Intein Proteins Split-Vector Gene Therapy

The patent introduces intein-mediated protein splicing as an intermediary mechanism to resolve the contradiction between cargo capacity and expression efficiency. By using inteins as self-splicing elements that facilitate the joining of protein fragments from multiple AAV vectors, the system achieves efficient reconstitution of full-length large proteins without relying on inefficient DNA concatemer formation or mRNA processing. The intein acts as a molecular mediator that enables reliable protein expression from segmented genetic material.

The patent replaces the mechanical system of DNA-level operations (concatemer formation, transcription of large multi-vector mRNAs) with a protein-level operation (intein-mediated splicing). Instead of relying on stable DNA concatemers and long mRNA transcripts, the system uses inteins to directly join protein fragments after translation, bypassing the instability issues inherent in multi-vector DNA and RNA systems.

The patent applies segmentation by dividing the coding sequence of large genes into multiple fragments, each fitting within the 5 kb capacity of a single AAV vector. These segmented coding sequences are distributed across multiple vectors, which are then co-delivered to target cells. The intein-mediated splicing system reconstructs the full-length functional protein from these segmented fragments, effectively overcoming the single-vector size limitation while maintaining expression efficiency.