scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
- University of Pittsburgh, United States
- Carnegie Mellon University, United States
- University of California, Berkeley, United States
- University of Pennsylvania, United States
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.
Article and author information
Author details
William R Stauffer
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).
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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scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
eLife 10:e64175.
https://doi.org/10.7554/eLife.64175
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Background: Several fields have described low reproducibility of scientific research and poor accessibility in research reporting practices. Although previous reports have investigated accessible reporting practices that lead to reproducible research in other fields, to date, no study has explored the extent of accessible and reproducible research practices in cardiovascular science literature.
Methods: To study accessibility and reproducibility in cardiovascular research reporting, we screened 639 randomly selected articles published in 2019 in three top cardiovascular science publications: Circulation, the European Heart Journal, and the Journal of the American College of Cardiology (JACC). Of those 639 articles, 393 were empirical research articles. We screened each paper for accessible and reproducible research practices using a set of accessibility criteria including protocol, materials, data, and analysis script availability, as well as accessibility of the publication itself. We also quantified the consistency of open research practices within and across cardiovascular study types and journal formats.
Results: We identified that fewer than 2% of cardiovascular research publications provide sufficient resources (materials, methods, data, and analysis scripts) to fully reproduce their studies. Of the 639 articles screened, 393 were empirical research studies for which reproducibility could be assessed using our protocol, as opposed to commentaries or reviews. After calculating an accessibility score as a measure of the extent to which an article makes its resources available, we also showed that the level of accessibility varies across study types with a score of 0.08 for Case Studies or Case Series and 0.39 for Clinical Trials (p = 5.500E-5) and across journals (0.19 through 0.34, p = 1.230E-2). We further showed that there are significant differences in which study types share which resources.
Conclusion: Although the degree to which reproducible reporting practices are present in publications varies significantly across journals and study types, current cardiovascular science reports frequently do not provide sufficient materials, protocols, data, or analysis information to reproduce a study. In the future, having higher standards of accessibility mandated by either journals or funding bodies will help increase the reproducibility of cardiovascular research.
Funding: Authors Gabriel Heckerman, Arely Campos-Melendez, and Chisomaga Ekwueme were supported by an NIH R25 grant from the National Heart, Lung and Blood Institute (R25HL147666). Eileen Tzng was supported by an AHA Institutional Training Award fellowship (18UFEL33960207).
