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

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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.