Development of a Gene Therapy for Usher Syndrome Type 1B (USH1B)

Usher综合征（USH）是一组以常染色体隐性遗传方式遗传的遗传性疾病，其特征为听力损失、前庭功能障碍和视网膜色素变性（RP）。临床上，USH分为三个主要组（USH 1 -3）。USH 1B是由编码非常规肌球蛋白马达蛋白的基因MYO 7A突变引起的。MYO 7A在视杆和视锥光感受器（PR）以及视网膜色素上皮（RPE）中表达。到目前为止，USH 1B患者还没有治疗方法。虽然基因补充方法主要适用于USH 1B，但MYO 7A编码序列（6.6 kb）超过了目前大多数基因治疗研究中使用的重组腺相关病毒（rAAV）载体（约4.7 kb）的负载能力。为了克服这个问题，我们在本发明中利用了一种新的双重rAAV载体方法（REVeRT），其中MYO 7A通过从两个单独的rAAV转录的两个mRNA的反式剪接在Myo 7a缺陷小鼠（Myo 7aretKO）中重组。作为第二种策略，缺失的Myo 7a功能将通过编码与Myo 7a同源但在成人视网膜中不表达的马达蛋白的Myo 7 b基因的转录激活来补偿。为了实现体内基因激活，将使用与转录激活因子融合的无催化活性的dCas 9（dCas 9-VPR）。生物化学、组织学和功能测定将用于监测不同基因治疗方法的治疗结果。最后，将在该联盟的项目SP3中产生的MYO 7A缺陷型视网膜类器官中检查MYO 7A的拯救以及MYO 7 B的反式激活。

Usher syndrome (USH) comprises a group of genetic diseases inherited in an autosomal recessive pattern that are characterized by hearing loss, vestibular dysfunction and retinitis pigmentosa (RP). Clinically, USH is classified into three principal groups (USH1-3). USH1B is caused by mutations in the gene MYO7A encoding an unconventional myosin motor protein. MYO7A is expressed in rod and cone photoreceptors (PR) and in retinal pigment epithelium (RPE). So far, there is no treatment for USH1B patients. While gene supplementation approaches are principally applicable to USH1B, the MYO7A coding sequence (6.6 kb) exceeds the cargo capacity of recombinant adeno-associated virus (rAAV) vectors (about 4.7 kb) that are used in most current gene therapy studies. To overcome this problem, we utilize in the present proposal a novel dual rAAV vector approach (REVeRT) in which MYO7A is recombined in Myo7a-deficient mice (Myo7aretKO) by trans-splicing of two mRNAs that are transcribed from two separate rAAVs. As a second strategy, missing Myo7a function will be compensated by transcriptional activation of the Myo7b gene encoding a motor protein that is homologous to Myo7a but is not expressed in the adult retina. To achieve in vivo gene activation catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) will be used. Biochemical, histological and functional assays will be used to monitor therapeutic outcome of the different gene therapy approaches. Finally, rescue of MYO7A as well as transactivation of MYO7B will be examined in MYO7A-deficient retinal organoids generated in the project SP3 of this consortium.