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AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset

Nature Neuroscience volume 25, pages 106–115 (2022)Cite this article

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Abstract

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood–brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

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Fig. 1: Capsid engineering locations and CAP-B library characterization.
Fig. 2: AAV.CAP-B10 tissue expression profile is biased toward the brain, with a significant decrease in liver targeting.
Fig. 3: Characterization of pooled capsid transgene expression in NHPs.
Fig. 4: Characterization of single-variant expression after delivery with each of AAV9, AAV-PHP.eB, AAV.CAP-B10 and AAV.CAP-B22 in marmosets.

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Data availability

The NGS datasets for capsid selection and marmoset pooled screening that are reported in this article are available under Sequence Read Archive accession code PRJNA769435. The following vector plasmids were deposited in Addgene for distribution, and viruses might be available for commonly packaged genomes (http://www.addgene.org): AAV.CAP-B10 (Addgene, 175004) and AAV.CAP-B22 (Addgene, 175005). All other constructs and tools will be available through the Beckman Institute CLOVER Center (https://clover.caltech.edu/). The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The codes used for M-CREATE data analysis were published previously33 and are available on GitHub: https://github.com/GradinaruLab/mCREATE.

References

  1. Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31, 317–334 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gaudet, D., Méthot, J. & Kastelein, J. Gene therapy for lipoprotein lipase deficiency. Curr. Opin. Lipidol. 23, 310–320 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Al-Zaidy, S. A. & Mendell, J. R. From clinical trials to clinical practice: practical considerations for gene replacement therapy in SMA type 1. Pediatr. Neurol. 100, 3–11 (2019).

    Article  PubMed  Google Scholar 

  4. Ameri, H. Prospect of retinal gene therapy following commercialization of voretigene neparvovec-rzyl for retinal dystrophy mediated by RPE65 mutation. J. Curr. Ophthalmol. 30, 1–2 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Evens, H., Chuah, M. K. & VandenDriessche, T. Haemophilia gene therapy: from trailblazer to gamechanger. Haemophilia 24, 50–59 (2018).

    Article  PubMed  Google Scholar 

  6. Ginn, S. L., Amaya, A. K., Alexander, I. E., Edelstein, M. & Abedi, M. R. Gene therapy clinical trials worldwide to 2017: an update. J. Gene Med. 20, e3015 (2018).

    Article  PubMed  Google Scholar 

  7. Srivastava, A. In vivo tissue-tropism of adeno-associated viral vectors. Curr. Opin. Virol. 21, 75–80 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Gray, S. J., Woodard, K. T. & Samulski, R. J. Viral vectors and delivery strategies for CNS gene therapy. Ther. Deliv. 1, 517–534 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Golebiowski, D. et al. Direct intracranial injection of AAVrh8 encoding monkey β-N-acetylhexosaminidase causes neurotoxicity in the primate brain. Hum. Gene Ther. 28, 510–522 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hocquemiller, M., Giersch, L., Audrain, M., Parker, S. & Cartier, N. Adeno-associated virus-based gene therapy for CNS diseases. Hum. Gene Ther. 27, 478–496 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kubes, P. & Jenne, C. Immune responses in the liver. Annu. Rev. Immunol. 36, 247–277 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Gao, B. Basic liver immunology. Cell. Mol. Immunol. 13, 265–266 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ronzitti, G., Gross, D.-A. & Mingozzi, F. Human immune responses to adeno-associated virus (AAV) vectors. Front. Immunol. 11, 670 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Verdera, H. C., Kuranda, K. & Mingozzi, F. AAV vector immunogenicity in humans: a long journey to successful gene transfer. Mol. Ther. 28, 723–746 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gray, S. J. et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum. Gene Ther. 22, 1143–1153 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. de Leeuw, C. N. et al. Targeted CNS delivery using human MiniPromoters and demonstrated compatibility with adeno-associated viral vectors. Mol. Ther. Methods Clin. Dev. 1, 5 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Allen, W. E. et al. Global representations of goal-directed behavior in distinct cell types of mouse neocortex. Neuron 94, 891–907 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Shima, Y. et al. A mammalian enhancer trap resource for discovering and manipulating neuronal cell types. eLife 5, e13503 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Graybuck, L. T. et al. Enhancer viruses for combinatorial cell-subclass-specific labeling. Neuron 109, 1449–1464 (2021).

    Article  CAS  PubMed  Google Scholar 

  21. Mich, J. K. et al. Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. Cell Rep. 34, 108754 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vormstein-Schneider, D. et al. Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans. Nat. Neurosci. 23, 1629–1636 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Keaveney, M. K. et al. A microRNA-based gene-targeting tool for virally labeling interneurons in the rodent cortex. Cell Rep. 24, 294–303 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hordeaux, J. et al. MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates. Sci. Transl. Med. 12, eaba9188 (2020).

    Article  CAS  PubMed  Google Scholar 

  26. Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zelikowsky, M. et al. The neuropeptide Tac2 controls a distributed brain state induced by chronic social isolation stress. Cell 173, 1265–1279 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zeng, J. et al. TRIM9-mediated resolution of neuroinflammation confers neuroprotection upon ischemic stroke in mice. Cell Rep. 27, 549–560 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Graybuck, L. T. et al. Enhancer viruses for combinatorial cell-subclass-specific labeling. Neuron 109, 1449–1464.e13 (2021).

    Article  CAS  PubMed  Google Scholar 

  30. Gao, G. et al. Adeno-associated virus-mediated gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses. Hum. Gene Ther. 20, 930–942 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mingozzi, F. & High, K. A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Calcedo, R., Chichester, J. A. & Wilson, J. M. Assessment of humoral, innate, and T-cell immune responses to adeno-associated virus vectors. Hum. Gene Ther. Methods 29, 86–95 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Kumar, S. et al. Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat. Methods 17, 541–550 (2020).

    Article  PubMed Central  Google Scholar 

  34. Matsuzaki, Y. et al. Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain. Neurosci. Lett. 665, 182–188 (2018).

    Article  CAS  PubMed  Google Scholar 

  35. Hordeaux, J. et al. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol. Ther. 26, 664–668 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kern, A. et al. Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J. Virol. 77, 11072–11081 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Girod, A. et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat. Med. 5, 1052–1056 (1999).

    Article  CAS  PubMed  Google Scholar 

  38. Michelfelder, S. et al. Peptide ligands incorporated into the threefold spike capsid domain to re-direct gene transduction of AAV8 and AAV9 in vivo. PLoS ONE 6, e23101 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Shen, S., Bryant, K. D., Brown, S. M., Randell, S. H. & Asokan, A. Terminal N-linked galactose is the primary receptor for adeno-associated virus 9. J. Biol. Chem. 286, 13532–13540 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Summerford, C., Johnson, J. S. & Samulski, R. J. AAVR: a multi-serotype receptor for AAV. Mol. Ther. 24, 663–666 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhang, R. et al. Adeno-associated virus 2 bound to its cellular receptor AAVR. Nat. Microbiol. 4, 675–682 (2019).

    Article  CAS  PubMed  Google Scholar 

  42. DiMattia, M. A. et al. Structural insight into the unique properties of adeno-associated virus serotype 9. J. Virol. 86, 6947–6958 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Foust, K. D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59–65 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Samaranch, L. et al. Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum. Gene Ther. 23, 382–389 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Flotte, T. R. & Büning, H. Severe toxicity in nonhuman primates and piglets with systemic high-dose administration of adeno-associated virus serotype 9-like vectors: putting patients first. Hum. Gene Ther. 29, 283–284 (2018).

    Article  CAS  PubMed  Google Scholar 

  46. Unzu, C. et al. Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates. J. Transl. Med. 10, 122 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Giannelli, S. G. et al. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery. Hum. Mol. Genet. 27, 761–779 (2018).

    Article  CAS  PubMed  Google Scholar 

  48. Schubert, R. et al. Virus stamping for targeted single-cell infection in vitro and in vivo. Nat. Biotechnol. 36, 81–88 (2017).

    Article  PubMed  Google Scholar 

  49. Morabito, G. et al. AAV-PHP.B-mediated global-scale expression in the mouse nervous system enables GBA1 gene therapy for wide protection from synucleinopathy. Mol. Ther. 25, 2727–2742 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hillier, D. et al. Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex. Nat. Neurosci. 20, 960–968 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Dayton, R. D., Grames, M. S. & Klein, R. L. More expansive gene transfer to the rat CNS: AAV PHP.EB vector dose–response and comparison to AAV PHP.B. Gene Ther. 25, 392–400 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Rincon, M. Y. et al. Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector. Gene Ther. 25, 83–92 (2018).

    Article  CAS  PubMed  Google Scholar 

  53. Yuste, R. et al. A community-based transcriptomics classification and nomenclature of neocortical cell types. Nat. Neurosci. 23, 1456–1468 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Krienen, F. M. et al. Innovations present in the primate interneuron repertoire. Nature 586, 262–269 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Coune, P. G., Schneider, B. L. & Aebischer, P. Parkinson’s disease: gene therapies. Cold Spring Harb. Perspect. Med. 2, a009431 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Piguet, F. et al. Rapid and complete reversal of sensory ataxia by gene therapy in a novel model of Friedreich ataxia. Mol. Ther. 26, 1940–1952 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Combs, B., Kneynsberg, A. & Kanaan, N. M. Gene therapy models of alzheimer’s disease and other dementias. Methods Mol. Biol. 1382, 339–366 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Challis, R. C. et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat. Protoc. 14, 379–414 (2019).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We wish to thank the entire Gradinaru laboratory for helpful discussions. We thank B. Deverman and S. Kumar for helpful discussions on M-CREATE design and implementation, as described in Kumar et al.33. We thank M. Borsos, A. Hori, T. Yan, X. Qu and Y. Chen for their technical assistance. We are grateful to I. Antoshechkin and the Millar and Muriel Jacobs Genetics and Genomics Core at the California Institute of Technology for assistance with next-generation sequencing. We thank M. Smith for helpful discussions and assistance with the bioinformatics pipeline. This work was primarily supported by Defense Advanced Research Projects Agency grant W911NF-17-2-0036 to V.G. and grants from the National Institutes of Health (NIH) to V.G.: Director’s New Innovator and PECASE DP2NS087949, BRAIN R01MH117069, BRAIN U01 UMH128336A and NIH Pioneer DP1OD025535. D.G was supported by the National Sciences and Engineering Research Council of Canada. Additional support for revision experiments was provided by K. L. Yang and the Hock E. Tan Center for Molecular Therapeutics in Neuroscience at MIT to G.F., the James and Patricia Poitras Center for Psychiatric Disorders Research at MIT to G.F., NIH grant U24 OD026638 to G.F., Guangdong Provincial Fund for Basic and Applied Basic Research (2019B1515130004) to Y.C., the Shenzhen Knowledge Innovation Program (JCYJ20170413165053031) to Y.C., the National Key R&D Program of China (2018YFE0203600) to Y.C., the Areas of Excellence Scheme of the University Grants Committee of Hong Kong (AoE/M-604/16) to N.Y.I. and the Hong Kong Center for Neurodegenerative Diseases (INNOHK18SC01) to N.Y.I. In addition, this work is funded by the Beckman Institute at the California Institute of Technology, the Vallee Foundation and the Moore Foundation. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: David Goertsen, Nicholas C. Flytzanis, Nick Goeden.

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    David Goertsen, Nicholas C. Flytzanis, Nick Goeden, Miguel R. Chuapoco & Viviana Gradinaru

  2. National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA

    Alexander Cummins & James Pickel

  3. Chinese Academy of Sciences Key Laboratory of Brain Connectome and Manipulation, Shenzhen Key Laboratory of Translational Research for Brain Diseases, The Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences; Shenzhen–Hong Kong Institute of Brain Science–Shenzhen Fundamental Research Institutions, Shenzhen, China

    Yijing Chen, Yingying Fan, Liping Wang & Yu Chen

  4. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA

    Qiangge Zhang, Jitendra Sharma & Guoping Feng

  5. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Qiangge Zhang & Guoping Feng

  6. Picower Institute of Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jitendra Sharma

  7. Tan & Yang Center for Autism Research, McGovern Institute of Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jitendra Sharma

  8. Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    Jitendra Sharma

  9. Division of Life Science, State Key Laboratory of Molecular Neuroscience and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    Yangyang Duan & Nancy Y. Ip

  10. Hong Kong Center for Neurodegenerative Diseases, Hong Kong Science Park, Hong Kong, China

    Nancy Y. Ip

  11. Guangdong Provincial Key Laboratory of Brain Science, Disease and Drug Development, Shenzhen–Hong Kong Institute of Brain Science, HKUST Shenzhen Research Institute, Shenzhen, Guangdong, China

    Yu Chen & Nancy Y. Ip

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  1. David Goertsen
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  2. Nicholas C. Flytzanis
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Contributions

D.G., N.C.F and N.G. analyzed all data and prepared all figures, with input from V.G. N.C.F and N.G. designed and performed the variant selection experiments, characterized the variants in mice and prepared the virus for the pooled marmoset experiments. J.P and A.C. performed National Institutes of Health (NIH) marmoset experiments and analyzed the associated data. J.P. supervised the NIH experiments and helped prepare the associated figure. During revision, the Massachusetts Institute of Technology (MIT), the Shenzhen Institute of Advanced Technology (SIAT) and the Hong Kong University of Science and Technology (HKUST) provided additional marmoset data on controls (AAV-PHP.eB) and to increase ‘n’ for all cohorts. D.G. and M.C. prepared the virus for single-variant characterization and performed marmoset single-variant characterization tissue analysis of MIT and NIH marmoset tissues. Q.Z. and J.S. assisted with the MIT AAV injections, animal perfusion and tissue sample collection, under the supervision of G.F. Yijing Chen and Y.F. performed single-variant characterization of the SIAT animals and analyzed the associated data, under the supervision of Yu Chen. L.W. supervised the marmoset breeding at the SIAT. Y.D. coordinated with D.G. for experiments at the SIAT, under the supervision of N.Y.I. N.C.F., N.G., D.G. and V.G. wrote the manuscript, with input from all authors. No experimental work was performed during the Los Angeles COVID-19 stay-at-home order. V.G. supervised all aspects of the work.

Corresponding author

Correspondence to Viviana Gradinaru.

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Competing interests

The California Institute of Technology has filed and licensed patent applications for the work described in this manuscript, with N.C.F., N.G. and V.G. listed as inventors (US Patent application no. 16/582,635, 2020). V.G. is a co-founder and board member and N.C.F. and N.G. are co-founders and employees of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company. Capsida Biotherapeutics did not provide funding and had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Subhojit Roy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Table 1 Systemic marmoset injections of a CNS-targeting engineered viral pool
Full size table
Extended Data Table 2 Systemic marmoset injections of CNS-targeting engineered viral variants
Full size table

Extended Data Fig. 1 Amino-acid contributions across the substitution library. (Related to Fig. 1.).

Amino-acid contributions across the 7-mer substitution to variants enriched in the brain and targeted away from the liver. The 1k variants with the highest enrichment in the brain of hSyn-Cre animals, the 1k variants with the lowest enrichment in the liver of Tek-Cre animals, and all 39,034 variants with positive enrichment in the brain and negative enrichment in the liver from round 2 were analyzed. Plotted is the z-score of all amino acids at each position. Amino acids found at a higher prevalence than average are displayed in yellow and those found at a lower prevalence are displayed in purple.

Extended Data Fig. 2 Quantification of decreased targeting from non-CNS organs in mice. (Related to Fig. 2.).

ssAAV9:CAG-NLSx2-EGFP, ssAAV-PHP.eB:CAG-NLSx2-EGFP, and ssAAV.CAP-B10:CAG-NLSx2-EGFP were intravenously injected into male adult mice at 1 × 1011 vg/mouse. GFP fluorescence was assessed after three weeks of expression. The number of cells expressing GFP after delivery with AAV.CAP-B10 in the spinal cord is significantly increased relative to AAV9 and AAV-PHP.eB (p = <0.0001 [AAV9 vs AAV-PHP.eB], < 0.0001 [AAV9 vs AAV.CAP-B10], 0.0016 [AAV-PHP.eB vs. AAV.CAP-B10]). The number of cells expressing GFP after delivery with AAV.CAP-B10 in the DRGs is significantly decreased relative to AAV9 and not significantly different from AAV-PHP.eB (p = 0.6488 [AAV9 vs AAV-PHP.eB], 0.0229 [AAV9 vs AAV.CAP-B10], 0.5956 [AAV-PHP.eB vs. AAV.CAP-B10]). In the myenteric and submucosal plexi of the intestines, the number of cells expressing GFP after delivery with AAV.CAP-B10 is significantly decreased relative to AAV9 and not significantly different relative to AAV-PHP.eB (Myenteric Plexi: p = 0.0165 [AAV9 vs AAV-PHP.eB], 0.0147 [AAV9 vs AAV.CAP-B10], 0.6461 [AAV-PHP.eB vs. AAV.CAP-B10]; Submucosal Plexi: p = 0.0066 [AAV9 vs AAV-PHP.eB], 0.0095 [AAV9 vs AAV.CAP-B10], > 0.9999 [AAV-PHP.eB vs. AAV.CAP-B10]). In the lungs, the number of cells expressing GFP after delivery with AAV.CAP-B10 is significantly decreased relative to AAV-PHP.eB and not significantly different from AAV9 (p = 0.5286 [AAV9 vs AAV-PHP.eB], 0.2805 [AAV9 vs AAV.CAP-B10], 0.0073 [AAV-PHP.eB vs. AAV.CAP-B10]). In the kidneys, spleen, pancreas, and testes, the number of cells expressing GFP after delivery with AAV.CAP-B10 transduction efficiency is significantly decreased relative to AAV9 and non-significantly changed relative to AAV-PHP.eB (Kidneys: p = 0.1118 [AAV9 vs AAV-PHP.eB], 0.0010 [AAV9 vs AAV.CAP-B10], 0.3894 [AAV-PHP.eB vs. AAV.CAP-B10]; Spleen: p = 0.1363 [AAV9 vs AAV-PHP.eB], 0.0024 [AAV9 vs AAV.CAP-B10], 0.5293 [AAV-PHP.eB vs. AAV.CAP-B10]; Pancreas: p = 0.0677 [AAV9 vs AAV-PHP.eB], 0.0452 [AAV9 vs AAV.CAP-B10], 0.2085 [AAV-PHP.eB vs. AAV.CAP-B10]; Testes: p = 0.2497 [AAV9 vs AAV-PHP.eB], 0.0005 [AAV9 vs AAV.CAP-B10], 0.1191 [AAV-PHP.eB vs. AAV.CAP-B10]). n = 6 mice per group except for the spinal cord and DRGs, which had n = 3 mice for the AAV9 and AAV-PHP.eB groups and n = 5 mice for the AAV.CAP-B10 group; mean ± SE; significance was determined using Ordinary one-way ANOVA with Tukey’s correction for spinal cord, Brown-Forsythe and Welch one-way ANOVA tests with Dunnett’s T3 correction for myenteric plexus and pancreas, Kruskal-Wallis one-way ANOVA test with Dunn’s correction for DRGs, submucosal plexus, lungs, kidneys, spleen, and testes.

Extended Data Fig. 3 Brain regions selected for cell-type quantification, depicted on brain expressing NLS-GFP after systemic delivery with AAV-PHP.eB. (Related to Fig. 2.).

Four areas of cortex (white), three areas of hippocampus (teal), four areas of thalamus (yellow), three areas of striatum (purple), one area of ventral midbrain (red), and three areas of cerebellum (dark blue) were selected from each brain section quantified. Each section was located roughly 1200 µm from the midline for consistency across animals. Scale bar is 1 mm.

Extended Data Fig. 4 Quantification of Purkinje cell expression. (Related to Fig. 2.).

AAV.CAP-B10 is significantly targeted away from Purkinje cells in the cerebellum. ssAAV-PHP.eB:CAG-NLSx2-EGFP and ssAAV.CAP-B10:CAG-NLSx2-EGFP were intravenously injected into male adult mice at 1 × 1011 vg/mouse. GFP fluorescence was assessed after three weeks of expression. (a, b) Quantification of the percentage of Purkinje cells displaying GFP expression in the cerebellum shows significantly fewer cells express EGFP after delivery by AAV.CAP-B10 than by AAV-PHP.eB (P = 0.0009). n = 6 mice per group, mean ± SE, two-tailed Welch’s t test. Scale bar is 200 µm.

Extended Data Fig. 5 Astrocyte specificity of AAV9, AAV.CAP-B10, and AAV.CAP-B22 characterized through immunostaining for the HA tag in conjunction with S100β. (Related to Fig. 4.).

Astrocyte specificity of AAV9, AAV.CAP-B10, and AAV.CAP-B22 characterized through immunostaining for the HA tag in conjunction with S100β. (a) Coronal sections from marmoset brains after injection of individual variants, showing the locations of the areas selected for magnification. Scale bar is 5 mm. (b) The number of astrocytes expressing FXN was compared in magnified frames taken from the cortex and corpus callosum of animals injected with single variants. AAV9 and AAV.CAP-B10 display very little FXN expression in astrocytes, whereas AAV.CAP-B22 displays FXN expression in astrocytes in both the cortex and the corpus callosum. Scale bar is 500 µm. (c) Further magnification indicates colocalization (examples marked with arrows) of HA and S100β immunostaining in both the cortex and corpus callosum of marmosets for AAV.CAP-B10 and AAV.CAP-B22. Scale bar is 100 µm.

Extended Data Fig. 6 Viral genome and RNA transcript measurements in cortex and liver tissue after single variant injection in marmosets. (Related to Fig. 4.).

(a) Viral genomes and RNA expression measurements in the liver corroborate protein expression data, indicating that AAV.CAP-B10 and AAV.CAP-B22 have decreased transduction and expression in the liver relative to AAV9. n = 1 for AAV9, 3 for AAV-PHP.eB, 2 for AAV.CAP-B10, 3 for AAV.CAP-B22. mean ± SE. (b) Viral genome and RNA expression measurements in the cortex indicate no significant difference between experimental conditions. n = 2 for AAV9, 3 for AAV-PHP.eB, 4 for AAV.CAP-B10, 4 for AAV.CAP-B22, mean ± SE, two-tailed Welch’s t-test. Values for both viral genomes and RNA transcripts were normalized by internal GAPDH control.

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Goertsen, D., Flytzanis, N.C., Goeden, N. et al. AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Nat Neurosci 25, 106–115 (2022). https://doi.org/10.1038/s41593-021-00969-4

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