AAV PHP.B-GFP

Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) is the ultimate goal for the treatment of neurological diseases. A variant of the neurotropic vector adeno-associated virus (AAV) serotype 9, AAV-PHP.B, has been shown to be highly efficient in delivering transgenes across the BBB of C57BL/6J mice. Based on the observed mouse strain dependence of this phenotype, the researchers whole-exome sequencing-based genetics to map this phenotype to a specific haplotype of lymphocyte antigen 6 complex, locus A (Ly6a) (stem cell antigen-1 [Sca-1]), which encodes a glycosylphosphatidylinositol (GPI)-anchored protein whose function is thought to be restricted to hematopoietic biology. Additional biochemical and genetic studies specifically linked high BBB transport to binding of AAV-PHP.B to LY6A (SCA-1). These studies identify for the first time a ligand for this GPI-anchored protein and suggest a role for it in BBB transport that could be hijacked by viruses in natural infections or by gene therapy vectors to treat neurological diseases.

The researchers identified 135 unique mutations within a ～4.5 Mbp segment of genomic DNA spanning the D3 and E3 karyotypic bands of mouse chromosome 15 that were most significantly associated with the observed phenotypes (Figure 1A and B). They hypothesized that the causative protein was LY6A, a GPI-anchored surface protein also known as SCA-1 that is highly expressed in brain microvessels. To test whether Ly6a is essential for the efficient delivery of AAV-PHP.B across the BBB, researchers i.v. injected AAV-PHP.B carrying the GFP transgene in Ly6a knockout mice (Ly6a−/−) in the C57BL/6J background and wild-type (WT) controls. Although AAV-PHP.B transduced the liver in both WT and Ly6a−/− mice, they observed minimal brain transduction in Ly6a−/− mice (Figure 1C). This suggests that LY6A is required for AAV-PHP.B transport across the BBB. After analysis of brain tissue by immunohistochemistry using a pan-LY6A antibody, high levels of expression were observed in microvascular endothelial cells of C57BL/6J animals. In contrast, LY6A expression was significantly reduced in BALB/cJ animals (Figure 1D). The expression of LY6A was not detected in the tissues of Ly6a−/− mice, which confirmed the specificity of the assay (Figure 1D). Either promoter mutations or mutations within the BALB/cJ LY6A open reading frame may lead to a dramatic decrease in LY6A expression on brain endothelium.

Figure 1. The Ly6a gene is associated with high AAV-PHP.B transduction across the BBB. (Hordeaux J, et al., 2019)