Scholarly article on topic '446. Inhibition of Hepatic Stellate Cell Activation and Liver Fibrosis by Expression of Cytoglobin In Vivo'

446. Inhibition of Hepatic Stellate Cell Activation and Liver Fibrosis by Expression of Cytoglobin In Vivo Academic research paper on "Biological sciences"

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Academic research paper on topic "446. Inhibition of Hepatic Stellate Cell Activation and Liver Fibrosis by Expression of Cytoglobin In Vivo"

444. Use of dsAAV8-Mediated Gene Transfer to Endogenous B-Cells To Investigate the Therapeutic Efficacy of Immunoregulatory Gene Products in Preventing the Onset of Diabetes in NOD Mice

Khaja K. Rehman,1 Zhong Wang,2 Xiao Xiao,2 Paul D. Robbins.1 1Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Orthopedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA.

The non-obese diabetic (NOD) mouse represents an animal model of human type 1 diabetes, developing spontaneous autoimmune diabetes. Previously, we have shown that intra-peritoneal (i.p.), intravenous (i.v.) or intra-ductal (i.d.) delivery of certain AAV serotypes stably transduces the pancreas and, in particular, the islets1. The endogenous B-cells within the islets were best transduced by AAV8 via the i.p. and i.v. routes, but by AAV6 via the pancreatic i.d. route. We also have been able to deliver and express genes specifically in B-cells for at least 6 months using an insulin promoter in a double-stranded, self-complementary AAV vector (dsAAV-Ins). This ability to deliver and express gene products specifically in B-cells using a dsAAV8-Ins vector allows for the testing of specific gene products in regulating progression of B-cell destruction and the onset of hyperglycemia in NOD mice. We have evaluated the effects of dsAAV8-Ins delivery of interleukin-4 (IL-4) and interleukin-10 (IL-10) to B-cells in NOD mice, using a dsAAV8-Ins-eGFP vector as a control. In female NOD mice, the extent of gene transfer to endogenous B-cells following i.p. delivery of dsAAV8-Ins-eGFP was comparable to normal Balb/C mice. Furthermore, following i.p. delivery of dsAAV8-Ins-IL-4 or IL-10, expression of IL-4 and IL-l0 cytokine was detected in islets isolated and cultured at different time points post-injection. Expression of IL-4 in B-cells prevented the onset of hyperglycemia in NOD mice whereas expression of IL-10 enhanced the time of onset of hyperglycemia. These results demonstrate the utility of using the dsAAV8-Ins vector to transfer immuno-regulatory agents to endogenous B-cells in NOD mice to examine their role in preventing or exacerbating capable the onset of type 1 diabetes.

445. Developmental Stage Determines Distribution of Organ Transduction after Intra-Amniotic Injection of Lentiviral Vector

Masayuki Endo, Tiago H. Coelho, Phil W. Zoltick, Alan W. Flake. 1The Children's Institute for Surgical Science, Department of Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA.

Gene transfer after intra-amniotic injection has in general been of low efficiency, and limited to epithelial cells in the skin, pulmonary, and gastrointestinal system. We hypothesized that early gestational administration might result in broader and more efficient gene transfer due to developmental accessibility of specific cell populations. To test this hypothesis, we injected lentiviral vector carrying the green fluorescent protein (GFP) reporter gene into the fetal murine amniotic space from the late head fold/early somite stage (E8) to E18. From E8 to E12 amniotic space injections were performed under ultrasound image guidance using a Visualsonics@ Vevo 660 system with a 40 mHz scanhead. After E12, injections were performed under direct vision using a stereoscopic microscope. We observed the injected mice under fluorescence stereo microscopy at sequential time points after birth and confirmed GFP expression by immunohistochemistry.

We injected 584 fetal mice from E8 to E18 and the total survival rate was 52.2% (305/584). In early gestation (E8-10), significant GFP expression was observed in multiple organs (Table). Remarkably, GFP expression was observed in tissues derived from

mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells after E11. The observed temporal patterns of gene expression correspond to the expected embryologie accessibility of organ specific cell populations.

We conclude from this study that early gestational intra-amniotic gene transfer has higher efficiency and a broader distribution of transduction than what has been previously observed later in gestation. This model may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

GFP positive organs by different gestational IAGT

Day of injection E08 E09 E10 E11 E12 E13-16 E17-18

Ectoderm-derived organs

Pineal gland ((+)) (-) (-) (-) (-) (-) (-)

Spinal cord ((+)) (-) (-) (-) (-) (-) (-)

Brain ((+)) ((+)) (-) (-) (-) (-) (-)

Olfactory bulb ((+)) ((+)) (-) (-) (-) (-) (-)

Pituitary ((+)) ((+)) ((+)) (-) (-) (-) (-)

Retina ((+)) (-) (-) (-) (-) (-) (-)

Lens ((+)) ((+)) ((+)) (-) (-) (-) (-)

Cornea ((+)) ((+)) ((+)) ((+)) ((+)) ((+)) (-)

Parotid gland ((+)) ((+)) ((+)) (-) (-) (-) (-)

Submaxillary gland ((+)) ((+)) ((+)) (-) (-) (-) (-)

Mammary gland ((+)) ((+)) (-) (-) (-) (-) (-)

Skin ((+)) ((+)) ((+)) ((+)) ((+)) (-) (-)

Inner ear ((+)) ((+)) (-) (-) (-) (-) (-)

Teeth ((+)) ((+)) ((+)) (-) (-) (-) (-)

Tongue ((+)) ((+)) ((+)) ((+)) (-) (-) (-)

Mesoderm-derived organs

Kidney ((+)) (-) (-) (-) (-) (-) (-)

Muscle ((+)) (-) (-) (-) (-) (-) (-)

Endoderm-derived organs

Thymus ((+)) ((+)) ((+)) (-) (-) (-) (-)

Thyroid ((+)) ((+)) ((+)) (-) (-) (-) (-)

Esophagus ((+)) ((+)) (-) (-) (-) (-) (-)

Paranasal sinus ((+)) ((+)) ((+)) ((+)) ((+)) ((+)) ((+))

Lung (-) (-) (-) (-) ((+)) ((+)) ((+))

446. Inhibition of Hepatic Stellate Cell Activation and Liver Fibrosis by Expression of Cytoglobin In Vivo

Ruian Xu,1,3 Phillip Harrison,2 Xinyan Li,1,3 Miao Chen,3 Hua Li,3 Pik-to Cheung,4 Weidong Xiao,1,5 Farzin Farzaneh.6 'Institute of Molecular Medicine, Huaqiao University, Fujian, China; 2Department of Hepatology and Transplantation, King's College London, London, United Kingdom; 3GRC, University of Hong Kong, Hong Kong, China; 4Department of Paediatric, University of Hong Kong, Hong Kong, China; 5Department of Paediatric, University of Pennsylvania, Philadelphia, PA; 6GKT School of Medicine, King's College London, London.

Activation of hepatic stellate cells (HSC) is critical to the development of liver fibrosis. Cytoglobin (Cygb), a hexacoordinate cytoplasmic globulin, is up-regulated in activated rat HSC. The aim of this study is to investigate its role in HSC activation and hepatic fibrogenesis.

The 570 bp cDNA encoding rat Cygb was cloned into a replication deficient adeno-associated virus-2 (rAAV-2), containing a CAG promoter and the woodchuck hepatitis B virus post-transcriptional regulatory element to facilitate expression. rAAV-2 vectors expressing Cygb (rAAV/Cygb) or eGFP (rAAV/eGFP) were packaged and then purified on heparin columns. Experimental animals were young adult male Sprague-Dawley (SD) rats, weighing around 150 grams. Liver injury was induced by biweekly administration of carbon tetrachloride (CCL4, 0.5 ml/kg), or by bile duct ligation (BDL). Two weeks before CCL4 administration, or 3 days before BDL, 5x1011 rAAV-2 vectors were delivered to male SD rats via portal vein (pv) injection. Following 8 weeks of CCL4 administration, liver histology demonstrated extensive fibrosis in rats given PBS or rAAV/eGFP, whereas those given rAAV/ Cygb had preserved liver architecture (n=10 for each group). Administration of rAAV/Cygb inhibited HSC differentiation, as evidenced by changes in procollagen 1, TGF-B1 and TIMP-1 mRNA, and a-

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright © The American Society of Gene Therapy

SMA and TGF-131 proteins. Twenty eight days after BDL, rats pretreated with rAAV/eGFP or PBS had significant cholestatic liver injury; whereas, rAAV/Cygb modulated HSC differentiation preserving liver architecture and reducing the rise in collagen synthesis, as assessed by hydroxyproline content of liver tissue. To explore the role of Cygb during progressive liver fibrosis, we administered rAAV-2 by pv injection to SD rats at week 8 during a 12 week course of CCL4 injections or day 12 after BDL. In both models, rAAV/eGFP did not prevent progression of liver fibrosis, whereas, rAAV/Cygb administration reduced expression of markers of HSC activation and improved fibrosis.

We demonstrate that Cygb, delivered to liver by rAAV-2 vector, reduces activation of HSC resulting in less extracellular matrix deposition in both toxic and cholestatic models of liver injury, even when given after the development of liver fibrosis. Manipulating Cygb expression is a potential novel therapeutic strategy for reducing hepatic fibrosis.

447. AP20187-Inducible Insulin-Like Effects in Diabetic Muscle and Liver Transduced with AAV

Gabriella Cotugno,1,2 Pietro Formisano,3 Ferdinando Giacco,3 Victor M. Rivera,4 Francesco Beguinot,3 Alberto Auricchio.1,5 1Gene Therapy, Telethon Institute of Genetics and Medicine, Naples, Italy; 2Genetica Medica, S.E.M.M. -European School of Molecular Medicine, Naples, Italy; 3Dept. of Cellular and Molecular Biology and Pathology, "Federico II" University, Naples, Italy;4Pharmacology, ARIAD Gene Therapeutics, Cambridge, MA; 5Dept. of Pediatrics, "Federico II" University, Naples, Italy.

Diabetes Mellitus, characterized by insulin deficiency (type I) or resistance (type II), derives from insulin action impairments in hormone target tissues: muscle, liver and adipocytes. Insulin regulates metabolism and glucose homeostasis through binding to a specific membrane receptor (IR) with tyrosine kinase activity. Induction of the insulin receptor signaling in hormone target cells may represent a tool to rescue glucose homeostasis in both insulin and insulin receptor deficiencies. Recently we have described that homodimerization of the chimeric insulin receptor LFv2IRE induced by the small dimerizer drug AP20187 results in insulin like actions in hepatocytes trasduced with adeno-associated viral vectors (AAV).

Here we show that AAV-mediated LFv2IRE expression in murine muscle and liver followed by systemic AP20187 administration results in reversible homodimerization and tyrosine-phosphorylation of the chimeric receptor which peaks 6 hours after drug administration. More importantly AAV vectors expressing LFv2IRE were administered to muscle and liver of the Non-Obese Diabetic (NOD) murine model of type I Diabetes. Significant increases in hepatic glycogen content were observed following AP20187 systemic administration to AAV-treated NOD mice. The analysis of glucose uptake from diabetic muscle transduced with AAV following AP20187 administration is in progress. The ability of the LFv2IRE-AP20187 system to induce insulin-like actions in hormone target tissues in diabetic mice can be further exploited to obtain glucose homeostasis thus representing a potential novel therapeutic strategy for pathological conditions due to either insulin deficiency or resistance.

Stem Cell Therapy

448. Suicide Gene-Mediated Conditional Ablation of Tumors Derived from Transplanted Embryonic Stem Cells

Juyeon Jung,1 Joanne Kim,1 Robert G. Pergolizzi,1 Ronald G. Crystal.1

'Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY.

Based on their capacity for self-renewal and pluripotency, embryonic stem cells (ESC) are being developed as regenerative medicines, but transplantation protocols using differentiated ESC have been hindered by the fact that even small numbers of undifferentiated ESC within a transplant can lead to the development of teratomas. Within the limitations of current technology, it has not been possible to ensure that a population of differentiated ESC is entirely free of undifferentiated ESC. In this context, the safety profile of ESC transplants would be enhanced if uncontrolled cell growth could be suppressed using external stimuli. To address this issue, we hypothesized that gene transfer to the ESC of conditional elements that could kill the transduced cells if activated, would permit control of inappropriate outgrowth of ESC in vivo. To assess this strategy, we developed a replication-defective recombinant lentiviral gene transfer vector containing the herpes simplex virus thymidine kinase (HSVtk) gene (a suicide gene able to convert the nontoxic prodrug nucleotide analog ganciclovir (GCV) to a toxic form), an internal ribosomal entry site (IRES) and green fluorescent protein (GFP) driven by the cytomegalovirus immediate early promoter, and used this vector to genetically modify murine ESC (CGR8 strain). Fluorescence microscopy and FACS analysis demonstrated that 100 % of these cells were killed in the presence of GCV (50 |JM) in vitro. To assess the ability of the HSVtk suicide strategy to eliminate ESC outgrowth, cloned undifferentiated ESC (105 cells) carrying the HSVtk-IRES-GFP transgene were administered subcutaneously into SCID mice to induce teratomas. Teratoma formation was verified by the histological identification of primitive ectoderm, endoderm and mesoderm within the tumor mass. On day 14, when the tumor was 261 ±52 mm3 in size, intraperitoneal GCV administration was initiated and continued for 4 wk (30 mg/kg, twice per day), while a control group received no GCV. Reduction of tumor mass in the group receiving GCV was evident by 2 wk and the tumors were completely eliminated by 4 wk (10/10 treated, 0/3 untreated). Residual fibrous, fat and cartilage tissue were observed at the site of the tumor in the GCV group, but histologic analysis showed no live tumor cells. Mice followed after cessation of GCV treatment did not show any recurrence of tumor growth, suggesting that the tumor-forming cells were killed and not simply arrested. The HSVtk gene is small (1.3 kb) and the use of an IRES avoids the need for a separate promoter, leaving sufficient space for most transgenes. These data demonstrate that this strategy can be used to deliver a therapeutic gene with additional conditional genetic elements that can be activated to control undifferentiated ESC outgrowth and transduced ESC that have escaped growth control due to insertional mutagenesis associated with the use of integrating vectors.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright © The American Society of Gene Therapy