Development of a Robust DNA Recombination Method for Large Gene Payload Delivery in Gene Therapy

The delivery of therapeutic genes is fundamental to gene therapy. Adeno-associated virus (AAV) has become the predominant vehicle for carrying gene payloads due to its superior flexibility in gene splitting and robust gene reconstruction efficiency. However, its limited packaging capacity remains a significant hurdle for the transduction of larger genes.

Recently, researchers developed AAV with translocation linking (AAVLINK), a technology that utilizes Cre/lox-mediated intermolecular DNA recombination to achieve the in vivo reassembly of large genes. The related research findings were published in the journal Cell.

Figure 1. AAVLINK drove expression of intact Shank3 or SCN1A and rescued behavior and seizure phenotypes of mutant mice, respectively. (Lin J, et al., 2026)

Compared to conventional methods, AAVLINK allows for flexible gene-splitting designs, enables highly efficient full-length gene reconstruction, and significantly minimizes the production of aberrant truncated proteins.

Utilizing animal models, researchers found that AAVLINK facilitates the robust expression of the full-length Shank3 gene and significantly rescues autism-like behavioral phenotypes in Shank3-deficient mice. Similarly, AAVLINK-mediated delivery of SCN1A, a large gene associated with epilepsy, restored gene expression and alleviated seizure phenotypes in mutant mice. These findings provide strong evidence that AAVLINK supports the functional delivery of large therapeutic genes within the nervous system.

Furthermore, the researchers developed AAVLINK 2.0 by integrating a destabilized Cre recombinase. This design offers stricter temporal control over recombination activity, maintaining high gene reconstruction efficiency while reducing potential safety concerns.

Using the AAVLINK strategy, the researchers constructed a vector library covering 193 large genes associated with genetic disorders, including autism and epilepsy, and validated the gene reconstruction capability of all constructs. The library also includes five CRISPR-based genetic tools, demonstrating the broad applicability of the AAVLINK platform.

This study introduces a strategy capable of delivering large gene payloads via AAV, thereby offering therapeutic possibilities for diseases previously considered inaccessible to AAV-based gene therapy.