Scott Ellis

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.

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.

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.

Assessment of Integration-defective HIV-1 and EIAV Vectors In Vitro and In Vivo

The interest in integrase-defective lentiviral vectors (IDLVs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. Here, we directly compared the transducing potential of IDLVs based on the equine infectious anemia virus (EIAV) to the more commonly described HIV-1 IDLVs. IDLVs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. We show that both the single and the triple mutant HIV-1 IDLVs transduce the PC12 cells, but not the C2C12 cells, with similar efficiency to their parental HIV-1 vector. In contrast, the single and triple EIAV IDLVs did not efficiently transduce either differentiated cell line. Moreover, this HIV-1 IDLV-mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. Four weeks following stereotactic administration into adult rat brains, only the single HIV-1 IDLV mutant displayed a comparable transduction profile to the parental HIV-1 vector. In contrast, neither EIAV IDLV mutants showed significant reporter gene expression. This work indicates that the transducing potential of IDLVs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector.

33. Advancing a State of the Art Gene Therapy Called OXB-202 That Resists Corneal Rejection in High Risk 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 100,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient corneal bed. In some of these patients the prognosis is extremely poor, with grafts failing at an accelerating rate to the point where patients are no longer considered suitable for further transplants and are left blind, despite an otherwise normally functioning visual system. The main reason for graft failure is irreversible immunological rejection and it is therefore unsurprising that neovascularization (both pre-and post-grafting) is a significant risk factor for subsequent graft failure. Neovascularization is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a human 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 Anaemia Virus (EIAV) called pONYK1EiA, which subsequently prevents rejection by suppressing neovascularization. Previously we have shown that rabbit corneas treated with pONYK1EiA substantially suppress corneal neovascularization, opacity and subsequent rejection in rabbit models of cornea graft rejection (Parker et al, 2014). We will present data from a 3-month GLP toxicology and biodistribution safety study of pONYK1EiA modified rabbit corneas in a rabbit corneal transplant model. In particular, the GLP study has been designed to include a number of high content in-life assessments that include regular slitlamp ophthalmic examinations, evaluation of corneal thickness and endothelial cell density using pachymetry and specular microscopy respectively and intraocular pressure measurements. We will present a summary of these data to show that there are no safety issues with pONYK1EiA modified corneas. The GLP safety study data to be presented supports the evaluation of OXB-202 corneas in a First-in-Man trial. The toxicology study, GMP manufactures and clinical development of OXB-202 has been supported by the UK Technology Strategy Board (Innovate UK).

31. EncorStat®: A Human Donor Cornea Genetically Engineered To Resist Rejection in High Risk Patients

Corneal transplantation is one of the most successful transplant procedures because of the relatively immune-privileged status of the eye and the avascularity of the cornea. However, normal corneal immune privilege can be eroded by neovascularization by providing a route of entry for immune-mediating cells, leading to subsequent irreversible immunological rejection of the corneal graft, the most common reason for graft failure. In high risk patients, which account for >20% of the 100,000 transplants carried out worldwide each year, the rejection rate can be very high (50-90%), particularly if there is pre-existing vascularization of the recipient corneal bed. In these patients the prognosis is extremely poor, with grafts failing at an accelerating rate to the point where patients are no longer considered suitable for further transplants and are left blind, despite an otherwise healthy eye. It is therefore not surprising that neovascularization (both pre- and post-grafting) is a significant risk factor for corneal graft failure. Neovascularization is thus an attractive target to prevent corneal graft failure due to rejection.EncorStat® is a human donor cornea modified prior to transplant by the ex vivo delivery of the genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin, by a lentiviral vector, derived from the Equine Infectious Anaemia Virus (EIAV), which prevents subsequent rejection by suppressing neovascularization.Modified rabbit corneas have been evaluated in two different models of corneal graft rejection, a highly aggressive model in which rejection is driven by the retention of thick graft sutures, and a less aggressive model in which rejection is driven by pre-vascularizing the recipient corneal bed prior to surgery. In this latter model thin sutures are used to secure the graft that are removed two weeks following surgery, which is more analogous to the clinical setting. The process to generate EncorStat® corneas has been optimized to secrete substantial and persistent levels of angiostatic proteins with very little shedding of residual vector. These corneas substantially suppress corneal neovascularization, opacity and subsequent rejection in both rabbit models of cornea graft rejection.The non-clinical data to be presented support the evaluation of EncorStat® corneas in a First-in-Man trial. With support from the UK Technology Strategy Board (Innovate UK), this trial will be conducted in 2016, following completion of non-clinical safety studies and GMP vector manufacture this year. An outline of this clinical trial design will also be presented.