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.

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.

Genetically modified macrophages expressing hypoxia regulated cytochrome P450 and P450 reductase for the treatment of cancer.

This study describes a combined gene and cell therapy based on the genetic modification of primary human macrophages, as a treatment for cancer. Here, we have utilised the tumour-infiltrating properties of macrophages as vehicles to deliver a gene encoding a prodrug-activating enzyme such as human cytochrome P450 2B6 (CYP2B6) inside tumours followed by killing the tumour cells with the prodrug cyclophosphamide (CPA). Macrophages were transduced with an adenoviral vector that expresses human cytochrome CYP2B6 via a synthetic hypoxia responsive promoter (OBHRE) and with human P450 reductase (P450R), via the CMV promoter. In the presence of CPA, these genetically modified macrophages showed increased cytotoxicity against various tumour cell lines compared to untransduced macrophages or macrophages transduced with CYP2B6 alone. In human ovarian carcinoma xenograft models, the median survival of mice treated with genetically modified macrophages plus CPA increased up to two-fold compared to the survival of mice treated with untransduced macrophages and CPA. Genetically modified autologous macrophages may be a feasible therapeutic option for the treatment of some solid tumours, such as ovarian cancer.

Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors.

BACKGROUND: We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. METHODS: Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. RESULTS: All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. CONCLUSIONS: It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target.

Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis.

The cellular response to hypoxia involves the promotion of angiogenesis, leading to increased blood flow and oxygenation. The macrophage has been identified as an orchestrator of this response in several pathologies, through the release of angiogenic factors in response to hypoxia. We have produced the first comprehensive transcriptome analysis of hypoxic primary human macrophages with respect to the regulation of angiogenesis. There is a marked induction of genes encoding factors known to stimulate angiogenesis, rather than factors that inhibit this process. We show that overexpression of the transcription factor EPAS1 using a recombinant adenoviral vector amplifies the induction of genes encoding angiogenic proteins in response to hypoxia. This defines a new strategy for enhancing transcriptome and proteome analyses by overexpressing disease-implicated genes using viral gene transfer methodologies.

Exploiting the hypoxia response.

Hypoxia (low oxygen) is a defining physiological feature of a number of diseases, including cancer, cardiovascular disease and retinopathy. Hypoxia plays an active role in the pathology of these diseases through its impact on gene expression, thereby making the hypoxia-signaling pathway a key target for the development of novel molecular therapies. This review focuses on how the elucidation of this pathway has led to the development of novel therapeutic strategies, including physiologically targeted gene therapy and the identification of novel therapeutic targets within the hypoxia-signaling pathway.

Long-term reversal of chronic anemia using a hypoxia-regulated erythropoietin gene therapy.

Anemia is a common clinical problem, and there is much interest in its role in promoting left ventricular hypertrophy through increasing cardiac workload. Normally, red blood cell production is adjusted through the regulation of erythropoietin (Epo) production by the kidney. One important cause of anemia is relative deficiency of Epo, which occurs in most types of renal disease. Clinically, this can be corrected by supplementation with recombinant Epo. Here we describe an oxygen-regulated gene therapy approach to treating homozygous erythropoietin-SV40 T antigen (Epo-TAg(h)) mice with relative erythropoietin deficiency. We used vectors in which murine Epo expression was directed by an Oxford Biomedica hypoxia response element (OBHRE) or a constitutive cytomegalovirus (CMV) promoter. Both corrected anemia, but CMV-Epo-treated mice acquired fatal polycythemia. In contrast, OBHRE-Epo corrected the hematocrit level in anemic mice to a normal physiologic level that stabilized without resulting in polycythemia. Importantly, the OBHRE-Epo vector had no significant effect on the hematocrit of control mice. Homozygous Epo-TAg(h) mice display cardiac hypertrophy, a common adaptive response in patients with chronic anemia. In the OBHRE-Epo-treated Epo-TAg(h) mice, we observed a significant reversal of cardiac hypertrophy. We conclude that the OBHRE promoter gives rise to physiologically regulated Epo secretion such that the hematocrit level is corrected to healthy in anemic Epo-TAg(h) mice. This establishes that a hypoxia regulatory mechanism similar to the natural mechanism can be achieved, and it makes EPO gene therapy more attractive and safer in clinical settings. We envisage that this control system will allow regulated delivery of therapeutic gene products in other ischemic settings.