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: 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 (1·9×10(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 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. FUNDING: Oxford BioMedica.

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 (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.

A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.

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.

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.

A stable producer cell line for the manufacture of a lentiviral vector for gene therapy of Parkinson's disease.

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.

Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity.

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.

Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy.

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.