ABSTRACT
An evaluation of the efficacy of 35% hydrogen peroxide vapor (HPV) against a novel Simian Immunodeficiency retrovirus (SIV) was conducted in a 624m3 GMP grade C manufacturing facility. SIV biological indicators, with an average titre of 7.11×108 TU/mL [equivalent to 1.3×108 Reverse transcriptase (RT) nanounits when analysed by F-PERT], were produced on-site using virus presented within an anticipated worst-case support matrix based on the manufacturers commercially confidential virus support media. SIV biological indicators were distributed around the manufacturing facility, including locations identified as key contamination control points by the manufacturer, and exposed to a HPV cycle. Commercially available biological indicators of 6 log10Geobacillus stearothermophilus were co-located with the SIV Biological Indicator (BIs). HPV was found to be efficacious in eliminating the SIV and commercial G. stearothermophilus BIs and offers a simple and robust method for biodecontamination of gene therapy facilities.
Subject(s)
Hydrogen Peroxide , Papillomavirus Infections , Humans , Genetic Vectors , Gases , Genetic Therapy , Manufacturing and Industrial FacilitiesABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Genetic Vectors , Infectious Anemia Virus, Equine/genetics , Macular Degeneration/congenital , Photoreceptor Cells, Vertebrate/metabolism , Transduction, Genetic , Animals , Blotting, Western , Body Fluids/metabolism , Cytomegalovirus/genetics , Electroretinography , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression , Genetic Therapy , Green Fluorescent Proteins/genetics , Intraocular Pressure , Macaca mulatta , Macular Degeneration/genetics , Macular Degeneration/metabolism , Macular Degeneration/physiopathology , Male , Polymerase Chain Reaction , Rabbits , Stargardt Disease , Tissue Distribution , TransfectionABSTRACT
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 â¼1 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.
