Nicholas D. Mazarakis

VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model

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 (SOD1G93A) (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.

Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1G93A) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date.

Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy

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.

Dopamine Gene Therapy for Parkinson’s Disease in a Nonhuman Primate Without Associated Dyskinesia

A gene therapy approach for the treatment of Parkinson’s disease. Several high-profile patients—fighter Muhammad Ali, Attorney General Janet Reno, Pope John Paul II, and Michael J. Fox—have thrust Parkinson’s disease (PD) into the popular press in the last decade. But it was nearly 50 years ago that l-dopa was introduced as a therapy for patients with PD, and this drug, with its troublesome side effects, remains the frontline treatment for this debilitating disease that has no cure. Now, an international team of researchers describe a potential treatment for PD that uses a multigene therapy approach designed to restore continuous synthesis of the neurotransmitter dopamine in the PD brain. PD arises from the destruction of a region of the midbrain called the substantia nigra, which is part of the basal ganglia—structures in the brain that control movement and motivation. Neurons in the substantia nigra produce the neurotransmitter dopamine, a key regulator of voluntary movement, cognition, and behavior. Currently, the basis of PD therapy is to replenish the brain’s dopamine stores, which is achieved through periodic oral administration of the drug l-dopa, a blood-brain barrier–crossing dopamine precursor. Although l-dopa treatment has restored motor function in millions of PD patients, this drug does not block the progressive neurodegeneration associated with the disease and, over time, can spur troublesome side effects, such as freezing and involuntary movement. These movement-related repercussions are caused by intermittent oral delivery of l-dopa, which gives rise to peaks and valleys in brain dopamine concentrations. Thus, scientists have sought treatment approaches that deliver dopamine in a continuous manner. To this end, Jarraya et al. have designed a gene therapy protocol in which the genes that encode the key dopamine biosynthetic enzymes are introduced directly into the brain to produce a perpetual, artificial dopamine factory in neurons of the striatum, the basal ganglia nucleus that receives most of the substantia nigra–released dopamine. In normal brains, the tyrosine hydroxylase enzyme converts the amino acid tyrosine to l-dopa, which is then turned into dopamine by aromatic l-amino acid decarboxylase. Another enzyme, guanosine 5′-triphosphate cyclohydrolase 1, produces a molecule that is reduced in PD brains and is needed for efficient dopamine synthesis. Because of vector-related size constraints, genes encoding these enzymes have previously been introduced into animal models of PD in three separate viral vectors and have delivered some benefits. However, for use in the clinic, it would be preferable to use one vector that encodes all three genes. Jarraya et al. used a lentiviral vector system to create such a vector and tested it in rhesus macaque monkeys artificially induced to have PD. The results of the experiments performed by Jarraya et al. reveal that one can achieve sustained, functional concentrations of dopamine in the brains of the parkinsonian monkeys and effect an improvement in mobility and a reduction in disability within the first 6 weeks after injection of the gene-carrying vector. Most encouraging is the fact that these effects were maintained, without the troublesome involuntary movements observed in l-dopa–treated patients, for more than a year in treated animals. Although these results are promising, a number of caveats remain, including the fact that the dopamine factory introduced by gene transfer resides in striatal neurons that do not normally produce dopamine. The ongoing phase 1 and 2 clinical trial conducted by the same group represents the ultimate test of the proof-of-concept findings described in this translational study. In Parkinson’s disease, degeneration of specific neurons in the midbrain can cause severe motor deficits, including tremors and the inability to initiate movement. The standard treatment is administration of pharmacological agents that transiently increase concentrations of brain dopamine and thereby discontinuously modulate neuronal activity in the striatum, the primary target of dopaminergic neurons. The resulting intermittent dopamine alleviates parkinsonian symptoms but is also thought to cause abnormal involuntary movements, called dyskinesias. To investigate gene therapy for Parkinson’s disease, we simulated the disease in macaque monkeys by treating them with the complex I mitochondrial inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which induces selective degeneration of dopamine-producing neurons. In this model, we demonstrated that injection of a tricistronic lentiviral vector encoding the critical genes for dopamine synthesis (tyrosine hydroxylase, aromatic l-amino acid decarboxylase, and guanosine 5′-triphosphate cyclohydrolase 1) into the striatum safely restored extracellular concentrations of dopamine and corrected the motor deficits for 12 months without associated dyskinesias. Gene therapy–mediated dopamine replacement may be able to correct Parkinsonism in patients without the complications of dyskinesias.

Retinoic acid receptor |[beta]|2 promotes functional regeneration of sensory axons in the spinal cord

The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor β2 (RARβ2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARβ2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARβ2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARβ2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARβ2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.

Lentivector-mediated delivery of GDNF protects complex motor functions relevant to human Parkinsonism in a rat lesion model

Although viral vector‐mediated delivery of glial cell‐line derived neurotrophic factor (GDNF) to the brain has considerable potential as a neuroprotective strategy in Parkinsons disease (PD), its ability to protect complex motor functions relevant to the human condition has yet to be established. In this study, we used an operant task that assesses the selection, initiation and execution of lateralized nose‐pokes in Lister Hooded rats to assess the efficacy with which complex behaviours are protected against neurotoxic lesions by prior injection of a lentiviral vector expressing GDNF. Unilateral injection of 6‐hydroxydopamine (6‐OHDA) into the medial forebrain bundle (MFB) caused rats to attempt fewer trials and to make more procedural errors. Lesioned rats also developed a pronounced ipsilateral bias, with a corresponding drop in contralateral accuracy. They were also slower to react to contralateral stimuli and to execute movements bilaterally. Rats that were pre‐treated 4 weeks prior to lesion surgery with an equine infectious anaemia virus (EIAV) vector carrying GDNF [EIAV‐GDNF, injected into the striatum and above the substantia nigra (SN)] performed significantly better on all of these parameters than control rats. In addition to the operant task, EIAV‐GDNF successfully rescued contralateral impairments in the corridor, staircase, stepping and cylinder tasks, and prevented drug‐induced rotational asymmetry. This study confirms that GDNF can protect against 6‐OHDA‐induced impairments in complex as well as simple behaviours, and reinforces the use of EIAV‐based vectors for the treatment of PD.

Lentiviral vectors for treating and modeling human CNS disorders

Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinsons disease (PD), Huntingtons disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral‐mediated overexpression of mutated human α‐synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non‐primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. Copyright

Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin

Due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the CNS has been difficult to fully assess. In this study, we have assessed the utility of a third-generation equine infectious anemia virus (EIAV) in the Brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (AVP) gene results in the production of nonfunctional mutant AVP precursor protein. Importantly, by using this model it is possible to monitor the success of the gene therapy treatment by noninvasive assays. Injection of an EIAV-CMV-AVP vector into the supraoptic nuclei of the hypothalamus resulted in expression of functional AVP peptide in magnocellular neurons. This was accompanied by a 100% recovery in water homeostasis as assessed by daily water intake, urine production, and urine osmolality lasting for a 1-year measurement period. These data show that a single gene defect leading to a neurological disorder can be corrected with a lentiviral-based strategy. This study highlights the potential of using viral gene therapy for the long-term treatment of disorders of the CNS.

Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors

Gene therapy holds great promise for the treatment of a wide range of inherited and acquired disorders. The development of viral vector systems to mediate safe and long-lasting expression of therapeutic transgenes in specific target cell populations is continually advancing. Gene therapy for the nervous system is particularly challenging due to the post-mitotic nature of neuronal cells and the restricted accessibility of the brain itself. Viral vectors based on lentiviruses provide particularly attractive vehicles for delivery of therapeutic genes to treat neurological and ocular diseases, since they efficiently transduce non-dividing cells and mediate sustained transgene expression. Furthermore, novel routes of vector delivery to the nervous system have recently been elucidated and these have increased further the scope of lentiviruses for gene therapy application. Several studies have demonstrated convincing therapeutic efficacy of lentiviral-based gene therapies in animal models of severe neurological disorders and the push for progressing such vectors to the clinic is ongoing. This review describes the key features of lentiviral vectors that make them such useful tools for gene therapy to the nervous system and outlines the major breakthroughs in the potential use of such vectors for treating neurodegenerative and ocular diseases.

Trophic activity of Rabies G protein-pseudotyped equine infectious anemia viral vector mediated IGF-I motor neuron gene transfer in vitro

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.