engraftment. This may in part be due to a less than optimal environment within the extracellular matrix. The aim of the present study was to assess the inuence of incorporating Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Fibroblast Growth Factor-2 (FGF) and brinogen (Fbg) within the implanted matrix in enhancing BOEC viability and function. BOECs were isolated from mouse peripheral blood and transduced with a lentiviral vector encoding the canine FVIII transgene under the transcriptional control of the CMV promoter. Two million transduced BOECs were resuspended in Matrigel, supplemented with either FGF, Fbg or both FGF and Fbg, and implanted subcutanenously into NOD/SCID mice. Canine FVIII antigen levels were assayed at weekly intervals and compared to control mice that received Matrigel implants consisting of the same numbers of cells without FGF and Fbg. FVIII antigen was assayed with the Asserachrom VIII:Ag ELISA that detects canine FVIII against a background of normal murine FVIII. After 4 weeks, the mean FVIII antigen in mice that received implants incorporating FGF, Fbg and FGF+Fbg was 36.9 mU/mL, 24.0 mU/ml and 52.3 mU/mL, respectively compared to 25.0 mU/ mL, for Matrigel alone. These therapeutic levels were sustained for up to 12 weeks as expected for cells engineered with a gene under the control of a viral promoter. To evaluate if the observed elevation of FVIII expression resulted from enhanced cellular viability resulting from inclusion of FGF or Fbg, mice were sacriced after 20 weeks and grafts were excised and processed for histological examination. Immuno-staining shows that the numbers of BOECs within Matrigel were similar between the 4 different groups of mice. However, the extent of the capillary architecture observed in the various retrieved implants was signicantly higher in those that contained FGF and FGF+Fbg compared to implants with Fbg and Matrigel without supplements. In conclusion, these results indicate that incorporation of adhesive proteins and vascular growth factors into the transplanted scaffolds enhances FVIII expression at least in part due to increased neo-vascularization.
768. Effects of Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanlippo Syndrome Type B Mice
Coy D. Heldermon,1 Elizabeth Qin,2 Kevin K. Ohlemiller,3 Erik Herzog,4 Carole Vogler,6 David F. Wozniak,5 Mark S. Sands.2 1 Department of Medicine, University of Florida, Gainesville, FL; 2 Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; 3Department of Otolaryngology, Washington University School of Medicine, St. Louis, MO; 4 Department of Biology, Washington University, St. Louis, MO; 5 Department of Psychiatry, Washington University School of Medicine, St. Louis, MO; 6Department of Pathology, St. Louis University School of Medicine, St. Louis, MO. Sanfilippo Syndrome type B (MPS IIIB) is a lysosomal storage disease (LSD) resulting from the deciency of N-acetyl glucosaminidase (NAGLU). We previously showed that intracranial AAV2/5-based gene therapy improves several aspects of the disease such as lifespan. However, there is growing evidence that LSDs will be most effectively treated by using a combination of approaches that target different aspects of disease. Therefore, we initially combined bone marrow transplantation (BMT) with intracranial AAV. Unfortunately, BMT provided little or no additional therapeutic benet. In fact, BMT actually interfered with the benet provided by AAV with respect to motor function and life span. In an attempt to discover a more effective combination of approaches to treat the disease in the mouse model of MPS IIIB, affected mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (AAV), intravenous lentiviralNAGLU (MND) or both (AAV/MND). Compared to untreated MPS IIIB animals, preliminary results indicate that all treatments resulted in some improvement in the clinical phenotype. AAV, MND and AAV/MND treatment results in improved motor function compared to untreated controls using a rocking rotarod paradigm. Early results from Auditory-evoked Brainstem Response (ABR) measurements of hearing also demonstrate improvement in all treatment groups. S299