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We previously discovered microRNAs (miRNAs) in cerebrospinal fluid (CSF) that differentiate Alzheimer’s disease (AD) patients from Controls. Here we examined the performance of 37 candidate AD miRNA biomarkers in a new and independent cohort of CSF from 47 AD patients and 71 Controls on custom TaqMan® arrays. We employed a consensus ranking approach to provide an overall priority score for each miRNA, then used multimarker models to assess the relative contributions of the top-ranking miRNAs to differentiate AD from Controls. We assessed classification performance of the top-ranking miRNAs when combined with apolipoprotein E4 (APOE4) genotype status or CSF amyloid-β(42) (Aβ(42)):total tau (T-tau) measures. We also assessed whether miRNAs that ranked higher as AD markers correlate with Mini-Mental State Examination (MMSE) scores. We show that of 37 miRNAs brought forth from the discovery study, 26 miRNAs remained viable as candidate biomarkers for AD in the validation study. We found that combinations of 6–7 miRNAs work better to identify AD than subsets of fewer miRNAs. Of 26 miRNAs that contribute most to the multimarker models, 14 have higher potential than the others to predict AD. Addition of these 14 miRNAs to APOE4 status or CSF Aβ(42):T-tau measures significantly improved classification performance for AD. We further show that individual miRNAs that ranked higher as AD markers correlate more strongly with changes in MMSE scores. Our studies validate that a set of CSF miRNAs serve as biomarkers for AD, and support their advancement toward development as biomarkers in the clinical setting.

Purpose: The purpose of this study is to determine whether bevacizumab is detectable in the breast milk of nursing mothers. Methods: Breast milk samples were collected from 2 patients receiving monthly intravitreal bevacizumab injections for choroidal neovascularization over the course of 16 months. Enzyme-linked immunosorbent assay and Western blot analysis was used to determine the levels of bevacizumab in the milk samples. Results: An enzyme-linked immunosorbent assay was developed using antibodies specific to bevacizumab in which the sensitivity threshold was 3 ng/mL. All breast milk samples assayed from the two patients actively undergoing treatment did not have detectable levels of bevacizumab. Samples collected 1.5 hours and 7 hours after an injection and 2 randomly chosen samples were negative by Western blot analysis. Conclusion: A sensitive assay to detect bevacizumab in breast milk samples assayed suggests that intravitreal injections do not result in detectable bevacizumab in breast milk.

Corneal transplantation is the oldest and one of the most successful transplant procedures with a success rate in many studies in excess of 90%. The high success rate is mainly attributable to the relatively immune-privileged status of the eye and the fact that the cornea is largely avascular. However, the success rate in patients with failed grafts is much lower such that regrafting is frequently the top indication for corneal transplantation in many centers. Neovascularization is the most important risk factor for rejection, as it allows access of the immune system to the donor tissue, compromising immune privilege of the graft/eye. We have developed a process to modify donor corneal tissue to prevent rejection by a single exposure to a gene therapy vector before surgery (EncorStat(®)). The vector used is based on clinically relevant equine infectious anemia virus (EIAV)-derived lentiviral platform and contains genes for two potently angiostatic genes, endostatin and angiostatin. We show that incubation of rabbit, primate, and human corneal tissue with the EIAV vector mediates strong, stable expression in the corneal endothelium. We have optimized this process to maximize transduction and, once this is complete, maximize the removal of free vector before transplant. Rabbit corneas treated with two different antiangiogenic expression vectors (EIAV-EndoAngio and to a lesser extent EIAV-Endo:k5) significantly suppressed neovascularization in a rabbit model of corneal rejection. As a result, corneal opacity, edema, and inflammatory infiltrates were reduced in these corneas. This study demonstrates that angiogenesis is a suitable target to prevent corneal rejection, and provides the first proof-of-concept data for the development of EncorStat, an ex vivo gene therapy treatment to prevent corneal rejection.

Ocular gene therapy involves the introduction of an exogenous gene product to a host's cellular and genetic machinery for endogenous production of a desired gene product. The eye represents an ideal target organ due to its easy visibility and accessibility, and several trials have demonstrated proof-of-principle safety and efficacy in a subtype of Leber's congenital amaurosis. There are numerous ongoing clinical trials exploring gene therapy in other retinal diseases. In autosomal recessively inherited retinal degenerations, the introduced gene product replaces a known genetically deficient gene product and provides restoration of function. In other disease states, such as neovascular age-related macular degeneration, the delivered gene product modulates existing proteins within a cell, such as vascular endothelial growth factor, for a desired therapeutic effect. This latter approach may have broader applications in other diseases such as diabetes and other retinal vascular diseases that are as yet unrealized. This review summarizes the current state of clinical research in ocular gene therapy focusing on those diseases in which the technology has reached clinical trials.

The soluble neuropilin-1 (sNRP-1) gene employs an extremely short dual function polyadenylation (pA) signal/stop codon that is efficient for termination of gene transcription and translation in vivo. However, the functionality and usefulness of this signal in regard to other genes is unknown. This quantitative study compares the levels of humanized Renilla green fluorescent protein (hrGFP) mRNA polyadenylated with either the sNRP-1 pA signal or the much larger, more widely used SV40 pA signal. We show that the overall starting copy number of hrGFP for equally loaded RNAs is equivalent between the two groups. Our data show little to no difference between levels of mRNA generated by the sNRP-1 polyadenylation signal and the SV40 polyadenylation signal despite a remarkable size difference in signal length. An extremely short polyadenylation signal could potentially alleviate gene insert size restrictions associated with cloning or with therapeutic vectors such as adeno-associated virus.

Proliferative ocular diseases encompass a wide variety of pathological processes with adverse cellular differentiation, proliferation and migration as common features. Pathologies may involve neovascular responses associated with diabetic retinopathy, retinopathy of prematurity or age-related macular degeneration. These diseases are quite prevalent and account for substantial visual impairment and blindness worldwide. Although treatment strategies are largely surgical, advances in our understanding of the proteins crucial to cell transdifferentiation, proliferation and migration, along with better gene transfer techniques, have greatly increased the potential for biological treatment options. In this report, the most common proliferative ocular vascular diseases and existing therapeutic modalities will be reviewed and an overview of possible gene therapy options will be discussed, along with potential candidate genes.

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4(216), which represses VEGF promoter activity. The TEAD4(216) isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4(216) protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4(216) isoform can competitively repress the stimulatory activity of the TEAD4(434) and TEAD4(148) enhancers. Synthesis of the native VEGF(165) protein and cellular proliferation is suppressed by the TEAD4(216) isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4(216) isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.

BACKGROUND: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP. METHODOLOGY/PRINCIPAL FINDINGS: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss. PRINCIPAL RESULTS: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. CONCLUSIONS/SIGNIFICANCE: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.

To describe the clinical features, histologic changes, and genetic analysis of Avellino corneal dystrophy in an African-American woman.Interventional case report.A 79-year-old African-American woman with corneal deposits consistent with Avellino corneal dystrophy was studied with histologic and genetic analysis.The patient had multiple crumb-like opacities in the anterior stroma of both eyes. Deep to these lesions were numerous faint, stellate lattice lesions. Corneal scraping confirmed the presence of Masson trichrome and Congo red positive subepithelial deposits. Genetic analysis revealed a heterozygous CGC/CAC change in exon 4 of the beta iG-H3 gene, resulting in an arginine to histidine substitution at codon 124.This case reveals several novel findings, including surface changes resembling vortex dystrophy and large granular deposits protruding through the anterior corneal surface. This is the first case described in an African-American patient.

Cell-based therapy rescues retinal structure and function in rodent models of retinal disease, but translation to clinical practice will require more information about the consequences of transplantation in an eye closely resembling the human eye. The authors explored donor cell behavior using human cortical neural progenitor cells (hNPC(ctx)) introduced into the subretinal space of normal rhesus macaques.hNPC(ctx) transduced with green fluorescent protein (hNPC(ctx)-GFP) were delivered bilaterally into the subretinal space of six normal adult rhesus macaques under conditions paralleling those of the human operating room. Outcome measures included clinical parameters of surgical success, multifocal electroretinogram (mfERG), and histopathologic analyses performed between 3 and 39 days after engraftment. To test the effects of GFP transduction on cell bioactivity, hNPC(ctx)-GFP from the same batch were also injected into Royal College of Surgeons (RCS) rats and compared with nonlabeled hNPC(ctx).Studies using RCS rats indicated that GFP transduction did not alter the ability of the cells to rescue vision. After cells were introduced into the monkey subretinal space by a pars plana transvitreal approach, the resultant detachment was rapidly resolved, and retinal function showed little or no disturbance in mfERG recordings. Retinal structure was unaffected and no signs of inflammation or rejection were seen. Donor cells survived as a single layer in the subretinal space, and no cells migrated into the inner retina.Human neural progenitor cells can be introduced into a primate eye without complication using an approach that would be suitable for extrapolation to human patients.

PURPOSE. Identification of transcription factors that regulate the transcription of the vascular endothelial growth factor (VEGF) gene may facilitate understanding of the etiology and progression of ocular neovascular diseases. The purpose of this study was to determine whether transcriptional enhancer factor 1-related (RTEF-1) was present within ocular vascular endothelial cells and whether it played a role in the control of the transcription of the VEGF gene. METHODS. Primary cultures of human retinal vascular endothelial cells (RVECs) were maintained under normoxic or hypoxic conditions before isolation of mRNA. RT-PCR was performed to detect RTEF-1 transcripts. Amplified products were cloned into an expression plasmid. Human VEGF promoter and deletion constructs were cloned into a pSEAP reporter vector. Various RTEF-1 isoforms and VEGF promoter constructs were coelectroporated into human cells, and reporter expression levels were determined. Retinal tissue from a mouse model of retinopathy of prematurity (ROP) was analyzed by RT-PCR for the presence of RTEF-1 transcripts. RESULTS. Full-length 1305-bp and novel 936-bp RTEF-1 transcripts were identified in cultured human RVECs under normoxic conditions. A novel 447-bp isoform was present in cells maintained in a hypoxic environment. Four of the 11 translated exons predicted to code for the 1305-bp product were spliced out of the 936-bp transcript. The 1305-bp product enhanced expression from the VEGF promoter 4-fold greater than background, whereas the 936-bp and the 447-bp isoforms enhanced expression 3X and 12X, respectively. Analysis with deletion promoter constructs determined that all isoforms required the presence of Spl elements for efficient activation and that the hypoxia response element (HRE) was not essential for enhancement. Transcripts for novel RTEF-1 isoforms were also identified in neural retinal tissue of mice. Different murine-specific isoforms were present at different stages of postnatal development. CONCLUSIONS. Novel RTEF-1 transcripts are present within human ocular vascular endothelial cells and mouse neural retina during normal and ROP development, and alternatively spliced products are produced under hyperoxic and hypoxic conditions. Alternative spliced variants of human RTEF-1 transcripts are able to potentiate expression from the VEGF 5' proximal promoter region.

Objective To demonstrate the progression of electroretinographic (ERG) findings in mucolipidosis IV. Methods Two patients with mucolipidosis IV were examined clinically and their condition was followed up for ophthalmic manifestations of the disease. Electroretinograms were performed on both patients, and conjunctival biopsy specimens were analyzed for characteristic ultrastructural inclusion bodies using light and electron microscopy. Genomic DNA isolated from peripheral blood was screened for 2 major founder mutations in the ML4 gene using polymerase chain reaction and restriction fragment length polymorphism analyses. Haplotypes were confirmed by automated sequencing of polymerase chain reaction products. Results In patient 1, an ERG obtained at 12 months of age showed mildly subnormal amplitude of rod-mediated and cone-mediated responses and significantly prolonged rod and cone b-wave implicit times. An ERG obtained when the patient was 6.6 years old disclosed marked progression with greater loss of b-wave than a-wave responses to rod-and-cone–mediated activity. Scotopic ERG at the highest intensity was electronegative in configuration. In patient 2, ERG showed minimal rod-mediated responses, severely subnormal cone-mediated responses, and prolonged cone b-wave implicit times. Again, electronegative configuration of the scotopic bright flash response indicated greater disturbance of b-wave generators. Conclusions Novel ERG findings in 2 cases of mucolipidosis IV are reported with associated clinical courses, histopathologic abnormality, and genetic studies. In both cases ERGs demonstrate an electronegative configuration, suggesting that the primary retinal disturbance in mucolipidosis IV may occur at or proximal to the photoreceptor terminals.

This study was designed to engineer a functional filaggrin (FLG) monomer linked to a cell-penetrating peptide (RMR) and to test the ability of this peptide to penetrate epidermal tissue as a therapeutic strategy for genetically determined atopic dermatitis (AD). A single repeat of the murine filaggrin gene (Flg) was covalently linked to a RMR motif and cloned into a bacterial expression system for protein production. Purified FLG+RMR (mFLG+RMR) was applied in vitro to HEK-293T cells and a reconstructed human epidermis (RHE) tissue model. Immunochemistry demonstrated RMR-dependent cellular uptake of FLG+RMR in a dose- and time-dependent manner in HEK cells. Immunohistochemical staining of the RHE model identified penetration of FLG+RMR to the stratum granulosum, the epidermal layer at which FLG deficiency is thought to be pathologically relevant. In vivo application of FLG+RMR to FLG-deficient flaky tail (ft/ft) mice skin demonstrated internalization and processing of recombinant FLG+RMR to restore the normal phenotype. These results suggest that topically applied RMR-linked FLG monomers are able to penetrate epidermal tissue, be internalized into the appropriate cell type, and be processed to a size similar to wild-type functional barrier peptides to restore necessary barrier function, and prove to be therapeutic for patients with AD.