Systemic delivery of scAAV9 in fetal macaques facilitates neuronal transduction of the central and peripheral nervous systems.

Correction of perinatally lethal neurogenetic diseases requires efficient transduction of several cell types within the relatively inaccessible CNS. Intravenous AAV9 delivery in mouse has achieved development stage-specific transduction of neuronal cell types, with superior neuron-targeting efficiency demonstrated in prenatal compared with postnatal recipients. Because of the clinical relevance of the non-human primate (NHP) model, we investigated the ability of AAV9 to transduce the NHP CNS following intrauterine gene therapy (IUGT). We injected two macaque fetuses at 0.9 G with 1 × 10(13) vg scAAV9-CMV-eGFP through the intrahepatic continuation of the umbilical vein. Robust green fluorescent protein (GFP) expression was observed for up to 14 weeks in the majority of neurons (including nestin-positive cells), motor neurons and oligodendrocytes throughout the CNS, with a significantly lower rate of transduction in astrocytes. Photoreceptors and neuronal cell bodies in the plexiform and ganglionic retinal layers were also transduced. In the peripheral nervous system (PNS), widespread transduction of neurons was observed. Tissues harvested at 14 weeks showed substantially lower vector copy number and GFP levels, although the percentage of GFP-expressing cells remained stable. Thus, AAV9-IUGT in late gestation efficiently transduces both the CNS and PNS with neuronal predilection, of translational relevance to hereditary disorders characterized by perinatal onset of neuropathology.

Effect of matrix metalloproteinases activity on outflow in perfused human organ culture.

PURPOSE: To test the hypothesis that extracellular matrix turnover, mediated by the matrix metalloproteinases, modulates aqueous humor outflow facility in a human outflow model. METHODS: Matrix metalloproteinase activity was manipulated and outflow facility evaluated using perfused human anterior segment organ culture. Purified matrix metalloproteinases, tissue inhibitors of metalloproteinases (TIMPs), and several families of synthetic inhibitors of matrix metalloproteinases were added to the perfusion medium. Matrix metalloproteinase expression was increased by adding recombinant interleukin (IL)-1alpha. Kinetic inhibition analysis was conducted for stromelysin, gelatinase A, and gelatinase B with the various inhibitors. Live-dead staining was used to evaluate culture viability. RESULTS: Increasing metalloproteinase activity, by adding purified metalloproteinases or by inducing their expression by IL-1alpha treatment, increased outflow facility. Inhibition of endogenous trabecular metalloproteinase activity using TIMP or several families of synthetic metalloproteinase inhibitors reduced outflow rates. The elevation and the reduction of outflow rates were reversible, with changes requiring 1 to 3 days. Kinetic enzyme inhibition analysis produced 50% inhibitory concentration values for these inhibitors that were compatible with the concentration ranges for outflow inhibition. CONCLUSIONS. The ability of several specific matrix metalloproteinase inhibitors to reduce outflow facility implies that endogenous extracellular matrix turnover by these enzymes was required for the maintenance of trabecular outflow resistance, at least in this human culture model. These observations provide support for the hypothesis that controlled extracellular matrix turnover is important in the regulation of aqueous humor outflow facility.

Histochemical demonstration of ornithine transcarbamylase activity in fetal liver.

A histochemical technique for demonstrating ornithine transcarbamylase (OTC) activity in human and rhesus monkey fetal liver is described. The proportion of OTC-positive cells increases in fetal monkey liver between 130 and 150 days of gestation. The histochemical assay can be used to verify the prenatal diagnosis of OTC deficiency in the human fetus.