title: Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors.
abstract: Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/-mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Conlon
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Cathryn S
authors.initials: CS
authors.lastname: Mah
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Pacak
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Mary B
authors.initials: MB
authors.lastname: Rucker Henninger
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Kirsten E
authors.initials: KE
authors.lastname: Erger
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Marda L
authors.initials: ML
authors.lastname: Jorgensen
authors.affiliation: 2 NHLBI Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases, California National Primate Research Center, University of California , Davis, California.|4 Department of Cell Biology and Human Anatomy, University of California , Davis, California.
authors.forename: C Chang I
authors.initials: CC
authors.lastname: Lee
authors.affiliation: 2 NHLBI Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases, California National Primate Research Center, University of California , Davis, California.|3 Department of Pediatrics, University of California , Davis, California.|4 Department of Cell Biology and Human Anatomy, University of California , Davis, California.
authors.forename: Alice F
authors.initials: AF
authors.lastname: Tarantal
authors.affiliation: 1 Powell Gene Therapy Center and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Byrne
mesh_terms: D000293:Adolescent; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D017321:Clinical Trials, Phase I as Topic; D017322:Clinical Trials, Phase II as Topic; D000229:Dependovirus / Q000235:genetics*; D003964:Diaphragm; D004353:Drug Evaluation, Preclinical; D016189:Dystrophin / Q000235:genetics*; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage*; D006009:Glycogen Storage Disease Type II / Q000235:genetics / Q000628:therapy*; D006801:Humans; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D000520:alpha-Glucosidases / Q000235:genetics*
chemical_list: C470011:CFAP91 protein, human; D002352:Carrier Proteins; D016189:Dystrophin; C509951:GAA protein, human; D000520:alpha-Glucosidases
keywords: AAV; Pompe disease; fetal gene transfer; neuromuscular disorders; rhesus monkey