Novel dual rAAV vectors for (epi)genome editing-based therapy of gain-of-function mutations

重组腺相关病毒载体是基因治疗的黄金标准。然而，许多基因超过了这些载体有限的DNA包装能力。这包括各种CRISPR/CAS模块，它们具有很高的治疗潜力。然而，原则上，这些模块可以分裂，包装在两个单独的rAAV载体中，然后可以使用所谓的双重rAAV载体方法在基因组、mRNA或蛋白质水平上完全重组。基因组或蛋白质水平的重组都是成熟的，但存在一些严重的缺陷，阻碍了它们的翻译潜力。相比之下，通过mRNA反式剪接在mRNA水平上的重建仍然是一种提供关键优势的双重rAAV策略，在很大程度上仍未被探索。在前期工作中，我们建立了一种体外mRNA反式剪接的方法，并证明了相应的双重剪接rAAVs可以在功能上重建小鼠视网膜中的Cas9-VPR模块。该模块可用于单个基因的激活，也可用于同时敲除和激活不同的基因。后一种策略是不依赖于突变的，可以治疗目前无法治疗的功能获得突变，在这种突变中，基因补充必须定期与突变等位基因的敲除相结合。在这项提案中，我们将应用我们独特的mRNA反式剪接技术，在小鼠模型中建立一种新的基因治疗方法，治疗由视紫红质最常见的功能获得突变(Pro23His)引起的常染色体显性遗传性视网膜色素变性。为此，CAS9-VPR模块将被应用于同时敲除视紫红质视紫红质，并激活视紫红质Pro23His小鼠模型视网膜中视锥特定功能的等价物M-视蛋白(Opn1mw)或S-视蛋白(Opn1sw)。后续实验将在分子、形态、功能和行为水平上解决治疗成功的问题。此外，这种方法的翻译潜力将在细胞培养和人类视网膜器官中得到解决。通过结合两种新的方法，该建议解决了基因治疗领域中仍未解决的两个关键问题：i)开发和实验验证了使用双rAAV载体重建大基因的安全和有效的方法；ii)开发和实验评估了目前无法治愈的功能获得突变的基因治疗的新策略。这两种方法都具有很高的翻译潜力，世界各地的许多患者都可以从中受益。

Recombinant adeno-associated viral (rAAV)-vectors are the gold standard for gene therapy. However, many genes exceed the limited DNA packaging capacity of these vectors. This includes various CRISPR/Cas modules which have a high therapeutic potential. In principle, however, these modules can be split, packaged in two separate rAAV vectors and can then be fully reconstituted at genome, mRNA or protein level using a so-called dual rAAV vector approach. Reconstitution at the genome or protein level are both well-established but harbor some important drawbacks hampering their translational potential. In comparison, reconstitution at the mRNA level via mRNA trans-splicing remains a largely unexplored dual rAAV strategy that offers key advantages. In preliminary work we developed an mRNA trans-splicing approach in vitro and show that the corresponding dual splicing rAAVs can functionally reconstitute the Cas9-VPR module in the mouse retina. This module can be used for activation of single genes or for simultaneous knockdown and activation of different genes. The latter strategy is mutation-independent and would allow for therapy of currently non-treatable gain-of-functions mutations where gene supplementation regularly has to be combined with the knockdown of the mutant allele. In this proposal, our unique mRNA trans-splicing technology will be applied to establish a novel gene therapy in the mouse model for autosomal dominant retinitis pigmentosa caused by the most common gain-of-function mutation in rhodopsin (Pro23His). For this purpose, the Cas9-VPR module will be applied to simultaneously knockdown the rod-specific rhodopsin and activate its cone-specific functional equivalents M-opsin (Opn1mw) or S-opsin (Opn1sw) in the retina of the rhodopsin Pro23His mouse model. The follow-up experiments will address the therapy success at the molecular, morphological, functional, and behavioral level. In addition, the translational potential of this approach will be addressed in cell culture and in human retinal organoids. By combining two novel approaches, this proposal thus addresses two still unsolved key problems in the field of gene therapy: i) The development and experimental validation of a safe and efficient method for reconstituting large genes using dual rAAV vectors ii) The development and experimental evaluation of a new strategy for gene therapy of currently untreatable gain-of-function mutations. Both approaches have a high translation potential from which many patients worldwide could benefit.