Development of a surgical approach for a wide-view suprachoroidal retinal prosthesis: evaluation of implantation trauma.

BACKGROUND: Our research goal is to develop a safe, reproducible surgical approach for implantation of a wide-field retinal stimulating array. The aim of this study was to evaluate the pathological response to acute implantation of a functional prototype electrode array in the suprachoroidal space. METHODS: The surgical techniques to implant a 72 platinum electrode array fabricated on 8 × 13 × 0.4 mm polyimide and silicone substrate were developed in a pilot study in anesthetized cats. For the main study, nine eyes were implanted in vivo and unoperated eyes were used as controls. Surgery consisted of a temporal approach with a full-thickness scleral incision 5 mm posterior to the limbus. A suprachoroidal "pocket" was created, the electrode array inserted to sit beneath the area centralis, and placement was confirmed visually. The eyes were collected subsequently for histopathology. RESULTS: The array was consistently inserted into the suprachoroidal space beneath the area centralis in nine eyes. There was a significant hemorrhage in two cases where implantation was complicated by choroidal congestion. Retinal folding occurred only when the array tip was within 2.6 mm of the optic disc (p < 0.01). There was choroidal incarceration at the incision in six eyes and scleral distortion at the array edges in five. No cases were found where the implant breached the retina, choroid, or sclera. CONCLUSIONS: A large stimulation array can be reliably inserted into the suprachoroidal space without trauma to the neuroretina. These findings suggest that this is an appropriate surgical approach for the placement of an electrode array for use in retinal stimulation.

Cytokine signalling by gp130 regulates gastric mucosal healing after ulceration and, indirectly, antral tumour progression.

The cytokines IL-6 and IL-11, which signal via the receptor gp130, have been implicated in various gut pathologies, including inflammation and wound healing. We used mouse cytokine signalling mutants to evaluate the role of gp130 pathways in gastric ulceration and healing and the effect of spatially remote fundic ulceration on antral tumour progression, since compromised wound healing may impact tumourigenesis. Glacial acetic acid applied to the serosal surface of stomachs from wild-type, gp130(757FF), IL-6(-/-) and IL-11 receptor (R)alpha(-/-) mice was used to induce discrete haemostasis/necrosis and resultant mucosal ulceration. Wound pathology and mRNA expression of key cytokine target genes were examined 2 and 14 weeks after ulcer induction. The outcome of fundic ulceration on antral tumour development in gp130(757FF) mice was also examined. Chemical haemostasis in gp130(7575FF) mice produces more severe gastric ulcers than in wild-type mice. Lack of IL-6 produces more severe ulceration, while loss of IL-11Ralpha less severe ulcers, suggesting a role for IL-11 in ulcer induction. Increased expression of ulcer-associated IL-11 and its established mitogenic target genes RegI, IIIbeta and IIIgamma paralleled severe ulceration in gp130(757FF) mice. In this model, coincident with fundic ulceration, antral tumour development was inhibited and correlated with decreased RegI, IIIbeta and IIIgamma and reduced MMP9 and 13 expression. IL-11-driven transcription via gp130 contributes to the gastric mucosal response to ulceration. Fundic mucosal ulceration modulates antral growth factor and metalloproteinase gene expression, thereby contributing to restricted tumour growth.

Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency.

Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1. Vectors incorporating an internal human elongation factor-1alpha regulatory element were capable of fully restoring the lymphoid differentiation potential of gammac-deficient lineage negative cells. Multilineage lymphoid reconstitution of a murine model was achieved at a similar level to that achieved by a conventional long-terminal repeat (LTR)-regulated vector used in previous clinical trials. Functional proliferative responses to mitogenic stimuli were also restored, and serum immunoglobulin levels were normalized. The reduced mutagenic potential conferred by SIN vector configurations and alternative non-LTR-based regulatory elements, together with proven efficacy in correction of cellular defects provides an important platform for development of the next phase of clinical trials for SCID-X1.

Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

Insights into the mechanisms of gastric adaptation to aspirin-induced injury: a role for regenerating protein but not trefoil peptides.

The phenomenon of reduced gastric mucosal injury despite repeated doses of a damaging agent is termed adaptation. Adaptation to nonsteroidal anti-inflammatory drug-induced injury has been clearly demonstrated in both humans and experimental animals; however, the precise mechanisms remain unclear. We hypothesized that mediators of adaptation might be the regenerating protein (RegI) and the trefoil peptides TFF1 and TFF2, because these proteins play pivotal roles in gastric mucosal protection and repair. The gene expression and the protein levels of these proteins were measured and compared in normal, aspirin-injured, and aspirin-adapted rat stomachs. TFF gene and protein expression levels were similar in all three groups, whereas RegI gene expression and protein levels in adapted stomach were increased. A time course analysis of RegI expression during the onset and offset of adaptation showed that mucosal RegI increased during the development of adaptation, was maintained during subsequent aspirin dosing, and returned to baseline levels once dosing had ceased and adaptation was lost-indicative of a causal role in the adaptation process. Colocalization of increased RegI with gastric epithelial areas showing increased proliferation also suggests that RegI may be an important mediator of the resolution of mucosal injury that is characteristic of gastric adaptation to aspirin.