Genomic loss of GPR108 disrupts AAV transduction in birds

Abstract

The G protein-coupled receptor 108 (GPR108) gene encodes a protein factor identified as critical for adeno-associated virus (AAV) entry into mammalian cells, but whether it is universally involved in AAV transduction is unknown. Remarkably, we have discovered that GPR108 is absent in the genomes of birds and in most other sauropsids, providing a likely explanation for the overall lower AAV transduction efficacy of common AAV serotypes in birds compared to mammals. Importantly, transgenic expression of human GPR108 and manipulation of related glycan binding sites in the viral capsid significantly boost AAV transduction in zebra finch cells. These findings contribute to a more in depth understanding of the mechanisms and evolution of AAV transduction, with potential implications for the design of efficient tools for gene manipulation in experimental animal models, and a range of gene therapy applications in humans.

Figure 1:. Genomic loss of GPR108 and link to AAV transduction efficiency in songbirds.

(A) Conservation of KIAA0319L (AAVR) PKD domains between human and zebra finch. (B) Simplified cladogram (left) depicting which amniote lineages lack GPR108 (in red), and diagrams showing the predicted GPR108 syntenic context of each lineage examined (right). (C) Comparison of the predicted peptide length and amino acid conservation between human and zebra finch for the enriched genes in the AAV entry screen in Pillay et al., 2016 (D) Images of in situ hybridization of AAV entry factors in the zebra finch brain. Green squares on sagittal sections on the left depict the areas shown on photomicrographs. The arcopallial (Arco) and nidopallium (Nido) areas shown are respectively analogous to deep motor cortical layers and superficial auditory cortical layers of mammals. These areas contain specific auditory or vocal-motor nuclei of the zebra finch song control circuitry. Images are 200 x 200 μm. (E) Quantification of AAV transduction by FACS analysis in CFS414-WT and CFS414-hGPR108 cells. Shown is change in percent of cells transduced in GPR108+ compared to WT cells. P-values for comparisons from two-tailed unpaired t tests: AAV9: p=0.4978; AAV2: p=0.7754; AAV5: 0.02345; AAV2G9: 3.122x10⁻⁴. Data presented as means +/− SD, n=4 replicates per condition. (F) Images of GFP-immunoenhanced AAV transduction in CSF414-WT and CSF414-hGPR108 cells for AAV2 at MOI of 10⁵ and AAV2G9 at MOI of 10⁴. Scale bar is 50 μ;m. (G) Schematic representing main stages in AAV transduction; both surface glycan binding and GPR108-dependent signaling appear to be critical in zebra finches.

Figure 2:. AAV Barcode-Seq reveals transduction efficiencies of capsids in the zebra finch brain.

(A) Transduction efficiency measured by AAV RNA Barcode-Seq. (B) Relative quantity of AAV vector genome DNA delivered by each capsid, measured by AAV DNA Barcode-Seq. For both B and C, phenotypic difference is normalized to AAV9. (C) Representative images of transduction efficiency of AAV9, Ad5, and VSV G pseudotyped lentivirus vectors stereotaxically injected into the nidopallium of zebra finches. Scale bar, 50 μm.