Achromatopsia - Disease Mechanisms and Cone-Directed Gene Therapy

描述(申请人提供)：视锥感光细胞负责中枢视力、色觉和明视，因此对人类日常生活中的视觉表现至关重要。因此，在设计视网膜疾病的治疗方法时，如色盲、视锥细胞营养不良和主要影响视锥细胞的视锥视杆细胞营养不良，靶向视锥细胞至关重要。然而，对于视锥细胞受到继发性影响的疾病，例如视网膜色素变性(RP)和大多数形式的老年性黄斑变性(AMD)，视锥细胞抢救更加重要，AMD是65岁以上人群视力丧失的主要原因。该方案的主要目的是建立两种自然犬色觉障碍模型，作为重组腺相关病毒(RAAV)介导的视锥导向基因治疗的平台。在这两种犬模型中，锥体环核苷酸门控通道β亚基(CNGB3)功能丧失突变导致与人类色盲相同的疾病表型。CNGB3基因突变是人类色觉障碍的最常见原因，使这两个犬种成为进行具有翻译潜力的视锥靶向基因替换研究的最佳动物模型。RAAV介导的视网膜基因治疗视网膜色素上皮(RPE)原发缺陷的长期成功已在包括狗在内的几个物种中得到证实。由于它们成功、稳定和明显安全的转基因表达，这些研究现在处于第一阶段临床试验。与RPE缺陷相比，原发性光感受器疾病的治疗更加困难，之前的基因治疗研究已经显示出不同的成功。在这项建议中要检验的假设是，在锥体特异性启动子的控制下，通过rAAV介导的锥体定向递送野生型CNGB3基因，可以恢复锥体的功能和结构，并防止退化。为了验证这一假设，我们特别提出了1)优化rAAV载体以实现靶向基因在两种犬模型中的锥体表达，以及2)在两种犬模型中最大限度地保持CNGB3基因锥体特异性表达后的视锥功能。前两个目标的结果将提供有关治疗的有效性、安全性和局限性的数据。在最终目标中，将对犬CNGB3突变的功能、结构和分子疾病相关性进行表征，并评估其在成功的基因治疗后的潜在逆转，以便更全面地了解疾病机制，并为建立最佳治疗时间窗口提供疾病指标。犬色盲模型为视锥导向基因治疗的原理验证和最终移植给患者提供了独特的机会。 与公共健康相关：视网膜中的视锥感光细胞负责日光和色觉。这些视锥细胞在许多视网膜疾病中丢失，导致严重的视力障碍，甚至失明。这项研究计划集中于开发一种新的基因疗法，以恢复患病的视锥细胞及其功能，并恢复白天视力。

DESCRIPTION (provided by applicant): Cone photoreceptors are responsible for central visual acuity, color vision, and photopic vision and are therefore critical for visual performance in daily human life. As such, it is of utmost importance to target cones when designing therapeutic treatment for retinal diseases such as achromatopsia, cone dystrophies, and cone-rod dystrophies that primarily affect cones. However, cone rescue is of even greater importance in diseases where cones are affected secondarily, such as in retinitis pigmentosa (RP) and most forms of age-related macular degeneration (AMD), the leading cause of vision loss in people over 65 years of age. The overarching aim of this proposal is to develop 2 natural canine achromatopsia models as a platform for recombinant adeno-associated virus (rAAV)-mediated cone-directed gene therapy. In both canine models, loss-of-function mutations in the cone cyclic nucleotide-gated channel beta subunit (CNGB3) lead to a disease phenotype identical to human achromatopsia. Mutations in CNGB3 are the most common cause for achromatopsia in man, making the 2 canine strains the optimal animal model in which to carry out cone-targeted gene replacement studies with translational potential. The long-term success of rAAV-mediated retinal gene therapy for primary defects of the retinal pigment epithelium (RPE) has been demonstrated in several species, including dogs. Because of their successful, stable, and apparently safe transgene expression, these studies are now in Phase 1 clinical trials. In contrast to RPE defects, the treatment of primary photoreceptor diseases is more difficult, and previous gene therapy studies have shown variable success. The hypothesis to be tested in this proposal is that cone function and structure can be restored, and degeneration prevented, using rAAV-mediated cone-directed delivery of wildtype CNGB3 cDNA under control of cone-specific promoters. To test this hypothesis, we specifically propose to 1) optimize rAAV vectors for targeted gene expression in cones and 2) maximize preservation of cone function following cone-specific expression of CNGB3 cDNA in the two canine models. Results from the first two aims will provide data regarding the efficiency, safety, and limitations of the treatment. In the final aim, functional, structural, and molecular disease correlates of the canine CNGB3 mutations will be characterized and their potential reversal following successful gene therapy assessed in order to more fully understand disease mechanisms and to provide disease metrics for establishing an optimal therapeutic time window. The canine achromatopsia models offer unique opportunities for proof-of-principle of cone-directed gene therapy and for eventual translation to patients. PUBLIC HEALTH RELEVANCE: The cone photoreceptors in the retina are responsible for day- and color vision. These cones are lost in many diseases of the retina which lead to severe vision deficit and even blindness. This research proposal focuses on the development of a new gene therapy to recover diseased cones and their function and to restore day-vision