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. 2021 Sep 27;95(20):e0116421.
doi: 10.1128/JVI.01164-21. Epub 2021 Aug 4.

Context-Specific Function of the Engineered Peptide Domain of PHP.B

R Alexander Martino  1 , Edwin C Fluck 3rd  2 , Jacqueline Murphy  1 , Qiang Wang  1 , Henry Hoff  1 , Ruth A Pumroy  2 , Claudia Y Lee  1 , Joshua J Sims  1 , Soumitra Roy  1 , Vera Y Moiseenkova-Bell  2 , James M Wilson  1
Affiliations

Context-Specific Function of the Engineered Peptide Domain of PHP.B

R Alexander Martino et al. J Virol. .

Abstract

One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.

Keywords: AAV; Ly6a; PHP.B; adeno-associated virus; capsid engineering; receptor-mediated transcytosis.

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Figures

FIG 1
FIG 1
The engineered peptide domain of AAV9-PHP.B does not confer Ly6a binding when grafted to HVR VIII of AAV1. (A) The AAV1-PHP.B vector was generated by grafting the engineered peptide from AAV9-PHP.B into an analogous position in AAV1’s HVR VIII. (B and C) SPR plots showing AAV1-PHP.B (B) and AAV9-PHP.B (C) binding to surface-immobilized Ly6a-hIgG1 protein. Colored lines show binding data for each vector at a range of vector concentrations. KD values were determined using a global 1:1 binding model. (D) AAV9-PHP.B and AAV1-PHP.B vectors were tested alongside WT control vectors in HEK293 and HEK293-Ly6a cells. All vectors packaged a CMV-ffLuc reporter gene cassette. Results show transduction efficiency normalized to the WT capsid transduction efficiency for each cell type. Horizontal bars represent group averages; standard deviations (SDs) are reported as error bars; n = 3. Groups were compared using ANOVA followed by post hoc two-sample, two-sided t tests (version R.4.0.0) for specific pairwise comparisons. The # symbols represent statistical significance for comparisons between Ly6a (−/+) cell lines; the * symbols represent statistical significance for comparisons between engineered vectors and WT vectors in a given cell type: #/*, P ≤ 0.05; ##/**, P ≤ 0.01; ###/***, P ≤ 0.001; ns, not significant. (E) C57BL/6J mice were treated intravenously with 1 × 1012 GC of the indicated vectors or a PBS injection control. Vectors packaged a CB7-EGFP reporter gene cassette. Brains were sectioned on day 21 post-vector administration for direct EGFP fluorescence analysis. We acquired images using a 4× objective and 2 s exposure. GC, genome copies; WT, wild type; KD, dissociation constant.
FIG 2
FIG 2
The flexible PHP.B peptide presents similarly in AAV9 and AAV1 contexts; peptide insertion has a minimal impact on capsid structure. (A and B) Electron density maps contoured at 1σ (gray mesh) and fitted model residues for HVR-VIII of the AAV9-PHP.B capsid (A, green) and AAV1-PHP.B capsid (B, salmon). Red text indicates residues from the PHP.B peptide insertion, and black text indicates residues from the parental capsid. (C and D) Solved structures of AAV9-PHP.B (C) or AAV1-PHP.B (D) superimposed onto previously reported structures for AAV9 (orange) or AAV1 (purple). All figures were rendered using PyMol.
FIG 3
FIG 3
Using linkers to increase PHP.B peptide flexibility does not enable Ly6a binding in AAV1-PHP.B and removes Ly6a binding in AAV9-PHP.B. (A and B) AAV1-PHP.B linker variants (A) or AAV9-PHP.B linker variants (B) were tested alongside WT control vectors in HEK293 and HEK293-Ly6a cells. All vectors packaged a CMV-ffLuc reporter gene cassette. Results show transduction efficiency normalized to the WT capsid transduction efficiency for each cell type. Transduction efficiencies for AAV1, AAV1-PHP.B, AAV9, and AAV9-PHP.B are included as reported in Fig. 1B for comparison. Horizontal bars represent group averages; SDs are reported as error bars; n = 3. Groups were compared using ANOVA followed by post hoc two-sample, two-sided t tests (version R.4.0.0) for specific pairwise comparisons. The # symbols represent statistical significance for comparisons between Ly6a (−/+) cell lines; the * symbols represent statistical significance for comparisons between engineered vectors and WT vectors in a given cell type: #/*, P ≤ 0.05; ##/**, P ≤ 0.01; ###/***, P ≤ 0.001; ns, not significant. NL, N-terminal linker; CL, C-terminal linker; WT, wild type.
FIG 4
FIG 4
Ly6a binding requires interactions with residues from the PHP.B peptide insertion and AAV9’s HVR VIII. (A) Alignment showing how the AAV1-PHP.B2 vector was generated by extending the grafted region to include the PHP.B peptide and surrounding residues from AAV9’s HVR VIII. (B and C) SPR plots showing AAV1-PHP.B2 (B) and AAV9-PHP.B (C) (reproduced from Fig. 1C) binding to surface-immobilized Ly6a-hIgG1 protein. Colored lines show binding data for each vector at a range of vector concentrations. KD values were determined using a global 1:1 binding model. (D) We tested AAV9-PHP.B and AAV1-PHP.B2 vectors alongside WT control vectors in HEK293 and HEK293-Ly6a cells. All vectors packaged a CMV-ffLuc reporter gene cassette. Results show transduction efficiency normalized to the WT capsid transduction efficiency for each cell type. Transduction efficiencies for AAV9 and AAV9-PHP.B are included as reported in Fig. 1D for comparison. Horizontal bars shown in (D) represent group averages; SDs are reported as error bars; n = 3. Groups were compared using ANOVA followed by post hoc two-sample, two-sided t tests (version R.4.0.0) for specific pairwise comparisons. The # symbols represent statistical significance for comparisons between Ly6a (−/+) cell lines; the * symbols represent statistical significance for comparisons between engineered vectors and WT vectors in a given cell type: #/*, P ≤ 0.05; ##/**, P ≤ 0.01; ###/***, P ≤ 0.001; ns, not significant. ddPCR, digital droplet PCR; WT, wild type; KD, dissociation constant.
FIG 5
FIG 5
AAV1-PHP.B2 localizes to, but does not cross, the BBB in vivo. AAV1-PHP.B2 and control vectors packaging a CB7-eGFP reporter gene were administered intravenously to C57BL/6J mice at a dose of 3 × 1011 GC per mouse. (A and B) Vector biodistribution in brain (A) and liver (B) tissue collected at necropsy on day 21 post-vector administration, reported as vector GCs per diploid cell. (C) Vector transcripts were also detected in brain samples using RT-qPCR and are reported as vector transcripts per μg total RNA. In A to C, individual measurements and a horizontal bar representing group averages are shown; SDs are reported as error bars; n = 4. Groups were compared using ANOVA followed by post hoc two-sample, two-sided t tests (version R.4.0.0) for specific pairwise comparisons. The * symbols represent statistical significance for comparisons between engineered vectors and WT vectors in a given tissue. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (D) Brain sections isolated at necropsy were analyzed for GFP reporter gene expression using anti-GFP IF. (E) AAV1-PHP.B samples were also treated with an RCA-1 lectin stain to assess colocalization of GFP signals with brain microvascular endothelial cells. We acquired the displayed images using a 20× objective and 1s exposure for both fluorophores. GC, genome copies; IF, immunofluorescence.
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