Observation of Silicone Oil Within the Vitreous and Sclera Following Intravitreal Administration of Biotherapeutics Using Insulin Syringes in Cynomolgus Monkeys.

Silicone oil droplets have been reported in the eyes of human patients following intravitreous (IVT) injections with several marketed biotherapeutic products. Intravitreous administration of a novel biotherapeutic in a 14-week cynomolgus monkey study using insulin syringes was associated with 2, non-test-article-related phenomena: "vitreous floater/clear sphere" on indirect ophthalmoscopy and intrascleral "foreign material near injection track" on histopathology. Retrospective analysis of 81 other preclinical studies of IVT administration of novel biotherapeutics found a greater frequency of clear spheres in monkey IVT studies using insulin syringes and formulations containing polysorbate. We were able to correlate microscopic findings of clear circular to oval areas in the sclera near the injection track with an energy-dispersive X-ray spectroscopy (EDS) signal for silicon at the same location in the sclera. These observations provide further evidence that silicone lubricant in insulin syringes/needles is the source of clear spheres noted in the vitreous and foreign material noted near the injection track in the sclera. Although considered inert and toxicologically insignificant, silicone deposition within the eye should form part of the risk-benefit equation in a clinical setting.

Spontaneous Background and Procedure-Related Microscopic Findings and Common Artifacts in Ocular Tissues of Laboratory Animals in Ocular Studies.

Identification of test article-related microscopic findings in ocular toxicology studies requires a working knowledge of the artifacts and procedure-related or background findings commonly encountered in such studies. The objective of this article is to provide a mini-atlas of the artifacts and procedure-related or spontaneous background findings commonly observed in ocular tissues from animals in toxicology studies of ocular drug candidates. Artifacts in the eye are often related to collection or fixation procedures and include swelling and vacuolation of lens fibers, separation of the neuroretina from the retinal pigment epithelium (RPE), and vacuolation of the optic nerve. Common in-life procedure-related findings include intravitreal injection needle tracks in the sclera and ciliary body pars plana and foci of RPE hypertrophy and/or hyperpigmentation at subretinal injection sites. Common background findings include corneal mineralization, uveal mononuclear cell infiltrates, and peripheral displacement of photoreceptor nuclei in the retina. A few uncommon spontaneous background findings that may be confused with test article-related findings, such as bilateral optic atrophy in macaques, are also included.

Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 µl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

Intraocular sarcomas in two rabbits.

Intraocular neoplasms are described in 2 adult rabbits. The left globe of an 8-year-old male rabbit was enucleated after chronic inflammatory disease resulted in a nonvisual eye. The left globe of a 5-year-old female rabbit also was enucleated after a history of lens-induced uveitis, cataract formation, and resultant glaucoma. In both rabbits, histopathology revealed a variably pleomorphic, poorly differentiated, invasive, intraocular spindle cell neoplasm closely associated with lens and lens capsular fragments. Gram stains failed to detect bacterial organisms or Encephalitozoon cuniculi. Polymerase chain reaction assays, used to amplify the 16S RNA gene of numerous bacteria and E. cuniculi, were also negative. Immunohistochemical staining demonstrated strong, diffuse expression for vimentin; however, staining for smooth muscle actin, cytokeratin, S100, and desmin were negative. Long-standing intraocular inflammation and/or traumatic insults to the eyes were considered as causes of these neoplasms. The histologic features of these intraocular neoplasms closely resemble post-traumatic ocular sarcomas in cats.

Asthma progression to airway remodeling and bone marrow eosinophil responses in genetically distinct strains of mice.

BACKGROUND: Patient factors that cause long-term airway remodeling are largely unidentified. This suggests that genetic differences may determine which asthmatic patients develop airway remodeling. A murine model with repeated allergen exposure leading to peribronchial fibrosis in complement factor 5 (C5)-deficient A/J mice has been used to study asthma progression. No studies have addressed the systemic effects of allergen sensitization or chronic allergen exposure on bone marrow eosinophilopoiesis in this mouse strain. OBJECTIVE: To investigate bone marrow eosinophil responses during acute sensitization and chronic allergen exposure using genetically distinct mouse strains differing in persistent airway reactivity and remodeling. METHODS: The C5-sufficient BALB/c and C5-deficient A/J mice were repetitively exposed to intranasal ovalbumin for 12 weeks. Subsequently, the mice were evaluated for airway eosinophilia, mucus-containing goblet cells, and peribronchial fibrosis. Both strains of mice were also acutely sensitized to ovalbumin. Bone marrow eosinophil progenitor cells and mature eosinophils were enumerated. RESULTS: BALB/c and A/J mice have similar bone marrow responses after acute allergen exposure, with elevations in bone marrow eosinophil progenitor cell and eosinophil numbers. After chronic allergen exposure, only C5-deficient A/J mice that developed peribronchial fibrosis exhibited bone marrow eosinophilia. BALB/c mice lacked peribronchial fibrosis and extinguished accelerated eosinophil production after long-term allergen challenge. CONCLUSIONS: Chronic airway remodeling after repeated allergen exposure in genetically different mice correlated with differences in long-term bone marrow eosinophilopoiesis. Preventing asthma from progressing to chronic airway remodeling with fibrosis may involve identifying genetically determined influences on bone marrow responses to chronic allergen exposure.

Identification of a functional domain within the essential core of histone H3 that is required for telomeric and HM silencing in Saccharomyces cerevisiae.

Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the alpha1 helix and L1 loop of the essential histone fold. Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter. Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.