ABSTRACT
The present study examines gene delivery to cultured motor neurons (MNs) with the Rabies G protein (RabG)-pseudotyped lentiviral equine infectious anemia virus (RabG.EIAV) vector. RabG.EIAV-mediated beta-galactosidase (RabG.EIAV-LacZ) gene expression in cultured MNs plateaus 120 h after infection. The rate and percent of gene expression observed are titer-dependent (P < 0.001). The rat IGF-I cDNA sequence was then cloned into a RabG.EIAV vector (RabG.EIAV-IGF-I) and was shown to induce IGF-I expression in HEK 293 cells. MNs infected with RabG.EIAV-IGF-I demonstrate enhanced survival compared to MNs infected with RabG.EIAV-LacZ virus (P < 0.01). In addition, IGF-I expression in cultured MNs induced profound MN axonal elongation compared to control virus (P < 0.01). The enhanced motor neuron tropism of RabG.EIAV previously demonstrated in vivo, together with the trophic effects of RabG.EIAV-IGF-I MN gene expression may lend this vector to therapeutic application in motor neuron disease.
Subject(s)
Gene Transfer Techniques , Genetic Vectors/genetics , Infectious Anemia Virus, Equine/genetics , Insulin-Like Growth Factor I/genetics , Motor Neurons/metabolism , Nerve Growth Factors/genetics , Animals , Cell Survival/genetics , Cells, Cultured , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Viral/genetics , Genetic Therapy/methods , Growth Cones/metabolism , Growth Cones/virology , Humans , Motor Neuron Disease/genetics , Motor Neuron Disease/metabolism , Motor Neuron Disease/therapy , Peptide Fragments/genetics , Promoter Regions, Genetic/genetics , Rats , Rats, Sprague-Dawley , Receptor, IGF Type 1/metabolism , Spinal Cord/embryology , Spinal Cord/metabolism , Spinal Cord/virology , Tumor Cells, Cultured , Viral Envelope Proteins/geneticsABSTRACT
Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-D-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing beta-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia.
Subject(s)
Hippocampus/drug effects , Hippocampus/physiopathology , Lentivirus/genetics , Nerve Growth Factors/metabolism , Neuroprotective Agents/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Cell Line , Genetic Vectors/genetics , Glial Cell Line-Derived Neurotrophic Factor , Hippocampus/metabolism , Hippocampus/pathology , Humans , N-Methylaspartate/toxicity , Nerve Growth Factors/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Rats , Tissue Culture TechniquesABSTRACT
Spinal muscular atrophy (SMA) is a frequent recessive autosomal disorder. It is caused by mutations or deletion of the telomeric copy of the survival motor neuron (SMN) gene, leading to depletion in SMN protein levels. The treatment rationale for SMA is to halt or delay the degeneration of motor neurons, but to date there are no effective drug treatments for this disease. We have previously demonstrated that pseudotyping of the nonprimate equine infectious anemia virus (using the lentivector gene transfer system) with the glycoprotein of the Evelyn-Rokitnicki-Abelseth strain of the rabies virus confers retrograde axonal transport on these vectors. Here, we report that lentivector expressing human SMN was successfully used to restore SMN protein levels in SMA type 1 fibroblasts. Multiple single injections of a lentiviral vector expressing SMN in various muscles of SMA mice restored SMN to motor neurons, reduced motor neuron death, and increased the life expectancy by an average of 3 and 5 days (20% and 38%) compared with LacZ and untreated animals, respectively. Further extension of survival by SMN expression constructs will likely require a knowledge of when and/or where high levels of SMN are needed.
Subject(s)
Lentivirus/genetics , Nerve Tissue Proteins/genetics , Animals , Cell Line , Cell Nucleus/metabolism , Cyclic AMP Response Element-Binding Protein , Disease Models, Animal , Fibroblasts/metabolism , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Humans , Immunohistochemistry , Lac Operon , Mice , Microscopy, Fluorescence , Motor Neurons/metabolism , RNA-Binding Proteins , SMN Complex Proteins , Time FactorsABSTRACT
Amyotrophic lateral sclerosis (ALS) causes adult-onset, progressive motor neuron degeneration in the brain and spinal cord, resulting in paralysis and death three to five years after onset in most patients. ALS is still incurable, in part because its complex aetiology remains insufficiently understood. Recent reports have indicated that reduced levels of vascular endothelial growth factor (VEGF), which is essential in angiogenesis and has also been implicated in neuroprotection, predispose mice and humans to ALS. However, the therapeutic potential of VEGF for the treatment of ALS has not previously been assessed. Here we report that a single injection of a VEGF-expressing lentiviral vector into various muscles delayed onset and slowed progression of ALS in mice engineered to overexpress the gene coding for the mutated G93A form of the superoxide dismutase-1 (SOD1(G93A)) (refs 7-10), even when treatment was only initiated at the onset of paralysis. VEGF treatment increased the life expectancy of ALS mice by 30 per cent without causing toxic side effects, thereby achieving one of the most effective therapies reported in the field so far.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/therapy , Axonal Transport , Disease Models, Animal , Infectious Anemia Virus, Equine/genetics , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/therapeutic use , Amyotrophic Lateral Sclerosis/pathology , Animals , Brain Stem/pathology , Disease Progression , Gene Expression , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Humans , Injections, Intramuscular , Mice , Mice, Transgenic , Motor Neurons/metabolism , Motor Neurons/pathology , Point Mutation/genetics , Spinal Cord/pathology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Survival Rate , Time Factors , Transgenes/genetics , Vascular Endothelial Growth Factor A/adverse effects , Vascular Endothelial Growth Factor A/metabolismABSTRACT
We have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (EIAV) for efficient gene transfer to the central and peripheral nervous systems. Previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. In the present study we have successfully produced high-titer EIAV vectors pseudotyped with envelope glycoproteins from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains); rabies virus [various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)]; Lyssavirus Mokola virus, a rabies-related virus; and Arenavirus lymphocytic choriomeningitis virus (LCMV). These vectors were delivered to the striatum or spinal cord of adult rats or muscle of neonatal mice by direct injection. We report that the lentiviral vectors pseudotyped with envelopes from the VSV Indiana strain, wild-type ERA, and CVS strains resulted in strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors exhibited moderate and weak transduction, respectively. Furthermore ERA- and CVS-pseudotyped lentiviral vectors demonstrated retrograde transport and expression in distal neurons after injection in brain, spinal cord, and muscle. The differences in transduction efficiencies and retrograde transport conferred by these envelope glycoproteins present novel opportunities in designing therapeutic strategies for different neurological diseases.
