James Miskin

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.

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.

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.

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.

Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV‐G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV‐G‐pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV‐G, and (2) the quantity of glycoprotein associated with virions. We measured production‐cell and virion‐associated quantities of two isoform variants of VSV‐G, which differ in their glycosylation status, VSV‐G1 and VSV‐G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV‐G at N336 allowed greater maximal expression of VSV‐G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50‐fold) by the degree of VSV‐G1 or VSV‐G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion‐associated VSV‐G1/2 quantities. These data indicate that the minimum required concentration of virion‐associated VSV‐G differs substantially between cell species/types. The implications of these data with regard to VSV‐G‐pseudotyped vector production, titration, and use in host‐cell restriction studies, are discussed. Copyright

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.

Not Reinventing the Wheel: Applying the 3Rs Concepts to Viral Vector Gene Therapy Biodistribution Studies

In pharmaceutical drug development, information on where a drug distributes within the body is helpful in understanding the overall chemical and/or biological characteristics of the compound. This is typically assessed preclinically in relevant animal species that are often used for the formal safety studies (toxicology, pharmacokinetics and drug metabolism. One accepted principle of animal studies is the application of the “3 R’s concept” (replacement, refinement and reduction http://www.nc3rs.org.uk/) to reduce animal use as far as makes sense. The focus of this article is to suggest that, for gene therapy biodistribution studies, reductions in animal use could be rationally achieved with no increase in clinical risk.

367. A Dopamine Gene Therapy for Advanced PD: 4 Years Phase I/II Clinical Update

Parkinsons disease (PD) is a neurodegenerative condition that results in a progressive degeneration of dopaminergic neurons. The dopamine (DA) precursor L-Dopa and dopamine agonists provide the primary standard of care and demonstrate good therapeutic benefit in the early stages of disease. However, their long term use is associated with severe motor side effects that are at least partially caused by the fluctuating nature of dopaminergic stimulation that arises from oral drug administration. As such, a therapy that provides a more continuous and local supply of dopamine to the site of pathology provides a potential approach for the development of new therapeutic strategies. ProSavin® is a gene therapy product that utilises a lentiviral vector to transfer three genes that are critical for de novo dopamine biosynthesis in the striatum, that is depleted of dopamine in PD. Fifteen advanced PD patients have received ProSavin® in three dose cohorts. ProSavin® has been demonstrated to be safe and well tolerated at all doses evaluated to date. No serious adverse events related to the study drug or surgical procedures were observed. All patients demonstrated improvement over baseline at both 6 and 12 months, which were sustained in some patients up to four years. Patients in the highest dose cohort demonstrated the greatest improvement in motor scores, a reduction in requirement for oral dopaminergic medication, and evidences of DA production by PET imaging. In summary, ProSavin® was safe and well tolerated in advanced PD patients. To increase the dose of ProSavin® by administering more vectors is not desirable due to physical constraints. Therefore we have generated OXB-102, an improved version of ProSavin®, that expresses the same enzymes but with an increased DA production per genetically modified cell. An update on OXB-102 will be presented.