Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Restoration of Vision and Retinal Responses After Adeno-Associated Virus-Mediated Optogenetic Therapy in Blind Dogs.

Purpose: Optogenetic gene therapy to render remaining retinal cells light-sensitive in end-stage retinal degeneration is a promising strategy for treatment of individuals blind because of a variety of different inherited retinal degenerations. The clinical trials currently in progress focus on delivery of optogenetic genes to ganglion cells. Delivery of optogenetic molecules to cells in the outer neural retina is predicted to be even more advantageous because it harnesses more of the retinal circuitry. However, this approach has not yet been tested in large animal models. For this reason, we evaluated the safety and efficacy of optogenetic therapy targeting remaining diseased cone photoreceptors in the Rcd1 dog model of retinitis pigmentosa. Methods: Imaging and measures of retinal function and functional vision were carried out, as well as terminal studies evaluating multi-electrode array recordings and histology. Results: Animals remained healthy and active throughout the study and showed improved retinal and visual function as assessed by electroretinography and visual-evoked potentials, improved navigational vision, and improved function of cone photoreceptors and the downstream retinal circuitry. Conclusions: The findings demonstrate that an optogenetic approach targeting the outer retina in a blind large animal model can partially restore vision. Translational Relevance: This work has translational relevance because the approach could potentially be extrapolated to treat humans who are totally blind because of retinal degenerative disease.

Treatment Potential for LCA5-Associated Leber Congenital Amaurosis.

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

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.

Safety of Same-Eye Subretinal Sequential Readministration of AAV2-hRPE65v2 in Non-human Primates.

We have demonstrated safe and effective subretinal readministration of recombinant adeno-associated virus serotype (rAAV) to the contralateral eye in large animals and humans even in the setting of preexisting neutralizing antibodies (NAbs). Readministration of AAV to the same retina may be desirable in order to treat additional areas of the retina not targeted initially or to boost transgene expression levels at a later time point. To better understand the immune and structural consequences of subretinal rAAV readministration to the same eye, we administered bilateral subretinal injections of rAAV2-hRPE65v2 to three unaffected non-human primates (NHPs) and repeated the injections in those same eyes 2 months later. Ophthalmic exams and retinal imaging were performed after the first and second injections. Peripheral blood monocytes, serum, and intraocular fluids were collected at baseline and post-injection time points to characterize the cellular and humoral immune responses. Histopathologic and immunohistochemical studies were carried out on the treated retinas. Ipsilateral readministration of AAV2-hRPE65v2 in NHPs did not threaten the ocular or systemic health through the time span of the study. The repeat injections were immunologically and structurally well tolerated, even in the setting of preexisting serum NAbs. Localized structural abnormalities confined to the outer retina and retinal pigmented epithelium (RPE) after readministration of the treatment do not differ from those observed after single or contralateral administration of an AAV carrying a non-therapeutic transgene in NHPs and were not observed in a patient treated with the nearly identical, FDA-approved, AAV2-hRPE65v2 vector (voretigene neparvovec-rzyl), suggesting NHP-specific abnormalities.

Evaluation of Dose and Safety of AAV7m8 and AAV8BP2 in the Non-Human Primate Retina.

Within the next decade, we will see many gene therapy clinical trials for eye diseases, which may lead to treatments for thousands of visually impaired people around the world. To target retinal diseases that affect specific cell types, several recombinant adeno-associated virus (AAV) serotypes have been generated and used successfully in preclinical mouse studies. Because there are numerous anatomic and physiologic differences between the eyes of mice and "men" and because surgical delivery approaches and immunologic responses also differ between these species, this study evaluated the transduction characteristics of two promising new serotypes, AAV7m8 and AAV8BP2, in the retinas of animals that are most similar to those of humans: non-human primates (NHPs). We report that while AAV7m8 efficiently targets a variety of cell types by subretinal injection in NHPs, transduction after intravitreal delivery was mostly restricted to the inner retina at lower doses that did not induce an immune response. AAV8BP2 targets the cone photoreceptors efficiently but bipolar cells inefficiently by subretinal injection. Additionally, transduction by both serotypes in the anterior chamber of the eye and the optic pathway of the brain was observed post-intravitreal delivery. Finally, we assessed immunogenicity, keeping in mind that these AAV capsids may be used in future clinical trials. We found that AAV8BP2 had a better safety profile compared with AAV7m8, even at the highest doses administered. These studies underscore the differences in AAV transduction between mice and primates, highlighting the importance of careful evaluation of therapeutic vectors in NHPs prior to moving to clinical trials.

Nonhuman primate models for diabetic ocular neovascularization using AAV2-mediated overexpression of vascular endothelial growth factor.

Neovascularization leads to blindness in numerous ocular diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and sickle cell disease. More effective and stable treatments for ocular neovascularization are needed, yet there are major limitations in the present animal models. To develop primate models of diabetic retinopathy and choroidal neovascularization, rhesus monkeys were injected subretinally or intravitreally with an adeno-associated virus (AAV)-2 vector carrying the cDNA encoding human vascular endothelial growth factor (VEGF). Overexpression of VEGF was measured by intraocular fluid sampling over time. Neovascularization was evaluated by ophthalmoscopy through angiography, optical coherence tomography, and ultimately histopathology. Overexpression of VEGF through AAV2 results in rapid development of features of diabetic retinopathy or macular edema, depending on the targeted cell type/mode of production of VEGF and diffusion of VEGF. Nonhuman primate models will be useful in testing long-term safety and efficacy of novel therapeutic agents for blinding neovascular diseases.

Spatial and temporal expression patterns of the choroideremia gene in the mouse retina.

PURPOSE: Choroideremia (CHM), an X-linked retinal disease, is caused by mutations affecting the CHM gene. This gene encodes REP-1, which functions in the covalent modifications of proteins involved in vesicle trafficking. The disease affects several cell types in the retina, but it is not known which cell types contribute directly or indirectly to disease progression. A study of the expression patterns of Chm and the related gene Chml in the mouse retina was undertaken in order to address this issue. METHODS: The expression patterns of Chm and Chml were determined by in situ hybridization. The localization of the Chm protein product, Rep-1, was determined spatially and temporally in the mouse retina by immunohistochemistry. RESULTS: Chm and Chml mRNA were found in every major layer of the retina in adult mice. During development, Rep-1 protein localization changes from a fairly diffuse pattern during embryogenesis to a more specific pattern at the time of retinal differentiation. In adulthood, Rep-1 localizes to distinct cellular compartments in multiple retinal cell types. CONCLUSIONS: Chm and Chml have the same broad expression profile in the mouse retina. In particular, the Chm transcript and corresponding protein are found in cell types other than those thought to be primarily affected in the human disease. These results have important implications for approaches with which to develop a relevant mouse model of choroideremia and for therapeutic strategies for this disease.

[Retrospective investigation on endemic situation of schistosomiasis in Hubei Province].

OBJECTIVE: To explore the changing rules of schistosomiasis infection and Oncomelania hupensis snail situation in areas where schistosomiasis transmission was controlled or interrupted or transmission rebounded after the interruption. METHODS: Daye City, Wuxue City and Jingshan County were selected and investigated retrospectively to collect the schistosomiasis epidemiological information 10 years before they reached the criteria of transmission controlled and the subsequent years until 2008. The database of retrospective investigation was established for analyzing the trends and rules of the changes of snail status and human infection status. RESULTS: In Daye City, there was no snail areas and schistosomiasis patients and cattle after reaching the criteria of transmission interrupted in 1987. The infection rate of residents were positively correlative with the infection rate of cattle and snail areas (r = 0.865, 0.843, P < 0.01). In Wuxue City, the proportion of snail areas occupying the historic snail areas kept a steady from the transmission controlled to transmission interrupted, but it began to rebound to the highest level in the history (8.93%) after the transmission interrupted for 3 years. In Jingshan County, the infection rates of residents and cattle were all stable from 1% to 2% after the transmission controlled. The proportion of snail areas occupying the historic snail areas was rebounded from 1.63% in 1985 to 21.50% in 2008, and the densities of living snails rebounded from 2005. CONCLUSIONS: The infected snails could be the sensitive indicator of the rebound of snail situation and human infection. The elimination of schistosomiasis still depends on the control of snail areas, including strengthening the infected snail control.

[Causes of re-emergence of Oncomelania snails in hardened ditches].

OBJECTIVE: To understand the impact factors of the re-emergence of Oncomelania snails in hardened ditches so as to find out the measures to improve the effect of snail eradication. METHODS: Thirteen infected townships from Jingmen City were randomly selected and investigated with the retrospective method and the field survey. All the data were analyzed for evaluating the effect of the hardened ditches on snail eradication and finding out the causes of the re-emergence of snails in hardened ditches RESULTS: After on-site investigation of 151 hardened ditches, the eradication rate of snail areas was 78.79%, and 71 ditches were founded with snails and the re-emergence rate was 47.02%. The major factors of the snail re-emergence were the spread of the snails from the upstream, the dilapidation of the hardened ditches, no using molluscicides before the hardened ditches, and the snails being brought by the engineering construction. CONCLUSIONS: The only hardened ditches can not eradicate the snails. We should manage an overall plan when processing the ditches hardening project, implementation with molluscicides before the project and strengthen the maintenance of the hardened ditches.

Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness.

Leber's congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65(-/-)) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4(+) T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials.

Accelerated mortality from hydrocephalus and pneumonia in mice with a combined deficiency of SPAG6 and SPAG16L reveals a functional interrelationship between the two central apparatus proteins.

SPAG6 and SPAG16L are proteins localized to the "9+2" axoneme central apparatus. Both are essential for sperm motility and male fertility. These two proteins are also expressed in other tissues containing ciliated cells, such as brain and lung. To study the effects of combined deficiency of these two proteins, a double mutant mouse model was created. The double mutant mice displayed a more profound phenotype of growth retardation and hydrocephalus compared to mice nullizygous for SPAG6 and SPAG16L alone. The double mutant mice died younger, and mortality was significantly higher than in single mutant mice. In addition, the double mutant mice demonstrated pneumonia and its complications, including hemorrhage, edema, and atelectasis, phenotypes not observed in mice nullizygous for mutations in the individual genes. No other cilia-related phenotypic change was detected in double mutant mice including lateralization defects. The ultrastructure of cilia in both the brain and lung of the double mutant mice appeared normal. This model of combined SPAG6 and SPAG16L deficiency provides a new platform to study primary ciliary dyskinesia. The findings also demonstrate that SPAG6 and SPAG16L have related roles in controlling the function of cilia in the brain and lung.

Deficiency of SPAG16L causes male infertility associated with impaired sperm motility.

The axonemes of cilia and flagella contain a "9+2" structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of M(r) approximately 71 x 10(3) (SPAG16L) and M(r) approximately 35 x 10(3) (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys-symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.

Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16.

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Heterogeneity of claudin expression by alveolar epithelial cells.

Claudins are proteins that participate in epithelial barrier function and regulate paracellular permeability. By immunohistochemistry of adult rat lung sections, claudin-3, claudin-4, and claudin-5 were found to be co-expressed by type II alveolar epithelial cells. Claudin-3 and claudin-4 were also co-expressed by some alveolar epithelial cells adjacent to type II cells. In contrast, claudin-5 was expressed throughout the alveolus. Isolated primary rat alveolar epithelial cells in culture also expressed claudin-3, claudin-4, and claudin-5, but showed little claudin-1 and claudin-2 expression. Claudin expression by isolated cells at both the mRNA and protein level varied with time in culture. In particular, claudin-3 and claudin-5 co-localized and were distributed around the alveolar cell periphery, but claudin-4 expression was heterogeneous. We also found that paracellular permeability was increased when cultured alveolar epithelial cells were treated with a fatty acid amide, methanandamide. Methanandamide did not alter cell viability. Claudin-3, claudin-4, claudin-5, occludin, and zona occludens 1 remained localized to cell-cell contact sites at the plasma membrane in methanandamide-treated cells, suggesting that plasma membrane localization of these junction proteins is not sufficient for maintaining barrier function. However, methanandamide-treated cells showed a 12-fold increase in claudin-5 expression and a 2- to 3-fold increase in claudin-3, consistent with the notion that specific changes in claudin expression levels may correlate with changes in alveolar epithelial barrier function.

In utero gene therapy rescues vision in a murine model of congenital blindness.

The congenital retinal blindness known as Leber congenital amaurosis (LCA) can be caused by mutations in the RPE65 gene. RPE65 plays a critical role in the visual cycle that produces the photosensitive pigment rhodopsin. Recent evidence from human studies of LCA indicates that earlier rather than later intervention may be more likely to restore vision. We determined the impact of in utero delivery of the human RPE65 cDNA to retinal pigment epithelium cells in a murine model of LCA, the Rpe65(-/-) mouse, using a serotype 2 adeno-associated virus packaged within an AAV1 capsid (AAV2/1). Delivery of AAV2/1-CMV-hRPE65 to fetuses (embryonic day 14) resulted in efficient transduction of retinal pigment epithelium, restoration of visual function, and measurable rhodopsin. The results demonstrate AAV-mediated correction of the deficit and suggest that in utero retinal gene delivery may be a useful approach for treating a variety of blinding congenital retinal diseases.