Tonia S. Rex

Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice

Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4(-/-) mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

A Novel Closed-Head Model of Mild Traumatic Brain Injury Caused by Primary Overpressure Blast to the Cranium Produces Sustained Emotional Deficits in Mice

Emotional disorders are a common outcome from mild traumatic brain injury (TBI) in humans, but their pathophysiological basis is poorly understood. We have developed a mouse model of closed-head blast injury using an air pressure wave delivered to a small area on one side of the cranium, to create mild TBI. We found that 20-psi blasts in 3-month-old C57BL/6 male mice yielded no obvious behavioral or histological evidence of brain injury, while 25–40 psi blasts produced transient anxiety in an open field arena but little histological evidence of brain damage. By contrast, 50–60 psi blasts resulted in anxiety-like behavior in an open field arena that became more evident with time after blast. In additional behavioral tests conducted 2–8 weeks after blast, 50–60 psi mice also demonstrated increased acoustic startle, perseverance of learned fear, and enhanced contextual fear, as well as depression-like behavior and diminished prepulse inhibition. We found no evident cerebral pathology, but did observe scattered axonal degeneration in brain sections from 50 to 60 psi mice 3–8 weeks after blast. Thus, the TBI caused by single 50–60 psi blasts in mice exhibits the minimal neuronal loss coupled to “diffuse” axonal injury characteristic of human mild TBI. A reduction in the abundance of a subpopulation of excitatory projection neurons in basolateral amygdala enriched in Thy1 was, however, observed. The reported link of this neuronal population to fear suppression suggests their damage by mild TBI may contribute to the heightened anxiety and fearfulness observed after blast in our mice. Our overpressure air blast model of concussion in mice will enable further studies of the mechanisms underlying the diverse emotional deficits seen after mild TBI.

Distribution of S- and M-Cones in Normal and Experimentally Detached Cat Retina

The lectin peanut agglutinin (PNA) and antibodies to short (S)‐ and medium to long wavelength (M/L)‐sensitive cones were utilized in order to define the relative distributions of the two spectral types of cone across the domestic cats retina. These values, in turn, were compared to those from retinas that had been experimentally detached from the retinal pigment epithelium. The pattern of cone distribution in the normal cats retina is established by the preponderance of M‐cones that constitute between 80% and 90% of all cones. Their peak density of over 26,000 cells/mm2 resides at the area centralis. Though M‐cone density decreases smoothly to the ora serrata where they have densities as low as 2,200 cells/mm2, the density decrease along the nasotemporal axis is slower,creating a horizontal region of higher cone density. S‐cones constitute between 10% and 20% of all cones, the number being quite variable even between individual animals of similar age. The highest S‐cone densities are found in three distinct locations: at the superior far periphery near the ora serrata, immediately at the area centralis itself, and in a broad zone comprising the central and lower half of the inferior hemiretina. S‐cones in the cat retina do not form a regular geometrical array at any eccentricity. As for the detached cat retina, the density of labeled S‐cone outer segments (OS) decreases rapidly as early as 1 day postdetachment and continues decreasing to day 28 when the density of cones labeling with anti‐S opsin has dropped to less than 10% of normal. This response points to a profound difference between rods and cones; essentially all rods, including those without OS, continue to express their opsin even in long‐term detachments. The implications of these results for visual recovery after retinal reattachment are discussed. J. Comp. Neurol. 430:343–356, 2001.

Neuroprotection of photoreceptors by direct delivery of erythropoietin to the retina of the retinal degeneration slow mouse.

The primary objectives of this study were to determine if erythropoietin (EPO) is neuroprotective to the photoreceptors in the retinal degeneration slow (rds) mouse in the absence of an increase in hematocrit and to determine if deglycosylated EPO (DEPO) is less neuroprotective. We performed subretinal injections of 10U EPO, DEPO or hyperglycosylated EPO (HEPO) in postnatal day 7 rds mice. Whole eye EPO levels were quantified by ELISA at specified time points post-injection. TUNEL analysis, hematocrit, and immunohistochemistry were performed at postnatal day 20. Half of the amount of EPO measured immediately after injection was detected less than 1 h later. Twenty four hours later, EPO levels were 1000 times lower than the amount originally detected. Uninjected rds mice contained 36 +/- 2 TUNEL-positive cells/mm retina and PBS injected mice contained 17 +/- 3 TUNEL-positive cells/mm retina. EPO, DEPO, and HEPO treated rds retinas contained 5 +/- 2, 9 +/- 2, and 3 +/- 1 TUNEL-positive cells/mm retina, respectively. The hematocrit was 43% in control and 41% in treated rds mice Previous studies have shown neuroprotection of the retina by treatment with as little as 24-39 mU EPO/mg total protein in the eye. In this study, we detected 40 mU/mg EPO in the eye 11 h after injection of 10 U EPO. Treatment with all forms of EPO tested was neuroprotective to the photoreceptors without a concomitant increase in hematocrit.

Delivery of Adeno-Associated Virus Vectors to the Fetal Retina: Impact of Viral Capsid Proteins on Retinal Neuronal Progenitor Transduction

ABSTRACT The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.

A mouse model of ocular blast injury that induces closed globe anterior and posterior pole damage

We developed and characterized a mouse model of primary ocular blast injury. The device consists of: a pressurized air tank attached to a regulated paintball gun with a machined barrel; a chamber that protects the mouse from direct injury and recoil, while exposing the eye; and a secure platform that enables fine, controlled movement of the chamber in relation to the barrel. Expected pressures were calculated and the optimal pressure transducer, based on the predicted pressures, was positioned to measure output pressures at the location where the mouse eye would be placed. Mice were exposed to one of three blast pressures (23.6, 26.4, or 30.4 psi). Gross pathology, intraocular pressure, optical coherence tomography, and visual acuity were assessed 0, 3, 7, 14, and 28 days after exposure. Contralateral eyes and non-blast exposed mice were used as controls. We detected increased damage with increased pressures and a shift in the damage profile over time. Gross pathology included corneal edema, corneal abrasions, and optic nerve avulsion. Retinal damage was detected by optical coherence tomography and a deficit in visual acuity was detected by optokinetics. Our findings are comparable to those identified in Veterans of the recent wars with closed eye injuries as a result of blast exposure. In summary, this is a relatively simple system that creates injuries with features similar to those seen in patients with ocular blast trauma. This is an important new model for testing the short-term and long-term spectrum of closed globe blast injuries and potential therapeutic interventions.

Gene Therapy and Animal Models for Retinal Disease

Those plagued by retinal diseases are often robbed of their vision, as often, effective treatments do not exist. Knowledge of the pathophysiology of retinal diseases stems from research on available animal models. Gene therapy may be useful for both genetic and acquired retinal diseases. This review will focus on retinal diseases for which gene therapy has demonstrated promise. The diseases are presented in order of the age at which they are generally first symptomatic and include retinopathy of prematurity, Leber congenital amaurosis, mucopolysaccharidoses, retinoblastoma, retinitis pigmentosa, diabetic retinopathy, glaucoma and age-related macular degeneration. We will describe the animal models used to study these disorders and emphasize the progress that has been made in using gene therapy for the treatment of retinal disease.

Systemic Adeno-Associated Virus-Mediated Gene Therapy Preserves Retinal Ganglion Cells and Visual Function in DBA/2J Glaucomatous Mice

A slow progressive death of neurons is the hallmark of neurodegenerative diseases, such as glaucoma. A therapeutic candidate, erythropoietin (EPO), has shown promise in many models of these diseases; however, it also causes polycythemia, a potentially lethal side effect. We have developed a novel mutant form of EPO that is neuroprotective but no longer erythropoietic by altering a single amino acid (arginine to glutamate at position 76; R76E). We hypothesized that a single intramuscular injection of recombinant adeno-associated virus carrying EpoR76E (rAAV2/5.CMV.EpoR76E) would protect retinal ganglion cells in a mouse model of glaucoma without inducing polycythemia. This systemic treatment not only protected the retinal ganglion cell somata located within the retina; it also preserved axonal projections within the optic nerve, while maintaining the hematocrit within normal limits. The rescued retinal ganglion cells retained their visual function demonstrated by flash visual evoked potentials. To our knowledge, this is the first demonstration of a therapy that protects neurons from death and prevents loss of visual function from the slow neurodegenerative effects of glaucoma. Because of its broad range of cellular targets, EpoR76E is likely to be successful in treating other neurodegenerative diseases as well.

Molecular Changes and Vision Loss in a Mouse Model of Closed-Globe Blast Trauma

PURPOSE To characterize retinal changes and assess vision after an eye-directed air blast. METHODS Adult C57Bl/6 mice were exposed to a blast directed at one eye. Optical coherence tomography and histology were performed to assess retina and optic nerve integrity. Cell death, oxidative stress, and glial reactivity were examined by immunohistochemistry. Visual changes were measured by ERG recordings and the optokinetic reflex. RESULTS In the outer retina, eye blast caused retinal pigment epithelium vacuoles and rare retinal detachments followed by regional cell death. Labeling for nitrotyrosine and markers of pyroptosis (caspase-1) and necroptosis (receptor-interacting protein kinases-1, -3) increased, primarily in the inner retina, after blast. Caspase-1 labeling was restricted primarily to the starburst amacrine cells. A few degenerating axons were detected at 28 days post blast. Despite a lack of substantial cell death or decreased ERG, there was a deficit in visual acuity after blast. CONCLUSIONS Oxidative stress, neuroinflammation, and cell death became increasingly prevalent, over time post blast suggestive of an ongoing neurodegenerative response. Outer retinal changes either resolved or remained focal. In contrast, inner retinal changes were more robust and spread from focal regions to the entire retina over time post blast. Our model of eye blast trauma causes molecular changes and a decrease in visual acuity within the first month post blast despite a lack of overt eye injury. This subtle response matches the delayed presentation of visual deficits in some blast-exposed Veterans.