Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys.

Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk-benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF.

Evaluation of Intrathecal Routes of Administration for Adeno-Associated Viral Vectors in Large Animals.

Delivery of adeno-associated viral (AAV) vectors into the cerebrospinal fluid (CSF) can achieve gene transfer to cells throughout the brain and spinal cord, potentially making many neurological diseases tractable gene therapy targets. Identifying the optimal route of CSF access for intrathecal AAV delivery will be a critical step in translating this approach to clinical practice. We previously demonstrated that vector injection into the cisterna magna is a safe and effective method for intrathecal AAV delivery in nonhuman primates; however, this procedure is not commonly used in clinical practice. More routine methods of administration into the CSF are now being explored, including intracerebroventricular (ICV) injection and injection through a lumbar puncture. In this study, we compared ICV and intracisternal (IC) AAV administration in dogs. We also evaluated vector administration via lumbar puncture in nonhuman primates, with some animals placed in the Trendelenburg position after injection, a maneuver that has been suggested to improve cranial distribution of vector. In the dog study, ICV and IC vector administration resulted in similarly efficient transduction throughout the brain and spinal cord. However, animals in the ICV cohort developed encephalitis associated with a T-cell response to the transgene product, a phenomenon that was not observed in the IC cohort. In the nonhuman primate study, transduction efficiency was not improved by placing animals in the Trendelenburg position after injection. These findings illustrate important limitations of commonly used methods for CSF access in the context of AAV delivery, and will be important for informing the selection of a route of administration for first-in-human studies.

Abnormal polyamine metabolism is unique to the neuropathic forms of MPS: potential for biomarker development and insight into pathogenesis.

The mucopolysaccharidoses (MPS) are rare genetic disorders marked by severe somatic and neurological symptoms. Development of treatments for the neurological manifestations of MPS has been hindered by the lack of objective measures of central nervous system disease burden. Identification of biomarkers for central nervous system disease in MPS patients would facilitate the evaluation of new agents in clinical trials. High throughput metabolite screening of cerebrospinal fluid (CSF) samples from a canine model of MPS I revealed a marked elevation of the polyamine, spermine, in affected animals, and gene therapy studies demonstrated that reduction of CSF spermine reflects correction of brain lesions in these animals. In humans, CSF spermine was elevated in neuropathic subtypes of MPS (MPS I, II, IIIA, IIIB), but not in subtypes in which cognitive function is preserved (MPS IVA, VI). In MPS I patients, elevated CSF spermine was restricted to patients with genotypes associated with CNS disease and was reduced following hematopoietic stem cell transplantation, which is the only therapy currently capable of improving cognitive outcomes. Additional studies in cultured neurons from MPS I mice showed that elevated spermine was essential for the abnormal neurite overgrowth exhibited by MPS neurons. These findings offer new insights into the pathogenesis of CNS disease in MPS patients, and support the use of spermine as a new biomarker to facilitate the development of next generation therapeutics for MPS.

Neuroinflammatory targets and treatments for epilepsy validated in experimental models.

A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. Most importantly, the same inflammatory pathways have also been found in surgically resected brain tissue from patients with treatment-resistant epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti-ictogenesis, antiepileptogenesis, and/or disease-modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof-of-concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries.

Delivery of an Adeno-Associated Virus Vector into Cerebrospinal Fluid Attenuates Central Nervous System Disease in Mucopolysaccharidosis Type II Mice.

Mucopolysaccharidosis type II (MPS II) is a rare X-linked genetic disorder caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), leading to impaired catabolism of ubiquitous polysaccharides and abnormal accumulation of these undegraded substrates in the lysosome. Like many lysosomal storage diseases, MPS II is characterized by both somatic and central nervous system (CNS) involvement. Intravenous enzyme replacement therapy can improve somatic manifestations of MPS II, but systemic IDS does not cross the blood-brain barrier and therefore cannot address CNS disease. In this study, an adeno-associated virus serotype 9 vector carrying the IDS gene was injected into the cerebrospinal fluid (CSF) of IDS deficient mice, a model of MPS II. Treated mice exhibited dose-dependent IDS expression and resolution of brain storage lesions, as well as improvement in long-term memory in a novel object recognition test. These findings suggest that delivery of adeno-associated virus vectors into CSF could serve as a platform for efficient, long-term enzyme delivery to the CNS, potentially addressing this critical unmet need for patients with MPS II and many related lysosomal enzyme deficiencies.

Neonatal tolerance induction enables accurate evaluation of gene therapy for MPS I in a canine model.

High fidelity animal models of human disease are essential for preclinical evaluation of novel gene and protein therapeutics. However, these studies can be complicated by exaggerated immune responses against the human transgene. Here we demonstrate that dogs with a genetic deficiency of the enzyme α-l-iduronidase (IDUA), a model of the lysosomal storage disease mucopolysaccharidosis type I (MPS I), can be rendered immunologically tolerant to human IDUA through neonatal exposure to the enzyme. Using MPS I dogs tolerized to human IDUA as neonates, we evaluated intrathecal delivery of an adeno-associated virus serotype 9 vector expressing human IDUA as a therapy for the central nervous system manifestations of MPS I. These studies established the efficacy of the human vector in the canine model, and allowed for estimation of the minimum effective dose, providing key information for the design of first-in-human trials. This approach can facilitate evaluation of human therapeutics in relevant animal models, and may also have clinical applications for the prevention of immune responses to gene and protein replacement therapies.

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits.

Neurogenesis takes place in the adult mammalian brain in three areas: Subgranular zone of the dentate gyrus (DG); subventricular zone of the lateral ventricle; olfactory bulb. Different molecular markers can be used to characterize the cells involved in adult neurogenesis. It has been recently suggested that a population of bone marrow (BM) progenitor cells may migrate to the brain and differentiate into neuronal lineage. To explore this hypothesis, we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells. Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells, then after several months in mature neurons and microglial cells, and thus without central nervous system (CNS) lesion. Most of transgene-expressing cells expressed NeuN, a marker of mature neurons. Thus, BM-derived cells may function as progenitors of CNS cells in adult animals. The mechanism by which the cells from the BM come to be neurons remains to be determined. Although the observed gradual increase in transgene-expressing neurons over 16 mo suggests that the pathway involved differentiation of BM-resident cells into neurons, cell fusion as the principal route cannot be totally ruled out. Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons. Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector. In addition to cells expressing markers of mature neurons, transgene-positive cells were also positive for nestin and doublecortin, molecules expressed by developing neuronal cells. These cells were actively proliferating, as shown by short term BrdU incorporation studies. Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40 vectors migrating to the hippocampus, and these cells were seen at earlier time points in the DG. We show that the cell membrane chemokine receptor, CCR5, and its ligands, enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity. SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells, suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS, both in the basal state and in response to injury. Furthermore, reduction in CCR5 expression in circulating cells provides profound neuroprotection from excitotoxic neuronal injury, reduces neuroinflammation, and increases neuronal regeneration following this type of insult. These results suggest that BM-derived, transgene-expressing, cells can migrate to the brain and that they become neurons, at least in part, by differentiating into neuron precursors and subsequently developing into mature neurons.

Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates.

The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.

Motor neuron transduction after intracisternal delivery of AAV9 in a cynomolgus macaque.

The image shows a section of the lumbar spinal cord from a cynomolgus macaque that had received AAV9.CB.EGFP via the cisterna magna. Expression of GFP in multiple motor neurons is visible. Injection into the cerebrospinal fluid has been shown to be an effective route of vector administration for neuron transduction.

Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I.

Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3-5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼ 30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.

Intrathecal gene therapy corrects CNS pathology in a feline model of mucopolysaccharidosis I.

Enzyme replacement therapy has revolutionized the treatment of the somatic manifestations of lysosomal storage diseases (LSD), although it has been ineffective in treating central nervous system (CNS) manifestations of these disorders. The development of neurotrophic vectors based on novel serotypes of adeno-associated viruses (AAV) such as AAV9 provides a potential platform for stable and efficient delivery of enzymes to the CNS. We evaluated the safety and efficacy of intrathecal delivery of AAV9 expressing α-l-iduronidase (IDUA) in a previously described feline model of mucopolysaccharidosis I (MPS I). A neurological phenotype has not been defined in these animals, so our analysis focused on the biochemical and histological CNS abnormalities characteristic of MPS I. Five MPS I cats were dosed with AAV9 vector at 4-7 months of age and followed for 6 months. Treated animals demonstrated virtually complete correction of biochemical and histological manifestations of the disease throughout the CNS. There was a range of antibody responses against IDUA in this cohort which reduced detectable enzyme without substantially reducing efficacy; there was no evidence of toxicity. This first demonstration of the efficacy of intrathecal gene therapy in a large animal model of a LSD should pave the way for translation into the clinic.

Oxidative Stress Is Associated with Neuroinflammation in Animal Models of HIV-1 Tat Neurotoxicity.

HIV-1 trans-acting protein Tat, an essential protein for viral replication, is a key mediator of neurotoxicity. If Tat oxidant injury and neurotoxicity have been described, consequent neuroinflammation is less understood. Rat caudate-putamens (CPs) were challenged with Tat, with or without prior rSV40-delivered superoxide dismutase or glutathione peroxidase. Tat injection caused oxidative stress. Administration of Tat in the CP induced an increase in numbers of Iba-1- and CD68-positive cells, as well as an infiltration of astrocytes. We also tested the effect of more protracted Tat exposure on neuroinflammation using an experimental model of chronic Tat exposure. SV(Tat): a recombinant SV40-derived gene transfer vector was inoculated into the rat CP, leading to chronic expression of Tat, oxidative stress, and ongoing apoptosis, mainly located in neurons. Intra-CP SV(Tat) injection induced an increase in microglia and astrocytes, suggesting that protracted Tat production increased neuroinflammation. SV(SOD1) or SV(GPx1) significantly reduced neuroinflammation following Tat administration into the CP. Thus, Tat-induced oxidative stress, CNS injury, neuron loss and inflammation may be mitigated by antioxidant gene delivery.

Role of Oxidative Stress in HIV-1-Associated Neurocognitive Disorder and Protection by Gene Delivery of Antioxidant Enzymes.

HIV encephalopathy covers a range of HIV-1-related brain dysfunction. In the Central Nervous System (CNS), it is largely impervious to Highly Active AntiRetroviral Therapy (HAART). As survival with chronic HIV-1 infection improves, the number of people harboring the virus in their CNS increases. Neurodegenerative and neuroinflammatory changes may continue despite the use of HAART. Neurons themselves are rarely infected by HIV-1, but HIV-1 infects resident microglia, periventricular macrophages, leading to increased production of cytokines and to release of HIV-1 proteins, the most likely neurotoxins, among which are the envelope glycoprotein gp120 and HIV-1 trans-acting protein Tat. Gp120 and Tat induce oxidative stress in the brain, leading to neuronal apoptosis/death. We review here the role of oxidative stress in animal models of HIV-1 Associated Neurocognitive Disorder (HAND) and in patients with HAND. Different therapeutic approaches, including clinical trials, have been used to mitigate oxidative stress in HAND. We used SV40 vectors for gene delivery of antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1), or glutathione peroxidase (GPx1) into the rat caudate putamen (CP). Intracerebral injection of SV (SOD1) or SV (GPx1) protects neurons from apoptosis caused by subsequent inoculation of gp120 and Tat at the same location. Vector administration into the lateral ventricle or cisterna magna protects from intra-CP gp120-induced neurotoxicity comparably to intra-CP vector administration. These models should provide a better understanding of the pathogenesis of HIV-1 in the brain as well as offer new therapeutic avenues.

Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna.

Adeno-associated virus serotype 9 (AAV9) vectors have recently been shown to transduce cells throughout the central nervous system of nonhuman primates when injected into the cerebrospinal fluid (CSF), a finding which could lead to a minimally invasive approach to treat genetic and acquired diseases affecting the entire CNS. We characterized the transduction efficiency of two routes of vector administration into the CSF of cynomolgus macaques-lumbar puncture, which is typically used in clinical practice, and suboccipital puncture, which is more commonly used in veterinary medicine. We found that delivery of vector into the cisterna magna via suboccipital puncture is up to 100-fold more efficient for achieving gene transfer to the brain. In addition, we evaluated the inflammatory response to AAV9-mediated GFP expression in the nonhuman primate CNS. We found that while CSF lymphocyte counts increased following gene transfer, there were no clinical or histological signs of immune toxicity. Together these data indicate that delivery of AAV9 into the cisterna magna is an effective method for achieving gene transfer in the CNS, and suggest that adapting this uncommon injection method for human trials could vastly increase the efficiency of gene delivery.

Relationship between the chemokine receptor CCR5 and microglia in neurological disorders: consequences of targeting CCR5 on neuroinflammation, neuronal death and regeneration in a model of epilepsy.

Chemokines may play a role in leukocyte migration across the blood-brain barrier (BBB) during neuroinflammation and other neuropathological processes, such as epilepsy. The CC chemokine receptor 5 (CCR5) is a member of CC-chemokine receptor family that binds several chemokines, including CCL3 (macrophage inflammatory protein-1alpha, MIP-1alpha), CCL4 (macrophage inflammatory protein-1beta, MIP-1beta) and CCL5 (RANTES). The current review examines the relationship between CCR5 and the microglia in different neurological disorders and models of CNS injury. CCR5 expression is upregulated in different neurological diseases, where it is often immunolocalized in microglial cells. A multistep cascade couples CCR5 activation by chemokines to Ca(2+) increases in human microglia. Because changes in [Ca(2+)] (i) affect chemotaxis, secretion, and gene expression, pharmacologic modulation of this pathway may alter inflammatory and degenerative processes in the CNS. Consequently, targeting CCR5 by using CCR5 antagonists may attenuate critical aspects of neuroinflammation in different models of neurological disorders. To illustrate the interaction between CCR5 and microglia in the CNS, we used a model of excitotoxicity, and demonstrate the intimate involvement of CCR5 in neuron injury and inflammation attendant to kainic acid (KA)-induced neurotoxicity. CCR5 participates in neuronal injury caused by the excitotoxin, KA, brings inflammatory cells to the sites of KA-induced CNS injury, defines the extent of tissue loss after KA exposure and limits reparative responses. We used a SV40-derived vector carrying an interfering RNA (RNAi) that targets CCR5. Delivered directly to the bone marrow, this vector decreased CCR5 expression in circulating cells. Animals so treated showed greatly reduced expression of CCR5 and its ligands (MIP-1alpha and RANTES) in the CNS, including in the brain vasculature, decreased BBB leakage, demonstrated greater KA-stimulated neurogenesis and increased migration of bone marrow-derived cells to the brain to become neurons. Thus, therapeutic targeting of CCR5 may allow control of potentially injurious neuroinflammatory responses, including decrease in microglial cells activation and proliferation, and facilitate neurogenic repair in seizure-induced and, potentially, other forms of CNS injury.

Ethanol protects from injury due to ischemia and reperfusion by increasing vascularity via vascular endothelial growth factor.

UNLABELLED: The cardioprotective effects of moderate ethanol consumption have been known for years and have generally been ascribed to long-term effects of alcohol on blood lipids. However, other mechanisms, particularly ethanol-induced increase in blood vessel density, may also be involved. Our goal was to understand the relationship between ethanol consumption, new blood vessel formation in vivo and protection from injury due to ischemia and ischemia/reperfusion. Using paired ethanol fed and control rats, we assessed capillary density in the heart, brain and skeletal muscle by immunostaining and quantified expression of vascular endothelial growth factor (VEGF) by Western blot analysis and immunocytochemistry. Numbers of vessels were significantly increased in the brain, heart and skeletal muscle of animals fed ethanol-rich diets. VEGF (and its receptors) were upregulated in these organs. These effects were very rapid: highly significantly increased vascularization was seen within 2 weeks of commencing alcohol feeding. A neutralizing VEGF antibody, bevacizumab, inhibited new blood vessel formation induced by moderate doses of ethanol. Ethanol consumption increased vascularization and promoted skeletal muscle regeneration following hindlimb ischemia; these effects were prevented by bevacizumab. Finally, ethanol consumption protected myocardium following experimental ischemia/reperfusion. CONCLUSION: Experimental ethanol ingestion rapidly increases VEGF production, significantly increasing the capillary bed in the heart, brain, and skeletal muscle. Moreover, the ethanol-induced increase of blood vessel density is protective against ischemic events (i.e., hindlimb ischemia and myocardium ischemia/reperfusion) and promotes skeletal muscle regeneration.

Blood-brain barrier abnormalities caused by HIV-1 gp120: mechanistic and therapeutic implications.

The blood-brain barrier (BBB) is compromised in many systemic and CNS diseases, including HIV-1 infection of the brain. We studied BBB disruption caused by HIV-1 envelope glycoprotein 120 (gp120) as a model. Exposure to gp120, whether acute [by direct intra-caudate-putamen (CP) injection] or chronic [using SV(gp120), an experimental model of ongoing production of gp120] disrupted the BBB, and led to leakage of vascular contents. Gp120 was directly toxic to brain endothelial cells. Abnormalities of the BBB reflect the activity of matrix metalloproteinases (MMPs). These target laminin and attack the tight junctions between endothelial cells and BBB basal laminae. MMP-2 and MMP-9 were upregulated following gp120-injection. Gp120 reduced laminin and tight junction proteins. Reactive oxygen species (ROS) activate MMPs. Injecting gp120 induced lipid peroxidation. Gene transfer of antioxidant enzymes protected against gp120-induced BBB abnormalities. NMDA upregulates the proform of MMP-9. Using the NMDA receptor (NMDAR-1) inhibitor, memantine, we observed partial protection from gp120-induced BBB injury. Thus, (1) HIV-envelope gp120 disrupts the BBB; (2) this occurs via lesions in brain microvessels, MMP activation and degradation of vascular basement membrane and vascular tight junctions; (3) NMDAR-1 activation plays a role in this BBB injury; and (4) antioxidant gene delivery as well as NMDAR-1 antagonists may protect the BBB.

HIV-1 Tat neurotoxicity: a model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes.

HIV-associated neurocognitive disorder (HAND) is an increasingly common, progressive disease characterized by neuronal loss and progressively deteriorating CNS function. HIV-1 gene products, particularly gp120 and Tat elicit reactive oxygen species (ROS) that lead to oxidant injury and cause neuron apoptosis. Understanding of, and developing therapies for, HAND requires accessible models of the disease. We have devised experimental approaches to studying the acute and chronic effects of Tat on the CNS. We studied acute exposure by injecting recombinant Tat protein into the caudate-putamen (CP). Ongoing Tat expression, which more closely mimics HIV-1 infection of the brain, was studied by delivering Tat-expression over time using an SV40-derived gene delivery vector, SV(Tat). Both acute and chronic Tat exposure induced lipid peroxidation and neuronal apoptosis. Finally, prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/zinc superoxide dismutase (SOD1) or glutathione peroxidase (GPx1), protected from Tat-induced apoptosis and oxidative injury. Thus, injection of recombinant HIV-1 Tat and the expression vector, SV(Tat), into the rat CP cause respectively acute or ongoing apoptosis and oxidative stress in neurons and may represent useful animal models for studying the pathogenesis and, potentially, treatment of HIV-1 Tat-related damage.

HIV-1 gp120 upregulates matrix metalloproteinases and their inhibitors in a rat model of HIV encephalopathy.

Matrix metalloproteinases (MMPs) are implicated in diverse processes, such as neuroinflammation, leakiness of the blood-brain barrier (BBB) and direct cellular damage in neurodegenerative and other CNS diseases. Tissue destruction by MMPs is regulated by their endogenous tissue inhibitors (TIMPs). TIMPs prevent excessive MMP-related degradation of extracellular matrix components. In a rat model of human immunodeficiency virus (HIV)-related encephalopathy, we described MMP-2 and MMP-9 upregulation by HIV-1 envelope gp120, probably via gp120-induced reactive oxygen species. Antioxidant gene delivery blunted gp120-induced MMP production. We also studied the effect of gp120 on TIMP-1 and TIMP-2 production. TIMP-1 and TIMP-2 levels increased 6 h after gp120 injection into rat caudate-putamen (CP). TIMP-1 and TIMP-2 colocalized mainly with neurons (92 and 95%, respectively). By 24 h, expression of these protease inhibitors diverged, as TIMP-1 levels remained high but TIMP-2 subsided. Gene delivery of the antioxidant enzymes Cu/Zn superoxide dismutase or glutathione peroxidase into the CP before injecting gp120 there reduced levels of gp120-induced TIMP-1 and TIMP-2, recapitulating the effect of antioxidant enzymes on gp120-induced MMP-2 and MMP-9. A significant correlation was observed between MMP/TIMP upregulation and BBB leakiness. Thus, HIV-1 gp120 upregulated TIMP-1 and TIMP-2 in the CP. Prior antioxidant enzyme treatment mitigated production of these TIMPs, probably by reducing MMP expression.

Lymphocyte adhesion to CCR5 ligands is reduced by anti-CCR5 gene delivery.

Immune-mediated damage to the central nervous system (CNS) is an important contributor to many CNS diseases, including epilepsy. Chemokines play a role in leukocyte recruitment to, and migration across, the blood-brain barrier (BBB) during many such processes. We previously investigated the role of the chemokine receptor CCR5 in a rat model of epilepsy based on intraperitoneal kainic acid (KA) administration. Before KA injection, rats were given intramarrow inoculations of SV(RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) that targets CCR5. Decreased CCR5 expression in blood cells after vector administration reduced expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, and strongly protected from BBB leakage, CNS loss and inflammation and facilitated CNS repair. We show here that rSV40-mediated downregulation of CCR5 in lymphocytes decreased cellular adhesion to surfaces carrying CCR5 ligands. These data suggest that reducing CCR5 in peripheral blood mononuclear cells (PBMCs) might alter their adhesion to the microvasculature and their participation in inflammatory processes.