Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal

Genetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vec...

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Main Authors: Xin Li, Lohra Mickelle Miller, Matthew Chrzanowski, Jiahe Tian, Martin F. Jarrold, Roland W. Herzog, Weidong Xiao, Benjamin Draper, Junping Zhang
Format: Article
Language:English
Published: Frontiers Media S.A. 2023-12-01
Series:Frontiers in Bioengineering and Biotechnology
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fbioe.2023.1327433/full
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author Xin Li
Lohra Mickelle Miller
Matthew Chrzanowski
Jiahe Tian
Martin F. Jarrold
Roland W. Herzog
Weidong Xiao
Benjamin Draper
Junping Zhang
author_facet Xin Li
Lohra Mickelle Miller
Matthew Chrzanowski
Jiahe Tian
Martin F. Jarrold
Roland W. Herzog
Weidong Xiao
Benjamin Draper
Junping Zhang
author_sort Xin Li
collection DOAJ
description Genetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vectors with four-genome lengths were produced using both adherent and suspension HEK293 cells to understand the 5′ITR termination. AAV8 vectors were produced from the human FVIII plasmid for a full-length cargo of 4,707 nucleotides with specific truncations, creating smaller genomes. Conventionally, rAAV is characterized by differentiating empty capsids from full capsids, but for this work, that description is incomplete. The small genomes in this study were characterized by charge detection-mass spectrometry (CD-MS). Using CD-MS, packaged genomes in the range conventionally attributed to partials were resolved and quantified. In addition, alkaline gels and qPCR were used to assess the identity of the packaged genomes. Together, these results showed a propensity for unit-length genomes to be encapsidated. Packaged genomes occurred as replication intermediates emanating from the 5′ITR, indicating that HEK293 cells prefer unit-length genomes as opposed to the 5′ITR termination and heterogeneous DNA packaging observed previously from Sf9 cell systems. As both manufacturing processes are used and continually assessed to produce clinical material, such an understanding will benefit rAAV design for basic research and gene therapy.
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spelling doaj.art-b644a5a27ba2402b86be15f164c43ac02023-12-20T07:56:40ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-12-011110.3389/fbioe.2023.13274331327433Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signalXin Li0Lohra Mickelle Miller1Matthew Chrzanowski2Jiahe Tian3Martin F. Jarrold4Roland W. Herzog5Weidong Xiao6Benjamin Draper7Junping Zhang8Herman B Wells Center for Pediatric Research, Indiana University IUSM, Indianapolis, IN, United StatesChemistry Department, Indiana University, Bloomington, IN, United StatesNikegen, Wynnewood, PA, United StatesDepartment of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United StatesChemistry Department, Indiana University, Bloomington, IN, United StatesHerman B Wells Center for Pediatric Research, Indiana University IUSM, Indianapolis, IN, United StatesHerman B Wells Center for Pediatric Research, Indiana University IUSM, Indianapolis, IN, United StatesMegadalton Solutions Inc, Bloomington, IN, United StatesHerman B Wells Center for Pediatric Research, Indiana University IUSM, Indianapolis, IN, United StatesGenetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vectors with four-genome lengths were produced using both adherent and suspension HEK293 cells to understand the 5′ITR termination. AAV8 vectors were produced from the human FVIII plasmid for a full-length cargo of 4,707 nucleotides with specific truncations, creating smaller genomes. Conventionally, rAAV is characterized by differentiating empty capsids from full capsids, but for this work, that description is incomplete. The small genomes in this study were characterized by charge detection-mass spectrometry (CD-MS). Using CD-MS, packaged genomes in the range conventionally attributed to partials were resolved and quantified. In addition, alkaline gels and qPCR were used to assess the identity of the packaged genomes. Together, these results showed a propensity for unit-length genomes to be encapsidated. Packaged genomes occurred as replication intermediates emanating from the 5′ITR, indicating that HEK293 cells prefer unit-length genomes as opposed to the 5′ITR termination and heterogeneous DNA packaging observed previously from Sf9 cell systems. As both manufacturing processes are used and continually assessed to produce clinical material, such an understanding will benefit rAAV design for basic research and gene therapy.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1327433/fulladeno-associated virusinvert terminal repeatsmall genomepackaging signalreplicative intermediates
spellingShingle Xin Li
Lohra Mickelle Miller
Matthew Chrzanowski
Jiahe Tian
Martin F. Jarrold
Roland W. Herzog
Weidong Xiao
Benjamin Draper
Junping Zhang
Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal
Frontiers in Bioengineering and Biotechnology
adeno-associated virus
invert terminal repeat
small genome
packaging signal
replicative intermediates
title Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal
title_full Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal
title_fullStr Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal
title_full_unstemmed Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal
title_short Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal
title_sort quantitative analysis of preferential utilization of aav itr as the packaging terminal signal
topic adeno-associated virus
invert terminal repeat
small genome
packaging signal
replicative intermediates
url https://www.frontiersin.org/articles/10.3389/fbioe.2023.1327433/full
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