Mimoun Azzouz

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.

Systemic Delivery of scAAV9 Expressing SMN Prolongs Survival in a Model of Spinal Muscular Atrophy

An adeno-associated virus vector expressing the survival motor neuron protein rescues mice with spinal muscular atrophy. Enough Protein to Reverse Spinal Muscular Atrophy A common neuromuscular disease, spinal muscular atrophy (SMA) causes ever-worsening muscle weakness, usually in babies or young children, almost always resulting in early death. The culprit is a defective gene—survival motor neuron (SMN)—that must be inherited from both parents for the child to be affected. A second SMN gene is usually incorrectly spliced and so is nonfunctional. To treat this disease, researchers have set their sights on delivering a replacement SMN to the motor neurons. The hope has been that gene therapy methods could be used to generate enough normal SMN protein to restore neuronal innervation of muscles in the affected children. Valori et al. have now improved on previous attempts to implement such a treatment in mice with an SMN-like disease. Their new gene therapy vector makes enough functional SMN protein to improve the agility of the affected animals and markedly increase their survival. To find gene therapy methods for SMA that work in animals before trying these treatments in humans, researchers had created mice with the disease. The mouse SMN gene was deleted (mice have only one copy) and replaced with the two human genes—the defective disease-causing form and its poorly spliced sibling. Valori et al. then treated these animals with a gene therapy vector that they had tested in fibroblasts. They coupled a fast, efficient virus (a modified self-complementary adeno-associated virus) to the coding sequence for the SMN protein, optimized for efficient codon usage. A single injection of this vector to new born mice with SMA improved their ability to move and perform physical tests. These mice, which usually die at 2 weeks of age, also showed markedly increased survival times. Subsequent immunohistochemistry showed that the introduced gene was expressed all over the body of the animals but particularly in the lumbar spinal cord, muscle, and liver. These results bring us one step closer to a successful gene therapy treatment for patients with SMA. The vector used here produces large amounts of replacement SMN protein, a goal that had not been achieved with previous approaches. Another noteworthy feature of this vector is that it increases SMN expression in numerous cell types, not just motor neurons, which may be a clue that the critical defects in SMA may be located in more than one kind of cell. Spinal muscular atrophy is one of the most common genetic causes of death in childhood, and there is currently no effective treatment. The disease is caused by mutations in the survival motor neuron gene. Gene therapy aimed at restoring the protein encoded by this gene is a rational therapeutic approach to ameliorate the disease phenotype. We previously reported that intramuscular delivery of a lentiviral vector expressing survival motor neuron increased the life expectancy of transgenic mice with spinal muscular atrophy. The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene therapy delivery to motor neurons to achieve a more clinically relevant effect. Here, we report that a single injection of self-complementary adeno-associated virus serotype 9 expressing green fluorescent protein or of a codon-optimized version of the survival motor neuron protein into the facial vein 1 day after birth in mice carrying a defective survival motor neuron gene led to widespread gene transfer. Furthermore, this gene therapy resulted in a substantial extension of life span in these animals. These data demonstrate a significant increase in survival in a mouse model of spinal muscular atrophy and provide evidence for effective therapy.

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.

Progressive Motor Neuron Impairment in an Animal Model of Familial Amyotrophic Lateral Sclerosis

Mutations of Cu,Zn superoxide dismutase cause an autosomal dominant form of familial amyotrophic lateral sclerosis. An animal model of the disease has been produced by expressing mutant human SOD1 in transgenic mice (G93A). In order to quantify the dysfunction of the motor unit in transgenic mice, electromyographic recordings were performed during the course of the disease. The first alterations in neuromuscular function appeared between P63 and P90. The deficits became even more striking after P100; compound muscle action potentials in the hindlimb decreased by 80% of initial value. Spontaneous fibrillation potentials were measured in more than 50% of transgenic mice. The number of motor units in the gastrocnemius muscle was progressively reduced over time, down to 18% of the control value at P130. Moreover, distal motor latencies increased after P120. These data suggest that the initial dysfunctions of motor unit are related to a severe motor axonal degeneration, which is followed at later periods by myelin alteration.

PTEN depletion rescues axonal growth defect and improves survival in SMN-deficient motor neurons

Phosphatase and tensin homolog (PTEN), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, is widely involved in the regulation of protein synthesis. Here we show that the PTEN protein is enriched in cell bodies and axon terminals of purified motor neurons. We explored the role of the PTEN pathway by manipulating PTEN expression in healthy and diseased motor neurons. PTEN depletion led to an increase in growth cone size, promotion of axonal elongation and increased survival of these cells. These changes were associated with alterations of downstream signaling pathways for local protein synthesis as revealed by an increase in pAKT and p70S6. Most notably, this treatment also restores beta-actin protein levels in axonal growth cones of SMN-deficient motor neurons. Furthermore, we report here that a single injection of adeno-associated virus serotype 6 (AAV6) expressing siPTEN into hind limb muscles at postnatal day 1 in SMNDelta7 mice leads to a significant PTEN depletion and robust improvement in motor neuron survival. Taken together, these data indicate that PTEN-mediated regulation of protein synthesis in motor neurons could represent a target for therapy in spinal muscular atrophy.

Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

The autosomal recessive neuromuscular disease spinal muscular atrophy (SMA) is caused by loss of survival motor neuron (SMN) protein. Molecular pathways that are disrupted downstream of SMN therefore represent potentially attractive therapeutic targets for SMA. Here, we demonstrate that therapeutic targeting of ubiquitin pathways disrupted as a consequence of SMN depletion, by increasing levels of one key ubiquitination enzyme (ubiquitin-like modifier activating enzyme 1 [UBA1]), represents a viable approach for treating SMA. Loss of UBA1 was a conserved response across mouse and zebrafish models of SMA as well as in patient induced pluripotent stem cell–derive motor neurons. Restoration of UBA1 was sufficient to rescue motor axon pathology and restore motor performance in SMA zebrafish. Adeno-associated virus serotype 9–UBA1 (AAV9-UBA1) gene therapy delivered systemic increases in UBA1 protein levels that were well tolerated over a prolonged period in healthy control mice. Systemic restoration of UBA1 in SMA mice ameliorated weight loss, increased survival and motor performance, and improved neuromuscular and organ pathology. AAV9-UBA1 therapy was also sufficient to reverse the widespread molecular perturbations in ubiquitin homeostasis that occur during SMA. We conclude that UBA1 represents a safe and effective therapeutic target for the treatment of both neuromuscular and systemic aspects of SMA.

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.