Clinical aspects

Description

	• Gene therapy approaches to cerebrovascular disease vary according to pathology or therapeutic aim.
	• Safety and efficacy of various approaches have been demonstrated by animal experimental studies.
	• Gene delivery may be carried out by intravascular or intraparenchymal injection.

Application of gene therapy in cerebrovascular disease can be considered under 5 headings: (1) gene transfer for therapeutic affects in cerebral blood vessels; (2) use of gene therapy for neuroprotection in cerebral ischemia; (3) promotion of regeneration of brain damage as a sequel of stroke; (4) therapy directed at the genetic basis of some stroke syndromes; (5) prevention and treatment of intracranial aneurysms; and (6) prevention of stroke by control of risk factors.

Gene transfer to cerebral blood vessels. Several studies have demonstrated the feasibility for transfer of genes into endothelial cells in vitro, as well as in vivo. Various methods of gene transfer to the blood vessels have been pursued. Gene transfer to cerebral blood vessels in vivo can be achieved by arterial delivery via a catheter. Viral vectors may be used; the most frequently used is adenoviral vector. One disadvantage of this method is that it may produce an inflammatory reaction. A nonviral vector, cationic liposome-DNA complex, is less efficient, but safer than adenoviral-mediated gene transfer. Expression of bacterial beta-galactosidase gene has been used as a marker. Another method of in vivo gene transfer to cerebral vessels is intracisternal injection.

For effect on pathology in the cerebral parenchyma, the delivery of genes is problematic. Injections can be made directly into the cerebral parenchyma by stereotaxic procedures or into cerebral ventricles and cisterns. Introduction of vectors into the bloodstream by intravenous or intra-arterial injection has the advantages of wider distribution, the ability to deliver large volumes, limited disturbance of neural tissue, and the feasibility of repeated administration.

Selected experiments in gene transfer to animal models of carotid artery restenosis are shown in Table 1.

Table 1. Selected Experiments in Gene Transfer to Carotid Artery Models of Restenosis

Experimental model	Technique/vector	Basis/objective	Remarks
Primary culture of rat vascular smooth muscle virus	Adenovirus vector coding a truncated form of fibroblast growth factor receptor-1 gene	Fibroblast growth factor is a potent trophic factor for vascular smooth muscle cells.	This technique is useful for the treatment of the diseases caused by excessive proliferation of vascular smooth muscle cells, such as restenosis after carotid angioplasty or endarterectomy.
Balloon-injured rat carotid arteries	Adenovirus vector expressing beta-galactosidase gene	Gene transfer to injured blood cells in vivo	This model of focal in vivo gene transfer into injured arteries is useful for evaluating treatment of localized vascular disorders, such as restenosis after angioplasty.
	Adenovirus-vector mediated herpes simplex virus-thymidine kinase gene transfer and ganciclovir	In vivo suppression of injury-induced vascular smooth muscle cell accumulation	20% to 46% reduction in neointima to media ratio
Rat carotid artery model of balloon angioplasty	Adenovirus-mediated p21 gene transfer	Overexpression of human p21 inhibits growth factor-stimulated vascular smooth muscle proliferation	Reduction in neointimal hyperplasia associated with inhibition of retinoblastoma gene product
Rat carotid artery balloon injury	Antisense oligonucleotides against proliferating cell nuclear antigen and cdc2 kinase transferred into injured arterial walls with hemagglutinating virus of Japan-liposome gene delivery system	Reduction of proliferating cell nuclear antigen and cdc2 messages with inhibition of neointima formation of the injured artery for 8 weeks	The c-NOS gene was also introduced into a similar injured rat carotid artery by hemagglutinating virus of Japan-liposome; neointima formation was inhibited by 70% for 2 to 4 weeks.
Rat and rabbit carotid arteries	Adenovirus-mediated administration of Gax transcription factor	Overexpression of Gax transcriptional factor inhibits neointimal formation	Gax transcriptional factor induces cell cycle arrest and apoptosis in the vascular smooth muscle cells that contribute to the neointimal layer.
Balloon injury of carotid artery in rats pre-immunized to adenovirus	Adenovirus-mediated delivery of fas ligand	Adenovirus-mediated delivery of fas ligand-induced apoptosis in medial vascular smooth muscle cells inhibits intimal hyperplasia.	Effectiveness in immunized animals is of practical importance.
Rat carotid artery stenosis model	Adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of betagamma signaling	Inhibition of betagamma subunits of heterotrimeric G proteins that play a critical role in physiological vascular smooth muscle proliferation	Targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.
Balloon angioplasty of the rabbit carotid artery	Endothelial progenitor cells were transduced with retroviral vectors expressing human endothelial nitric oxide synthase	Transplantation of genetically engineered cells to reduce intimal hyperplasia	Inhibition of intimal hyperplasia would reduce restenosis.

Vasospasm following subarachnoid hemorrhage is another problem that can be approached by gene therapy by providing a vasodilator in the smooth muscle cells. Antisense oligonucleotides, such as endothelin or collagen implicated in chronic narrowing of blood vessels, may be used to inhibit the vasoconstricting process. Some of these strategies are shown in Table 2.

Table 2. Gene Therapy Strategies for Vasospasm

Model	Technique/vector	Mode of action	Results/remarks
Canine basilar artery	Intracisternal injection of adenoviral vector carrying endothelial nitric oxide synthase gene	Vasodilation mediated by nitric oxide	Effects shown in vitro but not in vivo. Adenoviral-induced inflammation may aggravate vasospasm.
Rat femoral artery vasospasm model	Antisense oligonucleotide	Reduction of the transcription of procollagen type 1 gene	Inhibitory effect of antisense oligonucleotide on collagen formation reduces vasospasm and has potential application in cerebral vasospasm.
Basilar artery vasospasm in a canine model of subarachnoid hemorrhage	Intracisternal injection of antisense preproET-1 oligodeoxynucleotide plus clot lysis by recombinant tissue-type plasminogen activator	Reduction of vasospasm appeared to be related to reduced endothelin-1 synthesis.	These results support a causative role for endothelin-1 early in the course of vasospasm. Clot lysis allows better delivery of oligonucleotides to arteries within the subarachnoid space.
Vasospasm induced by injection of blood cisterna magna of rabbits	Injection of adenoviral vector that encodes prepro-calcitonin gene-related peptide into the cisterna magna of rabbits	Overexpression of calcitonin gene-related peptide, and extremely potent vasodilator attenuates arterial contraction.	Gene transfer of calcitonin gene-related peptide prevents cerebral vasoconstriction in vivo after experimental subarachnoid hemorrhage.

Cerebral ischemia. Neuronal death in cerebral ischemia may result, at least in part, from active processes mediated through the induction of death-promoting genes. Gene therapy strategies may work by blocking the effect of such genes. Herpes simplex viral vectors are preferred for gene therapy to the brain because of their neurotropic properties and these vectors have been used for transferring various neuroprotective genes to neurons. Some of the neuroprotective genes used for transfer in models of cerebral ischemia are shown in Table 3.

Table 3. Neuroprotective Gene Transfer in Models of Cerebral Ischemia

Gene	Vector(s)	Mode of action
Glucose transporter-1 gene	Herpes simplex virus	Protection from hypoglycemia and glutamate toxicity
N-methyl-D-aspartate-R1 gene	Adeno-associated virus	Antisense: downregulation of N-methyl-D-aspartate receptor density
Neuronal apoptosis inhibitory protein (NAIP)	Adenovirus	NAIP elevation in ischemia confers resistance to ischemic damage
Heat shock protein (HSP72)	Herpes simplex virus	Brain areas damaged by stroke express high levels of the HSP72, and increasing its expression has a neuroprotective effect
Glial cell line-derived neurotrophic factor (GDNF)	Adeno-associated virus, herpes simplex virus	GDNF prevents apoptosis
Hepatocyte growth factor	Adenovirus	Prevention of delayed neuronal death by inhibition of apoptosis through the blockade of Bax translocation from the cytoplasm to the nucleus
Bcl-2	Adeno-associated virus	Anti-apoptotic effect of Bcl-2
CXCL12 (CXC chemokine ligand 12): its receptor CXCR4 is needed for regeneration effect of oligodendrocyte progenitor cells	Retroviral	Neuroprotective: ameliorates ischemia-induced white matter injury and promotes oligodendrocyte progenitor cell proliferation and migration to the perifocal area in the ischemic mouse brain (13)
Vascular endothelial growth factor (VEGF)	Adeno-associated virus	VEGF can lead to the growth of new vessels in hypoxic or ischemic areas
VEGF, GDNF, and neural cell adhesion molecule- or gene-engineered umbilical cord cells.	Adenoviral vectors	Neuroprotective: intrathecal injection before distal occlusion of the middle cerebral artery (16).

Various strategies used for reducing cerebral infarction in animal stroke models are shown in Table 4.

Table 4. Gene Therapy Strategies for Cerebral Infarction in Various Models

Experimental model	Technique/vector	Mode of action	Remarks
Unilateral middle cerebral artery occlusion in the rat or mouse. Contralateral hemisphere served as the control	Transfer of glucose transporter gene to neurons with herpes simplex virus vector	Increased uptake of glucose by neurons protects against energy failure due to metabolic insults.	Partial protection of targeted striatal neurons but no reduction of volume of infarct
	Adenovirus vector coding for bFGF (1-154) may be used to induce angiogenesis in vivo.	Induction of angiogenesis to restore blood supply to ischemic tissues	This has potential application for revascularization.
Middle cerebral artery occlusion in the rat	Intracerebroventricular injection of antisense c-fos oligonucleotides	Suppression of ischemia-induced c-fos and AP-1 activity	Useful for dissecting the molecular mechanism of stroke, this model paves the way for clinical application.
Middle cerebral artery occlusion in the rat	Intracerebroventricular injection of oligonucleotide antisense to N-methyl-D-aspartate-Ra receptor	N-methyl-D-aspartate antagonism	Decrease in the size of the infarction
Middle cerebral artery occlusion in the mouse	Injection of lentivirus expressing matrix metalloproteinase-9 (MMP-9) under control of hypoxia response element (HRE) into the peri-infarct area on day 7	MMP-9 has beneficial effect in the subacute phase after ischemic stroke.	Improved behavioral recovery. Gene therapy obviates blood-brain damaging effect of unrestrained administration of MMP-9 (03).
Rat model of stroke. Middle cerebral artery occlusion after gene transfer	Intracerebroventricular injection	Adenovirus vector carrying a human IL-2 receptor antagonist protein	Reduction of infarct volume
	Injection into brain of dexamethasone-loaded R3V6 peptide micelles	Delivery of the heme oxygenase-1 gene to ischemic brain to reduce expression of an inflammatory cytokine, interleukin-6	Reduction of infarct volume (11)
Tandem occlusion of the middle cerebral artery and ipsilateral common carotid artery in the rat	Intraparenchymal injection of herpes simplex virus-packaged amplicon vector expressing bcl-2 into the brain	Prevention of programmed cell death	Decrease in the size of the infarct
Transient middle cerebral artery occlusion in rats	Adenovirus-mediated glial cell line-derived neurotrophic factor (Ad-GDNF) gene transfer	Neuroprotective effect of GDNF	Treatment with Ad-GDNF significantly reduced the infarct volume without affecting the regional cerebral blood flow (01).
	Intrastriatal injection of an adenoviral vector containing the apolipoprotein E (APOE) epsilon3 gene prior to induction of ischemia	ApoE protein plays an important role in modulating the outcome and regenerative processes after acute brain injury.	Treatment with APOE gene transfer reduces infarction and markedly improves outcome after induction of cerebral ischemia.
	Intraventricular injection of rAAV-transferring gene for BDNF	Neuroprotective effect of a neurotrophic factor	A single injection of rAAV-BDNF provided neuroprotection against cell death (25).
	Intracerebral injection of recombinant retrovirus vector with 5 copies of hypoxia-responsive elements (5HRE) and neurotrophin-3	Neuroprotective effect of a neurotrophic factor	Up-regulated NT-3 improved neurologic status (24)
	β-NGF microinjection delivered by pseudolentivirus vector	β-NGF overexpression improves neuronal survival by reducing cell apoptosis and increasing cell proliferation in the cerebral infarct.	Rescue of cognitive functional impairment through upregulation of GAP-43 protein expression (04)
Unilateral occlusion of the middle cerebral artery: post-stroke	AAV-mediated overexpression of netrin-1	Netrin-1 overexpression increases peri-infarct blood vessel density.	Improvement of motor function recovery after experimental stroke (21)
Transplantation into lesion site of a rat model of intracerebral hemorrhage	Human neural stem cells genetically modified with a mouse Akt1 gene using a retroviral vector	Differentiation of grafted stem cells at site of hemorrhage	Functional recovery and improved survival
Transplantation into focal ischemic injury model in the monkey cortex	AAV NeuroD1-based gene therapy	90% of the transfected astrocytes converted into neurons	In vivo cell conversion through NeuroD1-based gene therapy may be an effective approach to regenerate new neurons for tissue repair in adult primate brains (07).

Vectors for gene therapy of stroke. Several viral as well as nonviral vectors have been tested in animal models of stroke. Studies used stem cells that over-express different neurotrophic factors, such as BDNF, GDNF, or NT3, found that the delivery of these genetically-modified stem cells to animal models of ischemic stroke is safe and effective (05). Gene therapy involving rAAV (recombinant adeno-associated virus) vector seems effective in attenuation of ischemic damage in stroke and has greatly promising potential use for patients in the future (14).

Stem cell gene therapy for stroke. Beneficial effects of stem cell therapy in stroke have been attributed to release of neurotrophic factors from stem cells. Recombinant mesenchymal stem cells, transfected with BDNF gene and delivered with lentiviral vectors in animal models of stroke, migrate to areas surrounding the cerebral infarction, where they differentiate into neural cells. Synergistic effects of stem cells and BDNF may promote regeneration and recovery of the neurologic function. Bone marrow–derived mesenchymal stem cells, transduced with the engineered lentiviruses with co-overexpression of both BDNF and VEGF, were shown to significantly ameliorate brain pathology with improved neurofunctional performance after transplantation in rats subjected to global cerebral ischemia injury induced by cardiac arrest (26). This provides a proof of principle for future clinical application for cerebral ischemia, a condition that has poor neurologic outcome without an effective treatment.

Human umbilical cord blood CD34+ cells transfected with glial cell line-derived neurotrophic factor gene have been shown to reduced brain infarction volume and enhance functional recovery in spontaneous hypertensive rats subjected to transient middle cerebral artery occlusion (19). These findings support the use of combined gene and stem cell therapy for treating stroke. Transplantation of gene modified-stem cells overexpressing various neurotrophic factors has been demonstrated to significantly improve the functional recovery in stroke compared to stem cells only (09).

Angiogenic gene therapy. In patients with cerebral infarcts, angiogenic gene therapy may be beneficial, but it is limited by the difficulty of introducing genes into the brain. Ultrasound-targeted microbubble destruction for delivery of vascular endothelial growth factor gene has been shown to be safe and effective in a mouse model of middle cerebral artery occlusion (22).

Gene therapy with delivery of a neuroprotective protein. Heat shock proteins can have a neuroprotective effect, as they can promote cell survival following several types of stress. A reduction in lesion size was demonstrated following gene therapy with a viral vector expressing HSP27 in an experimental model of stroke (02).

Gene therapy using neurotrophic factors. Among the various neurotrophic factors, administration of GDNF to areas of ischemic brain injury has been shown to limit cerebral infarction and reduce damage to motor functions in animal models of stroke. Neurotrophic factor and antiapoptotic mechanisms, among others, have been suggested as the underlying factors for beneficial effects of GDNF.

Improved sensory and locomotor function in adult and old rats treated 24 hours following cortical ischemic stroke with human NT3 delivered using a clinically approved serotype of adeno-associated viral vector (AAV1) has been shown in 2 randomized, blinded preclinical trials (06). MRI and histological examination showed recovery due to sprouting of corticospinal axons from the less affected hemisphere. Triple gene therapy using intrathecal delivery of human umbilical cord blood mononuclear cells (UCB-MC) transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor, glial cell-derived neurotrophic factor, and neural cell adhesion molecule successfully rescued neurons resulted in reduction of infarct volume and attenuated neural cell death in a rat model of stroke (20).

Nerve growth factor has a neuroprotective action after cerebral ischemia, but clinical application is hindered by lack of a suitable method for systemic delivery of nerve growth factor into the ischemic region of the brain. Engineered exosomes with rabies viral glycoprotein (RVG) peptide on the surface for targeting neuron can be loaded with nerve growth factor for delivery into ischemic cortex after systemic administration (23). The preparation is stable and functions efficiently for a long time in vivo with a burst release of encapsulated nerve growth factor protein translated from the delivered mRNA, which has a neuroprotective function.

The effect of myoblast-mediated delivery of angiogenic genes, vascular endothelial growth factor-A (VEGF) together with platelet-derived growth factor-BB (PDGF-BB), for targeted induction of therapeutic collateralization was tested by implantation in a mouse model of chronic cerebral ischemia following internal carotid occlusion (17). Results showed the potential of this method as a novel treatment strategy for augmentation of collateral flow in the chronically hypoperfused brain.

Gene therapy using antioxidant genes. Reactive oxygen species contribute to neuronal death following cerebral ischemia and the antioxidant superoxide dismutase has been shown to be neuroprotective. In experimental animals, a neurotropic herpes simplex virus-1 vector containing the superoxide dismutase gene, injected into the striatum either before or after transient focal cerebral ischemia, improved the survival of neurons.

RNAi-based gene silencing for neuroprotection in cerebral ischemia. Invading T cells, via interferon-gamma secretion and neurotoxic cytokine production, are the main pathways for delayed post-ischemic tissue injury. Gene silencing of the endothelial very late antigen-4 by in vivo siRNA injection has been shown to reduce infarct volume and post-ischemic neuroinflammation in animal stroke models (15).

Combination of gene therapy with hypothermia. Use of hypothermia in stroke suppresses apoptotic death, including cytochrome c release, caspase activation, and DNA fragmentation, and has a synergistic effect with gene therapy. BCL-2 overexpression by gene transfer also inhibits aspects of apoptotic cell death cascades if given within 1.5 hours of onset of ischemia, which is impractical, but combination with hypothermia (33°C) can prolong the temporal therapeutic window for gene therapy to 5 hours (08).

Combination of gene therapy with drugs. Conjugation of plasmid DNA/dexamethasone with polyethylenimine is 150 nanometers in size and is taken up by cells easily. It has been shown to reduce infarction in animal model of transient cerebral ischemia and has potential for application in stroke patients (10).

Strokes with a genetic component. There is evidence of a genetic basis for stroke. Human strokes with a genetic component are listed in the article on molecular diagnosis of neurogenetic disorders. Identification of genes for stroke in humans will provide novel opportunities for developing gene therapy for patients with specific inherited susceptibility to stroke at a stage prior to development of clinical manifestations. Other genetic disorders wherein stroke occurs as a complication (eg, sickle cell anemia, protein C/S deficiency, antithrombin 3 deficiency, and Fabry disease) are possibly amenable to gene therapy.

Prevention of stroke by control of risk factors. Two important and well-known risk factors for stroke are hypercholesterolemia and hypertension, and they can be reduced by gene therapy.

Hypercholesterolemia. Hypercholesterolemia can be treated successfully by ex vivo gene transfer to infect hepatocytes with normal receptors and to transfuse the cells back into humans. Nitric oxide synthase gene therapy rapidly ameliorates several markers of atherosclerosis in the cholesterol-fed rabbit model.

Hypertension. Based on the observation that transgenic mice expressing human kallikrein develop sustained hypotension, significant reduction of blood pressure has been demonstrated by injecting a kallikrein gene construct into the skeletal muscle of spontaneously hypertensive rats. Kallikrein is a peptide that is involved in the production of kinin, a potent vasodilator. The experimental studies demonstrate the potential of kallikrein gene therapy for the treatment of human hypertension.

Gene therapy for intracranial aneurysms. Gene therapy may be used as an adjunct to endovascular treatment of intracranial arterial aneurysms by use of coils. Direct gene transfer may be enhanced in situ by coils carrying antibody-tethered adenovirus or by use of cell-specific or radiation-inducible promoters. Genes that may be of value in promoting healing and preventing recurrence after endovascular treatment include growth factors and metalloproteinase inhibitors.

Gene therapy of cerebral vasospasm. There is no satisfactory treatment for cerebral vasospasm following subarachnoid hemorrhage. Expression of the plasmid pVAX1-ADM (adrenomedullin) can significantly relieve cerebral vasospasm, increase the expression of serum ADM and nitric oxide synthase, and decrease the expression of serum endothelin (ET)-1 in a rat model of cerebral vascular spasm (12). It can also improve nervous system function.

Gene therapy for arteriovenous malformations of the brain. Intravenous delivery of an AAV vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) has been tested in a mouse model because FLT1 binds with high affinity to vascular endothelial growth factor, which is a risk factor for hemorrhage from arteriovenous malformations (27). AAV9-sFLT1 reduced the incidence of hemorrhage and has the potential for clinical development into a safe therapy for arteriovenous malformations of the brain.

Indications

Potential indications of gene therapy in cerebrovascular disease are:

(1) Prevention of restenosis after angioplasty of carotid arteries (2) Treatment of cerebral vasospasm (3) Neuroprotection in cerebral ischemia (4) Promotion of regeneration and recovery following cerebral infarction (5) Prevention of onset of clinical manifestations in patients with inherited stroke syndromes (6) Reduction of risk factors for stroke (ie, hypercholesterolemia and hypertension) (7) Treatment of intracranial aneurysms

Contraindications

None have been yet identified.

Results

Most of the gene therapy experiments done in animal models have demonstrated the safety and efficacy of gene therapy. Clinical trials in humans for other indications are in progress and have also demonstrated safety and efficacy. Clinical trials for applications in cerebrovascular diseases are anticipated in the future. Most early preclinical studies of gene therapy for cerebral infarction have involved direct gene transfer into the brain prior to induction of infarction for demonstration of neuroprotective effect. This is not practical in human patients. Because of the delay in gene expression following gene transfer, vector-based strategies may be important in the chronic poststroke phase of infarct recovery. Development of effective and safe delivery systems is enabling cerebrovascular gene therapy.

Adverse effects

Adverse effects of gene therapy are described in the article on gene therapy techniques.

Special considerations

Gene therapy for cerebrovascular diseases is still experimental and has not been introduced into clinical practice.

Scientific basis

The scientific basis of gene therapy has been discussed in the MedLink Neurology article, Gene therapy. Basis of application is also mentioned along with indications in a preceding section.