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Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector. Keywords: inner ear, AAV, non-human primate, cochlea, hair cells, adeno-associated virus vector, hereditary deafness, gene delivery

In a juvenile toxicology program, an unexpected finding of vacuolation of inner nuclear, ganglion cell, and nerve fiber layers of the retina was observed microscopically in routine Davidson’s fixed and hematoxylin and eosin–stained tissue sections of eyes in beagle dogs at approximately 5 weeks of age. There was no necrosis or degeneration of the affected cells and no associated inflammation. Fluorescein angiography revealed no vascular leakage. Optical coherence tomography (OCT) indicated swollen cells in the same layers of the retina as observed at light microscopic examination. Transmission electron microscopy revealed that the retinal vacuolation likely was consistent with intracellular swelling of amacrine, horizontal, and/or bipolar cells of the inner nuclear layer as affected cells had an expanded cytoplasm but contained normal nucleus and organelles. As assessed by animal behavior and full-field electroretinography, the retinal vacuolation appeared to have no impact on visual function. Retinal vacuolation was seen in approximately 40% of dogs at 5 weeks of age using OCT and/or light microscopic examination. Because the change was transient and age related, did not result in degenerative retinal changes, and was not present in dogs older than 5 weeks of age, it was considered a background developmental observation in beagle dogs.

Pathological choroidal neovascularization (CNV) is the common cause of vision loss in patients with age-related macular degeneration (AMD). Macrophages possess potential angiogenic function in CNV. We have demonstrated that human T lymphocyte-derived microparticles (LMPs) exert a potent antiangiogenic effect in several pathological neovascularization models. In this study, we investigated the alteration of proangiogenic properties of macrophages by LMPs treatment in vitro and in vivo models. LMPs regulated the expression of several angiogenesis-related factors in macrophages and consequently stimulated their antiangiogenic effects evidenced by the suppression of the proliferation of human retinal endothelial cells in co-culture experiments. The involvement of CD36 receptor in LMPs uptake by macrophages was demonstrated by in vitro assays and by immunostaining of choroidal flat mounts. In addition, ex vivo experiments showed that CD36 mediates the antiangiogenic effect of LMPs in murine and human choroidal explants. Furthermore, intravitreal injection of LMPs in the mouse model of laser-induced CNV significantly suppressed CNV in CD36 dependent manner. The results of this study suggested an ability of LMPs to alter the gene expression pattern of angiogenesis-related factors in macrophages, which provide important information for a new therapeutic approach for efficiently interfering with both vascular and extravascular components of CNV.

Retinoblastoma (Rb) is an aggressive childhood cancer of the developing retina. This disease is associated with epigenetic deregulation of several cancer pathways including upregulation of the proto-oncogene spleen tyrosine kinase (SYK). We have previously demonstrated that lymphocyte-derived microparticles (LMPs) possess strong cytotoxic effect on cancer cells. This report demonstrated that LMPs have potent pro-apoptotic properties on human Rb cells, which was associated with a strong reduction of SYK expression. Perturbing SYK activity in Rb cells induced cell apoptosis and upregulated expression of p53 and p21. Interestingly, inhibition of p53 or knockdown of p21, abolished LMP-induced caspase-3 activity and cell death. Blocking oxidized phospholipid-rich LMPs with a specific antibody significantly prevented LMP-induced Rb apoptosis and reversed the expression patterns of SYK, p53, p21. In summary, our results suggest that LMPs are important pro-apoptotic regulators for Rb cells through reduction of SYK expression and upregulation of the p53-p21 pathway which ultimately activates caspase-3. These data may open unexpected avenues for the development of LMPs as a novel therapeutic strategy that would be particularly useful and relevant for the treatment of Rb.

International audience; QueBall is a spherical robot capable of motion and equipped with touch sensors, multi-colored lights, sounds, and a wireless interface with an iOS device. While these capabilities may be useful in assisting the early diagnosis of autism, no detailed guidelines have yet been established to achieve this. In this report, we described the exploratory study conducted with an interdisciplinary research team to adapt QueBall's capabilities in order to have clinicians observe how children interact with QueBall. This is the preliminary phase in designing an experimental protocol to evaluate the use of QueBall in diagnosing autism for children from two to five years of age.

The rd8 mutation of the Crb1 gene, which can cause significant retinal degeneration, was found in homozygous form in the C57BL/6N mouse substrain. It is present in stocks from most major mouse vendors and in ES cells derived from the C57BL/6N substrain. Its presence can confound interpretation of ocular mutant phenotypes.

Interspecies differences in ocular anatomy can alter the way a drug interacts locally within the eye, whether administered directly to the eye or systemically. It is therefore important to understand these differences and how they can influence the outcome and interpretation of safety or efficacy data for ocular therapeutics. The eye is a complex system of tissues integrated into a functional sense organ. Oriented toward the toxicologist or ocular researcher, this chapter will discuss the individual ocular tissues in commonly used laboratory animals in comparison with humans and will provide a basic understanding of ocular anatomy including quantitative comparisons when possible in these species. It will also act as a reference to the terminology that will be encountered in subsequent chapters.

The pharmacokinetics of ophthalmic biotherapeutics are difficult to determine in human vitreous humor. Because of the high transparency of living tissue to near-infrared (NIR) light, the temporal changes in vitreous concentrations of a biomolecule labeled with an NIR fluorescent probe can be monitored in situ with a scanning laser ophthalmoscope (SLO).A humanized IgG was labeled with the NIR probe IRDye800CW (CVX-4164). Rabbits were given CVX-4164 intravitreally, and NIR fluorescence intensity was measured in the central plane of the vitreous humor with an SLO. Fluorescence intensities were converted to concentrations by using standard curves.Little background fluorescence was detected, and the minimum detectable concentration of CVX-4164 was10 nM. Vitreal concentrations of CVX-4164 determined in situ declined with time, with C(max) ≈ 1 μM and t½ = 145 hours (112-μg dose). The t½ of CVX-4164 was approximately three times greater than that of the IRDye800CW alone, whereas the vitreal clearance (CL) and volume of distribution (V(ss)) of the native dye were approximately 2000- and 550-fold greater than that of the conjugate. CVX-4164 concentrations determined in situ were 2.6 to 4.4 times higher than those determined by ex vivo NIR fluorescence or ELISA in homogenized vitreous humor, reflecting the greater spatial resolution of in situ imaging. Moreover, vitreal concentrations determined in situ were3 orders of magnitude greater than plasma concentrations of CVX-4164, as determined by ELISA, and had a different kinetic profile.This study demonstrates the feasibility of determining the pharmacokinetics of intraocular biotherapeutics labeled with NIR fluorescent probes by in situ monitoring.

Introduction The ICH S7A and S7B guidelines require that effects of test substances on the cardiovascular system be assessed with respect to blood pressure, heart rate and electrocardiogram intervals. Where adverse effects are identified additional supplemental studies, including ventricular contractility, should be conducted as deemed appropriate. However, there is an absence of definitive guidance regarding when to pursue supplementary studies, in part due to ill-defined criteria of what constitutes an adverse effect and to surgical/technical monitoring limitations of study designs. However with advances in technology it is now feasible to develop models for assessing LVP and contractility in conjunction with standard assessments. The objectives of this study were to 1) develop a model for chronic evaluation of LVP and contractility, 2) illustrate changes in LV contractility without concurrent proportional changes in heart rate and/or systemic blood pressure and 3) determine if the QA interval, the time between the Q on the ECG and the beginning of the upstroke on the arterial blood pressure, can be used as a indicator of altered LV contractility. Methods Dogs (N = 4) were implanted with a telemetry transmitter. LVP, contractility, ECG and BP were assessed prior to and up to 24 h following administration of Atenolol (10 mg/kg) and Pimobendan (0.45 mg/kg). Results Atenolol caused an ~ 30% decrease in HR, followed by a sustained decrease in maximum left ventricular contractility (+ dP/dt mmHg/s). No effects were noted on blood pressure. Pimobendan caused a 100% increase in contractility (+ dP/dt mmHg/s) which remained elevated for ~ 4 h. No effects were noted on blood pressure. Heart rate was highly variable initial decreasing, followed by a highly variable increase until 4 h postdose. Following administration of both compounds changes in maximum left ventricular contractility correlated with reverse changes in QA interval duration. Discussion This model demonstrates that evaluation of LV contractility complements measurements of heart rate and blood pressure as part of a more complete cardiovascular safety assessment strategy. Furthermore, we demonstrate an apparent correlation between dP/dt and QA interval and concluded that QA interval can be utilized as an indicator of a potential inotropic effect. However further confirmation should be assessed through additional in-vivo measurements of LVP and contractility.

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.