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.

Analysis of pre-treatment markers predictive of treatment benefit for the therapeutic cancer vaccine MVA-5T4 (TroVax).

Cancer vaccines such as MVA-5T4 (TroVax(®)) must induce an efficacious immune response to deliver therapeutic benefit. The identification of biomarkers that impact on the clinical and/or immunological efficacy of cancer vaccines is required in order to select patients who are most likely to benefit from this treatment modality. Here, we sought to identify a predictor of treatment benefit for renal cancer patients treated with MVA-5T4. Statistical modeling was undertaken using data from a phase III trial in which patients requiring first-line treatment for metastatic renal cell carcinoma were randomized 1:1 to receive MVA-5T4 or placebo alongside sunitinib, IL-2 or IFN-α. Numerous pre-treatment factors associated with inflammatory anemia (e.g., CRP, hemoglobin, hematocrit, IL-6, ferritin, platelets) demonstrated a significant relationship with tumor burden and patient survival. From these prognostic factors, the pre-treatment mean corpuscular hemoglobin concentration (MCHC) was found to be the best predictor of treatment benefit (P < 0.01) for MVA-5T4 treated patients and also correlated positively with tumor shrinkage (P < 0.001). Furthermore, MCHC levels showed a significant positive association with 5T4 antibody response (P = 0.01). The latter result was confirmed using an independent data set comprising phase II trials of MVA-5T4 in patients with colorectal, renal and prostate cancers. Retrospective analyses demonstrated that RCC patients who had very large tumor burdens and low MCHC levels received little or no benefit from treatment with MVA-5T4; however, patients with smaller tumor burdens and normal MCHC levels received substantial benefit from treatment with MVA-5T4.

MVA-5T4-induced immune responses are an early marker of efficacy in renal cancer patients.

Few immunotherapy compounds have demonstrated a direct link between the predicted mode of action of the product and benefit to the patient. Since cancer vaccines are thought to have a delayed therapeutic effect, identification of the active moiety may enable the development of an early marker of efficacy. Patients with renal cancer and requiring first-line treatment for metastatic disease were randomized 1:1 to receive MVA-5T4 (TroVax(®)) or placebo alongside Sunitinib, IL-2 or IFN-α in a multicentre phase III trial. Antibody responses were quantified following the 3rd and 4th vaccinations. A surrogate for 5T4 antibody response (the immune response surrogate; IRS) was constructed and then used in a survival analysis to evaluate treatment benefit. Seven hundred and thirty-three patients were randomized, and immune responses were assessed in 590 patients. A high 5T4 antibody response was associated with longer survival within the MVA-5T4-treated group. The IRS was constructed as a linear combination of pre-treatment 5T4 antibody levels, hemoglobin and hematocrit and was shown to be a significant predictor of treatment benefit in the phase III study. Importantly, the IRS was also associated with antibody response and survival in an independent dataset comprising renal, colorectal and prostate cancer patients treated with MVA-5T4 in phase I-II studies. The derivation of the IRS formed part of an exploratory, retrospective analysis; however, if confirmed in future studies, the results have important implications for the development and use of the MVA-5T4 vaccine and potentially for other similar vaccines.

Gene Therapy for Parkinson's Disease: Preclinical Evaluation of Optimally Configured TH:CH1 Fusion for Maximal Dopamine Synthesis.

A recent phase I-II, open-label trial of ProSavin, a lentiviral vector delivering the key enzymes in the dopamine biosynthetic pathway to non-dopaminergic striatal neurons, demonstrated safety and improved motor function in parkinsonian patients. However, the magnitude of the effect suggested that optimal levels of dopamine replacement may not have been achieved. OXB-102, a lentiviral vector with an optimized expression cassette for dopamine biosynthesis, has been shown to achieve a significantly higher dopamine yield than ProSavin. We assessed the efficacy of OXB-102 in the MPTP macaque model of Parkinson's disease (PD). At 6 months post-vector administration, all treated animals showed significant improvements in clinical scores and spontaneous locomotor activity compared to controls, with the highest recovery observed in the OXB-102 high-dose (HD) group. Positron emission tomography quantification of 6-[18F]-fluoro-L-m-tyrosine uptake showed a significant increase in amino acid decarboxylase activity for all treated animals, compared with controls, where the OXB-102 HD group showed the highest level of dopaminergic activity. A toxicology study in macaques demonstrated that the vector was safe and well tolerated, with no associated clinical or behavioral abnormalities and no immune response mounted against any transgene products. Overall, these data support the further clinical development of OXB-102 for the treatment of PD.

Safety and Efficacy of OXB-202, a Genetically Engineered Tissue Therapy for the Prevention of Rejection in High-Risk Corneal Transplant Patients.

Due to both the avascularity of the cornea and the relatively immune-privileged status of the eye, corneal transplantation is one of the most successful clinical transplant procedures. However, in high-risk patients, which account for >20% of the 180,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient cornea. The main reason for graft failure is irreversible immunological rejection, and it is therefore unsurprising that neovascularization (NV; both pre and post grafting) is a significant risk factor for subsequent graft failure. NV is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a donor cornea modified prior to transplant by ex vivo genetic modification with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the equine infectious anemia virus called pONYK1EiA, which subsequently prevents rejection by suppressing NV. Previously, it has been shown that rabbit donor corneas treated with pONYK1EiA substantially suppress corneal NV, opacity, and subsequent rejection in an aggressive rabbit model of cornea graft rejection. Here, efficacy data are presented in a second rabbit model, which more closely mirrors the clinical setting for high-risk corneal transplant patients, and safety data from a 3-month good laboratory practice toxicology and biodistribution study of pONYK1EiA-modified rabbit corneas in a rabbit corneal transplant model. It is shown that pONYK1EiA-modified rabbit corneas (OXB-202) significantly reduce corneal NV and the rate of corneal rejection in a dose-dependent fashion, and are tolerated with no adverse toxicological findings or significant biodistribution up to 13 weeks post surgery in these rabbit studies. In conclusion, angiogenesis is a valid target to prevent corneal graft rejection in a high-risk setting, and transplanted genetically modified corneas are safe and well-tolerated in an animal model. These data support the evaluation of OXB-202 in a first-in-human trial.

Lentiviral Vector Gene Transfer of Endostatin/Angiostatin for Macular Degeneration (GEM) Study.

Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat®). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 104 (n = 3), 2.4 × 105 (n = 3), or 8.0 × 105 transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 105 TU or 8.0 × 105 TU at 57-81 ng/mL for endostatin and 15-27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 105 TU. These data demonstrate that EIAV vectors provide a safe platform with robust and sustained transgene expression for ocular gene therapy.

EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat.

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

PURPOSE: StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. METHODS: Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. RESULTS: Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. CONCLUSIONS: In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

Safety and biodistribution of an equine infectious anemia virus-based gene therapy, RetinoStat(®), for age-related macular degeneration.

RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.

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.

Cross-trial analysis of immunologic and clinical data resulting from phase I and II trials of MVA-5T4 (TroVax) in colorectal, renal, and prostate cancer patients.

The attenuated vaccinia virus MVA has been engineered to deliver the tumor antigen 5T4 (MVA-5T4; TroVax), a surface glycoprotein expressed by most solid tumors. MVA-5T4 has been tested in 2 phase I/II and 7 phase II clinical trials in colorectal (4 trials), renal (4 trials), and prostate (1 trial) advanced cancer patients. Data have been collated from all 9 studies and used to investigate the magnitude and kinetics of 5T4-specific antibody responses after vaccination and to identify potential associations between the immune response and patient survival. Antibody responses specific for the 5T4 tumor antigen and the MVA viral vector were quantified in plasma samples taken from cancer patients before and after the treatment with MVA-5T4. Immunologic and survival data were analyzed using proportional hazards regression adjusting for age and gender. Both survival and immunologic response data were available for 189 patients with colorectal (n=73), renal (n=89), and prostate (n=27) cancer. Before the treatment with MVA-5T4, 5T4-specific antibody levels were significantly elevated in cancer patients compared with healthy donors. After MVA-5T4 administration, 5T4-specific antibody responses increased significantly and peaked after 3 to 4 vaccinations. Exploratory analyses showed significant associations between 5T4 antibody responses and overall survival across all 9 trials and in patients with colorectal cancer. The 5T4-specific antibodies were present at higher levels in cancer patients compared with healthy donors and increased significantly after treatment with MVA-5T4. Although the studies were uncontrolled, there were encouraging signs of activity which is associated with the magnitude of 5T4-specific antibody responses.