Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system.

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of beta-galactosidase-encoding vectors made from rAAV4 and rAAV5 compared with similarly designed rAAV2-based vectors. Injection of rAAV5 beta-galactosidase (betagal) or rAAV4betagal into the lateral ventricle resulted in stable transduction of ependymal cells, with approximately 10-fold more positive cells than in mice injected with rAAV2betagal. Major differences between the three vectors were revealed upon striatal injections. Intrastriatal injection of rAAV4betagal resulted again in striking ependyma-specific expression of transgene, with a notable absence of transduced cells in the parenchyma. rAAV2betagal and rAAV5betagal intrastriatal injections led to beta-gal-positive parenchymal cells, but, unlike rAAV2betagal, rAAV5betagal transduced both neurons and astrocytes. The number of transgene-positive cells in rAAV5betagal-injected brains was 130 and 5,000 times higher than in rAAV2betagal-injected brains at 3 and 15 wk, respectively. Moreover, transgene-positive cells were widely dispersed throughout the injected hemisphere in rAAV5betagal-transduced animals. Together, our data provide in vivo support for earlier in vitro work, suggesting that rAAV4 and rAAV5 gain cell entry by means of receptors distinct from rAAV2. These differences could be exploited to improve gene therapy for CNS disorders.

Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice.

Mucopolysaccharidosis (MPS) type VII patients lack functional beta-glucuronidase, leading to systemic and central nervous system dysfunction. In this study we tested whether recombinant adenovirus that encodes beta-glucuronidase (Adbetagluc), delivered intravenously and into the brain parenchyma of MPS type VII mice, could provide long-term transgene expression and correction of lysosomal distension. We also tested whether systemic treatment with the immunosuppressive anti-CD40 ligand antibody, MR-1, affected transgene expression. We found substantial plasma beta-glucuronidase activity for over 9 weeks after gene transfer in the MR-1- treated group, with subsequent decline in activity corresponding to a delayed anti-beta-glucuronidase antibody response. At 16 weeks, near wild-type amounts of beta-glucuronidase activity and striking reduction of lysosomal pathology were detected in livers from mice that had received either MR-1 cotreatment or control antibody. In the lung and kidney, beta-glucuronidase activity was markedly higher for the MR-1-treated group. beta-Glucuronidase activity in the brain persisted independently of MR-1 treatment. Activity was intense in the injected hemisphere and was also evident in the noninjected cortex and striatum, with dramatic improvements in storage deposits in areas of both hemispheres. These results indicate that prolonged enzyme expression from transgenes delivered to deficient liver and brain can mediate pervasive correction and illustrate the potential for gene therapy of MPS and other lysosomal storage diseases.

Systemic hyperosmolality improves beta-glucuronidase distribution and pathology in murine MPS VII brain following intraventricular gene transfer.

Mucopolysaccharidosis VII, a classical lysosomal storage disease, is caused by deficiency of the enzyme beta-glucuronidase. Central nervous system (CNS) manifestations are severe with accumulations of storage vacuoles in all cell types. Intraventricular gene transfer can lead to transduction of the ependyma, with production and secretion of beta-glucuronidase into the cerebral spinal fluid and underlying cortex resulting in reversal of disease pathology restricted to the periventricular areas. We tested if systemic hyperosmolality would increase the distribution of beta-glucuronidase in brain parenchyma after intraventricular virus injection. Mice were administered mannitol, intraperitoneally, 20 days after gene transfer and 1 day prior to sacrifice. Mannitol-induced systemic hyperosmolality caused a marked penetration of beta-glucuronidase into the brain parenchyma. If mannitol was administered at the time of the intraventricular injection of virus, there was penetration of vector across the ependymal cell layer, with infection of cells in the subependymal region. This also resulted in increased beta-glucuronidase activity throughout the brain. Sections of brains from beta-glucuronidase-deficient mice showed correction of cellular pathology in the subependymal region plus cortical structures away from the ventricular wall. These data indicate that virus-mediated gene transfer to the brain via the ventricles, coupled with systemic mannitol administration, can lead to extensive CNS distribution of beta-glucuronidase with concomitant correction of the storage defect. Our findings have positive therapeutic implications for the treatment of CNS disorders with gene transfer vectors and recombinant proteins.

Extensive beta-glucuronidase activity in murine central nervous system after adenovirus-mediated gene transfer to brain.

Mucopolysaccharidosis type VII (MPS VII), caused by beta-glucuronidase deficiency, is a classic lysosomal storage disease. In the central nervous system (CNS), there is widespread pathology with distention of vacuoles in neurons and glia. An approach to therapy for MPS VII would require extensive delivery of enzyme to the CNS and subsequent uptake by the affected cells. In this study we show that intrastriatal injection of recombinant adenovirus encoding beta-glucuronidase (Ad betagluc) to MPS VII or wild-type mice results in focal, intense beta-glucuronidase mRNA expression near the injection site. Further, histochemical staining for enzyme activity showed that beta-glucuronidase activity extended well beyond transduced cells. Activity was detected throughout the ipsilateral striatum as well as in the corpus callosum, ventricles, and bilateral neocortex. Similarly, after injection into the right lateral ventricle or cisterna magna, enzyme activity was present in the ependymal cells of the ventricles, in the subarachnoid spaces, and also in the underlying cortex (150-500 microm from ependyma). The distribution of enzyme was most extensive 21 days after gene transfer to normal mouse brain, with more than 50% of the hemisphere positive for beta-glucuronidase activity. Eighty-four days after adenovirus injection a substantial level of enzyme expression remained (>40% of hemisphere positive for beta-glucuronidase activity). Histological sections from striatum of beta-glucuronidase-deficient mice injected with Ad betagluc showed a marked reduction in the number of distended vacuoles in both neurons and glia, as compared with uninjected striatum. Importantly, correction was noted in both hemispheres. Our finding that a relatively small number of transduced cells produce enzyme that reaches a large proportion of the CNS has favorable implications in developing direct gene transfer therapies for lysosomal storage disorders.

In vitro release of endothelium-derived relaxing factor by acetylcholine is increased during the guinea pig pregnancy.

The relaxation of isolated blood vessels by acetylcholine is dependent on the presence of intact endothelium and its release of a smooth muscle relaxing nitroso-like compound. Pregnancy is associated with altered vascular responsiveness to a variety of agents. Because many of these agents stimulate the release of endothelium-derived relaxing factor, we investigated in vitro the effect of pregnancy on acetylcholine-mediated relaxation of guinea pig uterine and carotid artery rings. The presence of intact and functional endothelium was confirmed both by examination under the scanning electron microscope and by vessel relaxation after the addition of acetylcholine. The addition of acetylcholine to the vessel bath produced dose-dependent relaxation of both carotid and uterine artery segments obtained from pregnant and nonpregnant animals after they had been submaximally preconstricted with phenylephrine. There was a significant increase in both response and efficacy to acetylcholine during pregnancy for both uterine and carotid arteries (p less than 0.0001 for each). The concentrations of endothelial cells (cells per square micrometer) were similar in uterine arteries from pregnant and nonpregnant animals. We conclude that the most likely explanation for these findings is a pregnancy-mediated enhancement of endothelium-derived relaxing factor activity.

Effect of pregnancy on uterine and carotid artery response to norepinephrine, epinephrine, and phenylephrine in vessels with documented functional endothelium.

The effect of pregnancy on arterial sensitivity to vasoconstrictors is controversial. Some of the controversy may reflect methodologic differences. Vessel reactivity in vitro is altered by both the tension placed on the segment and the presence or absence of functional endothelium. We investigated the effect of pregnancy on guinea pig uterine and carotid arteries to norepinephrine, epinephrine, and phenylephrine. Each vessel segment was stretched to the optimal point along its length-tension curve, and functional endothelium was documented by acetylcholine-stimulated relaxation. A significant dose-response relationship was observed in each vessel for each agent (each p less than 0.0001). Pregnancy was demonstrated to be associated with a significant reduction in both uterine artery response and sensitivity to norepinephrine, epinephrine, and phenylephrine. However, there was no consistent pregnancy-associated effect on carotid artery response and sensitivity.

Intermittent maple syrup urine disease: a case report.

A case of intermittent maple syrup urine disease (branched chain keto-aciduria) in an 8-year-old girl is reported. The early detection of this rare disorder is important since control of branched chain amino acid intake may control metabolic abnormalities and prevent neurological damage.