It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Background Parkinson's 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 Parkinson's 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 Parkinson's 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 x 10(7) transducing units [TU]); mid dose (4.0 x 10(7) TU); and high dose (1 x 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 Parkinson's 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 Parkinson's disease. Improvement in motor behaviour was observed in all patients.

ProSavin is an equine infectious anemia virus vector-based gene therapy for Parkinson's disease for which inducible HEK293T-based producer cell lines (PCLs) have been developed. These cell lines demonstrate stringent tetracycline-regulated expression of the packaging components and yield titers comparable to the established transient production system. A prerequisite for the use of PCL-derived lentiviral vectors (LVs) in clinical applications is the thorough characterization of both the LV and respective PCL with regard to identity and genetic stability. We describe the detailed characterization of two ProSavin PCLs (PS5.8 and PS46.2) and resultant ProSavin vector. The two cell lines demonstrate stable production of vector over a time period sufficient to allow generation of master and working cell banks, and subsequent large-scale vector production. ProSavin generated from the PCLs performs comparably in vivo to that produced by the standard transient transfection process with respect to transduction efficiency and immunogenicity. The development of ProSavin PCLs, and the detailed characterization described here, will aid the advancement of ProSavin for clinical application.

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm,notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord 1 . Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase ( SOD1 ) 2 , 3 , 4 . The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases 5 , 6 , 7 . 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 ( SOD1 G93A ) 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.

Top of pageAbstract Lentiviral vectors hold great promise for the treatment of chronic diseases. Vectors based on the equine infectious anemia virus EIAV have shown long-term stable correction in models for Parkinson's disease, spinal muscular atrophy, spinal injury, motor neuron disease and diabetes insipidus. They have also been used to create transgenic chickens and pigs where gene expression is stable over several generations. A notable feature in all of these models is the long term expression and the long term safety (see references below). We were concerned to learn of a study where a very early generation EIAV vector (SMART2 with the pONY3.1 packaging system) had been injected in utero in mice and the resulting pups had developed liver tumours (http://www.advisorybodies.doh.gov.uk/genetics/gtac/lentiviruses-1104.pdf). We compared the properties and vector genome structure with other vectors, in particular HIV vectors, which had not had any side effects or tumours reported. In general, transduction efficiencies reported in mice with HIV vectors are lower than with EIAV, possibly due to the known host cell restriction. One hypothesis was therefore that the EIAV vectors had a higher number of integrations per cell providing an increased opportunity for insertional mutagenesis. There is nothing unusual about the integration properties of EIAV vectors. The findings to date show a preference for active regions of the chromosome but no particular bias to a location or to positions relative to a transcription unit. Furthermore some tumours had only one or two integrations. At present we believe that a role for insertional mutagenesis alone seems unlikely. The SMART series of EIAV vectors contain an open reading frame in the WPRE that could in theory express a truncated form of the oncogenic X protein from the woodchuck hepatitis virus. This could potentially act as a weak oncogene (Kingsman et al 2005), particularly in the rapidly proliferating fetal tissues. Certain vector integrations could contribute to the promotion of the tumour and so it remains to be seen whether this truncated protein is either necessary and/or sufficient to cause tumours in this model. We will present data on the integration characteristics of EIAV in cell lines and in transduced livers and liver tumours from the in utero studies. Our data lead us to favour the hypothesis that the X-like protein acts as an oncogene in this model. Clinical EIAV vectors do not express this protein and lack all extraneous viral sequences. Further studies are required to test all of these hypotheses in order to inform the further development of these vectors for in utero gene therapy.

We have developed minimal vectors based on the non-primate lentivirus Equine Infectious Anaemia Virus (EIAV) for use in the clinic. This vector system fulfils a number of the requirements necessary for effective gene therapy for haemophilia A including: adequate coding capacity, long term expression, absence of a pre-existing immune response and the ability to transduce non-dividing cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in selective death of motor neurons of the cerebral cortex, brain stem and spinal cord. Some familial cases are caused by missense mutations in the gene encoding the Cu/Zn superoxide dismutase 1 (SOD-1). conferring a toxic gain of function to this protein. The precise mechanisms of these neurodegenerative effects are unknown. Gene therapy-based strategies for treating ALS have mainly utilised viral vectors to overexpress neurotrophic genes in motor neurons of transgenic mouse models. One potential alternative approach to alleviate ALS symptoms (caused by mutations in SOD-1) would be to specifically silence expression of the mutant form of this gene. Interfering RNA (RNAi) provides us with a powerful tool for mediating such therapeutic gene silencing approaches. Viral vectors have provided an efficient method for delivering interfering RNA by overexpression of short hairpin loop structures (shRNAs), that are processed into functional double stranded RNAi molecules in the host cell.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in death of motor neurons from the motor cortex and spinal cord. Some familial cases of ALS are caused by missense mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). The deleterious effects of mutant SOD1 expression result from a toxic gain of function, however, the precise mechanisms of neurodegeneration are unknown. Gene therapy strategies for ALS have used viral vectors to deliver neurotrophic genes to vulnerable motor neurons. One alternative approach to treat SOD1-linked ALS would be to silence expression of mutant SOD1. Interfering RNA (RNAi) provides a powerful tool for investigating such therapeutic gene silencing approaches. Viral vectors generated to overexpress short hairpin RNA (shRNA) species provide an effective method for delivering RNAi. Lentiviral vectors based on the equine infectious anaemia virus (EIAV) mediate efficient and sustained transgene expression in the nervous system. Furthermore, these vectors have the capacity to be retrogradely transported along axons and this makes them of particular use for targeting motor neuron populations. We investigated the potential of EIAV vectors for delivering shRNAs targeted against mutant SOD1. Vectors carrying optimised RNAi target sequences were tested in primary neurons derived from SOD1 transgenic mice and mediated efficient ablation of mutant SOD1 expression. Further work will uncover the efficiency of such vectors in vivo and for alleviating disease phenotypes in transgenic mouse models of ALS.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.

Oxidative stress is thought to play an important role in the pathogenesis of Parkinson's disease (PD). Glutathione (GSH), a major cellular antioxidant, is decreased in the substantia nigra pars compacta of PD patients. The aim of the present study was to investigate whether deprenyl and its desmethyl metabolite, putative neuroprotective agents in the treatment of PD, could protect cultured rat mesencephalic neurons from cell death caused by GSH depletion due to treatment with L-buthionine-(S,R)-sulfoximine (BSO). BSO (10 microM) caused extensive cell death after 48 hr, as demonstrated by disruption of cellular integrity and release of lactate dehydrogenase into the culture medium. Both deprenyl and desmethylselegiline, at concentrations of 5 and 50 microM, significantly protected dopaminergic neurons from toxicity without preventing the BSO-induced loss in GSH. Protection was not associated with monoamine oxidase type B inhibition in that pargyline, a potent MAO inhibitor, was ineffective and pretreatment with pargyline did not prevent the protective effects of deprenyl. Protection was not associated with inhibition of dopamine uptake by deprenyl because the dopamine uptake inhibitor mazindol did not diminish BSO toxicity. The antioxidant ascorbic acid (200 microM) also protected against BSO-induced cell death, suggesting that oxidative events were involved. This study demonstrates that deprenyl and its desmethyl metabolite can diminish cell death associated with GSH depletion.

Abstract: l‐Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)‐B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO‐B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µM) and the NMDA receptor blocker MK‐801 (10 µM) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µM) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µM) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.