TY - JOUR
T1 - AAV-p40 Bioengineering Platform for Variant Selection Based on Transgene Expression
AU - Westhaus, Adrian
AU - Cabanes-Creus, Marti
AU - Jonker, Timo
AU - Sallard, Erwan
AU - Navarro, Renina Gale
AU - Zhu, Erhua
AU - Baltazar Torres, Grober
AU - Lee, Scott
AU - Wilmott, Patrick
AU - Gonzalez-Cordero, Anai
AU - Santilli, Giorgia
AU - Thrasher, Adrian J.
AU - Alexander, Ian E.
AU - Lisowski, Leszek
N1 - Funding Information: This work was supported by project grants from the Australian National Health and Medical Research Council (NHMRC) (APP1108311, APP1156431, and APP1161583) to Leszek Lisowski and Ian E. Alexander, and the Paediatric Precision Medicine Program (PPM1 K5116/RD274) to Leszek Lisowski, as well as funding from LogicBio Therapeutics. Anai Gonzalez-Cordero is supported by Luminesce Alliance (PPM1 K5116/RD274)—Innovation for Children’s Health for its contribution and support. Luminesce Alliance—Innovation for Children’s Health, is a not-for-profit cooperative joint venture between the Sydney Children’s Hospitals Network, the Children’s Medical Research Institute, and the Children’s Cancer Institute. It has been established with the support of the NSW Government to coordinate and integrate pediatric research. Luminesce Alliance is also affiliated to the University of Sydney and the University of New South Wales Sydney. Publisher Copyright: © 2022, by Mary Ann Liebert, Inc., publishers.
PY - 2022/6/1
Y1 - 2022/6/1
N2 - The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.
AB - The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.
KW - AAV
KW - Adeno-associated viral vectors
KW - Capsid bioengineering
KW - Directed evolution
KW - Novel vectors
KW - Vector development
UR - http://www.scopus.com/inward/record.url?scp=85131902560&partnerID=8YFLogxK
U2 - https://doi.org/10.1089/hum.2021.278
DO - https://doi.org/10.1089/hum.2021.278
M3 - Article
C2 - 35297686
SN - 1043-0342
VL - 33
SP - 664
EP - 682
JO - Human gene therapy
JF - Human gene therapy
IS - 11-12
ER -