Mechanisms of AAV neutralization by human alpha-defensins

Antiviral immunity compromises the efficacy of adeno-associated virus (AAV) vectors used for gene therapy. This is well understood for the adaptive immune response. However, innate immune effectors like alpha-defensin antimicrobial peptides also block AAV infection, although their mechanisms of action are unknown. To address this gap in knowledge, we investigated AAV2 and AAV6 neutralization by human neutrophil peptide 1 (HNP1), a myeloid alpha-defensin, and human defensin 5 (HD5), an enteric alpha-defensin. We found that both defensins bind to AAV2 and inhibit infection at low micromolar concentrations, similar to our prior studies of AAV6. While HD5 prevents AAV2 and AAV6 from binding to cells, HNP1 does not. However, AAV2 and AAV6 exposed to HD5 after binding to cells are still neutralized, indicating an additional block to infection. Accordingly, both HD5 and HNP1 inhibit externalization of the VP1 unique domain of both AAV2 and AAV6, which contains a phospholipase A2enzyme required..., , # Mechanisms of AAV neutralization by human alpha-defensins

## Description of the data and file structure

Files are provided for each figure as follows:

**Figure 1A:** 2025_AAVDefensinMechanism_Fig01A_analysis.prism

Scans of 96-well plates of HeLa cells infected with green fluorescent protein-expressing AAV2 were obtained using a multi-mode plate scanner from 4 independent replicates. The experimental variable was human defensin 5 (HD5). From these images, total well fluorescence was enumerated using Fiji by applying a rolling ball background subtraction and measuring integrated density limited to threshold. Sample wells were normalized to control wells (no HD5), and the normalized data from all replicates was analyzed using GraphPad Prism 10.4.1 (.prism).

**Figure 1B:** 2025_AAVDefensinMechanism_Fig01B_analysis.prism

Scans of 96-well plates of HeLa cells infected with green fluorescent protein-expressing AAV2 were obtained using a multi-mode plate scanner from 3 independent repli...,