Brittain, T. J. | bioRxiv Science

bioRxiv2024

Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

bioengineering↗

bioRxiv2020

Isolation and biophysical characterization of GSU0105, a triheme c-type cytochrome from Geobacter sulfurreducens

C-type cytochromes play an important role in respiration of dissimilatory metal-reducing bacteria. They form extended conduits for charge transfer between the cellular metabolism and external electron acceptors such as particles of iron oxide, metal ions, and humic substances. Out of more than a hundred c-type cytochromes in Geobacter sulfurreducens, only a small fraction has been previously characterized. Here we present our results on expression and biophysical characterization of GSU0105, a novel 3-heme cytochrome, important for Fe(III) respiration in G. sulfurreducens. We successfully cloned the gene and achieved ~3 mg/L of culture GSU0105 expression in E.coli. Despite a similar size (71 amino acids) and the same number of c-type hemes to the members of the cytochrome (cyt) c7 family, multiple sequence alignment suggests that GSU0105 does not belong to the cyt c7 family. UV-Vis spectroscopy revealed typical c-type cytochrome spectral features, including a weak iron-sulfur charge transfer band suggesting that at least one heme is ligated with a methionine residue. Far UV circular dichroism studies demonstrate approximately 35% content of -helices and {beta}-sheets, each, as well as thermal aggregation occurring above 60 {degrees}C. A combination of SAXS and analytical size exclusion chromatography data shows that GSU0105 is monomeric in solution. Finally, affinity pull-down assays demonstrate high binding affinity to PpcD and weaker binding to the other members of the cyt c7 family.

biochemistry↗