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- [F-18]-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.

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.

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).

The primary standard of care for Parkinson's disease (PD) is oral dopaminergic treatments and although these are initially highly efficacious, over time they lead to debilitating long term side effects that seriously impact on the quality of life and restrict the long-term effectiveness of such treatments. OXB-102 is a lentiviral-based vector that delivers the genes encoding the three key enzymes in the dopamine (DA) biosynthetic pathway, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP-cyclohydrolase (CH1), to nondopaminergic striatal neurons of the sensorimotor putamen, thus providing these cells with the ability to synthesise and release their own DA. The effectiveness of this strategy has already been demonstrated in rodents, non-human primates and Parkinson's (PD) patients (Palfi et. al, Lancet 2014) with a precursor gene therapy vector called ProSavin®. OXB-102 is an improved version of ProSavin® that expresses the same enzymes but with an increased DA production per genetically modified cell. In non-clinical studies the efficacy of a full-strength and a 1/5th dose of OXB-102 has been compared to the efficacy of full-strength ProSavin® in the 'gold standard' MPTP NHP model of PD; a vector that does not express any genes was used as a negative control. The longitudinal follow-up consisted of recording clinical rating scores and video-based quantification of locomotor activity before and after vector administration. The full-strength and the 1/5th dose of OXB-102 were as efficacious as the full-strength dose of ProSavin®, whereas the control-treated macaques maintained a significant PD phenotype, indicating improved dopamine production from OXB-102. Positron emission tomography (PET) was also carried out at baseline and at 3 and 6 months following vector administration using 18F-FMT, a presynaptic biomarker that acts as a substrate of AADC. There was a significant increase in the FMT signal in both of the OXB-102 treatment groups that was greater than in the ProSavin® treated animals whilst there was no significant change in the FMT signal in the control treatment group. Results from a 6-month toxicology and biodistribution study in the NHP indicate that the OXB-102 vector is safe and well tolerated following stereotactic administration into the putamen and the vector does not significantly spread beyond the site of administration GMP manufacture of OXB-102 for a planned clinical trial in PD patients is currently in progress.

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.

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.

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.

Pancreatic ductal adenocarcinoma is characterized by a paucity of neoplastic cells embedded in a densely desmoplastic stroma. Therefore, laser capture microdissection was performed to obtain homogeneous populations of normal and neoplastic ductal cells. These were subjected to a comparative study of gene expression utilizing human cDNA arrays. A variety of dysregulated genes were identified, comprising cell cycle and growth regulators, invasion regulators, signalling and developmental molecules. In addition to genes already found to be overexpressed in pancreatic cancer, such as TIMP1 , MMP7 , CD59 , rhoC and NDKA , we present evidence to implicate genes which have not previously been reported in this tumour type. These include the overexpressed genes ABL2 , Notch4 and SOD1 , as well as XRCC1 , a DNA repair gene whose transcript was found downregulated. Quantitative real-time RT–PCR (QRT–PCR) was employed to confirm differential expression of ABL2 , Notch4 and SOD1 and immunohistochemical analysis was used to verify decreased protein expression of XRCC1 using a custom-built pancreatic tissue array. Combining microarray-derived gene expression profiles of pure pancreatic cell populations, QRT–PCR and pancreas-specific tissue arrays therefore proved to be highly informative in elucidating the molecular pathology of this highly malignant tumour type.