The liver plays a pivotal role in numerous biochemical processes, including intermediary metabolism, synthesis and secretion of serum proteins, energy metabolism, and detoxification of drugs and other xenobiotics. Hepatocytes are, therefore, an important target for gene therapy, and important target for gene therapy in many genetic diseases that disrupt these processes. In the current application, our group is seeking to optimize the conditions for gene transfer into hepatocytes using recombinant adeno-associated virus (rAAV) vectors, which possess the inherent advantages of long-term stability, safety, and low immunogenicity. rAAV-mediated gene transfer is very efficient in myofibers and some other cell types, but previous reports have indicated that long-term rAAV transduction of the liver may be limited to maximum of approximately 5% of hepatocytes using current methods. This may not be sufficient for many genetic/metabolic disorders. The primary goal of the current application is to identify and circumvent the rate-limiting steps in rAAV-mediated transduction of hepatocytes and thereby increase the efficiency of rAAV-mediated transduction of hepatocytes to a point where it is sufficient for widespread clinical use. The optimization of hepatocyte gene transfer will be accomplished in the context of three gene therapy projects, each of which typifies a defect in one of the major hepatic functions: Project 1: gene therapy for phenylketonuria (a disorder of amino acid metabolism), Project 2: gene therapy for alpha 1-anti-trypsin deficiency (a defect in hepatic synthesis and secretion), and Project 3: gene therapy for glycogen storage (that affect a key process in energy metabolism, the release of free glucose from glycogen). In addition, a pilot study will investigate the feasibility of gene therapy in the murine mdr-2 knock-out mouse (a model defect in bile salt secretion analogous to progressive familial Intrahepatic cholestasis), in a paradigm whereby corrected cells could selectively repopulate the liver due to their survival advantage. In order to fully develop rAAV transduction of the liver, it may be necessary to enhance delivery to the hepatocyte, increase attachment to the cell membrane, improve internalization, nuclear entry and uncoating, maximize the transcriptional activity of vector genomes, and optimize conditions for therapeutic protein secretion. Since these disorders may ultimately require therapy in the newborn period or during child-bearing years, biological safety issues related to long-term genetic stability and biodistribution of rAAV genomes will also be addressed. These studies will be supported by the UF Vector Core Facility, for the production of high-titer, highly purified rAAV stocks, and the UF Immunology/Pathology Core. It is anticipated that by the end of this grant period, these basic improvements in rAAV-mediated delivery of genes to the liver will have improved the changes for successful gene therapy in one or more of these disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK058327-03
Application #
6524309
Study Section
Special Emphasis Panel (ZDK1-GRB-2 (M1))
Program Officer
Mckeon, Catherine T
Project Start
2000-09-25
Project End
2005-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
3
Fiscal Year
2002
Total Cost
$992,040
Indirect Cost
Name
University of Florida
Department
Pediatrics
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Smith, Barbara K; Martin, A Daniel; Lawson, Lee Ann et al. (2017) Inspiratory muscle conditioning exercise and diaphragm gene therapy in Pompe disease: Clinical evidence of respiratory plasticity. Exp Neurol 287:216-224
Fu, Dongtao A; Campbell-Thompson, Martha (2017) Periodic Acid-Schiff Staining with Diastase. Methods Mol Biol 1639:145-149
Fu, Dongtao A; Campbell-Thompson, Martha (2017) Immunohistochemistry Staining for Human Alpha-1 Antitrypsin. Methods Mol Biol 1639:139-143
Ling, Chen; Yin, Zifei; Li, Jun et al. (2016) Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors. Mol Ther Methods Clin Dev 3:16029
Conlon, Thomas J; Mah, Cathryn S; Pacak, Christina A et al. (2016) Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors. Hum Gene Ther Clin Dev 27:152-159
Ling, Chen; Wang, Yuan; Lu, Yuan et al. (2015) The Adeno-Associated Virus Genome Packaging Puzzle. J Mol Genet Med 9:
Ling, Chen; Wang, Yuan; Lu, Yuan et al. (2015) Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo. J Virol 89:952-61
Li, Baozheng; Ma, Wenqin; Ling, Chen et al. (2015) Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo. Hum Gene Ther Methods 26:211-20
Gruntman, Alisha M; Flotte, Terence R (2015) Progress with Recombinant Adeno-Associated Virus Vectors for Gene Therapy of Alpha-1 Antitrypsin Deficiency. Hum Gene Ther Methods 26:77-81
Nayak, Sushrusha; Doerfler, Phillip A; Porvasnik, Stacy L et al. (2014) Immune responses and hypercoagulation in ERT for Pompe disease are mutation and rhGAA dose dependent. PLoS One 9:e98336

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