Development of an Usher Gene Therapy

总结 USH 2A基因突变是Usher综合征II型（USH 2）的原因，其特征在于 视网膜色素变性（RP）和听力障碍。USH 2是联合失明的主要遗传原因 和耳聋。它影响了全世界约40万名受试者。USH 2A没有治愈或治疗方法- 相关的光感受器变性。最常见的USH 2A突变位于框内外显子 13.尽管临床观察表明， 外显子13跳跃可能是致病的。使用CRISPR/Cas9机制的基因修复代表了另一种 纠正外显子13突变的方法尽管在富集校正的细胞后离体高效， 目前还不知道可能25%的细胞的原位同源定向修复是否足够。基因转移 载体通过其转导速率控制其功效。它解决了所有致病突变的整体内， USH 2A基因（>470个突变，NHLBI/NIH外显子组测序计划）。USH 2A编码cDNA是 长度为15.6kb。它不能被常规的腺相关病毒（AAV）和慢病毒所容纳 载体，其有效载荷分别限制为5 kb和10 kb。我们开发了一个完全删除的 腺病毒载体（GreGT），其避免了辅助病毒和复制能力的污染 腺病毒（RCA）。由于有效载荷高达33 kb，我们假设它将有效地提供全长 USH 2A编码的cDNA与其光感受器靶点结合。我们期望这些研究将建立GreGT载体 作为治疗其他大基因缺陷的平台。我们的应用程序建立在 UD Staerz（Greffex，Inc.），作为一名经验丰富的免疫学家和分子生物学家， 生物学家指导了不同载体系统的开发，杨军（John A Moran眼科中心， 犹他州大学），他在视网膜变性领域拥有独特的专业知识。

SUMMARY Mutations in the USH2A gene are the cause of the Usher syndrome type II (USH2) that is characterized by retinitis pigmentosa (RP) and hearing impairments. USH2 is the leading genetic cause of combined blindness and deafness. It affects about 400,000 subjects worldwide. There is no cure or therapy for USH2A- associated photoreceptor degeneration. The most common USH2A mutations are located in in-frame exon 13. Skipping of exon 13 has been investigated as therapy, even though a clinical observation suggested that exon 13 skipping may be pathogenic. Gene repair using the CRISPR/Cas9 machinery represents another approach to correct exon 13 mutations. Although highly efficient ex vivo after enrichment for corrected cells, it is not known whether the in situ homology-directed repair of possibly 25% of cells will suffice. A gene transfer vector controls its efficacy by its transduction rate. It addresses all pathogenic mutations within the entirety of the USH2A gene (>470 mutations, NHLBI/NIH Exome Sequencing Project). The USH2A coding cDNA is 15.6kb in length. It cannot be accommodated by conventional adeno-associated virus (AAV) and lentivirus vectors, whose payloads are limited to 5kb and 10kb, respectively. We have developed a fully deleted adenoviral vector (GreGT) that avoids contaminations with helper viruses and replication competent adenoviruses (RCA). With a payload of up to 33kb, we hypothesize that it will efficiently deliver a full-length USH2A coding cDNA to its photoreceptor targets. We expect that these studies will establish the GreGT vector as platform for the treatment of other defects afflicting large genes. Our application builds upon a collaboration between UD Staerz (Greffex, Inc.), who as an experienced immunologist and molecular biologist has guided the development of different vector systems, and Jun Yang (John A Moran Eye Center, University of Utah), who has developed a unique expertise in the area of retinal degeneration.