Poster O14 - Characterization of adeno-associated virus interactions with alginate

Introduction: Adeno-associated viruses (AAVs) are non-enveloped viruses often used in gene therapy for their ability to deliver DNA. To be extend therapeutically relevant dosages, encapsulation has been explored. One potential candidate for encapsulating AAV for later release is alginate hydrogels. Alginate is an anionic polysaccharide derived from brown algae. Initially, AAV was loaded into the alginate hydrogel and released over time was characterized. However, during release testing it was noticed less AAV than predicted released from hydrogels. Alginate has a similar structure to heparin, a glycosaminoglycan often used as a blood anticoagulant and known to bind to certain AAV serotypes. It was hypothesized that interactions similar to AAV-heparin binding were occurring between alginate and AAV. The serotypes chosen to test were AAV1, known to not bind to heparin, AAV2, known to bind to heparin, and AAV8 and AAV9 whose ability to bind heparin had not been as thoroughly studied. Our goal in this research is to understand alginate-AAV interactions and to see if they are similar to that of heparin-AAV binding.

Materials and

Methods: Atomic force microscopy (AFM) was used to explore the interactions between AAV and alginate and heparin. Either biotinylated heparin or alginate were coated onto the surface of a functionalized glass surface with streptavidin on the surface. AAV was attached to the surface of an AFM cantilever (MLCT-Bio-DC: Bruker Nano) via heterobifunctional PEG linkers. Four stereotypes of AAV were used: AAV1, AAV2, AAV8, and AAV9. The unbinding forces between AAV and either heparin or alginate was measured, and the binding efficiency was recorded. Binding measurements were taken at two speeds using force spectroscopy mode with a custom-built AFM. The data was processed using IGOR Pro software to extract the unbinding forces as a function of the loading rate of each unbinding event. AAV release from alginate hydrogels were further explored to determine if the nanoscale forces had macroscale observable effects. AAV release from alginate hydrogel data was taken using a BD DUAL LSR Fortessa flow cytometer and analyzed in FlowJo.

Results, Conclusions, and Discussions: Unbinding events were observed between AFM tip bound AAV and alginate on a glass surface. The unbinding efficiency and average force of unbinding were recorded and compared to the AAV release measurements from the alginate hydrogels. AAV1 showed both the highest release and the lowest binding efficiency, as well as not showing high binding to heparin confirming it does not bind to heparin. It also showed the lowest unbinding forces for both alginate and heparin with forces being around 200pN and 150pN respectively. AAV2, known for heparin binding, showed higher release than AAV8 and AAV9 and similar unbinding force, with an average unbinding force for heparin around 320pN and 450pN for alginate. AAV8 showed lower release and higher binding efficiency for alginate than the other serotypes but had unbinding forces between AAV1 and AAV2 with both heparin and alginate averaging around 300pN. AAV9 showed the lowest release as well as the highest unbinding force for heparin with an unbinding force of around 700pN. The unbinding force for alginate was comparable to that of other serotypes at around 250pN. AAV9 also showed the largest difference in binding efficiency between alginate and heparin. The binding efficiency of each serotype supports the claim that both AAV1, AAV2 and AAV8 interact with alginate in similar ways as they do with heparin while AAV9 behaves differently. On the macroscale we observed AAV1 and AAV2 was able to release from an alginate hydrogel, while AAV8 and AAV9 were not. Taken together this data supports the hypothesis that AAV1, AAV2, and AAV8 bind to alginate similarly to how it binds to heparin. Interestingly, the data suggests that AAV9 might bind to alginate differently than alginate.

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