AAVLINK: Potent DNA-recombination method for large cargo delivery in gene therapy

Delivery of therapeutic genes is essential for gene therapy. Adeno-associated viruses (AAVs) are a prime vector for carrying gene cargoes because of their superior gene segmentation flexibility and robust gene reconstitution efficiency. However, their limited packaging capacity is a major challenge for large gene transduction.

In a study published in Cell on Jan. 27, Prof. LU Zhonghua's team from the Shenzhen Institutes of Advanced Technology of the Chinese Academy of Sciences, and collaborators from Peking University First Hospital, developed the AAV with translocation LINKage (AAVLINK), which harnesses Cre/lox-mediated intermolecular DNA recombination to enable in vivo reassembly of large genes.

AAVLINK allows flexible gene segmentation design, achieves high-efficiency full-length gene reconstitution, and markedly reduces the production of aberrant truncated proteins compared with conventional approaches.

Using animal models, the researchers found that AAVLINK enabled robust expression of full-length Shank3 and significantly rescued autism-like behavioral phenotypes in Shank3-deficient mice, and the delivery of the large epilepsy-associated gene SCN1A using AAVLINK restored gene expression and alleviated seizure phenotypes in mutant mice. The findings provided strong evidence that AAVLINK supported functional delivery of large therapeutic genes in the nervous system.

Furthermore, the researchers introduced a destabilized Cre recombinase, and developed AAVLINK 2.0. This design allowed tighter temporal control of the recombination activity, reducing potential safety concerns while preserving high gene reconstitution efficiency.

Utilizing AAVLINK strategy, the researchers constructed a vector bank covering 193 large genes associated with inherited disorders including autism and epilepsy. They validated the gene reconstitution capacity of all constructs. Also, this vector bank comprised five CRISPR-based genetic tools, offering insights into the wide application of AAVLINK.

The study proposes a strategy to facilitate the delivery of large gene cargoes by using AAVs, which provides therapeutic possibilities for diseases that have been considered inaccessible to AAV-based gene therapy.