Expression of Escherichia coli β-Galactosidase and Rat HPRT in the CNS of Macaca mulatta Following Adenoviral Mediated Gene Transfer

Abstract

Adenoviral-mediated gene transfer to the caudate nucleus of Macaca mulatta was accomplished using stereotactic injection of two distinct recombinant Ad5 vectors containing the gene for Escherichia coli beta-galactosidase and the cDNA for rat hypoxanthine-guanine phosphoribosylphosphotransferase (HPRT), respectively. Multiple analyses (including immunohistochemistry, histochemistry, transmission electron microscopy, RNA in situ hybridization, nucleotide pool analysis, and enzyme assay) confirmed efficient expression of beta-galactosidase and rat HPRT. Transgene expression was evident in both neurons and glia. Clinically, no evidence of meningitis or cerebritis was observed and no focal neurological deficits were detected in the animal. These preliminary studies indicate that recombinant adenovirus is capable of mediating high level transgene expression to the brains of higher order mammals.

A simple method for the rapid generation of recombinant adenovirus vectors

“…5 Core facilities housed within academic institutions or commercial 'kits' are widely used to streamline the process and avoid the time commitment for an individual laboratory. However, the time required to generate the vectors can range from a best-case scenario of 2 months to many months.…”

“…Our previous work 5 and others' indicate that vector preparations are typically contaminated with varying amounts of wild-type virus when standard methods of homologous recombination between adenovirus backbones (digested to remove the packaging signal and E1-containing sequences) and shuttle plasmids are used. The wild-type is probably a result of the input adenovirus-DNA backbone being incompletely digested.…”

Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe

“…However, success to date has been limited. 27 Comparable problems were observed during adenovirusmediated gene transfer to the CNS of rhesus monkeys: transgene expression was variable and declined over time, 28,29 in part due to inflammatory responses to adenovirus-transduced cells. 30 Vectors derived from adeno-associated viruses (AAV) elicit little, if any, damage or inflammatory response after intracerebral injection, and may deliver long-term transgene expression to non-dividing cells in multiple tissues, including the brain in rodents and in monkeys.…”

β-Galactosidase marker genes to tag and track human hematopoietic cells

“…LacZ was transferred to the cotton rat or to nonhuman primate airway epithelium using adenoviral vectors in preparation for a clinical trial of cystic fibrosis. 34 -36 The gene encoding ␤-gal was also transferred to neuronal [37][38][39][40] and retinal cells, [41][42][43] keratinocytes, 44,45 bladder 46 and gallbladder cells, 47 mesangial and tubular cells, 48,49 endothelial and other vascular cells, 50 -54 skeletal and heart muscle cells, [55][56][57][58][59] hepatocytes, 60 -63 salivary gland cells, 64 and even thyroid follicular cells 65 using different, mostly adenoviral vectors in different species. In these experiments, limited side effects were observed and were believed to be related to the vector itself, 34,51,57,65,66 rather than to the LacZ gene or its product.…”