TY - JOUR
T1 - High-Throughput in Vitro, Ex Vivo, and in Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction
AU - Westhaus, Adrian
AU - Cabanes-Creus, Marti
AU - Rybicki, Arkadiusz
AU - Baltazar, Grober
AU - Navarro, Renina Gale
AU - Zhu, Erhua
AU - Drouyer, Matthieu
AU - Knight, Maddison
AU - Albu, Razvan F.
AU - Ng, Boaz H.
AU - Kalajdzic, Predrag
AU - Kwiatek, Magdalena
AU - Hsu, Kenneth
AU - Santilli, Giorgia
AU - Gold, Wendy
AU - Kramer, Belinda
AU - Gonzalez-Cordero, Anai
AU - Thrasher, Adrian J.
AU - Alexander, Ian E.
AU - Lisowski, Leszek
N1 - Publisher Copyright:
© Adrian Westhaus et al., 2020; Published by Mary Ann Liebert, Inc.
PY - 2020/5
Y1 - 2020/5
N2 - Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.
AB - Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.
KW - AAV
KW - Adeno-associated virus
KW - Gene therapy
KW - IPSC
KW - Next-generation sequencing
KW - Viral vectors
UR - http://www.scopus.com/inward/record.url?scp=85084721725&partnerID=8YFLogxK
U2 - 10.1089/hum.2019.264
DO - 10.1089/hum.2019.264
M3 - Article
C2 - 32000541
AN - SCOPUS:85084721725
SN - 1043-0342
VL - 31
SP - 575
EP - 589
JO - Human Gene Therapy
JF - Human Gene Therapy
IS - 9-10
ER -