In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9.
Journal
Laboratory investigation; a journal of technical methods and pathology
ISSN: 1530-0307
Titre abrégé: Lab Invest
Pays: United States
ID NLM: 0376617
Informations de publication
Date de publication:
12 2021
12 2021
Historique:
received:
22
10
2020
accepted:
02
08
2021
revised:
30
07
2021
pubmed:
22
8
2021
medline:
30
12
2021
entrez:
21
8
2021
Statut:
ppublish
Résumé
Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.
Identifiants
pubmed: 34417549
doi: 10.1038/s41374-021-00654-x
pii: S0023-6837(22)00390-7
doi:
Substances chimiques
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1627-1636Informations de copyright
© 2021. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.
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