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

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 anima...

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Autores principales:	Bilge E Öztürk, Molly E Johnson, Michael Kleyman, Serhan Turunç, Jing He, Sara Jabalameli, Zhouhuan Xi, Meike Visel, Valérie L Dufour, Simone Iwabe, Luis Felipe L Pompeo Marinho, Gustavo D Aguirre, José-Alain Sahel, David V Schaffer, Andreas R Pfenning, John G Flannery, William A Beltran, William R Stauffer, Leah C Byrne
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	macaca fascicularis callithrix jacchus scRNA-Seq retinal degeneration adeno-associated virus Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/2f839da7c7b643a2acc12975971ae2e2
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spelling	oai:doaj.org-article:2f839da7c7b643a2acc12975971ae2e22021-11-24T16:34:29ZscAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution10.7554/eLife.641752050-084Xe64175https://doaj.org/article/2f839da7c7b643a2acc12975971ae2e22021-10-01T00:00:00Zhttps://elifesciences.org/articles/64175https://doaj.org/toc/2050-084XBackground: 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.Bilge E ÖztürkMolly E JohnsonMichael KleymanSerhan TurunçJing HeSara JabalameliZhouhuan XiMeike ViselValérie L DufourSimone IwabeLuis Felipe L Pompeo MarinhoGustavo D AguirreJosé-Alain SahelDavid V SchafferAndreas R PfenningJohn G FlanneryWilliam A BeltranWilliam R StaufferLeah C ByrneeLife Sciences Publications Ltdarticlemacaca fasciculariscallithrix jacchusscRNA-Seqretinal degenerationadeno-associated virusMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	macaca fascicularis callithrix jacchus scRNA-Seq retinal degeneration adeno-associated virus Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	macaca fascicularis callithrix jacchus scRNA-Seq retinal degeneration adeno-associated virus Medicine R Science Q Biology (General) QH301-705.5 Bilge E Öztürk Molly E Johnson Michael Kleyman Serhan Turunç Jing He Sara Jabalameli Zhouhuan Xi Meike Visel Valérie L Dufour Simone Iwabe Luis Felipe L Pompeo Marinho Gustavo D Aguirre José-Alain Sahel David V Schaffer Andreas R Pfenning John G Flannery William A Beltran William R Stauffer Leah C Byrne scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
description	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.
format	article
author	Bilge E Öztürk Molly E Johnson Michael Kleyman Serhan Turunç Jing He Sara Jabalameli Zhouhuan Xi Meike Visel Valérie L Dufour Simone Iwabe Luis Felipe L Pompeo Marinho Gustavo D Aguirre José-Alain Sahel David V Schaffer Andreas R Pfenning John G Flannery William A Beltran William R Stauffer Leah C Byrne
author_facet	Bilge E Öztürk Molly E Johnson Michael Kleyman Serhan Turunç Jing He Sara Jabalameli Zhouhuan Xi Meike Visel Valérie L Dufour Simone Iwabe Luis Felipe L Pompeo Marinho Gustavo D Aguirre José-Alain Sahel David V Schaffer Andreas R Pfenning John G Flannery William A Beltran William R Stauffer Leah C Byrne
author_sort	Bilge E Öztürk
title	scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
title_short	scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
title_full	scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
title_fullStr	scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
title_full_unstemmed	scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
title_sort	scaavengr, a transcriptome-based pipeline for quantitative ranking of engineered aavs with single-cell resolution
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/2f839da7c7b643a2acc12975971ae2e2
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scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution

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