EFS-LbCpf1 AAV (Serotype 9)

Adeno-associated viruses (AAV) are small (25 nm in diameter), non-enveloped viruses belonging to the genus Dependovirus in the family Parvoviridae with an icosahedral structure. They are single-stranded DNA viruses with a carrying capacity of approximately 4.7 kilobases and their infectious life cycle is dependent on a helper virus, such as adenovirus, herpes virus, or papillomavirus. Recombinant AAV vectors have low immunogenicity and an excellent safety profile and provide long-term therapeutic gene expression, which is important for the clinical application of gene therapy. Not surprisingly, AAV is currently the vector of choice and has been successfully used to treat hemophilia and retinal degeneration.

AAV has two open reading frames (ORFs) consisting of the Rep and Cap genes. The Rep gene encodes four different replication proteins (Rep78, Rep 68, Rep 52, and Rep 40), and the Cap gene encodes three AAV8 capsid proteins (VP) (VP1, VP2, and VP3), which are translated from different start codons of the same open reading frame (ORF). AAP has also been identified and promotes AAV packaging. All three VPs are expressed from the same ORF but are generated by alternative splicing, maintaining a common C-terminal domain. VP1 contains an additional N-terminal sequence compared to VP2, which contains an additional amino acid sequence at its N-terminus relative to VP3. The capsid proteins of AAV assemble to form T1 icosahedral virions, with VP1, VP2, and VP3 in a 1:1:10 ratio, containing a total of sixty units. The structure of the intact capsid was studied using cryo-electron microscopy, X-ray crystallography, and image reconstruction, revealing that the N-terminal regions of VP1 and VP2 fold within the capsid structure, thereby blocking the binding activity of the N-terminal regions of VP1 and VP2. The common C-terminal VP3 region (~534 aa) determines the receptor binding of the virus.