Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFlt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFlt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFlt-1 prevented the development of laser photocoagulation-induced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.
The aim of this study was to develop a fast and reliable analytical procedure for the display of the protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear protein components following a corneal wound in the rabbit. Calibrated 10-microL glass, fire-polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6-mm diameter centrally placed anterior keratectomy. Tear proteins were eluted by a reversed-phase high-performance liquid chromatography (RP-HPLC) column and the tear protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (TIC) chromatography. Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of protein components. The molecular weight of each protein component was determined by on-line ESI. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and beta2-microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, beta2-microglobulin was found to be glycosylated with N-acetylhexosamine. ESI-positive TIC chromatograms and mass spectra revealed comparative differences in the tear protein spectra after corneal wounding. One day after wounding, rabbit lysozyme with a molecular weight of 14,717 Da was found to be 8-fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear protein with the molecular weight of 16,060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI-MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and proteins (<80 kDa) in tears.
The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1-5) following a standard unilateral 6 mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13 peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1- 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability.
We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (Po0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.
Non-infectious anterior uveitis (AU) is a potentially sight threatening inflammatory condition. The current gold standard for treatment is topical steroids, but low ocular bioavailability and compliance issues with the intensive dosing regimen limit the efficacy of this treatment. Liposomes as a drug delivery system may help to overcome these problems. We studied the efficacy of a PEG-liposomal formulation of liposomal steroids, administered as a single subconjunctival dose, in the treatment of experimental uveitis in rabbit eyes. Rabbits that received subconjunctival liposomal triamcinolone acetonide phosphate (LTAP) or liposomal prednisolone phosphate (LPP) had significantly lower mean inflammatory scores than untreated controls on Day 4 after induction of uveitis (LPP vs controls, p = 0.049) and 8 (LPP vs controls, p = 0.007; LTAP vs controls, p = 0.019), and lower scores than rabbits given topical PredForte1% 4 times a day on Day 8 (p = 0.03). After antigen rechallenge, the subconjunctival liposomal steroid groups continued to have greater suppression of inflammation than untreated controls on Day 11 (p = 0.02). Localization of liposomes in inflamed ocular tissue was confirmed by histology and immunostaining, and persisted in the eye for at least one month. Our study demonstrates that a single subconjunctival injection of liposomal steroids induces effective and sustained anti-inflammatory action.
Inherited retinal diseases (IRDs) are a heterogenous group of orphan eye diseases that typically result from monogenic mutations and are considered attractive targets for gene-based therapeutics. Following the approval of an IRD gene replacement therapy for Leber’s congenital amaurosis due to RPE65 mutations, there has been an intensive international research effort to identify the optimal gene therapy approaches for a range of IRDs and many are now undergoing clinical trials. In this review we explore therapeutic challenges posed by IRDs and review current and future approaches that may be applicable to different subsets of IRD mutations. Emphasis is placed on five distinct approaches to gene-based therapy that have potential to treat the full spectrum of IRDs: 1) gene replacement using adeno-associated virus (AAV) and nonviral delivery vectors, 2) genome editing via the CRISPR/Cas9 system, 3) RNA editing by endogenous and exogenous ADAR, 4) mRNA targeting with antisense oligonucleotides for gene knockdown and splicing modification, and 5) optogenetic approaches that aim to replace the function of native retinal photoreceptors by engineering other retinal cell types to become capable of phototransduction.
ILM appears to be the predominate barrier to AAV transfection. An efficacious and safe method of AAV2 gene delivery, taking into account the potential need for repeat treatments, appears to be the surgical removal of ILM and layering of AAV under air.v.
Real-time PCR analysis showed that the expression levels of GAPDH, beta-actin, and HPRT were higher in the first postnatal week and then declined. However, from 2 to 8 weeks, the mRNA levels of these three genes underwent significant variations (P < 0.01) in their levels of expression. In contrast, the expression level of 18S rRNA showed no significant variation (P >or= 0.5) over this time period. Conclusions. The present study shows that GAPDH, beta actin and HPRT gene were differentially expressed in early postnatal scleral development. It also suggests that these gene products could be implicated in the developmental process and have a crucial role in the early postnatal period. This study demonstrates that 18S rRNA may be preferable to normalize genes of interest in studies of early development.
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