A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR.

Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs.

Antibody Binding to Recombinant Adeno Associated Virus Monitored by Charge Detection Mass Spectrometry.

Recombinant adeno-associated virus (rAAV) is a leading gene therapy vector. However, neutralizing antibodies reduce its efficacy. Traditional methods used to investigate antibody binding provide limited information. Here, charge detection mass spectrometry (CD-MS) was used to investigate the binding of monoclonal antibody ADK8 to AAV serotype 8 (AAV8). CD-MS provides a label-free approach to antibody binding. Individual binding events can be monitored as each event is indicated by a shift of the antibody-antigen complex to a higher mass. Unlike other methods, the CD-MS approach reveals the distribution of antibodies bound on capsids, allowing AAV8 subpopulations with different affinities to be identified. The charge state generated by the electrospray of large ions is normally correlated with the structure, and the charge is expected to increase when an antibody binds to the capsid exterior. Surprisingly, binding of the first ADK8 to AAV8 causes a substantial decrease in the charge, suggesting that the first antibody binding event causes a significant structural change. The charge increases for subsequent binding events. Finally, high ADK8 concentrations cause agglutination, where ADK8 links AAV capsids to form dimers and higher order multimers.