1. Medicine
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scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution

  1. Bilge E Öztürk
  2. Molly E Johnson
  3. Michael Kleyman
  4. Serhan Turunç
  5. Jing He
  6. Sara Jabalameli
  7. Zhouhuan Xi
  8. Meike Visel
  9. Valérie L Dufour
  10. Simone Iwabe
  11. Felipe Pompeo Marinho
  12. Gustavo D Aguirre
  13. José-Alain Sahel
  14. David V Schaffer
  15. Andreas R Pfenning
  16. John G Flannery
  17. William A Beltran
  18. William R Stauffer
  19. Leah C Byrne  Is a corresponding author
  1. University of Pittsburgh, United States
  2. Carnegie Mellon University, United States
  3. University of California, Berkeley, United States
  4. University of Pennsylvania, United States
Research Article
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Cite this article as: eLife 2021;10:e64175 doi: 10.7554/eLife.64175

Abstract

Background:

Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging.

Methods:

Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal.

Results:

To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart and liver following systemic injection.

Conclusions:

These results validate scAAVengr as a powerful method for development of AAV vectors.

Funding:

This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research.

Data availability

Data, including count matrix files, raw fastq files as well as AAV/cell barcode tables generated from read quantification, have been uploaded to GEO under accession code GSE161645.

The following data sets were generated

Article and author information

Author details

  1. Bilge E Öztürk

    Ophthalmology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5117-077X
  2. Molly E Johnson

    Ophthalmology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  3. Michael Kleyman

    Computational Biology, Carnegie Mellon University, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  4. Serhan Turunç

    Ophthalmology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  5. Jing He

    Neurobiology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  6. Sara Jabalameli

    Ophthalmology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  7. Zhouhuan Xi

    Ophthalmology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  8. Meike Visel

    Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    Meike Visel, MV is an inventor on AAV capsid variants (US patent IDs: 10,214,785, 10,745,453). MV has also received royalty payments from UC Berkeley. The author has no other competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5033-3730
  9. Valérie L Dufour

    Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, University of Pennsylvania, Philadelphia, United States
    Competing interests
    No competing interests declared.
  10. Simone Iwabe

    Department of Clinical Sciences & Advanced Medicine, University of Pennsylvania, Philadelphia, United States
    Competing interests
    No competing interests declared.
  11. Felipe Pompeo Marinho

    Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, University of Pennsylvania, Philadelphia, United States
    Competing interests
    No competing interests declared.
  12. Gustavo D Aguirre

    Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, University of Pennsylvania, Philadelphia, United States
    Competing interests
    No competing interests declared.
  13. José-Alain Sahel

    Neurobiology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    José-Alain Sahel, JAS has served as a consultant (with no consulting fee) for Pixium Vision, GenSight Biologics and SparingVision. Personal financial interests: Pixium Vision, GenSight Biologics, Prophesee and Chronolife, SparingVision, SHARPEYE, Vegavect, Newsight Therapeutics. The author has no other competing interests to declare..
  14. David V Schaffer

    Chemical Engineering, University of California, Berkeley, Berkeley, United States
    Competing interests
    David V Schaffer, DS is named as an inventor on patent applications on AAV capsid variants (U.S. Patent Applications No. 16/315,032, 16/486,681). DS is also a co-founder of 4D Molecular Therapeutics, and DS performs consultancy and owns stock options in this company. The author has no other competing interests to declare..
  15. Andreas R Pfenning

    Computational Biology, Carnegie Mellon University, Pittsburgh, United States
    Competing interests
    Andreas R Pfenning, AP has received an honorarium from the University of Rhode Island, and has applied for patents on specific Nuclear-Anchored Independent Labeling System (PCT/US2020/038520 and PCT/US2020/038528). The author has no other competing interests to declare..
  16. John G Flannery

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    John G Flannery, JGF is an inventor on patent application on AAV capsid variants (U.S. Patent Application No. 16/315,032, 16/486,681). The author has no other competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0720-8897
  17. William A Beltran

    Department of Clinical Sciences & Advanced Medicine, University of Pennsylvania, Philadelphia, United States
    Competing interests
    William A Beltran, WAB is an inventor on patent application on AAV capsid variants(16/315,032). The author has no other competing interests to declare..
  18. William R Stauffer

    Neurobiology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    William R Stauffer, WRS in an inventor on a patent application for methods of AAV capsid development (PCT/US2019/068489). The author has no other competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1031-8824
  19. Leah C Byrne

    University of Pittsburgh, Pittsburgh, PA, United States
    For correspondence
    lctbyrne@gmail.com
    Competing interests
    Leah C Byrne, LB is named as an inventor on patent applications on AAV capsid variants and AAV screening methods (U.S. Patent Applications No. 16/315,032, 16/486,681, PCT/US2019/068489). LB has consulted on AAV-mediated gene therapy for Vedere Therapeutics, and is a named founder of Vegavect and Newsight Therapeutics. The author has no other competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3229-4993

Funding

Ford Foundation

  • Leah C Byrne

UPMC Immune Transplant and Therapy Center

  • Leah C Byrne

Van Sloun Fund for Canine Genetic Research

  • Gustavo D Aguirre

National Eye Institute (F32EY023891)

  • Leah C Byrne

National Eye Institute (R24EY-022012)

  • David V Schaffer
  • John G Flannery
  • William A Beltran

National Eye Institute (R01EY017549)

  • Gustavo D Aguirre
  • William A Beltran

National Eye Institute (P30EY001583)

  • Gustavo D Aguirre
  • William A Beltran

National Institute of Mental Health (UG3MH120094)

  • Andreas R Pfenning
  • William A Beltran
  • Leah C Byrne

National Institute of Mental Health (DP2MH113095)

  • William R Stauffer

Research to Prevent Blindness

  • Leah C Byrne

Foundation Fighting Blindness

  • Leah C Byrne

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All procedures were performed in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, and for canine studies with approval by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC # 803813), and for the NHP and mouse studies with approval from the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC #18042326).

Reviewing Editor

  1. Brandon K Harvey, NIDA/NIH, United States

Publication history

  1. Preprint posted: October 2, 2020 (view preprint)
  2. Received: October 20, 2020
  3. Accepted: October 11, 2021
  4. Accepted Manuscript published: October 19, 2021 (version 1)
  5. Version of Record published: November 24, 2021 (version 2)

Copyright

© 2021, Öztürk et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

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    Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules (MTs) affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that MT stability in the β-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable MTs. Consistently, MT hyper-stabilization prevents, and MT depolymerization promotes the capacity of single β-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that MT depolymerization activates otherwise dormant β-cells via initiation of secretion clusters (hot spots). MT depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without MTs, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca2+ influx was not affected by MT depolymerization yet required for secretion under these conditions, indicating that MT-dependent regulation of secretion hot spots acts in parallel with Ca2+ signaling. Our findings uncover a novel MT function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional β-cell heterogeneity.

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    Richard K Nakamura et al.
    Research Article Updated

    Background:

    Blinding reviewers to applicant identity has been proposed to reduce bias in peer review.

    Methods:

    This experimental test used 1200 NIH grant applications, 400 from Black investigators, 400 matched applications from White investigators, and 400 randomly selected applications from White investigators. Applications were reviewed by mail in standard and redacted formats.

    Results:

    Redaction reduced, but did not eliminate, reviewers’ ability to correctly guess features of identity. The primary, preregistered analysis hypothesized a differential effect of redaction according to investigator race in the matched applications. A set of secondary analyses (not preregistered) used the randomly selected applications from White scientists and tested the same interaction. Both analyses revealed similar effects: Standard format applications from White investigators scored better than those from Black investigators. Redaction cut the size of the difference by about half (e.g. from a Cohen’s d of 0.20–0.10 in matched applications); redaction caused applications from White scientists to score worse but had no effect on scores for Black applications.

    Conclusions:

    Grant-writing considerations and halo effects are discussed as competing explanations for this pattern. The findings support further evaluation of peer review models that diminish the influence of applicant identity.

    Funding:

    Funding was provided by the NIH.