Promising Intravitreal Retina-targeting and Immune-evading Adeno-associated Virus Capsid Variant: Preliminary Study

Introduction/objective Retinal diseases are an important focus of in vivo gene therapy, for which adeno-associated virus (AAV) is a preferred vector. Intravitreal injections are a highly desirable delivery route because it is simpler, safer, and less expensive than the main alternative, subretinal injection. However, existing capsids have low efficiency, require high doses, and are susceptible to neutralization by antibodies that are present in the vitreous. Methods We developed a novel AAV capsid with improved properties to address these issues. Using AAV5 as starting material, we applied a combination of directed evolution and rational design to develop PT1, a novel capsid capable of transducing the retina after intravitreal injection. Results Compared to its parent capsid AAV5, PT1 has 15 surface amino acid substitutions in five variable regions and has its VP1-unique region swapped with that of AAV8. Its production yield is similar to that of another AAV5-derived control capsid and superior to that of AAV2. PT1 could transduce the retina-derived cell line ARPE-19 as efficiently as control retinal capsids and with better specificity than AAV2. PT1 performed exceptionally well in an in vitro immune evasion assay, largely surpassing control capsids. In vivo transduction following intravitreal injection in mice suggested strong specificity for Müller glia cells. Discussion PT1’s most striking property is its ability to evade preexisting neutralizing antibodies (NABs), apparently thanks to its multiple mutations spread over large areas of the capsid surface, disrupting several epitopes. Another interesting property is its apparent specificity for Müller glial cells, an important therapeutic target. Both of these properties are based on limited evidence and will require further experimental confirmation. Conclusion The results of our preliminary study suggest that PT1 could become a useful addition to the current retinal AAV capsid toolkit, should future studies confirm our findings.