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Optimizing Gene Therapies in Large Animal Models of Retinal Degeneration

优化大型视网膜变性动物模型的基因治疗

基本信息

批准号：
8716945
负责人：
Leah Byrne
金额：
$ 5.33万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8716945
关键词：
Address Adverse effects Affect Animal Model Animals Area Behavioral Canis familiaris Cells Clinical Trials Cloning DNA delivery Data Development Disease Electrophysiology (science)Frequencies Future Gene Delivery General Anesthesia Genes Genetic Goals Histology Human Immunohistochemistry Individual Infection Knowledge Leber&apos s amaurosis Libraries Location Macaca mulatta Macular Hole Measures Mediating Modeling Modification Molecular Profiling Monkeys Mus Mutate Mutation Needles Outcome Patients Photoreceptors Population Process RPE65 protein Relative (related person)Retina Retinal Retinal Degeneration Retinal Detachment Retinal Diseases Route Serotyping Structure Structure of retinal pigment epithelium Techniques Technology Testing Therapeutic Training Tropism Variant Viral Vector Vision research Vitreous humor adeno-associated viral vector base behavior test career cell type cellular transduction deep sequencing directed evolution experience fovea centralis gene therapy improved in vivo imaging inherited retinal degeneration intravitreal injection mutant nonhuman primate novel pre-clinical preclinical safety protein expression public health relevance research study retinal damage retinal rods safety study skills subretinal injection success therapeutic DNA transgene expression vector

项目摘要

7. Project Summary/Abstract Photoreceptors (PRs) and retinal pigment epithelium (RPE) are the primary location of mutations underlying many forms of inherited retinal degeneration for which there is currently no treatment. Therefore, a major goal in the field of vision research is to develop improved technology for gene delivery to these cells. This proposal aims to address this problem by creating novel adeno-associated (AAV) vectors capable of targeting PRs and RPE when delivered into the canine vitreous. We have selected the dog as it is a relevant large animal model that shares similar globe volume and retinal structure with humans, as well as naturally- occurring mutations that result in phenotypically similar retinal diseases as those identified in patients. Naturally occurring serotypes of AAVs are unable to penetrate structural barriers of the retina, and therefore therapeutic delivery of DNA to PRs and RPE has required until now that the viral vector be injected into the subretinal space. Subretinal injections entail the passage of a needle through the retina as well as a transient retinal detachment that separates the RPE from the PRs. This microsurgical technique requires general anesthesia, delivers the viral vector to a limited area of the retina/RPE, and has been associated in preclinical safety studies and human clinical trials with retinal thinning under the fovea and the formation of macular holes. Recently, AAV vectors have been optimized for increased targeting of PRs and RPE following intravitreal delivery, which would provide easier, safer and more extensive targeting of the PRs and RPE than vectors delivered via the subretinal route. Successful results have been achieved in mice, however, when tested in large animals these vectors were inefficient due to differences in retinal structure. Based on strong initial results in mice, we will use a similar directed evolution approach to select, from a mixture of highly diverse libraries of mutated AAVs, individual variants with the novel ability to traverse structural barriers and infect the dog outer retina from the vitreous. Resulting vectors will be characterized by packaging GFP and analyzing expression profiles. We will then test whether the genetic features that increase transduction in dogs are also sufficient to improve infection of the non-human primate outer retina. Furthermore, we will use deep sequencing to analyze rounds of selection, in order to elucidate at which steps diversity is reduced and to determine if the final outcome of directed evolution can be predicted from the initial rounds. The identified novel variants will be used to test corrective gene therapies in the rcd1 dog, a model of PR disease caused by a mutation in the rod-specific PDE6¿ gene, as well as in the RPE65 mutant dog, a model of Leber's congenital amaurosis due to a genetic defect in the RPE. We will then measure whether intravitreal injections provide equivalent or better rescue compared to subretinal injections by evaluating retinal structure and function. Together, these experiments will provide a diverse training experience and valuable preclinical data for novel gene therapy treatments in the retina.

7. 项目总结/文摘