Abstract 1739 Generation of AAV9 Gene Therapy Capsid Variants Capable of Evading Neutralizing anti-AAV9 Antibodies derived from Patients Post-Zolgensma® Treatment

Adeno-associated viruses (AAVs) are extensively studied as promising vectors for gene therapy applications. Following the FDA approval of Zolgensma® (an AAV9 based biologic used to treat children under the age of two diagnosed with Spinal Muscular Atrophy Type 2), AAV9 has been identified as an attractive vector for central nervous system (CNS) delivery as it can cross the blood-brain barrier. However, a major challenge for AAV gene therapy is that a high proportion of the general population possesses pre-existing neutralizing antibodies (NAbs) against AAV9 capsids, leading to vector inactivation and the exclusion of patients that can enroll in gene therapy trials. One strategy to circumvent this immunity is the generation of capsid variants capable of evading the pre-existing NAbs which requires the characterization of the antigenic regions of the AAV capsids. Towards this end, we recovered 21 unique, monoclonal antibodies (mAbs) from three patients post-Zolgensma® treatment. Using cryo-electron microscopy and 3D image reconstruction, we have determined the structures of these 21 mAbs in complex with the AAV9 capsid at high resolution which allowed the identification of the contact residues of the antibodies on the capsid surface. This information was utilized in our capsid engineering pipeline to generate variants capable of escaping the mAbs. The escape phenotype was analyzed by native immuno-dot-blot followed by neutralization assay experiments. Additionally, the new capsid variants were screened for productivity and for their ability to maintain transduction efficiency. Successful variants were combined to obtain an engineered AAV9 capsid which is not recognized by as many mAbs as possible. Presently, we have developed two AAV9 capsid variants which differ from wild-type AAV9 by six amino acids and are capable of evading 18 out of the 21 patient mAbs. Furthermore, both variants maintained their ability to be purified by AAV9 Capture Select Affinity Ligand® purification and showed improved transduction efficiency compared to the parental AAV9 capsid. These engineered AAV9 variants could expand the patient cohort eligible for AAV9-mediated gene therapies and may even allow the possibility of re-dosing of the patients if the need arises. This project is funded by NIH R01 GM082946.