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Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood–brain barrier in rodents and primates

Nature Biomedical Engineering volume 6, pages 1257–1271 (2022)Cite this article

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Abstract

The development of gene therapies for the treatment of diseases of the central nervous system has been hindered by the limited availability of adeno-associated viruses (AAVs) that efficiently traverse the blood–brain barrier (BBB). Here, we report the rational design of AAV9 variants displaying cell-penetrating peptides on the viral capsid and the identification of two variants, AAV.CPP.16 and AAV.CPP.21, with improved transduction efficiencies of cells of the central nervous system on systemic delivery (6- to 249-fold across 4 mouse strains and 5-fold in cynomolgus macaques, with respect to the AAV9 parent vector). We also show that the neurotropism of AAV.CPP.16 is retained in young and adult macaques, that this variant displays enhanced transcytosis at the BBB as well as increased efficiency of cellular transduction relative to AAV9, and that it can be used to deliver antitumour payloads in a mouse model of glioblastoma. AAV capsids that can efficiently penetrate the BBB will facilitate the clinical translation of gene therapies aimed at the central nervous system.

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Fig. 1: Capsid engineering of BBB-penetrant AAV variants in mice.
Fig. 2: Systemic CNS gene delivery in NHPs by AAV.CPP.16 and AAV.CPP.21.
Fig. 3: Types of CNS cells transduced by AAV.CPP.16.
Fig. 4: Effects of AAV.CPP.16 on BBB integrity, transcytosis and cellular transduction.
Fig. 5: AAV.CPP.16-based systemic PD-L1 antibody therapy against GBM.
Fig. 6: Systemic HSV-TK1 suicide gene therapy mediated by AAV.CPP.16.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. All raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank K. J. Smith and H. Hirai for providing feedback on the manuscript, G. Freeman for providing the PD-L1 antibody, A. Charest and R. Piranlioglu for providing the GBM1694 cells, H. Jin for providing AAV reagents and D. Feng, Y. Ni and B. J. Scott for technical assistance. This study was supported by a Brigham and Women’s Hospital sundry fund (to F.B.).

Author information

Author notes

  1. These authors contributed equally: Yizheng Yao, Jun Wang, Yi Liu, Yuan Qu.

Authors and Affiliations

  1. Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Yizheng Yao, Jun Wang, Yi Liu, Yuan Qu, Kaikai Wang, Yang Zhang, Yuxin Chang, Zhi Yang, Jie Wan, Junfeng Liu, Hiroshi Nakashima, Sean E. Lawler, E. Antonio Chiocca, Choi-Fong Cho & Fengfeng Bei

  2. Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan, China

    Jun Wang

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Contributions

F.B. conceived and supervised the study, with input from E.A.C.; Y.Y., J.Wang, Y.L. and Y.Q. performed experiments and collected data, with inputs from K.W., Y.Z., Y.C., Z.Y., J.Wan and J.L.; H.N., S.E.L. and C.-F.C. contributed to methodology; F.B., Y.Y., J.W., Y.L. and Y.Q. analysed data. F.B. and Y.Y. wrote the manuscript with input from all the authors.

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Correspondence to Fengfeng Bei.

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Competing interests

F.B. and E.A.C. receive royalties from patents generated by this study (Patent applications: PCT/US19/41386, PCT/US21/12746 and PCT/US22/73051). F.B. is a co-founder and scientific advisor of Brave Bio Inc., an AAV gene therapy start-up. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Initial round of in vivo selection revealing potential candidates for further optimization.

a, Fluorescence images of sagittal brain sections in mice 21 days after intravenous injection of 1 × 1010 vg of single-stranded AAV-EF1α-H2B-RFP. White dots indicate RFP-positive cells. Scale bars: 1000 μm for whole brain section and 30 μm for insets. b, Fluorescence images of sagittal brain sections in mice 21 days after intravenous injection of 1 × 1011 vg of selected AAVs-EF1α-H2B-RFP. Scale bars: 1,000 μm for whole brain section and 300 μm for insets. c, Percentage of transduced brain cells and relative fold change of AAV transduction as quantified by counting transduced RFP-positive cells using ImageJ. n = 3 mice per group, mean ± s.e.m., one-way ANOVA with Tukey’s post-test.

Source data

Extended Data Fig. 2 Sequence optimization of Bip inserts yields additional AAV variants with further enhanced brain tropism.

a, Sequences of standard 5-mer Bips, as well as derivatives during optimization. b, Fluorescence images of sagittal brain sections in mice 21 days after intravenous injection of 1 × 1011 vg of AAVs-EF1α-H2B-RFP. Scale bars: 1000 μm for whole brain section and 300 μm for insets. c, Percentage of transduced brain cells and relative fold change of AAV transduction in the brain as quantified by counting RFP-positive cells using ImageJ. d, Percentage of transduced liver cells by AAVs relative to DAPI counter-staining. White dots indicate RFP-positive cells. n = 3 mice per group, mean ± s.e.m., one-way ANOVA with Tukey’s post-test.

Source data

Extended Data Fig. 3 Brain transductions by AAV.CPP.16 and AAV.CPP.21 in four common mouse strains.

a-l, Representative fluorescence images of brain regions in C57BL/6J, BALB/cJ, FVB/NJ and 129S1/SvlmJ mice 21 days after intravenous injection of 1 × 1011 vg of AAVs-EF1α-H2B-RFP (AAV9, a-d; AAV.CPP.16, e-h; AAV.CPP.21, i-l). White dots indicate RFP-positive cells. Scale bars: 500 μm. m, quantification of transduced brain cells in C57BL/6J at a high dose of AAVs (1 × 1012 vg, IV). n = 3 mice per group, mean ± s.e.m., one-way ANOVA with Tukey’s post-test.

Source data

Extended Data Fig. 4 Viral genome distribution of AAV.CPP.16 vs. AAV9 in BALB/cJ.

5 × 1011 vg AAVs-CAG-GFP-WPRE were intravenously injected to BALB/cJ mice and AAV genomic DNA was isolated 21 days later. AAV viral genomes in selected CNS regions (a) and peripheral organs (b) were measured by qPCR using primers targeting WPRE and normalized to mouse genome (targeting glucagon). n = 4 mice per group, mean ± s.e.m., Student’s t-test.

Source data

Extended Data Fig. 5 Transduction of oligodendrocytes, motor neurons and peripheral tissues by AAV.CPP.16 and AAV9 in BALB/cJ.

a-e, Transduced oligodendrocytes (Olig2+) in the brain and quantification on percentage. Scale bar for a, b: 25 μm. Scale bar for c, d: 10 μm. n = 3 mice per group, mean ± s.e.m. f, Immunostaining of spinal cord sections. Motor neurons were visualized using ChAT antibody. Scale bars: 250 μm for the left two and 50 μm for the right two. g, Transduction of peripheral tissues and quantification on relative fold change. BALB/cJ mice were examined 3 weeks after intravenous injection of 1× 1012 vg AAV.CPP.16-CAG-H2B-GFP or AAV9-CAG-H2B-GFP. Mean ± s.e.m., n = 4-8, Student’s t-test.

Source data

Extended Data Fig. 6 Imaging, western blot and histological analysis for AAV.CPP.16-aPD-L1-treated GBM-bearing mice.

a, Timeline for procedures in a mouse glioblastoma (GBM) model. b, Example of bioluminescence imaging for verification of tumor formation at day 7 post tumor implantation. c, aPD-L1-HA expression in the liver and muscle as measured by western blot. d-f, Representative images of hematoxylin-eosin-stained brain sections in a long-term survivor mouse treated with AAV.CPP.16-aPD-L1. Little residual tumor tissue was detectable suggesting eradication of GBM. g, Immunostaining showing AAV.CPP.16-mediated expression of aPD-L1-HA and astrogliosis in one long-term-surviving animal. Scale bar in g: 25 μm. n = 3-5 mice per group, mean ± s.e.m., one-way ANOVA with Tukey’s post-test.

Source data

Extended Data Fig. 7 Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs).

a, Gating strategies for all panels. Lymphocyte populations were selected based on SSC-H and FSC-H parameters, followed by FSC-H and FSC-A gating to eliminate doublets. CD45 marks for all immune cells. b, Representative flow cytometry plots and quantification of T cells (CD3+/CD45+) and their subtypes (CD4+/CD3+ T cells, CD8+/CD3+ T cells, IFNg+/CD3+ T cells, Granzyme B+/CD8+ T cells, CD25+/Foxp3 + /CD4+ Tregs). n = 3-11 mice per group, mean ± s.e.m., one-way ANOVA with Tukey’s post-test.

Source data

Extended Data Fig. 8 Pro-survival effect by monoclonal antibodies against PD-L1 in GL261 mouse GBM model.

a, Experimental timeline for studying the effect of PD-L1 mAB in the GL261 GBM model. A large dose of PD-L1 mAB (339.6A2, mouse IgG1, κ; 2.25 mg in total) was administered. b, Kaplan–Meier survival curves showing survival outcome of PD-L1 mAB or control in the GL261 GBM model. n = 6 each group, Log-rank test used to define statistical significance.

Source data

Extended Data Fig. 9 Pro-survival effect of systematic therapy of AAV.CPP.16-aPD-L1 in a genetic GBM tumor model.

a, Experimental timeline. GBM1694 tumor cells with human wild-type EGFR over-expression and Ink4a/Arf-PTEN co-deletion were implanted into BALB/c mice to model GBM. 1 × 1012 vg of AAV.CPP.16-CAG-aPD-L1-HA were administered at day 5 post tumor implantation. b, Kaplan–Meier survival curves showing survival times of AAV.CPP.16-aPD-L1 or PBS control. n = 6 each group, Log-rank test used to define statistical significance.

Source data

Supplementary information

Supplementary Information

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Supplementary Tables

Supplementary Tables 1–4.

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Yao, Y., Wang, J., Liu, Y. et al. Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood–brain barrier in rodents and primates. Nat. Biomed. Eng 6, 1257–1271 (2022). https://doi.org/10.1038/s41551-022-00938-7

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