In a recent publication by Elvir Becirovic’s team at the Laboratory for Retinal Gene Therapy, University Hospital Zurich, Switzerland, a groundbreaking technology named REVeRT is introduced. This dual AAV vector system relies on mRNA trans-splicing, allowing the functional reconstitution of large genes or CRISPR modules that exceed the AAV capacity.

The conventional Adeno-associated virus (AAV)-mediated gene transfer, widely used for in vivo gene delivery, faces limitations due to its restricted genome packaging capacity, hampering the expression of substantial genes like CRISPR-Cas9 modules. Various strategies have been developed using dual AAV vectors, involving gene splitting and reconstitution at different levels upon co-delivery of dual AAVs. Nevertheless, the existing DNA trans-splicing method, while efficient, presents limitations such as potential immunogenicity.
To overcome these challenges, a novel approach involving mRNA trans-splicing AAVs has been introduced through the REVeRT (Reporter Gene Reconstitution via Trans-splicing) technology. This technology has been meticulously developed and optimized for reporter gene reconstitution in vitro. It utilizes a split-fluorophore assay, resulting in cerulean expression upon the successful reconstitution of two split fragments. A specific split site derived from splice site consensus sequences is employed, and reconstitution takes place via mRNA trans-splicing. This process incorporates various elements, including promoters, complementary binding domains, splice sites, and polyadenylation signals. The identification and utilization of optimal binding domains, splice acceptor sites, and specific sequences for split sites ensure highly efficient reconstitution with minimal off-target effects.

The functional performance of REVeRT is validated in a dual AAV vector setup in vivo, particularly in the retina, a common target for many AAV gene therapies. Injection of split fluorophore variants into the eyes of mice using AAV8(Y733F) vectors results in robust and specific fluorophore expression in photoreceptors and retinal pigment epithelial cells. Additionally, a split luciferase reporter system is developed, showcasing high reconstitution efficiency in various organs after intraperitoneal application of dual AAV9 vectors, with the heart exhibiting the most significant signal.

The translational potential of dual REVeRT AAVs is explored further in human retinal organoids. These organoids demonstrate a reconstituted luciferase signal at 20.2% of control organoids transduced with single AAVs expressing full-length luciferase. This exemplifies the efficacy of dual REVeRT AAVs in highly differentiated human tissue cultures. In a therapeutically relevant context, the technology is applied to “dead” (d)Cas9 fused to VP64, p65, and Rta (VPR), a potent CRISPR activation (CRISPRa) module. Successful activation of the Myo7b gene is observed in mouse cells and various organs after different routes of administration, indicating REVeRT’s potential for functional gene activation in diverse applications.

Moreover, REVeRT showcases the functional reconstitution of prime editors (PEs) in vitro, efficiently editing both mouse and human genomic loci with comparable efficiency to full-length PE2. The technology also enables the simultaneous knockout and activation of different genes in vivo, a strategy referred to as CONNACT, proving effective and demonstrating potential therapeutic applications.

In a study targeting Stargardt disease using a mouse model, dual REVeRT AAV vectors are used to deliver full-length ABCA4 into the retinas of affected mice. This study reveals a potential therapeutic approach for this inherited macular degeneration, which currently lacks effective treatment. Intravitreal injection of these vectors leads to the expression and proper localization of the ABCA4 protein. The efficiency of reconstitution ranges from 34.0% to 64.3%, depending on the promoter used, indicating the potential of dual REVeRT AAVs for addressing genetic diseases associated with large genes.


The REVeRT technology represents a significant advancement, allowing the functional reconstitution of large genes or CRISPR modules that surpass the AAV’s packaging capacity. It offers efficiency comparable to widely-used dual AAV systems while providing advantages such as flexible split site selection, independence from protein folding for reconstitution, and a reduced risk of immune responses. REVeRT demonstrates promise in various applications, from gene activation to multiplexing for concurrent gene manipulation, and particularly in gene supplementation therapy, as demonstrated in the context of Stargardt disease.

This breakthrough finding can help you overcome the gene length limit of AAV-based gene delivery. If you are interested in this approach, we can help! At PackGene, we offer RUO to GMP AAV vector design and packaging, mRNA, and plasmid services to meet your research and clinical experiment needs. See more information

Riedmayr, L.M., Hinrichsmeyer, K.S., Thalhammer, S.B. et al. mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy. Nat Commun 14, 6578 (2023).
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