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

eLife

Michael Kleyman¹  Andreas R Pfenning¹  William R Stauffer²  Jing He²  José-Alain Sahel³  Sara Jabalameli³  Molly E Johnson³  Bilge E Öztürk³  Serhan Turunç³  Leah C Byrne⁴  Zhouhuan Xi⁵  William A Beltran⁶  Valérie L Dufour⁶  Simone Iwabe⁶  Gustavo D Aguirre⁶  Luis Felipe L Pompeo Marinho⁶  Meike Visel⁷  David V Schaffer⁸  John G Flannery⁹ 

[1] Computational Biology, School of Computer Science, Carnegie Mellon University, Pittsburgh, United States;Department of Neurobiology, University of Pittsburgh, Pittsburgh, United States;Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States;Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States;Department of Neurobiology, University of Pittsburgh, Pittsburgh, United States;Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States;Department of Bioengineering, University of Pittsburgh, Pittsburgh, United States;Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States;Eye Center of Xiangya Hospital, Hunan Key Laboratory of Ophthalmology, Central South University, Changsha, China;Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States;Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States;Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States;Chemical Engineering, University of California, Berkeley, Berkeley, United States;Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States;Vision Science, Herbert Wertheim School of Optometry, University of California Berkeley, Berkeley, United States;

关键词: macaca fascicularis;    callithrix jacchus;    scRNA-Seq;    retinal degeneration;    adeno-associated virus;    Rhesus macaque;    Other;   

DOI  :  10.7554/eLife.64175

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY