Adeno-Associated Virus (AAV) is widely recognized as one of the safest and most efficient viral vectors for in vivo gene therapy. However, its natural packaging capacity is strictly restricted to approximately 4.7 kilobases (kb). This structural threshold poses a major hurdle for delivering large, high-value therapeutic payloads—such as full-length Duchenne Muscular Dystrophy (DMD), Dysferlin (DYSF), Stargardt disease genes (ABCA4), or bulky genome editing tools like Prime Editors (PE, ~6.3 kb) and TwinPE.
To solve this bottle-neck, Protheragen has scalable, robust dual-vector large-gene delivery platforms. By engineering split transgene systems packaged into two complementary AAV capsids, we achieve precise intracellular full-length gene reconstitution with exceptional efficiency and minimal off-target effects.
Our platform utilizes advanced protein-level and RNA-level reconstitution strategies to deliver large genetic material.
Fig.1 Design strategies for dual-vector large-gene delivery. (Tasfaout H., et al., 2024)
Intein-Based Split AAV
The coding sequence of a large protein (or base/prime editor) is split into N-terminal and C-terminal fragments, each fused with a engineered split-intein domain and packaged into separate AAV vectors.
Reconstitution
Upon co-infection of the target cell, the two halves are expressed and spontaneously associate via split-intein non-covalent interactions. A fast, traceless trans-splicing reaction auto-catalyzes, yielding the intact functional protein while splicing out the intein moieties.
Trans-Splicing Systems
Uses engineered pre-mRNA splicing signals (SD/SA) to join split transcript fragments at the pre-mRNA level inside the cell nucleus.
Overlapping / HR Dual-AAV Systems
Utilizes homologous DNA regions in both vectors to drive cell-mediated homologous recombination, yielding full-length transcription units.
Protheragen offers end-to-end, customized development services to help transform high-potential gene candidates into viable AAV therapeutic products.
Our dual-vector large-gene delivery technology unlocks pipeline possibilities for formerly "undruggable" monogenic and complex genetic diseases:
At Protheragen, we combine deep expertise in split-intein engineering with an extensive tissue-specific AAV serotype library to overcome the long-standing payload constraints of conventional AAV vectors. Backed by end-to-end CRO/CDMO capabilities—spanning custom construct optimization and high-titer dual-vector production to comprehensive translational animal testing—we deliver reliable, scalable dual-AAV solutions designed to accelerate your large-gene therapy pipeline from initial design through preclinical proof-of-concept.
Talk to our AAV delivery scientists today to custom-design your dual-vector system or request a quote for high-titer dual-AAV packaging.
Reference
Our AAVLinkTM platform is at the forefront of adeno-associated viral (AAV) vector development, leveraging our extensive expertise and state-of-the-art technologies to drive innovation in gene therapy.
