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Figure 7 Use of a tetracycline-regulated lentiviral vector expressing ciliary neurotrophic factor (CNTF) in the quinolinic acid rat model of Huntington's disease has a dose-dependent neuroprotective effect. Rats received intrastriatal injections of a tetracycline-regulated lentiviral vector expressing either CNTF (TRE-CNTF) or the reporter protein GFP (TRE-GFP). The vector-injected rats were treated with saline (''off'') or doxycycline (''on'') to regulate transgene expression. Quinolinic acid was infused into the striatum of these mice and they were subsequently sacrificed and brains analyzed by immunocytochemistry for the striatal marker DARPP-32. Representative photomicrographs showing DARPP-32-immunostained striatal (A) and nigral sections (B). The quinolinic acid-induced lesion is clearly indentified by the loss of DARPP-32 staining in the GFP- and CNTF-off groups, whereas a significant protective effect is observed when CNTF expression is switched on. (From Ref. 253, © 2002 Mary Ann Liebert, Inc.)

Figure 7 Use of a tetracycline-regulated lentiviral vector expressing ciliary neurotrophic factor (CNTF) in the quinolinic acid rat model of Huntington's disease has a dose-dependent neuroprotective effect. Rats received intrastriatal injections of a tetracycline-regulated lentiviral vector expressing either CNTF (TRE-CNTF) or the reporter protein GFP (TRE-GFP). The vector-injected rats were treated with saline (''off'') or doxycycline (''on'') to regulate transgene expression. Quinolinic acid was infused into the striatum of these mice and they were subsequently sacrificed and brains analyzed by immunocytochemistry for the striatal marker DARPP-32. Representative photomicrographs showing DARPP-32-immunostained striatal (A) and nigral sections (B). The quinolinic acid-induced lesion is clearly indentified by the loss of DARPP-32 staining in the GFP- and CNTF-off groups, whereas a significant protective effect is observed when CNTF expression is switched on. (From Ref. 253, © 2002 Mary Ann Liebert, Inc.)

apoptosis, is capable of producing damage equivalent to the acute necrotic lesion observed soon after severe ischemia. Cells undergoing apoptosis exhibit characteristic fingerprints, including chromosome condensation, internucleosomal DNA fragmentation, and membrane blebbing. A thorough understanding of the gene expression profiles and signaling events that occur in the prenumbra immediately following an ischemic event is likely to yield several molecular/cellular targets that are amenable to pharmacological and gene-based therapeutics.

2. Possible Mechanisms of Disease

Ischemia promotes adaptive and pathologic responses in the neuronal compartment. As illustrated in Fig. 8, extrinsic and intrinsic perturbations in the ischemic brain appear to activate a neuronal ischemic sensor, which in turn promotes adaptive and pathologic gene expression. It is the complex interplay among the numerous molecular signaling cascades and time sensitivity of the disorder that makes development of gene-based approaches for stroke both an exciting and daunting task. The ischemic sensor activates a series of transcription factors that, depending their respective stoichiometry and sub-cellular localization, act to initiate either an adaptive or pathologic signaling cascade. Insights into candidate gene products that confer protection against ischemic exposure can be gleaned from prior preconditioning studies (176). The gene products involved in adaptive processes include those that inactivate reactive oxygen species (ROS), and those that participate in DNA repair and cytoplasmic calcium regulation (177,178). Hypoxic insult also stimulates the secretion of factors like erythropoietin (EPO) and vascular endothelial growth factor (VEGF), which can act via a paracrine manner to effect neuroprotection (179,180). Other gene products, including heat shock protein 72 (HSP 72) and glucose transporter Glut-1, have been identified that block the initiation of pathologic gene expression (181-184). Such factors may provide sufficient protection during the early stages of cellular responses to ischemia in order to subvert potential downstream apoptotic signaling.

3. Potential Gene-based Therapies for Stroke

Many early experimental gene therapy approaches targeted either intermediate or the late stages of the apoptotic process. The commitment to apoptosis, which is believed to involve mitochondrial permeability transition, can be averted by overexpression of a subset of Bcl-2 protein family members and a neuronal apoptosis inhibitory protein (NAIP). HSV-based vectors overexpressing Bcl-2 reduces the incidence of apoptosis and improves neuronal survival both in vitro and in vivo (167,185). Adenoviral vector-mediated expression of NAIP resulted in reduced levels of activated caspase-3 and diminished neuronal degeneration following transient fore-brain ischemia (186,187). Other strategies involve inhibition of cellular caspases that act during later stages of apoptosis through the use of cell-permeable peptides to block the morphologic features of apoptosis (173). In addition, vector-mediated expression of anti-inflammatory molecules (i.e., in-

terleukin-1 receptor antagonist) has been employed to minimize poststroke inflammatory responses within the brain (188-190). However, because ischemia induces global disruptions of cellular processes upstream of mitochondrial commitment, caspase activation, and resultant inflammation, it is unclear whether these strategies can effectively interrupt the initiation of apoptosis or restore function to neurons endangered by this cellular process (191,192). It is also important to note that many of the stroke-related gene transfer approaches performed to date have been employed prior to the onset of experimental ischemia. For gene transfer to be clinically applicable for ischemic disorders, treatments must be assessed following the ischemic event (realistically, at times 1-2 postinjury) (193).

Gene-based neuroprotective approaches that employ neu-rotrophic and angiogenic factors have also been extensively examined. Tsai and colleagues have used AAV vectors to express GDNF in the setting of experimental stroke as an approach to minimize neuronal damage caused by transient ischemia (194). Rats receiving AAV-GDNF immediately following bilateral common carotid artery ligation and middle cerebral artery occlusion exhibited significantly reduced in-farct volumes as compared with animals receiving a p-galac-tosidase-expressing AAV control vector (Fig. 9). Zhang et al. performed studies using adenovirus-expressed GDNF to examine the therapeutic window following transient middle cerebral artery occlusion (MCAO) in rats (193). The protective effect afforded by GDNF overexpression (i.e., reduced infarct size and inhibition of caspase-3 expression) was evident only if Ad-GDNF was administered at the time of reperfusion, but the effects of the gene transfer were minimal at 1 postreperfusion. These results underscore the importance of early postischemia interventions in minimizing neurodegeneration.

Restoration of energy stores in hypoxic neurons via overexpression of the rat glucose transporter, GLUT-1, has been an approach that has shown promise in experimental models of ischemia and brain injury. HSV vector-mediated delivery of GLUT-1 in 3 models of injury (transient focal cerebral ischemia, kainic acid, and 3-acetylpyridine) led to localized increased uptake of glucose, reduced neuron loss, blunted decline in ATP concentrations and metabolism, and decreased glutamate release and cytosolic calcium levels (183,184,195). Approaches that more directly address the detrimental cyto-solic calcium excess observed following ischemia include overexpression of calcium-binding proteins. Viral vector-mediated delivery of the calcium-binding protein calbindin D28K exhibits calcium-buffering activity and is protective in conditions of hypoglycemia and experimental stroke (196-198).

During times of cellular stress, resident proteins can become misfolded, a process that can result in diminution of protein function/activity and potentially lead to intracellular aggregation [reviewed in (199)]. Stress response factors, such as heat shock proteins, have been shown to act as molecular chaperones to assist in protein folding and may represent another approach to support a cell under ischemic attack. Several reports of vector-mediated overexpression of heat shock pro

Figure 8 Ischemia promotes adaptive and pathologic responses in the neuronal compartment. Extrinsic and intrinsic perturbations in the ischemic brain activate a neuronal ischemic sensor, which in turn promotes adaptive and pathologic gene expression (solid lines). In addition, select stimuli can activate neuronal death independent of de novo gene expression (dashed lines). Specific gene products, which have demonstrated the ability to block the initiation, commitment, or execution phases of the programmed death pathway, are also included (boxed items). (From Ref. 254, © 1999 Elsevier Science B.V.)

Figure 8 Ischemia promotes adaptive and pathologic responses in the neuronal compartment. Extrinsic and intrinsic perturbations in the ischemic brain activate a neuronal ischemic sensor, which in turn promotes adaptive and pathologic gene expression (solid lines). In addition, select stimuli can activate neuronal death independent of de novo gene expression (dashed lines). Specific gene products, which have demonstrated the ability to block the initiation, commitment, or execution phases of the programmed death pathway, are also included (boxed items). (From Ref. 254, © 1999 Elsevier Science B.V.)

teins (e.g., HSP72) in ischemic animal models demonstrate a neuroprotective role for these factors (200,201). HSV ampli-con-mediated delivery of HSP72 to rats 30 min after MCAO resulted in higher numbers of surviving neuron numbers as compared with animals receiving a p-galactosidase-express-ing amplicon (200).

In aggregate, gene transfer applications for stroke have shown preclinical promise. For such approaches to gain merit as viable treatment options in humans, major issues need to be addressed. One consideration relates to means of vector delivery. Focal administration of viral vectors following an ischemic event will likely have minimal benefit due to the large areas of the brain that are typically affected. Convection-enhanced delivery may provide the means in which to widely distribute a given therapeutic vector, but remains largely untested in ischemic paradigms (11). A second issue that is not entirely unrelated to the first concerns the window of therapeutic opportunity. As described above, a multitude of signaling events occurs immediately after ischemia that determine whether the compromised neuron follows an adaptive or a pathologic set of molecular instructions. This time window is extremely limited (1-2 poststroke), which makes the implementation of stereotactic means of gene therapy vector delivery nearly improbable. A comprehensive understanding of the molecular signals and their temporal expression profiles will likely identify targets at the earliest of times within this restricted therapeutic window. Until these gaps in disease process knowledge are filled and technical hurdles overcome,

Figure 9 Injection of rAAV-GDNF markedly reduces cortical infarction induced by middle cerebral arterial ligation in rats. The right middle cerbral artery and bilateral common carotid arterial were occluded for 90 minutes. Animals received a PBS, rAAV-lacZ (1010 viral particles), or rAAV-GDNF (1010 viral particles) unilateral infusion during arterial occlusion, were sacrificed 72 hours later, and their brains were coronally sectioned (2-mm thickness) for TTC staining. White areas represent infarcted zones in the cerebral cortex. Rats receiving rAAV-GDNF exhibited a marked reduction in infarct size as compared to animals receiving the rAAV-lacZ or PBS control. (From Ref. 255, © 2000 Elsevier Science B.V.) See the color insert for a color version of this figure.

Figure 9 Injection of rAAV-GDNF markedly reduces cortical infarction induced by middle cerebral arterial ligation in rats. The right middle cerbral artery and bilateral common carotid arterial were occluded for 90 minutes. Animals received a PBS, rAAV-lacZ (1010 viral particles), or rAAV-GDNF (1010 viral particles) unilateral infusion during arterial occlusion, were sacrificed 72 hours later, and their brains were coronally sectioned (2-mm thickness) for TTC staining. White areas represent infarcted zones in the cerebral cortex. Rats receiving rAAV-GDNF exhibited a marked reduction in infarct size as compared to animals receiving the rAAV-lacZ or PBS control. (From Ref. 255, © 2000 Elsevier Science B.V.) See the color insert for a color version of this figure.

clinical gene therapy interventions for stroke will remain an impractical potential alternative to pharmacologic compound-based therapeutics.

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