ABSTRACT
Neurogenetic diseases typically have globally distributed lesions, and pathology usually develops early in life, requiring early diagnosis and treatment. We investigated the effects of transferring a corrective gene into the fetal brain before the onset of pathology in the mucopolysaccharidosis (MPS) type VII mouse, a model of a lysosomal storage disease. A single adeno-associated virus serotype 1 vector injection into the ventricle at 15.5 days of gestation resulted in widespread distribution and lifelong expression of the normal gene in the brain and spinal cord. The normal enzyme was distributed to neighboring cells (as expected) and completely prevented the development of storage lesions throughout the central nervous system (CNS). No vector transfer was found outside the CNS, including the gonads, but a small amount of enzyme was present in visceral tissues, consistent with transfer from cerebrospinal fluid to venous circulation. The enzyme was present peripherally in such low amounts that it did not result in the severe skeletal dysmorphology that occurs readily when systemic treatment is used in neonates. However, the survival probability of the treated animals was significantly increased. The results suggest that the nervous system disease may contribute to the overall physiologic health of the animal in this type of disease.
Subject(s)
Dependovirus/genetics , Glucuronidase/metabolism , Glycosaminoglycans/metabolism , Mucopolysaccharidosis VII/therapy , Animals , Brain/embryology , Brain/enzymology , Brain/metabolism , Central Nervous System/embryology , Central Nervous System/enzymology , Central Nervous System/metabolism , Female , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genetic Vectors/pharmacokinetics , Glucuronidase/genetics , In Situ Hybridization , Lysosomal Storage Diseases/enzymology , Lysosomal Storage Diseases/genetics , Lysosomal Storage Diseases/therapy , Lysosomes/enzymology , Lysosomes/metabolism , Male , Mice , Mice, Mutant Strains , Mucopolysaccharidosis VII/enzymology , Mucopolysaccharidosis VII/genetics , Pregnancy , Survival Analysis , Tissue DistributionABSTRACT
We have inoculated a herpes simplex virus type 1 (HSV-1) vector into a variety of sites in the mouse brain and assayed the regions of latency and expression of a beta-glucuronidase (GUSB) cDNA from the latency-associated transcript promoter. Injection sites used were somatosensory cortex, visual cortex, striatum, dorsal hippocampus, and CSF spaces. Latent vector was detected in regions at a distance from the respective injection sites, consistent with axonal transport of vector. Regions of GUSB activity varied by injection site and included cerebral cortex, striatum, thalamus, hypothalamus, substantia nigra, hippocampus, midbrain, pons, medulla, cerebellum, and spinal cord. After a single injection, GUSB enzymatic activity reached wild-type levels in several brain regions. GUSB was found in some areas without any detectable vector, indicative of axonal transport of GUSB enzyme. GUSB-deficient mice, which have the lysosomal storage disease mucopolysaccharidosis (MPS) VII, have lysosomal storage lesions in cells throughout the brain. Adult MPS VII mice treated by injection of vector into a single site on each side of the brain had correction of storage lesions in a large volume of brain. The potential for long-term, widespread correction of lysosomal storage diseases with HSV-1 vectors is discussed.
Subject(s)
Genetic Therapy , Genetic Vectors/therapeutic use , Glucuronidase/metabolism , Herpesvirus 1, Human/genetics , Lysosomes/enzymology , Mucopolysaccharidosis VII/therapy , Animals , Brain/metabolism , Brain/pathology , Brain/virology , Chlorocebus aethiops , Female , Genetic Vectors/administration & dosage , Glucuronidase/analysis , Mice , Mice, Inbred C3H , Mice, Mutant Strains , Models, Anatomic , Mucopolysaccharidosis VII/genetics , Mucopolysaccharidosis VII/metabolism , Tissue Distribution , Vero CellsABSTRACT
Recombinant viral vectors have been used to study a variety of fundamental issues in developmental neurobiology, as well as pathogenesis and treatments for various neurodegenerative diseases. Lentiviral vectors are valuable tools for neurobiology research owing to their ability to transduce nondividing cells, such as neurons, and to introduce therapeutic or reporter genes into central nervous system (CNS) cells in vivo and in vitro.
Subject(s)
Central Nervous System/metabolism , Genetic Vectors , Lentivirus/genetics , Animals , Central Nervous System/cytology , MiceABSTRACT
A number of different transfection reagents have been used for lentiviral vector production. We directly compared transfection buffers, DNA purification methods, chemical facilitators, and DNA concentrations to optimize production. The use of N,N-bis (2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), sodium butyrate, and one fourth the total amount of DNA used in standard transient transfection protocols were the best conditions for virus production. These reagents were combined into a single protocol and scaled-up to produce liter quantities of virus in a multitray tissue culture vessel.
