Kyriacos Mitrophanous

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.

Efficient production of germline transgenic chickens using lentiviral vectors

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100‐fold higher than any previously published method, with no detectable silencing of transgene expression between generations.

Stable gene transfer to the nervous system using a non-primate lentiviral vector.

We have constructed a non-primate lentiviral vector system based on the equine infectious anaemia virus (EIAV). This system is able to transduce both dividing and non-dividing cells, including primary cultured hippocampal neurons and neurons and glia in the adult rat central nervous system (CNS), at efficiencies comparable with HIV-based vectors. We demonstrate that the only EIAV proteins required for this activity are gag/pol and that the only accessory protein required for vector production is rev. In addition, we show that the pol encoded dUTPase activity that is found in all non-primate lentiviruses is not required. The vectors can be pseudotyped with a range of envelopes including rabies G and MLV 4070A and can be concentrated to high titres. The ability of EIAV to infect mitotically inactive cells makes this vector an attractive alternative to the immunodeficiency viruses for gene therapy.

A Rev-Independent Human Immunodeficiency Virus Type 1 (HIV-1)-Based Vector That Exploits a Codon-Optimized HIV-1 gag-pol Gene

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.

Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial

BACKGROUND Parkinsons disease is typically treated with oral dopamine replacement therapies; however, long-term treatment leads to motor complications and, occasionally, impulse control disorders caused by intermittent stimulation of dopamine receptors and off-target effects, respectively. We aimed to assess the safety, tolerability, and efficacy of bilateral, intrastriatal delivery of ProSavin, a lentiviral vector-based gene therapy aimed at restoring local and continuous dopamine production in patients with advanced Parkinsons disease. METHODS We undertook a phase 1/2 open-label trial with 12-month follow-up at two study sites (France and UK) to assess the safety and efficacy of ProSavin after bilateral injection into the putamen of patients with Parkinsons disease. All patients were then enrolled in a separate open-label follow-up study of long-term safety. Three doses were assessed in separate cohorts: low dose (1·9×10(7) transducing units [TU]); mid dose (4·0×10(7) TU); and high dose (1×10(8) TU). Inclusion criteria were age 48-65 years, disease duration 5 years or longer, motor fluctuations, and 50% or higher motor response to oral dopaminergic therapy. The primary endpoints of the phase 1/2 study were the number and severity of adverse events associated with ProSavin and motor responses as assessed with Unified Parkinsons Disease Rating Scale (UPDRS) part III (off medication) scores, at 6 months after vector administration. Both trials are registered at ClinicalTrials.gov, NCT00627588 and NCT01856439. FINDINGS 15 patients received ProSavin and were followed up (three at low dose, six mid dose, six high dose). During the first 12 months of follow-up, 54 drug-related adverse events were reported (51 mild, three moderate). Most common were increased on-medication dyskinesias (20 events, 11 patients) and on-off phenomena (12 events, nine patients). No serious adverse events related to the study drug or surgical procedure were reported. A significant improvement in mean UPDRS part III motor scores off medication was recorded in all patients at 6 months (mean score 38 [SD 9] vs 26 [8], n=15, p=0·0001) and 12 months (38 vs 27 [8]; n=15, p=0·0001) compared with baseline. INTERPRETATION ProSavin was safe and well tolerated in patients with advanced Parkinsons disease. Improvement in motor behaviour was observed in all patients. FUNDING Oxford BioMedica.

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.

Continuous high-titer HIV-1 vector production

Human immunodeficiency virus type 1 (HIV-1)–based vectors are currently made by transient transfection, or using packaging cell lines in which expression of HIV-1 Gag and Pol proteins is induced. Continuous vector production by cells in which HIV-1 Gag-Pol is stably expressed would allow rapid and reproducible generation of large vector batches. However, attempts to make stable HIV-1 packaging cells by transfection of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only low levels of p24 antigen (20–80 ng/ml), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. Here we report that expression of HIV-1 Gag-Pol by a murine leukemia virus (MLV) vector allows constitutive, long-term, high-level (up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were constructed using codon-optimized HIV-1 Gag-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 107 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.

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.

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV‐1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV‐1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.